GREEN Citric Acid CIR EXPERT PANEL MEETING SEPTEMBER 26-27, 2011

Transcription

1 GREEN Citric Acid CIR EXPERT PANEL MEETING SEPTEMBER 26-27, 2011

2 Memorandum To: CIR Expert Panel Members and Liaisons From: Monice M. Fiume MMF Senior Scientific Analyst/Writer Date: September 8, 2011 Subject: Citric Acid and Its Inorganic Salts and Alkyl and Glycol Esters as Used in Cosmetics Draft Safety Assessment Included is the draft report on Citric Acid and Its Inorganic Salts and Alkyl and Glycol Esters as Used in Cosmetics. There are a total of 46 ingredients included in this draft report. This is the first time the Panel is seeing this document. The Scientific Literature Review was issued on June 9, As determined by FDA, citric acid, calcium citrate, ferric citrate, manganese citrate, potassium citrate, sodium citrate, diammonium citrate, and triethyl citrate are generally recognized as safe (GRAS) direct food additives. Based on the FDA GRAS determination, we have taken the approach that the oral safety of these 8 ingredients has been substantiated and we have not included the voluminous literature addressing ingestion of these ingredients. Rather, this report will focus on the dermal application of these 8 ingredients. Arguably, citric acid is an α-hydroxy acid (AHA), but because it is a tricarboxylic acid, it was not included when CIR conducted its massive review of the safety of AHAs (i.e., glycolic and lactic acid, and their various salts and simple esters) and published its findings back in For your information, since the AHA report was written, the National Toxicology Program conducted a photocarcinogenesis study of glycolic acid. A cosmetic formulation containing 4% or 10% glycolic acid (ph 3.5) was not photocarcinogenic in SKH-1 mice. The glycolic and lactic acid report is available online at Also referenced in the Introduction is the US Food and Drug Administration (FDA) recommended Guidance for Industry: Labeling for Topically Applied Cosmetic Products Containing Alpha Hydroxy Acids as Ingredients. The link to this document is This information concerning the glycolic and lactic acid report is only being provided as background because citric acid can be considered an AHA; data on glycolic and lactic acid are not included in the safety assessment on citric acid.

3 CCR Citric Acid page The following unpublished data are included in the data tab: 1. Unpublished Information on Citric Acid and Potassium Citrate. Memo dated Nov, 19, 2010 a. Archer Daniels Midland Co Citric Acid Anhydrous, USP/FCC. b. Archer Daniels Midland Co Material safety data sheet: Citric Acid Anhydrous. (not referenced in the safety assessment) c. Archer Daniels Midland Co ph of Citric Acid-Sodium Citrate solutions. d. Archer Daniels Midland Co Solubility of Citric Acid in water. e. Archer Daniels Midland Co Potassium Citrate, USP/FCC. f. Archer Daniels Midland Co Material safety data sheet: Potassium Citrate granular. (not referenced in the safety assessment) g. Archer Daniels Midland Co Process stages: Potassium Citrate, USP/FCC. 2. Consumer Product Testing Co Repeated insult patch test of C-SAT (Tristearyl Citrate). Experiment Reference Number: C Memo dated Dec 1, Information on Triisostearyl Citrate and Trioctyldodecyl Citrate. Memo dated Jan. 4, Lubrizol G-66 Guerbet ester toxicology studies (INCI: Trioctyldodecyl Citrate). Memorandum Jan. 4, Safety Studies of Triisostearyl Citrate. Memo Jan 4, a. Consumer Product Testing Co The MatTek Corporation EpiOcular tissue model in vitro toxicity testing system: Triisostearyl Citrate. Experiment Reference No.: V b. Product Investigations, Inc Determination of the irritating and sensitizing propensities of EX-1028 (Triisostearyl Citrate) on human skin. Report: P11 No c. NOTOX B.V Evaluation of the mutagenic activity of EX-1028 (Triisostearyl Citrate) in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). NOTOX Project Updated Concentration of Use by FDA Product Category: Citric Acid and its Salts and Esters. Memo dated Jan 28, Concentration of Use Additional Citrate Ingredients. Memo dated March 15, Unpublished Information on Laureth-7 Citrate. Memo dated Feb. 22, a. Cognis Data profile Plantapon LC 7 (INCI name: Laureth-7 Citrate) (C-SAT and BHL-Fo H are identical) b. RCC Salmonella typhimurium reverse mutation assay of C-SAT (Laureth-7 Citrate). RCC - Study Number c. Consumer Product Testing Co Repeated Insult Patch Test of C-SAT (Laureth-7 Citrate) (tested as a 10% dilution). Experiment Reference Number C d. Henkel KgaA In-vitro-test: NET-CAM [HCI] Reaction time method of BHL- Fo H (H is Laureth-7 Citrate) (tested at 5% active substance [AS]). Report No. R Clinical Data on Products Containing Citric Acid, Triethyl Citrate or Triisostearyl Citrate. Memo dated June 21, 2011.Clinical Research Laboratories, Inc a. Repeated insult patch test of a cuticle cream containing 4% Citric Acid. CRL Study Number: CRL b. Consumer Product Testing Co Repeated insult patch test of a powder blush containing 4.8% Triethyl Citrate. Experiment Reference Number: C c. Consumer Product Testing Co Repeated insult patch test of a lip gloss containing 15.5% Triisostearyl Citrate. Experiment Reference Number: C

4 Citric Acid page Data submission received from RIFM. 11. Morflex. Citroflex. Citric Acid Esters. (Previously submitted unpublished data pulled from an existing report file.) 12. Fouda HG. November Safety assessment of citroflex plasticizers in vitro hydrolysis by serum, liver, and intestinal enzymes. (Previously submitted unpublished data pulled from an existing report file.) 13. Hill Top Research Repeated insult patch test, Test # A-71. (Previously submitted unpublished data pulled from an existing report file.) 14. Unilever Sensitization potential of Citroflex A2, Citroflex A4 and Citroflex 2 tested in guinea pigs. (Previously submitted unpublished data pulled from an existing report file.) If there are no additional data needs, the Panel should be prepared to formulate a tentative conclusion, with the rationale provided for the Discussion, and issue a Tentative Report for public comment. If the data are not sufficient for making a determination of safety, then an Insufficient Data Announcement should be issued, listing the additional data that are needed.

5 Distributed for Comment Only -- Do Not Quote or Cite CIR Panel Book Page 1

6 CCR Distributed for Comment Only -- Do Not Quote or Cite CITRIC ACID REPORT June 9, 2011: SLR Issued September 26-27, 2011: Draft Report The following unpublished data were submitted by the Council, following the announcement of the SLR, and are included in the draft report: 1. Unpublished Information on Citric Acid and Potassium Citrate. Memo dated Nov, 19, 2010 a. Archer Daniels Midland Co Citric Acid Anhydrous, USP/FCC. b. Archer Daniels Midland Co Material safety data sheet: Citric Acid Anhydrous. (not referenced in the safety assessment) c. Archer Daniels Midland Co ph of Citric Acid-Sodium Citrate solutions. d. Archer Daniels Midland Co Solubility of Citric Acid in water. e. Archer Daniels Midland Co Potassium Citrate, USP/FCC. f. Archer Daniels Midland Co Material safety data sheet: Potassium Citrate granular. (not referenced in the safety assessment) g. Archer Daniels Midland Co Process stages: Potassium Citrate, USP/FCC. 2. Consumer Product Testing Co Repeated insult patch test of C-SAT (Tristearyl Citrate). Experiment Reference Number: C Memo dated Dec 1, Information on Triisostearyl Citrate and Trioctyldodecyl Citrate. Memo dated Jan. 4, Lubrizol G-66 Guerbet ester toxicology studies (INCI: Trioctyldodecyl Citrate). Memorandum Jan. 4, Safety Studies of Triisostearyl Citrate. Memo Jan 4, a. Consumer Product Testing Co The MatTek Corporation EpiOcular tissue model in vitro toxicity testing system: Triisostearyl Citrate. Experiment Reference No.: V b. Product Investigations, Inc Determination of the irritating and sensitizing propensities of EX-1028 (Triisostearyl Citrate) on human skin. Report: P11 No c. NOTOX B.V Evaluation of the mutagenic activity of EX-1028 (Triisostearyl Citrate) in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). NOTOX Project Updated Concentration of Use by FDA Product Category: Citric Acid and its Salts and Esters. Memo dated Jan 28, Concentration of Use Additional Citrate Ingredients. Memo dated March 15, Unpublished Information on Laureth-7 Citrate. Memo dated Feb. 22, a. Cognis Data profile Plantapon LC 7 (INCI name: Laureth-7 Citrate) (C-SAT and BHL-Fo H are identical) b. RCC Salmonella typhimurium reverse mutation assay of C-SAT (Laureth-7 Citrate). RCC - Study Number c. Consumer Product Testing Co Repeated Insult Patch Test of C-SAT (Laureth- 7 Citrate) (tested as a 10% dilution). Experiment Reference Number C d. Henkel KgaA In-vitro-test: NET-CAM [HCI] Reaction time method of BHL-Fo H (H is Laureth-7 Citrate) (tested at 5% active substance [AS]). Report No. R Clinical Data on Products Containing Citric Acid, Triethyl Citrate or Triisostearyl Citrate. Memo dated June 21, 2011.Clinical Research Laboratories, Inc a. Repeated insult patch test of a cuticle cream containing 4% Citric Acid. CRL Study Number: CRL b. Consumer Product Testing Co Repeated insult patch test of a powder blush containing 4.8% Triethyl Citrate. Experiment Reference Number: C c. Consumer Product Testing Co Repeated insult patch test of a lip gloss containing 15.5% Triisostearyl Citrate. Experiment Reference Number: C Data submission received from RIFM. CIR Panel Book Page 2

7 The following unpublished data from the report on Acetyl Tributyl Citrate that had data applicable to this report: 1. Morflex. Citroflex. Citric Acid Esters. (Previously submitted unpublished data pulled from an existing report file.) 2. Fouda HG. November Safety assessment of citroflex plasticizers in vitro hydrolysis by serum, liver, and intestinal enzymes. (Previously submitted unpublished data pulled from an existing report file.) 3. Hill Top Research Repeated insult patch test, Test # A-71. (Previously submitted unpublished data pulled from an existing report file.) 4. Unilever Sensitization potential of Citroflex A2, Citroflex A4 and Citroflex 2 tested in guinea pigs. (Previously submitted unpublished data pulled from an existing report file.) CIR Panel Book Page 3

8 CITRIC ACID SEARCH STRATEGY Keep Me Posted Results are updated weekly SciFinder Search: March 17, 2011 Session began March 17, 2011 at 10:57 AM March 17, :13 AM Explore substances by ID: , , , , , , , , , , , , , , , , , , , , , , initiated Explore complete Explore results Answer set 1 created with 23 answers from REGISTRY Detailed display from Answer set 1 of Retrieve reference information in 23 substances of Answer set 1 Adverse Effect, including toxicity Answer set 2 created with 652 answers from CAPLUS 768 answers from MEDLINE Refine Answer set 2 by document type Book, Clinical Trial, Journal, Report, Review Answer set 3 created with 511 answers from CAPLUS 728 answers from MEDLINE Saved 1239 reference answers from Answer set 3 as 'Citric Acid Search 1' Saved 23 substance answers from Answer set 1 as 'Citric Acid 1 -substance detail' Retrieve reference information in 23 substances of Answer set 1 Biological Study, Uses Answer set 4 created with 82,582 answers from CAPLUS 7,001 answers from MEDLINE Refine Answer set 4 by document type CIR Panel Book Page 4

9 Book, Clinical Trial, Journal, Report, Review Answer set 5 created with 32,088 answers from CAPLUS 6,803 answers from MEDLINE Saved reference answers from Answer set 5 as 'Citric Acid 1 - biol study & use' Combine the current reference answer set with saved answer sets initiated: Citric Acid 1 - biol study & use OR Citric Acid Search 1 at March 17, :54 AM Answer set 6 created with 32,088 answers from CAPLUS 6,803 answers from MEDLINE Retrieve reference information in 23 substances of Answer set 1 Adverse Effect, including toxicity, Biological Study, Uses Answer set 7 created with 82,582 answers from CAPLUS 7,001 answers from MEDLINE Refine Answer set 7 by document type Book, Clinical Trial, Journal, Report, Review Answer set 8 created with 32,088 answers from CAPLUS 6,803 answers from MEDLINE CIR Panel Book Page 5 Saved reference answers from Answer set 8 as 'Citric Acid 1 - adverse effect, biol effect, use' Retrieve reference information in 21 substances of Answer set 1 Answer set 9 created with 25,706 answers from CAPLUS 738 answers from MEDLINE Retrieve reference information in 21 substances of Answer set 1 rse Effect, including toxicity er set 10 created with 23 answers from CAPLUS 9 answers from MEDLINE e Answer set 10 by document type

10 , Clinical Trial, Journal, Report, Review er set 11 created with nswers from CAPLUS swers from MEDLINE 1 reference answers from Answer set 11 as 'Citric Acid 1 - no Citric Acid - tox, refined' eve reference information in 2 substances of Answer set 1 Retrieve reference information in 2 substances of Answer set 1 Adverse Effect, including toxicity Answer set 12 created with 448 answers from CAPLUS 694 answers from MEDLINE Refine Answer set 12 by document type Book, Clinical Trial, Journal, Report, Review Answer set 13 created with 403 answers from CAPLUS 662 answers from MEDLINE Retrieve reference information in 2 substances of Answer set 1 CIR Panel Book Page 6 Answer set 14 created with History Session began April 8, 2011 at 10:17 AM April 8, :17 AM Explore substances by ID: PEG-5 Tricetyl Citrate initiated Explore complete Explore results No answers April 8, :18 AM Explore references by research topic: PEG-5 tricetyl citrate initiated, resulting in 2 candidates Explore complete Candidates Selected 1 reference was found containing all of the concepts "PEG", "5" and "tricetyl citrate". Explore results

11 Answer set 2 created with 1 answer from CAPLUS Detailed display from Answer set 2 of Inclusion compounds containing macromolecules Copyright 2011 American Chemical Society. All Rights Reserved. Copyright Sun Microsystems, Inc. All Rights Reserved. (Java runtime environment) Copyright 2011, Yahoo! Inc. All Rights Reserved. (YUI Base, Fonts, Reset CSS) Copyright , The Dojo Foundation. All Rights Reserved Copyright 2011, Exadel, Inc. All Rights Reserved. (JBoss RichFaces) Copyright 2002 InfoChem GmbH. All Rights Reserved. (InfoChem s reaction classification program CLASSIFY) CAplus SM : Copyright 2011 American Chemical Society. All Rights Reserved. (The U.K. patent material in this product/service is U.K. Crown copyright and is made available with permission. Copyright Crown Copyright. The French (FR) patent material in this product/service is made available from Institut National de la Propriete Industrielle (INPI).) CAS REGISTRY SM : Copyright 2011 American Chemical Society. All Rights Reserved. (Some records contain information from GenBank. See also: Benson D.A., Karsch-Mizarachi I., Lipman D.J., Ostel J., Rapp B.A., Wheeler D.L. Genbank. Nucl. Acids Res. 28(1):15-18 (2000). Property values tagged with IC are from the ZIC/VINITI data file provided by InfoChem.) CAS Registry is a service mark of the American Chemical Society. GenBank is a registered trademark of the U.S. Library of Medicine. CASREACT : Copyright 2011 American Chemical Society. All Rights Reserved. CASREACT contains reactions from CAS and from: ZIC/VINITI database ( ) provided by InfoChem; INPI data prior to 1986; Biotransformations database compiled under the direction of Professor Dr. Klaus Kieslich; organic reactions, portions copyright John Wiley & Sons, Ltd., John Wiley and Sons, Inc., Organic Reactions Inc., and Organic Synthesis Inc. Reproduced under license. All Rights Reserved. CHEMCATS : Copyright 2011 American Chemical Society. All Rights Reserved. Chemical supplier information is supplied on an "as is" basis. Full information regarding substance availability, price, etc., is provided when you request supplier information. CHEMLIST : Copyright 2011 American Chemical Society. All Rights Reserved. MEDLINE : is produced by the U.S. National Library of Medicine. MEDLINE is a registered trademark of the U.S. National Library of Medicine. Toxline Search (March 20, 2011) ((CITRIC AND ACID) OR OR ) AND (TOXICITY OR TOXIC OR REPRODUCTI* OR FETOTOXI* OR EMBRYOTOXI* OR TERATOGENIC* OR TERATOGEN) (SINCE 1990) ((CITRIC AND ACID) OR OR ) AND (SENSITISAT* OR SENSITIZAT* OR SENSITZER OR IRRITAT* OR IRRITANT) (SINCE 1990) - 72 ((CITRIC AND ACID) OR OR ) AND (CARCINOGENIC* OR CARCINOGENIC OR CANCER OR GENOTOXIC* OR MUTAGEN* OR CLASTOGEN* OR TUMOR) (SINCE 1990) ((CITRIC AND ACID) OR OR ) AND (METABOLISM OR METABOLITE OR ABSORP* OR ABSORB* OR DISTRIBUTION OR DISTRIBUTED OR EXCRETE* OR EXCRETION OR PHARMACOKINETIC) (SINCE 1990) ((CITRATE AND (ALUMINUM OR CALCIUM OR COPPER OR (DISODIUM AND CUPRIC) OR FERRIC OR MAGNESIUM OR MANGANESE OR *SODIUM OR POTASSIUM OR ZINC OR DIAMMONIUM)) OR OR OR OR CIR Panel Book Page 7

12 11-5 OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR ) AND (TOXICITY OR TOXIC OR REPRODUCTI* OR FETOTOXI* OR EMBRYOTOXI* OR TERATOGENIC* OR TERATOGEN) ((CITRATE AND (ALUMINUM OR CALCIUM OR COPPER OR (DISODIUM AND CUPRIC) OR FERRIC OR MAGNESIUM OR MANGANESE OR *SODIUM OR POTASSIUM OR ZINC OR DIAMMONIUM)) OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR ) AND (SENSITISAT* OR SENSITIZAT* OR SENSITZER OR IRRITAT* OR IRRITANT) 37 ((CITRATE AND (ALUMINUM OR CALCIUM OR COPPER OR (DISODIUM AND CUPRIC) OR FERRIC OR MAGNESIUM OR MANGANESE OR *SODIUM OR POTASSIUM OR ZINC OR DIAMMONIUM)) OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR ) AND (CARCINOGENIC* OR CARCINOGENIC OR CANCER OR GENOTOXIC* OR MUTAGEN* OR CLASTOGEN* OR TUMOR) ((CITRATE AND (ALUMINUM OR CALCIUM OR COPPER OR (DISODIUM AND CUPRIC) OR FERRIC OR MAGNESIUM OR MANGANESE OR *SODIUM OR POTASSIUM OR ZINC OR DIAMMONIUM)) OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR ) AND (METABOLISM OR METABOLITE OR ABSORP* OR ABSORB* OR DISTRIBUTION OR DISTRIBUTED OR EXCRETE* OR EXCRETION OR PHARMACOKINETIC) ((STEARYL OR ISOPROPYL OR ISODECYL OR DILAURYL OR DISTEARYL OR TRIETHYL OR TRIBUTYL OR TRICAPRYLYL OR TRILAURYL OR ALKYL OR TRISTEARYL OR TRIISOPROPYL OR TRIETHYLHEXYL OR TRIHEXYLDECYL OR TRIISOCETYL OR TRIISOSTEARYL OR TRIOCTYLDODECYL OR TRIOLEYL OR ETHYL) AND CITRATE) OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR (((PROPYLENE AND GLYCOL) OR LAURETH OR DILAURETH OR ((PEG OR (POLYETHYLENE AND GLYCOL)) AND (TRICAPRYL OR TRILAURYL OR TRILAURETH OR TRIMYRISTYL OR TRISTEARYL OR (TRIPROPYLENE AND GLYCOL))) AND CITRATE) OR OR OR OR ) AND (TOXICITY OR TOXIC OR REPRODUCTI* OR FETOTOXI* OR EMBRYOTOXI* OR TERATOGENIC* OR TERATOGEN) CIR Panel Book Page 8

13 (((PROPYLENE AND GLYCOL) OR LAURETH OR DILAURETH OR ((PEG OR (POLYETHYLENE AND GLYCOL)) AND (TRICAPRYL OR TRILAURYL OR TRILAURETH OR TRIMYRISTYL OR TRISTEARYL OR (TRIPROPYLENE AND GLYCOL))) AND CITRATE) OR OR OR OR ) AND (SENSITISAT* OR SENSITIZAT* OR SENSITZER OR IRRITAT* OR IRRITANT) (((PROPYLENE AND GLYCOL) OR LAURETH OR DILAURETH OR ((PEG OR (POLYETHYLENE AND GLYCOL)) AND (TRICAPRYL OR TRILAURYL OR TRILAURETH OR TRIMYRISTYL OR TRISTEARYL OR (TRIPROPYLENE AND GLYCOL))) AND CITRATE) OR OR OR OR ) AND (CARCINOGENIC* OR CARCINOGENIC OR CANCER OR GENOTOXIC* OR MUTAGEN* OR CLASTOGEN* OR TUMOR) (((PROPYLENE AND GLYCOL) OR LAURETH OR DILAURETH OR ((PEG OR (POLYETHYLENE AND GLYCOL)) AND (TRICAPRYL OR TRILAURYL OR TRILAURETH OR TRIMYRISTYL OR TRICETYL OR TRISTEARYL OR (TRIPROPYLENE AND GLYCOL))) AND CITRATE) OR OR OR OR ) AND (METABOLISM OR METABOLITE OR ABSORP* OR ABSORB* OR DISTRIBUTION OR DISTRIBUTED OR EXCRETE* OR EXCRETION OR PHARMACOKINETIC) CIR Panel Book Page 9

14 GROUPED ABSTRACTS Citric Acid (since 1990) salts esters derm effects; includes alkyl esters (all): 598 references Citric Acid - tox and carc (sine 1990) 912 references Salts and esters tox and carc: 2864 references Citric Acid ADME: 1411 references Salts and esters ADME: 3438 references CIR Panel Book Page 10

15 Citric Acid Search Info # uses conc data FDA ChemPortal NTIS Registry Merck EU SCCS SIDS IARC NTP EAFUS OTC HPV IUCLID data date searched Citric Acid X X X X X - - X X X X - Aluminum Citrate X - X X Calcium Citrate NR X X X X X - - X Copper Citrate NR X X X - Disodium Cupric Citrate NR X - X Ferric Citrate X X X X X Magnesium Citrate X X X X X - - X Manganese Citrate NR X - X X X X - X Monosodium Citrate X - X X - X X - Potassium Citrate X X X X X Sodium Citrate X X X X X Zinc Citrate X X X Diammonium Citrate X X X X X Stearyl Citrate X - X X Isopropyl Citrate X - X X X Isodecyl Citrate X - X Dilauryl Citrate X - X Distearyl Citrate NR X - X X Triethyl Citrate X X X X X Tributyl Citrate X X X X Tricaprylyl Citrate X - X Trilauryl Citrate NR X - X Tri-C12-13 Alkyl Citrate X - X Tri-C14-15 Alkyl Citrate X - X Tristearyl Citrate NR X - X X Triisopropyl Citrate NR X - X Triethylhexyl Citrate X - X Trihexyldecyl Citrate NR - - X X Triisocetyl Citrate X - X Triisostearyl Citrate X - X Trioctyldodecyl Citrate X - X AHA opinion paper WHO/ JECFA CIR Panel Book Page 11

16 # uses conc data FDA ChemPortal NTIS Registry Merck EU SCCS SIDS IARC NTP EAFUS OTC HPV IUCLID data date searched Trioleyl Citrate NR X - X Ethyl Citrates NR X - X Propylene Glycol Citrate NR X - X Laureth-6 Citrate NR X - X Laureth-7 Citrate X - X Disodium Laureth-7 Citrate NR - - X Dilaureth-7 Citrate NR - - X Sodium Dilaureth-7 Citrate NR X - X PEG-5 Tricapryl Citrate NR - - X PEG-5 Trilauryl Citrate NR - - X Trilaureth-9 Citrate - - X PEG-5 Trimyristyl Citrate NR - - X PEG-5 Tricetyl Citrate NR - - x PEG-5 Tristearyl Citrate NR - - X Tripropylene Glycol Citrate - - X WHO/ JECFA CIR Panel Book Page 12

17 Citric Acid and Its Inorganic Salts and Alkyl and Glycol Esters Data Profile* Sept 2011 Writer, Monice Fiume Reported Use Impurities/ Method of Mfg Toxicokinetics Acute Tox - Dermal Acute Tox - Oral Repeated Dose - Oral Repro/Dev Tox Genotoxicity Dermal Irritation Non-Human Dermal Sens Non-Human Dermal Irritation- Human Dermal Sens Human Ocular Irritation Citric Acid X X X X X X X X X Aluminum Citrate X X X X X Calcium Citrate Copper Citrate Diammonium Citrate X Dilaureth 7 Citrate Dilauryl Citrate X Disodium Cupric Citrate Disodium Laureth 7 Citrate Distearyl Citrate X Ethyl Citrates X Ferric Citrate X X Isodecyl Citrate X Isopropyl Citrate X X X Laureth 6 Citrate Laureth 7 Citrate X X X X Magnesium Citrate X Manganese Citrate Monosodium Citrate X X PEG 5 Tricapryl Citrate PEG 5 Tricetyl Citrate PEG 5 Trilauryl Citrate PEG 5 Trimyristyl Citrate PEG 5 Tristearyl Citrate Potassium Citrate X X Propylene Glycol Citrate Sodium Citrate X X X Sodium Dilaureth 7 Citrate Stearyl Citrate X X X X Tributyl Citrate X X X X X Tri C Alkyl Citrate X Tri C14 15 Alkyl Citrate X Tricaprylyl Citrate X Triethyl Citrate X X X X X X X X Triethylhexyl Citrate X Trihexyldecyl Citrate Triisocetyl Citrate X Triisopropyl Citrate Triisostearyl Citrate X X X X X Trilaureth 9 Citrate Trilauryl Citrate Trioctyldodecyl Citrate X X X X X X Trioleyl Citrate Tripropylene Glycol Citrate X Tristearyl Citrate X X Zinc Citrate X X indicates that data were available in the category for that ingredient 1 CIR Panel Book Page 13

18 Report

19 Draft Report Citric Acid and Its Inorganic Salts and Alkyl and Glycol Esters as Used in Cosmetics September 26, 2011 All interested persons are provided 60 days from the above date to comment on this Draft Report and to identify additional published data that should be included or provide unpublished data which can be made public and included. Information may be submitted without identifying the source or the trade name of the cosmetic product containing the ingredient. All unpublished data submitted to CIR will be discussed in open meetings, will be available at the CIR office for review by any interested party and may be cited in a peer-reviewed scientific journal. Please submit data, comments, or requests to the CIR Director, Dr. F. Alan Andersen. The 2011 Cosmetic Ingredient Review Expert Panel members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is F. Alan Andersen, Ph.D. This report was prepared by Monice Fiume, Senior Scientific Analyst/Writer. Cosmetic Ingredient Review th Street, NW, Suite 412 " Washington, DC " ph " fax " cirinfo@cir-safety.org CIR Panel Book Page 14

20 TABLE OF CONTENTS Introduction... 1 Chemistry... 2 Definition, Structure, and Properties... 2 Production... 3 Use... 3 Cosmetic... 3 Non-Cosmetic... 4 Toxicokinetics... 4 In Vitro... 4 Oral... 5 Effect on Transdermal Absorption... 5 Toxicological studies... 5 Single Dose (Acute) Toxicity... 5 Repeated Dose Toxicity... 6 Oral... 6 Intraperitoneal... 7 Reproductive and Developmental Toxicity... 7 Oral... 7 In-Vitro... 7 Spermicidal Effects... 8 Genotoxicity... 8 Anti-Mutagenic Effects... 8 Carcinogenicity... 8 Irritation and Sensitization... 8 Skin Irritation/Sensitization... 9 Ocular Irritation... 9 Case Reports... 9 Miscellaneous studies... 9 Effects in Skin... 9 Lipid Bilayer Permeation Cough Reflex Anesthetic Effects Summary Discussion Conclusion Tables Table 1. Definitions and structures of citric acid, salt and esters Table 2. Chemical and physical properties Table 3. Impurities and Composition Table 4. Ingredient-Specific Methods of Manufacture Table 5. Reported functions of citric acid and its salts and esters Table 6a. Frequency and concentration of use according to duration and type of exposure Table 6b. Ingredients not reported to be used Table 7. Examples of non-cosmetic uses Table 8. Acute toxicity studies Table 9. Genotoxicity studies Table 10. Dermal irritation and sensitization Table 11. Ocular irritation studies References ii CIR Panel Book Page 15

21 INTRODUCTION This safety assessment reviews the safety of citric acid, an α (or β)-hydroxytricarboxylic acid, as used in cosmetics. The following 12 inorganic salts, 20 alkyl esters, and 13 glycol esters of citric acid also are included in this safety assessment, for a total of 46 ingredients: Inorganic Salts Aluminum Citrate Calcium Citrate Copper Citrate Diammonium Citrate Disodium Cupric Citrate Ferric Citrate Magnesium Citrate Manganese Citrate Monosodium Citrate Potassium Citrate Sodium Citrate Zinc Citrate Alkyl Mono-, Di-, and Triesters Isodecyl Citrate Isopropyl Citrate Stearyl Citrate Dilauryl Citrate Distearyl Citrate Tributyl Citrate Tri-C Alkyl Citrate Tri-C14-15 Alkyl Citrate Tricaprylyl Citrate Triethyl Citrate Triethylhexyl Citrate Trihexyldecyl Citrate Triisocetyl Citrate Triisopropyl Citrate Trilauryl Citrate Trioctyldodecyl Citrate Trioleyl Citrate Triisostearyl Citrate Tristearyl Citrate Ethyl Citrates Glycol Mono-, Di-, and Triesters Disodium Laureth-7 Citrate Laureth-6 Citrate Laureth-7 Citrate Propylene Glycol Citrate Dilaureth-7 Citrate Sodium Dilaureth-7 Citrate PEG-5 Tricapryl Citrate PEG-5 Trilauryl Citrate PEG-5 Trimyristyl Citrate PEG-5 Tricetyl Citrate PEG-5 Tristearyl Citrate Trilaureth-9 Citrate Tripropylene Glycol Citrate Citric acid is reported to function in cosmetics as a chelating agent, ph adjuster, or fragrance ingredient. While some of the inorganic salts of citric acid are also reported to function as a ph adjuster or chelating agent, there are many other reported functions, including skin conditioning agent, buffering agent, cosmetic astringent, oral care agent, cosmetic biocide, or pesticide. The alkyl esters are reported to function primarily as skin conditioning agents, but a few have other possible functions reported, including plasticizer, solvent, and fragrance ingredient. The glycol esters of citric acid are reported to function mostly as skin conditioning agents or surfactants. As determined by the Food and Drug Administration (FDA), citric acid, calcium citrate, ferric citrate, manganese citrate, potassium citrate, sodium citrate, diammonium citrate, and triethyl citrate are generally recognized as safe (GRAS) direct food additives. Since these 8 ingredients have been shown to be safe for ingestion, this report will focus on the dermal application of these ingredients. For the other ingredients, all available data will be included. Because citric acid, arguably, is an α-hydroxy acid (AHA), it may be relevant to consider the earlier safety assessment of glycolic and lactic acids and their salts and simple esters. CIR reviewed these AHA ingredients (i.e., the report on Glycolic Acid, Ammonium, Calcium, Potassium, and Sodium Glycolates, Methyl, Ethyl, Propyl, and Butyl 1 CIR Panel Book Page 16

22 Glycolates, Lactic Acid, Ammonium, Calcium, Potassium, Sodium, and TEA-Lactates, and Lauryl, Myristyl, and Cetyl Lactates) and published the findings in Distributed for Comment Only -- Do Not Quote or Cite Therein, the Expert Panel concluded that those ingredients are safe for use in cosmetic products at concentrations 10%, at final formulation ph 3.5, when formulated to avoid increasing sun sensitivity or when directions for use include the daily use of sun protection. These ingredients are safe for use in salon products at concentrations 30%, at final formulation ph 3.0, in products designed for brief, discontinuous use followed by thorough rinsing from the skin, when applied by trained professionals, and when application is accompanied by directions for the daily use of sun protection. In the FDA Guidance for Industry: Labeling for Topically Applied Cosmetic Products Containing Alpha Hydroxy Acids as Ingredients, from 2005, 2 the Administration specifically mentions citric acid containing products, for which the following labeling may be warranted: Sunburn Alert: This product contains an alpha hydroxy acid (AHA) that may increase your skin's sensitivity to the sun and particularly the possibility of sunburn. Use a sunscreen, wear protective clothing, and limit sun exposure while using this product and for a week afterwards. See: CHEMISTRY Definition, Structure, and Properties Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid), is a common metabolite of plants and animals, and is well known for its part in the Krebs cycle. 3 It precipitates as white, translucent crystals of monoclinic holohedra form. Citric acid is a polyprotic AHA. However, citric acid can also be classified as a β-hydroxy acid, as two of the carboxylic acid functional groups of citric acid are two carbons removed from the hydroxy group. (Figure 1). Figure 1. Citric Acid Citric acid differs structurally from the AHAs reviewed previously (i.e., glycolic and lactic acid) by having three carboxylic functional groups, instead of just one. Due to these three carboxylic acid functional groups, citric acid has three different pk a s, making it a prime buffer component. Even the most acidic of these carboxylates, the center acid functional group, is only a weak acid, with a pka of 3.1. Citric acid is soluble in water and soluble in some organic liquids, with an octanol/water partition coefficient around -1. Citric acid, and the citrate salts, are solids. The citrate alkyl esters and citrate glycol esters, however, vary from oily liquids (for shorter chain analogues like ethyl) to powdery solids (for longer chain analogues like stearyl). Directly dependent on chain length and degree of substitution, these esters are less soluble in water and more soluble in organic liquids, with octanol/water partition coefficients estimated between 1 and 12. The definitions and structures of the ingredients included in this review are provided in Table 1. It is worth noting that the terminology is not always intuitive, in that monosodium citrate, for example, is the monosodium salt, but sodium citrate is the trisodium salt. The available physical and chemical property information is found in Table 2. Impurities and composition data are provided in Table 3. 2 CIR Panel Book Page 17

23 Production Industrial, large scale production of citric acid is accomplished, most commonly, via mycological fermentation of crude sugar stocks (e.g., molasses), historically by strains of Aspergillus niger. 4 A common problem associated with these fermentation methods is the co-synthesis of isocitric acid (1-hydroxy-1,2,3-propanetricarboxylic acid). However, isocitric acid can be separated using a variety of crystallization techniques. Careful control of the trace element content is very important for high production. 3,5 (While citric acid can also be extracted from citrus fruits, over 99% of the world s citric acid output is produced by microbial fermentation. 5 ) The citrate salts are produced by the same fermentation process, but are simply crystallized in the presence of appropriate alkaline solutions (e.g., citric acid can be crystallized with sodium hydroxide to produce sodium citrate). Citrate alkyl esters are typically produced via the condensation of the appropriate alcohol (e.g., utilize butyl alcohol to produce tributyl citrate) with citric acid. 6 Similarly, the citrate glycol esters are produced via the condensation of the appropriate, previously ethoxylated, alcohol (e.g., utilize laureth-9 to produce trilaureth-9 citrate) with citric acid. Some ingredient-specific methods of manufacture are described in Table 4. USE Cosmetic Citric acid is reported to function in cosmetics as a chelating agent, ph adjuster, or fragrance ingredient. 7 Some of the inorganic salts of citric acid are reported to function as a ph adjuster or chelating agent; these salts also have many other reported functions, including skin conditioning agent, buffering agent, cosmetic astringent, oral care agent, cosmetic biocide, or pesticide. The alkyl esters are reported to function primarily as skin conditioning agents, but a few of these have other reported functions, including plasticizer, solvent, and fragrance ingredient. The glycol esters of citric acid are reported to function mostly as skin conditioning agents or surfactants. The various cosmetic functions of these ingredients are provided in Table 5; some ingredients have more than one reported function. Voluntary Cosmetic Registration Program (VCRP) data obtained from the FDA in 2011, 8 and concentration of use information received in response to a survey conducted by the Personal Care Products Council (Council), 9-11 indicate that 24 of the 46 citrates named in this report are currently used in cosmetic formulations. Citric acid is used in almost every category of cosmetic ingredients, with 6795 reported uses 8 at concentrations up to 35% in leave-on formulations, 10% in rinse-off formulations, and 39% in products diluted for use. 10 Sodium, tributyl, and triethyl citrate are reported to be used in 980, 331, and 244 cosmetic formulations, respectively. 8 All other in-use ingredients have less than 50 uses. The ingredient with the highest concentration of use is triisostearyl citrate; it is used at up to 80% in lipstick formulations. 10 Trioctyldodecyl citrate is used at up to 30% in leave-on formulations; it is used at up to 21% in products applied to the eye area and 19% in lipstick formulations. Tricaprylyl citrate is used at up to 27% in leave-on formulations. All other in-use ingredients are used at concentrations of 12%. Frequency and concentration of use data are provided in Table 6a. The ingredients not in use, according to the VCRP and Council survey, are listed in Table 6b. Products containing citric acid and some of its salts and esters may be applied to baby skin, used near the eye area or mucous membranes, or could possibly be ingested or inhaled. Since some of these ingredients are reported to be in products that could be inhaled, effects on the lungs that may be induced by aerosolized products containing these ingredients are of concern. The particle size of aerosol hair sprays and in pump hair sprays is around 38 μm and >80 μm, respectively, and is large compared to respirable particle sizes ( 10 μm). Therefore, because of their size, most aerosol particles are deposited in the nasopharyngeal region and are not respirable. 3 CIR Panel Book Page 18

24 All of the ingredients included in this review are listed in the European Union inventory of cosmetic ingredients. 12 Water-soluble zinc compounds are listed in Annex III of the Cosmetic Directive, with a maximum authorized concentration in the finished cosmetic product of 1% calculated as zinc; therefore, zinc citrate has a maximum authorized concentration of use of 1%, calculated as zinc, in finished cosmetic products in the European Union. 13 Non-Cosmetic The following 8 ingredients are GRAS direct food additives, restricted only by good manufacturing practices: citric acid; calcium citrate; ferric citrate; manganese citrate; potassium citrate; sodium citrate; diammonium citrate; and triethyl citrate. 14 Additionally, the following are allowed as indirect food additives: citric acid; magnesium citrate; monosodium citrate; potassium citrate; sodium citrate; diammonium citrate; stearyl citrate; isopropyl citrate; distearyl citrate; triethyl citrate; tributyl citrate; tristearyl citrate (an additional 6 ingredients). 15 Magnesium, potassium, and sodium citrate are used in over-the counter drug products. 7 Examples of other non-cosmetic uses of citric acid and some of the citrates are provided in Table 7. TOXICOKINETICS Citric acid is well absorbed and largely metabolized when administered orally; it is an intermediate in the Krebs cycle. Oral administration of aluminum citrate to male Sprague-Dawley rats, 6 days/wk for 4 wks, resulted in a statistically significant increase in levels of aluminum in the brain in one study. In another study in which Sprague-Dawley rats were given aluminum citrate in the drinking water for 8 mos, aluminum levels were increased in other parts of the body but not in the brain. Distearyl citrate, when added to the diet of rats, was poorly absorbed. Nearly complete absorption was observed when isopropyl citrate was administered in the diet of rats. Orally administered citric acid is well absorbed and largely metabolized. 16 Exogenous and endogenous citric acid can be completely metabolized and serve as a source of energy. Citric acid is an intermediate in the Krebs (or tricarboxylic acid) cycle. 17 Citric acid completes the breakdown of pyruvate, formed from glucose through glycolysis, and it liberates carbon dioxide. Approximately 2 kg of citric acid are formed and metabolized every day in humans. Citrate is thought to be freely filterable at the glomerulus of the kidney, and 65-90% of filtered citrate is reabsorbed in humans. 18 Ten to 35% of filtered citrate is excreted in the urine. The normal blood citrate level in humans is approximately 25 mg/l. 19 In Vitro Trihexyl Citrate Trihexyl citrate is not a cosmetic ingredient. This information is presented because trihexyl citrate is structurally similar to cosmetic ingredients included in this review, and may provide read-across data. Trihexyl citrate was incubated with rat serum, an intestinal cytosolic fraction, and a liver cytosolic fraction obtained from Sprague-Dawley rats to determine the hydrolysis of trihexyl citrate in each of these preparations. 20 Dimethyl sulfoxide (DMSO) was used as the vehicle; the volume of DMSO did not exceed 1% of the total volume of the incubation medium. A concentration of 50 nmol/l was used with all three preparations; a concentration of 1000 nmol/l was also used with rat serum. In rat serum, at concentrations of 50 and 1000 nmol/l, the half -life of trihexyl citrate hydrolysis was 4 and 90 min, respectively. Hexanol was produced as a product of hydrolysis. Dihexyl citrate is formed as an intermediate. Hydrolysis was concentration dependent, being faster at lower concentrations. Hydrolysis did not occur with 5 µmol/ml of serum. Hydrolysis in the rat intestinal and rat liver preparations was much slower that in the serum. The half- life of hydrolysis for 50 nmol/ml trihexyl citrate in the rat liver cytosolic fraction was 1.2 min. (The half-life was not given for the intestinal fraction.) 4 CIR Panel Book Page 19

25 Oral Aluminum Citrate Male Sprague-Dawley rats were gavaged with 100 mg aluminum/kg bw, as aluminum citrate, 6 days/wk for 4 wks. 21 A control group was given tap water. Half of the animals were killed at the termination of dosing; the remaining animals were killed after a 5-wk non-treatment period. The levels of aluminum in the brain were statistically significantly increased after 4 wks of dosing with aluminum citrate, and there was no major difference between the animals killed at the termination of dosing or 5 wks later. Ten female Sprague-Dawley rats were given drinking water with 80 mmol/l aluminum citrate for 8 mos; a control group of 8 rats was given untreated water. 22 After 8 mos of dosing, aluminum concentrations were statistically significantly increased in bone, the spleen, liver, and kidneys, but not the brains, of treated animals. Stearyl/Distearyl Citrate Stearyl citrate is hydrolyzed readily to stearyl alcohol and citric acid in dogs, and to a lesser extent, in rats. 23 Stearyl citrate, predominantly as distearyl citrate, was added to the feed of rats at a concentration of %. 16 Stearyl citrate was poorly absorbed. (Additional details were not provided.) Isopropyl Citrate Isopropyl citrate, mostly as the monoisopropyl ester, was administered in the diet of 6 rats in a mono- and diglycerides vehicle at concentrations of 10%. 16 Isopropyl citrate was nearly completely absorbed. (Additional details were not provided.) Effect on Transdermal Absorption Triethyl Citrate Triethyl citrate inhibited the transdermal absorption of viprostol, a synthetic prostaglandin E 2, through the skin of male hypertensive rats. 24 This effect was demonstrated by the statistically significant decrease in blood radioactivity levels following the topical application of [ 14 C]viprostol in triethyl citrate compared to those found with the use of petrolatum or silicone as the vehicle. Comparison of metabolic profiles also demonstrated slower hydrolysis of viprostol to free acid with the use of triethyl citrate as the vehicle. TOXICOLOGICAL STUDIES The dermal LD 50 values for citric acid and triethyl citrate were >5 g/kg in rabbits. Results of oral, inhalation, and other parenteral single-dose studies with various citrates did not indicate any notable toxic effects in mice, rats, rabbits, or dogs. Administration of 80 mmol/l aluminum citrate in water for 6 wks did not affect the body weights of rats. Repeated oral dosing with an isostearyl citrate ester mixture or a distearyl citrate ester mixture did not produce adverse effects in rats, rabbits, or dogs. Repeated oral dosing with tributyl citrate did not have an adverse effect on rats or cats. Single Dose (Acute) Toxicity Acute toxicity studies are summarized in Table 8. Acute toxicity testing did not raise any toxicological concerns. 5 CIR Panel Book Page 20

26 Repeated Dose Toxicity Oral Aluminum Citrate In a toxicokinetics study described previously in this report, a group of 10 female Sprague-Dawley rats were given aluminum citrate in the drinking water at a concentration of 80 mmol/l for 8 mos. 22 Final body weights of animals of the test group were statistically significantly decreased compared to the controls. Kidney function was not affected by dosing. Isostearyl Citrate Ester Mixture A 6-wk feeding study of an isopropyl citrate ester mixture, consisting of 27% isostearyl citrate, 9% diisopropyl citrate, and 2% triisostearyl citrate, in a vehicle consisting of mono- and diglycerides (1:1) vegetable oil was performed using rats. 25 Male rats had an average daily intake of 0.78 g and females 0.54 g, and no adverse effects were observed. (Additional details were not provided). Groups of 10 rats were fed diets containing 0, 0.28, 0.56, or 2.8% of the above isopropyl citrate ester mixture in the same vehicle (corresponding to 0, 0.11, 0.21, and 1.06% isopropyl citrate ester, respectively) for 2 yrs. 25 Again, no signs of toxicity were observed. Microscopic examination of select tissues did not reveal any test-article related changes. Six-wk dietary and 6-wk gavage studies were performed in rabbits using the same isopropyl citrate ester mixture in the same vehicle. 25 Signs of toxicity were not observed in groups of 1-8 rabbits given feed containing % of the isopropyl citrate ester mixture or in groups of 1-3 rabbits gavaged daily with 0, 2.2, 4.4, or 9.2% of the isopropyl citrate ester mixture. Select tissues of the 8 high-dose males used in the feeding study were examined microscopically, and no abnormalities were found. Groups of 2 cocker puppies and 2 adult mongrel dogs were also fed a diet containing the isopropyl citrate ester mixture in vehicle. 25 Adverse effects were not observed when dogs were fed a diet containing 0.06% of the test article for 12 wks. Distearyl Citrate Ester Mixture A 6-wk feeding study of a distearyl citrate ester mixture, consisting of 12.5% stearyl citrate, 75% distearyl citrate, and 12.5% tristearyl citrate, was performed using rats. 25 Male rats had an average daily intake of 1.32 g and females 1.06 g of the mixture, and no adverse effects were observed. (Additional details were not provided). Groups of 10 rats were fed diets containing 0, 0.5, 2.0, or 10.0% of the distearyl citrate ester mixture for 2 yrs. 25 No signs of toxicity were observed. Microscopic examination of select tissues did not reveal any test-article related changes. In a 6-wk dietary study in rabbits with the same distearyl citrate ester mixture, two groups of 8 rabbits were given feed containing 2 or 10 % of the mixture. 25 No signs of toxicity were observed. Select tissues of the rabbits of the 10% group were examined microscopically, and no abnormalities were found. Groups of 2 cocker puppies and 2 adult mongrel dogs were also fed a diet containing the distearyl citrate ester mixture. 25 Adverse effects were not observed when dogs were fed a diet containing 3.0% of the test article for 12 wks. Tributyl Citrate Groups of three or four rats, number per sex not specified, were fed a diet containing 0, 5, or 10% tributyl citrate for 6 wks. 26 No effect on body weight gain was observed in the 5% group. Body weight gains in the 10% group were decreased; the decrease may have been attributable to frequent diarrhea. No effects on blood counts were reported, and no microscopic lesions were observed. 6 CIR Panel Book Page 21

27 Two cats were dosed daily by gavage with 5 ml/kg tributyl citrate daily for 2 mos, and two cats were used as negative controls. 26 No significant effects were observed. Intraperitoneal Tributyl Citrate A test group of 20 mice (sex not stated) was dosed by intraperitoneal (i.p.) injection with 580 mg/kg tributyl citrate in 3% acacia for 14 days, while a group of 20 control mice was dosed with vehicle only. 27 Two animals per group were killed at the end of the study. Body weight gains were decreased in the test animals, and the decrease was significant after 7 days. No significant changes in blood counts were observed, and no microscopic lesions were observed. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Oral administration of 1064 mg/kg bw aluminum citrate concurrent with 62 mg/kg bw citric acid to rats was not maternally-, embryo-, or fetotoxic; the aluminum concentration was statistically significantly increased in the liver, bone, and placenta of the test animals, but no aluminum was detected in the fetus. Oral administration of a 2.8% isopropyl citrate ester mixture or up to 9.5% of a diisostearyl citrate ester mixture did not produce any reproductive or developmental effects in multi-generation studies. Oral Aluminum Citrate A group of 20 presumed-pregnant rats were dosed daily by gavage with 1064 mg/kg bw aluminum citrate and 62 mg/kg bw citric acid, concurrently, on days 6-15 of gestation, and a negative control group of 20 gravid rats received distilled water only. 28 All animals were killed on day 20 of gestation. The actual numbers of gravid test and control rats were 15 and 17, respectively. Administration of aluminum citrate with citric acid was not maternally-, embryo-, or fetotoxic. A statistically significant increase in the absence of xiphoids was the only skeletal variation reported. The aluminum concentration in the maternal liver, kidney, brain, bone (femur), and placenta, as well as in the whole fetus, was determined. The aluminum concentration was statistically significantly increased in the liver, bone, and placenta of the test animals compared to controls; however, no aluminum was detected in the whole fetuses of treated animals. Isopropyl Citrate A multi-generation study was performed in which 5 generations of rats were fed a diet containing 0 or 2.8% of the isopropyl citrate ester mixture in vehicle (equivalent to 1.1% of isopropyl citrate esters) that was described earlier in this report. 25 Administration of the test article did not result in any reproductive or developmental effects or any general signs of toxicity. Diisostearyl Citrate A multi-generation study was performed in which 4 generations of rats were fed a diet containing 0, 1.9, or 9.5% of the diisostearyl citrate ester mixture that was described earlier in this report. 25 Administration of the test article did not result in any reproductive or developmental effects or any general signs of toxicity. In-Vitro Sodium Citrate The embryotoxic potential of sodium citrate was evaluated in a whole rodent embryo culture system using 9.5-dayold embryos from female Han Wistar rats without metabolic activation. 29 The no-effect concentration for all parameters evaluated, including crown-rump length and abnormalities, was >115 µm sodium citrate. 7 CIR Panel Book Page 22

28 Spermicidal Effects Citric Acid The spermicidal effect of citric acid was determined by suspending human sperm in a solution of citric acid. 30 Addition of 0.1% citric acid to human sperm reduced ph and rendered sperm immotile within 30 min, while 1% was almost instantly spermicidal. The effect on sperm penetration of cervical mucus was also evaluated by adding the acid to human cervical mucus in capillary tubes. Addition of 0.01% citric acid reduced, and addition of 0.1% completely abolished, sperm penetration. GENOTOXICITY Citric acid and its salts and esters were mostly negative in in vitro and in vivo genotoxicity tests. Exceptions were weakly positive results for in vitro and in vivo host-mediated assays with citric acid, equivocal results in an Ames test with aluminum citrate, and a weak dose-related response in a suspension test in S. typhimurium TA1537 that was not reproducible. Citric acid had anti-mutagenic effects, inhibiting the mutagenicity of 4- nitro-o-phenylenediamine and sodium azide. Genotoxicity studies are summarized in Table 9. Anti-Mutagenic Effects Citric Acid The anti-mutagenic effect of citric acid was evaluated in an Ames test, with 4-nitro-o-phenylenediamine and sodium azide used as mutagens. 31 Using S. typhimurium strain TA97, concentrations of µg/0.1 ml/plate citric acid inhibited the mutagenicity of 20 µg/0.1 ml/plate 4-nitro-o-phenylenediamine by % without metabolic activation and % with metabolic activation. Using strain TA100, concentrations of µg/0.1 ml/plate citric acid inhibited the mutagenicity of 1.5 µg/0.1 ml/plate sodium azide by % without metabolic activation and % with metabolic activation. CARCINOGENICITY Aluminum Citrate The National Toxicology Program (NTP) has planned toxicity/carcinogenicity testing for aluminum citrate. 32 rationale for testing is that aluminum is listed by the EPA as a drinking water contaminant with a high health research priority. The IRRITATION AND SENSITIZATION In irritation studies in rabbits, 30% citric acid was not a primary irritant, 60% produced some erythema and edema that subsided with time, and undiluted citric acid produced mild to severe erythema and mild to moderate edema. Triethyl citrate, at concentrations up to 100%, was not an irritant in guinea pigs or rabbits, and trioctyldodecyl citrate applied neat was not a primary skin irritant in rabbits. In human studies, citric acid was not a dermal irritant at concentrations up to 5% aq., and 20% triethyl citrate was not irritating in humans. Sodium citrate did not produce any immediate (non-immunologic contact urticaria) reactions. In sensitization testing, a cuticle cream containing 4% citric acid was not an irritant or a sensitizer in humans; 2.5% aq. citric acid produced positive results in skin prick test in 3 of 91 urticaria or anigoedema patients. Triethyl citrate, applied undiluted during epidermal induction, was a strong sensitizer in a guinea-pig maximization test, but 20% in pet. was not a primary irritant or sensitizer in human studies. Trioctyldodecyl citrate was a mild sensitizer in a local lymph node assay when applied neat, but the same concentration was not an irritant or sensitizer in human studies. Tributyl citrate (concentration not stated) was not a sensitizer in animal studies. In human studies, 25% tristearyl citrate,100% triisostearyl citrate, and 10% laureth-7 citrate were not irritants or sensitizers in repeated insult patch tests. Citric acid was predicted to be a moderate/severe to severe/extreme ocular irritant in in vitro studies, and was minimally to mildly irritating at concentrations of 10 and 30%, respectively, in studies using rabbits. In in vitro studies, triisostearyl citrate was predicted to be non-irritating and laureth-7 citrate was predicted to 8 CIR Panel Book Page 23

29 be slightly irritating to eyes. Triethyl citrate, 33.3%, did produce irritation in rabbit eyes, and undiluted trioctyldodecyl citrate was non-irritating. Skin Irritation/Sensitization Non-human and human skin irritation and sensitization studies are summarized in Table 10. In non-human studies, irritation and sensitization study results varied, while no irritation or sensitization was reported in human studies. Ocular Irritation Ocular irritation studies are summarized in Table 11. In Draize tests, citric acid and triethyl citrate produced some irritation and trioctyldodecyl citrate was non-irritating to rabbit eyes. Case Reports Citric Acid A woman reported difficulty breathing and severe facial pain 4 h after a professionally-administered cosmetic peel procedure with a product containing 10% citric acid (and other compounds that were not identified). 33 The facial peel was applied for 4 h. The patient also had first and second degree burns to the face and anterior neck. Permanent facial and neck scars, but no airway pathology, resulted. MISCELLANEOUS STUDIES Citric acid increased cell renewal and epidermal thickness in human skin, and there appeared to be a greater increase at higher concentrations and/or lower ph of citric acid. Citric acid is a tussive agent. The cough reflex to citric acid is produced by irritation of the larynx and the trachea, and is thought to be mediated by receptors that are distributed mainly in the larynx and upper airways. Triethyl and tributyl citrate had an anesthetic effect in rabbit eyes. Effects in Skin Citric Acid The effect of 1M citric acid on skin cell renewal and irritation (as stinging) was determined at phs of 3, 5, and The dansyl chloride method was used to determine skin cell renewal and irritation was evaluated subjectively as stinging in the nasal fold area; stinging was scored on a scale of 0-4 every minute for 15 min. Two mg/cm 2 of the citric acid test product were applied to the test area on the volar forearm of human subjects 2x/daily. The vehicle consisted of 15% ethanol (SD 40), 5% ethoxydiglycol, 5% butylene glycol, and water. Cell renewal was measured in at least 8 subjects; citric acid increased cell renewal by 16.1, 12.8, and 3% at ph 3, 5, and 7, respectively. Using a minimum of 10 subjects, the irritation scores for 1 M citric acid at ph 3, 5, and 7 were 38, 35.4, and 23.6, respectively. The effect of 5% citric acid on skin cell renewal and irritation was also evaluated at the same phs. 35 Cell renewal was greater at this higher concentration; 18, 14, and 8% increases were seen with 5% citric acid at ph 3, 5, and 7, respectively. Irritation scores (as stinging) were 2.3, 2.1, and 1.1 (on a scale of 1-5) at ph 3, 5, and 7, respectively. (Details of application were not provided.) Five male subjects participated in a 30-day study to evaluate the effects of citric acid on skin morphology. 36 Cream formulations containing 10, 20, or 25% citric acid were evaluated, and 0.2 ml of each cream were applied to a 2 cm x 2 cm area of the ventral forearm of each subject. A fourth site on the forearm was used as an untreated control. Occlusive patches, 3x/wk, were applied during wk 1 and non-occlusive patches, 3x/wk, were applied during wks 2-3. Open applications were made daily during wk 4. At the end of dosing, a 3 mm punch biopsy was taken from each site. Irritation was observed with the 20 and 25% formulations. (Details as to the extent of irritation was not provided, other than it was visible ). Microscopically, an increase in viable epidermal thickness that increased with dose was observed at all dose 9 CIR Panel Book Page 24

30 levels, a substantial increase in Langerhans cells was observed with the 20 and 25% citric acid creams, and glycosaminoglycan (GAG) content was markedly increased at the sites dosed with 20 and 25% citric acid compared to that seen at the untreated and 10% citric acid sites. A 20% citric acid lotion, ph 3.5, was applied twice daily for 3 mos to photodamaged skin of the forearm of 6 female subjects. 37 The lotion vehicle without citric acid was applied to the contralateral arms as a control. A 4-mm punch biopsy specimen was taken from each site after 3 mos of application. Application of the lotion containing citric acid produced a statistically significant increase in skin-fold thickness, with a 16.3% increase from baseline recorded. The skin fold thickness of the vehicle-treated skin decreased slightly. Viable epidermis thickness also increased in a statistically significant manner, increasing 40% as compared to untreated skin. A statistically significant increase in GAG content was evidenced by a 2.5-fold increase in epidermal hyaluronic acid staining, a 57% increase in dermal hyaluronic acid staining, and a 66% increase in dermal chondroitin sulfate staining, as compared to skin treated with vehicle only. (While the % increase in staining was greater for chondroitin sulfate; staining for hyaluronic acid was approximately double that of chondroitin sulfate in both vehicle and citric-acid treated sites.) Seven subjects with moderate to severe photoaged skin applied a lotion containing 25% citric acid, ph 3.5, to one forearm and a placebo lotion to the other forearm twice daily for 6 mos. 38 (Similar lotions containing glycolic or lactic acid were also evaluated.) Skin thickness measurements were performed in triplicate throughout the study. The two-skin-layer thickness of the forearm treated with citric acid (and the other AHAs) increased 25%, while the thickness of the control forearm decreased 2%; the difference between the citric acid and control sites was statistically significant. (There was no statistically significant difference in skin thickness among the three AHAs tested.) Microscopically, the mean epidermal thickness of skin and the mean thickness of papillary dermis in samples of skin treated with the citric acid lotion were statistically significantly greater than controls. (Total number of samples examined microscopically was not given). There was no indication of inflammation. The amount of ground substance was variably increased in the citric acid-treated samples. Collagen fibers appeared to be increased in treated skin samples, but there was not a statistically significant difference in collagen fiber density in the papillary dermis between AHA-treated and untreated sites. It has been hypothesized that AHAs have the following mechanism of action. 39 In the stratum corneum, a low concentration of AHAs diminish corneocyte cohesion. In keratinocytes, AHAs stimulate epidermal proliferation, possibly by improving energy and redox status of the keratinocytes. In fibroblasts, high concentrations of AHA in an appropriate vehicle are thought to induce epidermolysis and epidermal separation, and impact the papillary dermis and reticular dermis, leading to dermal changes that include the synthesis of new collagen. Lipid Bilayer Permeation Aluminum Citrate The lipid bilayer permeation of neutral aluminum citrate was determined by measuring the flux across unilamellar phospholipid vesicles, or liposomes, using two independent procedures. 40 The permeation of aluminum citrate was then compared to that of citric acid (as well as malic and lactic acids). Lipid bilayer permeation of 1.82 mm aluminum citrate was slow; the permeability coefficient was, at most, 2 x cm/s. Comparison of permeation of aluminum citrate to the acids indicated that the flux of aluminum citrate is limited by diffusion across the water/lipid interface. (The permeability coefficient for 6.0 mm citric acid was 3.1 x cm/s.) 10 CIR Panel Book Page 25

31 Cough Reflex Citric Acid Citric acid is used as a tussive agent in cough challenge testing. 41 Ten human subjects were exposed to incremental doses of citric acid ( mm) using an air-driven nebulizer. Using the mean cough frequency, a statistically significant dose-response relationship was observed. Individuals had different threshold and maximum tolerable concentrations; using interpolated values, the concentration that caused five coughs was mm citric acid. Using 10 Dunkin Hartley guineapigs exposed to 0.9% saline and then, 10 min later, a single challenge of mm citric acid for 2 min, the calculated concentration producing five coughs (in 10 min) was 74.1 mm citric acid. The cough reflex to citric acid is produced by irritation of the larynx and the trachea, and thought to be mediated by receptors that are distributed mainly in the larynx and upper airways. 42 In human subjects, the cough reflex was reduced with higher inspiratory flow rates as opposed to lower rates. The researchers were not able to definitively state a reason the decrease was seen, but did state an important factor may be laryngeal deposition of the aerosol. Anesthetized guinea pigs were administered 10% w/v aq. citric acid for 1 min; airway resistance increased 79% and lung compliance decreased 68%. 43 In anesthetized guinea pigs in which the vagi had been cut, a 5% increase in resistance and compliance was seen following exposure to citric acid. In conscious guinea pigs exposed to a 10% w/v aq. aerosol of citric acid for 2 min using a glass nebulizer (particle size, µm), the animals coughed 1-2 times in the first 30 sec, and then a short period of hyperventilation was observed. The researchers theorized that the bronchoconstriction was due to an increase in airway resistance and involved parasympathetic innervation. Anesthetic Effects Triethyl and Tributyl Citrate The corneal reflex in rabbit eyes was temporarily eliminated upon instillation of 3 drops of a 5% suspension of triethyl or tributyl citrate in 3% acacia; the number of animals used was not stated. 27 The anesthetic effect was confirmed by the intradermal administration of 0.1 ml of a 2% solution of triethyl or tributyl citrate into an area of the shaved back of guinea pigs; the number of animals used was not stated. Triethyl citrate resulted in insensitivity to pricking of the area lasting min, while tributyl citrate produced a deadened area for a period greater than 2 h. SUMMARY Citric acid is an α-hydroxy tricarboxylic acid that is reported to function in cosmetics as a chelating agent, ph adjuster, or fragrance ingredient. Citric acid can also be classified as a β-hydroxy acid. The 12 inorganic salts are reported to have many diverse functions, while the 33 alkyl and glycol esters are reported to function mostly as skin conditioning agents, although they can have other functions. Citric acid is used in almost every category of cosmetic ingredient, with 6795 reported uses 88 at concentrations up to 39%. With the exception of sodium, tributyl, and triethyl citrate, all other in-use ingredients have less than 50 uses. Triisostearyl citrate is used at up to 80% in lipstick formulations. Trioctyldodecyl and tricaprylyl citrate are used at concentrations of 30 and 27%, respectively; all other in-use ingredients are used at 12%. Citric acid, calcium citrate, ferric citrate, manganese citrate, potassium citrate, sodium citrate, diammonium citrate, and triethyl citrate are GRAS direct food additives. These ingredients, plus 6 others (magnesium citrate; isopropyl citrate; distearyl citrate; stearyl citrate; tristearyl citrate; and tributyl citrate) are FDA-approved indirect food additives. Citric acid is ubiquitously found in nature in virtually all organisms as an intermediate of the Krebs cycle. Orally administered citric acid is well absorbed and largely metabolized. Oral administration of aluminum citrate to male Sprague- Dawley rats, 6 days/wk for 4 wks, resulted in a statistically significant increase in levels of aluminum in the brain in one 11 CIR Panel Book Page 26

32 study. In another study in which Sprague-Dawley rats were given aluminum citrate in the drinking water for 8 mos, aluminum levels were increased in other parts of the body, but not in the brain. Distearyl citrate, when added to the diet of rats, was poorly absorbed, while nearly complete absorption was observed when isopropyl citrate was administered in the diet of rats. The dermal LD 50 values for citric acid and triethyl citrate were >5 g/kg in rabbits. Results of oral, inhalation, and other parenteral single-dose studies with various citrates did not indicate any notable toxic effects in mice, rats, rabbits, or dogs. Administration of 80 mmol/l aluminum citrate in water for 6 wks did not affect the body weights of rats. Repeated oral dosing with an isostearyl citrate ester mixture or a distearyl citrate ester mixture did not have adverse effects on rats, rabbits, or dogs. Repeated oral dosing with tributyl citrate did not have an adverse effect on rats or cats. Oral administration of 1064 mg/kg bw aluminum citrate concurrent with 62 mg/kg bw citric acid to rats was not maternally-, embryo-, or fetotoxic; the aluminum concentration was statistically significantly increased in the liver, bone, and placenta of the test animals, but no aluminum was detected in the fetus. Oral administration of a 2.8% isopropyl citrate ester mixture or up to 9.5% of a diisostearyl citrate ester mixture did not produce any reproductive or developmental effects in multigenerational studies. Citric acid and its salts and esters gave mostly negative reports in in vitro and in vivo genotoxicity tests. Exceptions were weakly positive results in in vitro and in vivo host-mediated assays with citric acid, equivocal results in an Ames test with aluminum citrate, and a weak dose-related response in a suspension test with sodium citrate in S. typhimurium TA1537 that was not reproducible. Citric acid had anti-mutagenic effects, inhibiting the mutagenicity of 4- nitro-o-phenylenediamine and sodium azide. In irritation studies in rabbits, 30% citric acid was not a primary irritant, 60% produced some erythema and edema that subsided with time, and undiluted citric acid produced mild to severe erythema and mild to moderate edema. Triethyl citrate, at concentrations up to 100%, was not an irritant in guinea pigs or rabbits, and trioctyldodecyl citrate applied neat was not a primary skin irritant in rabbits. In human studies, citric acid was not a dermal irritant at concentrations up to 5% aq., and 20% triethyl citrate was not irritating in humans. Sodium citrate did not produce any immediate (non-immunologic contact urticaria) reactions. In sensitization testing, a cuticle cream containing 4% citric acid was not an irritant or a sensitizer in humans; 2.5% aq. citric acid produced positive results in skin prick test in 3 of 91 urticaria or anigoedema patients. Triethyl citrate, applied undiluted during epidermal induction, was a strong sensitizer in a guinea-pig maximization test, but 20% in pet. was not a primary irritant or sensitizer in human studies. Trioctyldodecyl citrate was a mild sensitizer in a local lymph node assay when applied neat, but the same concentration was not an irritant or sensitizer in human studies. Tributyl citrate (concentration not stated) was not a sensitizer in animal studies. In human studies, 25% tristearyl citrate,100% triisostearyl citrate, and 10% laureth-7 citrate were not irritants or sensitizers in repeated insult patch tests. Citric acid was predicted to be a moderate/severe to severe/extreme ocular irritant in in vitro studies, and was minimally to mildly irritating at concentrations of 10 and 30%, respectively, in studies using rabbits. In in vitro studies, triisostearyl citrate was predicted to be non-irritating and laureth-7 citrate was predicted to be slightly irritating to eyes. Triethyl citrate, 33.3%, did produce irritation in rabbit eyes, and undiluted trioctyldodecyl citrate was non-irritating. Citric acid increased cell renewal and epidermal thickness in human skin, and there appeared to be a greater increase at higher concentrations and/or lower ph of citric acid. Citric acid is a tussive agent used in inhalation challenge tests. The cough reflex to citric acid is produced by irritation of the larynx and the trachea, and is thought to be mediated by 12 CIR Panel Book Page 27

33 receptors that are distributed mainly in the larynx and upper airways. Triethyl and tributyl citrate had an anesthetic effect in rabbit eyes. Distributed for Comment Only -- Do Not Quote or Cite To be determined. To be determined. DISCUSSION CONCLUSION 13 CIR Panel Book Page 28

34 TABLES Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure Citric Acid and inorganic salts Citric Acid [hydrate] an α-hydroxy tricarboxylic acid Aluminum Citrate a complex salt of aluminum hydroxide and citric acid Calcium Citrate the calcium salt of citric acid Copper Citrate (hemitrihydrate) the complex copper (II) salt of citric acid. Herein, copper complexes with the carboxylates and the hydroxyl group. Disodium Cupric Citrate the disodium salt of the complex formed between copper (II) and citric acid. Herein, copper complexes with the hydroxyl group and one of the carboxylates. Ferric Citrate [hydrate] the iron (III) salt of citric acid Magnesium Citrate the magnesium salt of citric acid Manganese Citrate the manganese (II) salt of citric acid Monosodium Citrate the monosodium salt of citric acid 14 CIR Panel Book Page 29

35 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure Potassium Citrate the tripotassium salt of citric acid Sodium Citrate (anhydrous) (dihydrate) the trisodium salt of citric acid Zinc Citrate the zinc (II) salt of citric acid Diammonium Citrate the diammonium salt of citric acid -Monoesters Stearyl Citrate [CAS No. is not specific to monoester] Isopropyl Citrate [CAS No. is not specific to monoester] the ester of stearyl alcohol and citric acid the ester of isopropanol and citric acid Alkyl Esters Isodecyl Citrate [CAS No. is not specific to monoester] the ester of branched chain decyl alcohols and citric acid one example of an iso -Diesters Dilauryl Citrate the diester of lauryl alcohol and citric acid Distearyl Citrate the diester of stearyl alcohol and citric acid 15 CIR Panel Book Page 30

36 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure -Triesters Triethyl Citrate the triester of ethyl alcohol and citric acid CH 3 O O O O H 3 C O OH O CH 3 Tributyl Citrate the triester of butyl alcohol and citric acid Tricaprylyl Citrate the triester of capryl alcohol and citric acid Trilauryl Citrate the triester of lauryl alcohol and citric acid Tri-C12-13 Alkyl Citrate the triester of C12-13 alcohols and citric acid wherein R is a 12 or 13 carbon chain Tri-C14-15 Alkyl Citrate the triester of C14-15 alcohols and citric acid wherein R is a 14 or 15 carbon chain Tristearyl Citrate the triester of stearyl alcohol and citric acid Triisopropyl Citrate the triester of isopropyl alcohol and citric acid 16 CIR Panel Book Page 31

37 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure Triethylhexyl Citrate Distributed for Comment Only -- Do Not Quote or Cite the triester of 2-ethylhexanol and citric acid Trihexyldecyl Citrate the triester of 2-hexyldecanol and citric acid. Triisocetyl Citrate the triester of isocetyl alcohol and citric acid one example of an iso Triisostearyl Citrate the triester of isostearyl alcohol and citric acid one example of an iso Trioctyldodecyl Citrate the triester of 2-octyldodecanol and citric acid 17 CIR Panel Book Page 32

38 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure Trioleyl Citrate Distributed for Comment Only -- Do Not Quote or Cite the triester of oleyl alcohol and citric acid Ethyl Citrates a mixture of mono-, di- and triesters of ethanol and citric acid -Monoesters Propylene Glycol Citrate [CAS No. is not specific to monoester] Glycol Esters the ester of citric acid and propylene glycol wherein R is a hydrogen atom or an ethyl group Laureth-6 Citrate [CAS No. is generic to any laureth-n citrate] the ester of citric acid and laureth-6 Laureth-7 Citrate [CAS No. is generic to any laureth-n citrate] the ester of citric acid and laureth-7 Disodium Laureth-7 Citrate the disodium salt of the ester of citric acid and laureth-7 -Diesters Dilaureth-7 Citrate the diester of citric acid and laureth-7 Sodium Dilaureth-7 Citrate the sodium salt of the diester of citric acid and laureth-7 18 CIR Panel Book Page 33

39 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure -Triesters PEG-5 Tricapryl Citrate Distributed for Comment Only -- Do Not Quote or Cite the triester of citric acid and PEG-5 decanol alcohol PEG-5 Trilauryl Citrate the triester of citric acid and PEG-5 dodecanol Trilaureth-9 Citrate the triester of laureth-9 and citric acid PEG-5 Trimyristyl Citrate the triester of citric acid and PEG-5 myristyl alcohol PEG-5 Tricetyl Citrate the triester of citric acid and PEG-5 cetyl alcohol 19 CIR Panel Book Page 34

40 Table 1. Definitions and structures of citric acid, salt and esters Ingredient CAS No. Definition Formula/structure PEG-5 Tristearyl Citrate the triester of citric acid and PEG-5 stearyl alcohol Tripropylene Glycol Citrate a triester of propylene glycol and citric acid Table 2. Chemical and physical properties Property Description Reference Citric Acid molecular weight monohydrate: appearance and form monoclinic holohedrism crystals 4 monohydrate: orthorhombic crystals free-flowing, colorless, translucent crystals or as a white granular to fine 44 powder melting point 153 C 4 monohydrate: 100 C boiling point decomposes above 175 C 17 log P ±0.396 (at 25 C) 45 log K ow vapor pressure <0.001 mm Hg (20 C) 3.7 x 10-9 mm Hg (25 C) solubility solubility in water increases with temperature (from 54% w/w/ at 10 C to 84%) 4 at 100 C; freely soluble in alcohol; very slightly soluble in ether 44 in water: 162 g/100 ml (at 25 C); in alcohol: 59.1 g/100 ml (at 25 C) solubility in water increases with temperature from ~54 wt percentage at 10 C 48 to ~88 wt percentage at 100 C density monohydrate: pk a pk 1 = 3.128; pk 2 = 4.761; pk 3 = (25 C) 4 ph (citric acid-sodium citrate solution) ph of water solutions with equal percentages of citric acid and sodium citrate ranged from 4.15 (0.25% each chemical) to 3.54 (15% of each chemical) CIR Panel Book Page 35

41 Table 2. Chemical and physical properties Property Description Reference Aluminum Citrate density 1.5 g/cm 3 4 molecular weight appearance and form solubility molecular weight appearance and form solubility appearance and form solubility Calcium Citrate fine white, odorless powder molecular weight dibasic: tribasic: molecular weight melting point solubility soluble in 1050 parts cold water, somewhat soluble in hot water; insoluble in alcohol Copper Citrate green or bluish-green crystalline powder; odorless slightly soluble in water; soluble in ammonia, diluted acids, and cold alkali citrate solutions; freely soluble in hot alkali citrate solutions Ferric Citrate garnet-red transparent scales or pale brown powder slowly but completely soluble in cold water; readily soluble in hot water, practically insoluble in alcohol decomposes molecular weight monohydrate: appearance and form boiling point log K ow (calculated) Magnesium Citrate Monosodium Citrate 570 g/l (at 25 C); insoluble in ethanol and ether Potassium Citrate monohydrate: white crystals, granules, or powder; odorless monohydrate: white coarse powder 211 C (calculated) vapor pressure 2.09 x mm Hg (25 C) solubility stability molecular weight dihydrate: appearance and form melting point 1 g dissolves slowly in 0.65 ml water; practically insoluble in alcohol monohydrate: 190 g/100 ml water (at 25 C); insoluble in alcohol and ether monohydrate: very hygroscopic; readily deliquesces in moist air Sodium Citrate dihydrate: white crystals, granules, or powder; odorless anhydrous: >300 C dihydrate: 150 C density monohydrate: log K ow (calculated) vapor pressure solubility molecular weight x mm Hg (25 C) soluble in water, ~425 g/l (25 C) monohydrate: soluble in 1.3 parts water; insoluble in alcohol Zinc Citrate CIR Panel Book Page 36

42 Table 2. Chemical and physical properties Property Description Reference appearance and form solubility molecular weight appearance and form solubility melting point powder; odorless slightly soluble in water; soluble in diluted mineral acids and alkali hydroxides granules or crystals Diammonium Citrate soluble in 1 part water; slightly soluble in alcohol C molecular weight appearance and form melting point Distearyl Citrate Triethyl Citrate clear, colorless, oily liquid -55 C boiling point 294 C vapor pressure density refractive index 6.4 x 10-3 mm Hg (20 C) (20 C) (@25 C/D) solubility 6.5 g/100 ml water (25 C) 5.5 g/100 ml water (25 C); insoluble in hexane miscible with alcohol, ether 59 4 log K ow 1.3 (35 C) (measured) (calculated) Tributyl Citrate molecular weight appearance and form colorless or pale yellow liquid; odorless 4 melting point -20 C 4 boiling point 170 C (1 mm Hg) C (22 mm Hg) 4 vapor pressure 9.6 x 10-2 mm Hg (20 C) 59 density (20 C) 4 refractive index (@25 C/D) 59 solubility insoluble in water; miscible with most organic liquids 4 log P (predicted) ± (25 C) 45 pk a (predicted) 11.3 ± 0.29 (25 C) 45 Tricaprylyl Citrate molecular weight boiling point C (6-7 mm Hg) 60 density g/cm 3 60 log P (predicted) ± (25 C 45 pk a ± Trilauryl Citrate molecular weight boiling point (predicted) C 45 density (predicted) g/cm 3 (20 C) 45 log P (predicted) (25 C) 45 pk a (predicted) (25 C) CIR Panel Book Page 37

43 Table 2. Chemical and physical properties Property Description Reference Tristearyl Citrate molecular weight boiling point (predicted) C density (predicted) g/cm 3 (20 C) log P (predicted) (25 C) pk a (predicted) (25 C) Triisopropyl Citrate molecular weight boiling point (predicted) 331 C density (predicted) g/cm 3 (20 C) log P (predicted) (25 C) pk a (predicted) (25 C) Triisostearyl Citrate molecular weight 944 appearance clear viscous liquid Trioctyldodecyl Citrate molecular weight 1032 boiling point (predicted) C density (predicted) g/cm 3 (20 C) log P (predicted) (25 C) pk a (predicted) (25 C) Trioleyl Citrate molecular weight boiling point (predicted) C density (predicted) g/cm 3 (20 C) log P (predicted) (25 C) pk a (predicted) (25 C) Laureth-7 Citrate molecular weight 677 appearance and form clear, slightly yellow liquid acid number saponification value ph value (10%) CIR Panel Book Page 38

44 Table 3. Impurities and Composition Ingredient Impurities/Composition Reference Stearyl Citrate 10-15% monostearyl, 70-80% distearyl, and 10-15% tristearyl derivatives 16 Isopropyl Citrate 65-80% monoisopropyl, 15-30% diisopropyl, and 5-10% triisopropyl citrate 16 Triisostearyl Citrate supplied as >90% triisostearyl citrate 61 impurities include residual isostearyl alcohol (<10%) and citric acid (<0.5%) Trioctyldodecyl Citrate supplied as ~100% trioctyldodecyl citrate (according to one supplier) 61 impurities include residual octyldodecyl alcohol (<5%) and citric acid Laureth-7 Citrate approximately 0.05% of a mixture of tocopherol and hydrogenated palm glycerides citrate, included as an antioxidant, may be present in laureth-7 citrate (according to one supplier); no residual preservatives or solvents are reported to be present 63 Table 4. Ingredient-Specific Methods of Manufacture Ingredient Method of Manufacture Reference Calcium Citrate Copper Citrate Ferric Citrate Manganese Citrate Potassium Citrate Zinc Citrate Diammonium Citrate Triethyl Citrate Tributyl Citrate Triisostearyl Citrate Trioctyldodecyl Citrate Laurth-7 Citrate neutralization of citric acid with calcium hydroxide or calcium carbonate prepared by the interaction of hot aqueous solutions of copper sulfate and sodium citrate prepared from reaction of citric acid with ferric hydroxide obtained by precipitating manganese carbonate from manganese sulfate and sodium carbonate solutions. The filtered and washed precipitate is digested first with sufficient citric acid solution to form manganous citrate and then with sodium citrate to complete the reaction crystallizing and drying of a potassium citrate solution that is prepared using a citric acid solution and potassium hydroxide prepared from zinc carbonate and citric acid partial neutralization of citric acid with ammonia esterification of ethyl alcohol with citric acid synthesized from n-butyl alcohol and citric acid manufactured from isostearyl alcohol and citric acid in a proprietary esterification process, without the use of heavy metal catalysts manufactured from octyldodecyl alcohol and citric acid in a proprietary esterification process, without the use of heavy metal catalysts esterification of a fatty alcohol ethoxylate (laureth-7) with citric acid CIR Panel Book Page 39

45 Table 5. Reported functions of citric acid and its salts and esters Distributed for Comment Only -- Do Not Quote or Cite ph adjuster Citric Acid Calcium Citrate Monosodium Citrate Potassium Citrate Sodium Citrate Chelating Agent Citric Acid Diammonium Citrate Potassium Citrate Sodium Citrate Fragrance Ingredient Citric Acid Sodium Citrate Triethyl Citrate Buffering Agent Diammonium Citrate Potassium Citrate Sodium Citrate Skin Conditioning Agent Emollient Dilauryl Citrate Distearyl Citrate Isodecyl Citrate PEG-5 Tricapryl Citrate PEG-5 Tricetyl Citrate PEG-5 Trilauryl Citrate PEG-5 Trimyristyl Citrate PEG-5 Tristearyl Citrate Stearyl Citrate Tri-C12-13 Alkyl Citrate Tri-C14-15 Alkyl Citrate Triethylhexyl Citrate Triisopropyl Citrate Trioleyl Citrate Skin Conditioning Agent Humectant Propylene Glycol Citrate Skin Conditioning Agent Occlusive Tricaprylyl Citrate Trihexyldecyl Citrate Triisocetyl Citrate Triisostearyl Citrate Trilauryl Citrate Trioctyldodecyl Citrate Tristearyl Citrate Skin Conditioning Agent Miscellaneous Dilaureth-7 Citrate Ferric Citrate Magnesium Citrate Tripropylene Glycol Citrate Surfactant Cleansing Agent Disodium Laureth-7 Laureth-6 Citrate Laureth-7 Citrate Sodium Dilaureth-7 Citrate Trilaureth-9 Citrate Surfactant Emulsifying Agent Dilaureth-7 Citrate Disodium Laureth-7 Citrate PEG-5 Tricapryl Citrate PEG-5 Tricetyl Citrate PEG-5 Trilauryl Citrate PEG-5 Trimyristyl Citrate PEG-5 Tristearyl Citrate Trilaureth-9 Citrate Hair Fixative Ethyl Citrates Plasticizer Isodecyl Citrate Isopropyl Citrate Tributyl Citrate Triethyl Citrate Triethylhexyl Citrate Cosmetic Astringent Aluminum Citrate Oral Care Agent Zinc Citrate Cosmetic Biocide Zinc Citrate Pesticide Copper Citrate Solvent Isopropyl Citrate Tributyl Citrate Not Reported Disodium Cupric Citrate Manganese Citrate Reference 7 25 CIR Panel Book Page 40

46 Table 6a. Frequency and concentration of use according to duration and type of exposure # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 Citric Acid Aluminum Citrate Diammonium Citrate Totals* NR 6 NR Duration of Use Leave-On NR 2 NR Rinse-Off NR 4 NR Diluted for (Bath) Use NR NR NR NR Exposure Type Eye Area NR NR 1 NR Incidental Ingestion NR NR NR NR Incidental Inhalation-Spray 171 a,b a,b NR NR NR NR Incidental Inhalation-Powder NR NR Dermal Contact NR 2 NR Deodorant (underarm) NR NR NR NR Hair - Non-Coloring NR NR 3 NR Hair-Coloring NR NR NR NR Nail NR NR NR NR Mucous Membrane NR 1 NR Baby Products NR NR NR NR Dilauryl Citrate Ethyl Citrates Ferric Citrate Totals* 1 NR NR Duration of Use Leave-On NR NR NR NR 4 NR Rinse Off 1 NR NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR NR NR NR NR NR Incidental Inhalation-Powder NR NR NR NR NR Dermal Contact 1 NR NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR 2 NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR 1 NR 0.5 Baby Products NR NR NR NR NR NR Isodecyl Citrate Laureth-7 Citrate Magnesium Citrate Totals* 4 NR 1 NR Duration of Use Leave-On 4 NR NR NR NR Rinse-Off NR NR 1 NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR NR NR NR NR NR Incidental Inhalation-Powder NR NR NR NR NR Dermal Contact 4 NR NR NR NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR 1 NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR Baby Products NR NR NR NR NR NR 26 CIR Panel Book Page 41

47 Table 6a. Frequency and concentration of use according to duration and type of exposure # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 Monosodium Citrate Potassium Citrate Sodium Citrate Totals* Duration of Use Leave-On NR Rinse Off Diluted for (Bath) Use 14 5 NR NR Exposure Type Eye Area NR NR 1 NR Incidental Ingestion NR NR Incidental Inhalation-Spray NR NR NR a,b a,b Incidental Inhalation-Powder NR 5 NR Dermal Contact Deodorant (underarm) NR NR NR NR Hair - Non-Coloring NR NR Hair-Coloring NR NR NR NR Nail NR NR NR NR Mucous Membrane Baby Products NR 5 NR NR 9 NR Stearyl Citrate Tributyl Citrate Tri-C12-13 Alkyl Citrate Totals* NR Duration of Use Leave-On NR Rinse-Off NR NR Diluted for (Bath) Use NR NR NR NR Exposure Type Eye Area NR 1-2 NR NR 1 NR Incidental Ingestion NR 12 NR NR NR NR Incidental Inhalation-Spray NR 1-3 a,b NR NR Incidental Inhalation-Powder NR4 NR 1 NR NR NR Dermal Contact < NR Deodorant (underarm) NR 3 NR NR NR NR Hair - Non-Coloring NR NR NR Hair-Coloring NR NR 55 NR NR NR Nail NR NR NR NR Mucous Membrane NR NR Baby Products NR NR 1 NR NR NR Tri-C14-15 Alkyl Citrate Tricaprylyl Citrate Triethyl Citrate Totals* Duration of Use Leave-On Rinse-Off NR NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area Incidental Ingestion NR NR Incidental Inhalation-Spray 13 b b NR NR 144 a,b a Incidental Inhalation-Powder NR NR 1 NR NR 3 Dermal Contact Deodorant (underarm) NR NR NR NR 48 2 Hair - Non-Coloring NR NR Hair-Coloring NR NR NR NR NR 0.5 Nail NR NR NR NR NR NR Mucous Membrane NR NR CIR Panel Book Page 42

48 Table 6a. Frequency and concentration of use according to duration and type of exposure # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 # of Uses 8 Conc of Use (%) 10 Baby Products NR NR NR NR NR Triethylhexyl Citrate Triisocetyl Citrate Triisostearyl Citrate Totals* 1 NR Duration of Use Leave-On 1 NR Rinse Off NR NR NR NR 3 NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR 1 NR Incidental Ingestion NR NR 7 NR Incidental Inhalation-Spray NR NR NR NR NR NR Incidental Inhalation-Powder NR 11 2 NR NR Dermal Contact 1 NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR 3 NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR 7 NR Baby Products NR NR NR NR NR NR Trioctyldodecyl Citrate Tripropylene Glycol Citrate Zinc Citrate Totals* NR Duration of Use Leave-On NR Rinse-Off NR NR NR NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR Incidental Ingestion NR NR Incidental Inhalation-Spray b NR NR 4 NR Incidental Inhalation-Powder NR NR 0.05 Dermal Contact NR Deodorant (underarm) NR NR NR NR 4 a NR Hair - Non-Coloring NR NR NR NR NR NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR Baby Products NR NR NR NR NR NR * Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses. a Includes deodorants, in that it is not known whether or not the product is a spray. b Includes suntan products, in that it is not known whether or not the reported product is a spray. NR no reported uses 28 CIR Panel Book Page 43

49 Table 6b. Ingredients not reported to be used Calcium Citrate Copper Citrate Citrate Dilaureth-7 Disodium Cupric Citrate Disodium Laureth-7 Citrate Distearyl Citrate Isopropyl Citrate Laureth-6 Citrate Manganese Citrate PEG-5 Tricapryl Citrate PEG-5 Tricetyl Citrate* *not yet included in an industry survey PEG-5 Trilauryl Citrate PEG-5 Trimyristyl Citrate PEG-5 Tristearyl Citrate Propylene Glycol Citrate Sodium Dilaureth-7 Citrate Trihexyldecyl Citrate Triisopropyl Citrate Trilaureth-9 Citrate Trilauryl Citrate Trioleyl Citrate Tristearyl Citrate Table 7. Examples of non-cosmetic uses Ingredient Non-Cosmetic Use Reference Citric Acid used in the food, beverage, and pharmaceutical industries; active ingredient in pesticide products; 3,5,44,68 manufacture of ecologically compatible detergents; chemical cleaning; metal cleaning; concrete admixtures; plasticizers; photography Calcium Citrate calcium fortifier in foods; anti-caking agent in dry mixes 4 Copper Citrate as an astringent or antiseptic 4 Diammonium Citrate determination of phosphate, especially in fertilizers 4 Potassium Citrate as a replacement for sodium citrate in foods; as a buffering agent in foods; as a source of potassium 4,52 ion in a nutritional supplement; sequestering or emulsifying agent Sodium Citrate anticoagulant; acidulant in beverages, confectionery, effervescent salts, powders, and tablets, 4 pharmaceutical syrups, and elixirs; ph adjuster in food; as an synergistic oxidant; in processing cheese; in the manufacture of alkyd resins; in the manufacture of citric acid salts as a sequestering agent to remove trace metals; in electroplating; in special inks Triethyl Citrate plasticizer for cellulose derivatives and natural resins; plasticizer in pharmaceutical excipients 59,69 Tributyl Citrate plasticizer and solvent for nitrocellulose lacquers; in polishes, inks, and similar preparations; 4 plasticizer in pharmaceutical excipients; as an anti-foam agent Zinc Citrate used in toothpaste and mouthwash 4 29 CIR Panel Book Page 44

50 Table 8. Acute toxicity studies Ingredient Animals* No./Group Dose LD 50 Reference DERMAL Citric Acid Citric Acid rabbits 10 5 g/kg tested >5 g/kg 47 Triethyl Citrate Triethyl Citrate rabbits 4 not stated >5 g/kg 57 Triethyl Citrate guinea pig not stated not stated >10 ml/kg 70 ORAL Isopropyl Citrate Isopropyl Citrate Esters, rats 6M/4-11F M: g/kg M: >20.7 g/kg (>7.0 g/kg ## ) 25 mostly mono- + vehicle # F: g/kg F: 18.8 g/kg (7.2 g/kg) as above, in cottonseed rats 9-10M/ M: g/kg M: >17.2 g/kg (>6.5 g/kg) 25 oil (2:1) 7.5% Isopropyl Citrate Esters, mostly mono-, in 10% ethanol 15% Isopropyl Citrate Esters, mostly mono-, in 10% ethanol 75% aq. Isopropyl Citrate Esters, mostly mono-, Isopropyl Citrate Esters, mostly mono- + vehicle Isopropyl Citrate Esters, mostly mono- Stearyl Citrate Esters, predominantly 20% in cottonseed oil Stearyl Citrate Esters, predominantly distearyl Distributed for Comment Only -- Do Not Quote or Cite F F: g/kg F: 19.2 g/kg (7.3 g/kg) rats 10 M g/kg 3.7 g/kg rats 10F g/kg 2.8 g/kg rats 10F g/kg 3.6 g/kg dogs 4 12 g/kg >12 g/kg; not fatal at this dose dogs g/kg >2.25 g/kg; not fatal at this dose Stearyl Citrate rats 2-13M/ M/F: g/kg M/F: >5.4 g/kg 2-13 F dogs 4 5 g/kg >5 g/kg; not fatal at this dose Tributyl Citrate Tributyl Citrate rats ml/kg no deaths reported Tributyl Citrate cats ml/kg no deaths reported Trioctyldodecyl Citrate Trioctyldodecyl Citrate rats 10 (5/sex) 5 g/kg no deaths reported INHALATION Triethyl Citrate rats no stated 6-h exposure to vapor ppm INTRAPERITONEAL Monosodium Citrate Monosodium Citrate white mice not stated M solution 7.6 mmol/kg Monosodium Citrate albino rats not stated M solution 6.3 mmol/kg Tributyl Citrate Tributyl Citrate Swiss albino not stated chosen from a logarithmic scale 2900 mg/kg 27 mice INTRAVENOUS Monosodium Citrate white mice not stated M; rapid administration 0.23 mmol/kg 72 Monosodium Citrate white mice M administered at rate of 2.01 mmol/kg mmol/min (6 ml/min) Monosodium Citrate rabbits not stated M; administered at a rate 1.76 mm/kg of mmol/min (0.75 ml/min) 72 *unless it is given, the sex of the animals was not stated # - this test material is composed of 27% isopropyl citrate, 9% diisopropyl citrate, and 2% triisopropyl citrate; when + vehicle - vehicle consisting of monoand diglycerides (1:1) of vegetable oil ## - when available, the isopropyl citrate ester content without vehicle is given in ( - the test material is composed of 12.5% stearyl citrate, 75% distearyl citrate, and 12.5% tristearyl citrate CIR Panel Book Page 45

51 Table 9. Genotoxicity studies Concentration Vehicle Procedure Test System Results Reference IN VITRO Citric Acid µg/plate distilled water Ames test, in triplicate; negative and positive controls 5000 µg/plate phosphate buffer Ames test S. typhimurium TA97, TA98, TA100, TA104,+/- met act S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537, +/- met act negative negative 1000 µg/ml saline chromosome aberration assay Chinese hamster fibroblast cells negative µg/ml saline cytogenetic study human embryonic lung cultures, negative WI-38 not given saline host-mediated assay S. typhimurium TA1530, G46; S. cerevisiae D3 negative in S. typhimurium; weakly positive in S. cerevisiae 1.0 mg/ml not stated RK bacterial assay; was used as a non-mutagenic control E. coli CHY832 negative Aluminum Citrate 10-10,000 µg/plate water Ames test S. typhimurium TA100, TA1535, TA97, TA98, TA102, TA104 +/- met act; TA1537, without met act Ferric Citrate 25,000 µg/plate phosphate Ames test S. typhimurium TA92, TA94, negative buffer TA98, TA100, TA1535, TA1537, equivocal in TA97 w/met act +/- met act 500 µg/ml sodium CMC chromosome aberration assay Chinese hamster fibroblast cells negative 2 mm not stated DNA strand break Chinese hamster V79 cells no reduction in double-stranded DNA Monosodium Citrate 5000 µg/plate phosphate buffer Ames test S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537, +/- met act negative 3000 µg/ml saline chromosome aberration assay Chinese hamster fibroblast cells negative Potassium Citrate % DMSO Ames test S. typhimurium TA1535, TA1537, negative TA1538; +/- met act % (S. typhimurium) % (S. cerevisiae) DMSO suspension test S. typhimurium TA1535, TA1537, TA1538, S. cerevisiae D4; +/- met act Sodium Citrate (Dihydrate) 6.25 x x 10-4 % DMSO Ames test S. typhimurium TA1535, TA1537, TA1538, +/- met act 6.25 x x DMSO suspension test S. typhimurium TA1535, TA1537, 10-4 % TA1538, S. cerevisiae D4 Triethyl Citrate % DMSO Ames test S. typhimurium TA1535, TA1537, TA1538; +/- met act % (S. typhimurium) % (S. cerevisiae) Distributed for Comment Only -- Do Not Quote or Cite DMSO suspension test S. typhimurium TA1535, TA1537, TA1538, S. cerevisiae D4; +/- met act negative negative weak dose-related response in S. typhimurium TA1537 without activation, repeat trial neg; neg in S. cerevisiae; negative w/activation negative negative Tributyl Citrate not given not given Ames test not given negative CIR Panel Book Page 46

52 Table 9. Genotoxicity studies Concentration Vehicle Procedure Test System Results Reference not given not given chromosome aberration assay human peripheral blood negative 81 lymphocytes Triisostearyl Citrate 10-10,000 µg/plate ethanol Ames test, in triplicate; negative S. typhimurium TA1535, TA1537, negative 82 and positive controls TA98, TA100,+/- met act Laureth-7 Citrate µg/plate deionized water Ames test, in triplicate; negative and positive controls S. typhimurium TA98, TA100,+/- met act negative 83 IN VIVO Citric Acid mg/kg saline cytogenetic assay, oral rats negative , 3500 mg/kg saline cytogenetic assay, oral rats negative 75 (acute); 300, 3000 mg/kg (subacute) mg/kg saline host-mediated assay, oral Saccharomyces D3 weakly positive 75 (acute & subacute) 3500 mg/kg (acute & subacute) 1.2, 12, 120 mg/kg (subacute) 500, 3500 mg/kg (acute); 300, 3000 mg/kg (subacute) mice saline host-mediated assay, oral S. typhimurium TA1530 and G46 mice saline Distributed for Comment Only -- Do Not Quote or Cite dominant lethal assay, oral, 1x/day for 5 days saline dominant lethal assay, oral, 1 dose (acute) or 1x/day for 5 days (subacute) Abbreviations: CMC carboxymethyl cellulose; DMSO dimethyl sulfoxide; met act metabolic activation rats rats neg. (acute); weakly pos. (subacute) sig. increase in preimplantation loss at wk 4 in high dose group negative Table 10. Dermal irritation and sensitization Test Article Concentration Test Pop. Procedure Results Reference NON-HUMAN IRRITATION Citric Acid Citric Acid 30% aq. 3 NZW rabbits Draize test, 0.5 ml applied for 4 h to intact and abraded; occlusive patch Citric Acid not stated rabbits acute dermal irritation/corrosion study Citric Acid 60% pure NZW rabbits, 5M/3F 0.5 ml; applications to 1 animal for 3 min, to 1 for 60 min, to the remainder for 4 h Citric Acid 100% 10 rabbits 5 g/kg were applied in an acute study (details not provided) Citric Acid 15% 32 male Wistar rats Evan s blue test: 2% Evan s blue was injected i.v. into the tail of rats; 0.1 ml was then injected intradermally to a site on the back; animals were killed after 0.5, 1, 3,and 6 h 32 not a primary irritant; PII=84 slightly irritating; avg erythema score = min: very slight erythema 60 min: very slight erythema 4-hr: very slight-moderate to severe erythema, very slightmoderate edema, subsided to well-defined erythema and no edema after 48 h mild (n=3), moderate (n=4), and severe (n=2) erythema; mild (n=8) and moderate (n=2) edema statistically significantly more dye was extracted with Citric Acid compared to saline CIR Panel Book Page 47

53 Table 10. Dermal irritation and sensitization Test Article Concentration Test Pop. Procedure Results Reference Triethyl Citrate Triethyl Citrate 40, 70, 100% in ethanol Triethyl Citrate % in 0.01% DBS/ saline 4 F guinea pigs/gp guinea pigs, 4 M/gp 24 h, 8 mm occlusive patch; test sites scored 24 and 48 h after patch removal intradermal injection, 0.1 ml; test sites scored after 24h Triethyl Citrate 100% 4 rabbits 5 g/kg were applied in an acute study (details not provided) Triethyl Citrate 15 and 33.3% in alcohol SDA 39C 3 albino rabbits Triethyl Citrate 33.3% in pet 3 albino rabbits Trioctyldodecyl Citrate Triethyl Citrate neat induction: intradermal, 2.5% in 0.01% DBS/ saline; epidermal, 100% challenge: 50% in absolute eth. 6 rabbits (sex not specified) 9 guinea pigs 0.5 ml applied to a 2x2 (unites not given area of intact and abraded skin for 24 h with an occlusive covering 33 barely perceptible erythema at 24 h in 1 animal of the 100% group; no irritation with 40 or 70% faint pink reaction at all test sites with all concentrations no irritation not a primary irritant; PII = 0 as above not a primary irritant; PII = 0 Trioctyldodecyl Citrate 0.5 ml applied to intact and abraded skin for 24 h under an occlusive patch SENSITIZATION Triethyl Citrate Magnusson-Kligman GPMT; FCA was used at intradermal induction; occlusive patches were used during intradermal induction and at challenge Tributyl Citrate Tributyl Citrate not provided not provided GPMT or LLNA (add l details not provided) Trioctyldodecyl Citrate Trioctyldodecyl Citrate Citric Acid 0, 10, 50, 100% w/v in acetone/ olive oil (4:1, v/v) 0.3N solution (vehicle not specified) 5 mice LLNA; 25 µl/ear were applied daily for 3 days; untreated and positive (α-hexylcinnamic aldehyde) control were used HUMAN IRRITATION Citric Acid not specified Citric Acid 5% aq., ph 2 20 subjects, 14F/6M Distributed for Comment Only -- Do Not Quote or Cite stinging potential was evaluated by applying 0,1-0.2 ml to an abraded site on the forearm for 5 min; sig. change measured as difference from first to last day of dosing 50 µl applied to the back using 12 mm occlusive patch each AM; each PM, either the same patch or 0.5% aq SLS was applied; procedure repeated for 4 days; irritation was measured by visual scoring, TEWL, and skin color reflectance not a primary skin irritant; PII = 0.00 strong sensitizer; 9/9 animals sensitized after 2 challenges; primarily intense erythema, with some moderate and diffuse erythema, was observed negative neat material was considered a mild sensitizer; the SI for the concentrations tested ranged from 1.1 to 3.1 citric acid produced the most painful stinging response: citric, acetic >> aconitic>tartaric>ascorbic; citric acid has scored quite low when intercompared to other acids for primary irritancy no irritation with citric acid alone; exposure with SLS caused a clear irritant reaction, however, this reaction was less than that seen 1x daily exposure to SLS CIR Panel Book Page 48

54 Table 10. Dermal irritation and sensitization Test Article Concentration Test Pop. Procedure Results Reference Citric Acid 5% aq., ph 4 as above as above no irritation with citric acid alone; exposure with SLS caused a clear irritant reaction, however, this reaction was less than that seen 1x daily exposure to SLS Citric Acid, in hand cleansers (A and B; % Citric Acid not given) hand cleansers as above (A&B), plus a 3 rd cleanser (not def.) hand cleansers as above (A&B), 2 addl. cleanser (not def.) neat neat 12 subjects/ group 8 subjects/ group use test; product was applied 20/day for 2 wks; s.c. hydration was measured with a corneometer; TEWL measured with an evaporation meter; sig. determined as above forearm wash test; each group received 2 products to apply simultaneously; forearms were washed for 1 min 2x, then rinsed for 30 sec; sig. changes measured as above 10% 40 subjects patch test; 50 µl of each cleanser applied using 12 mm Finn chambers; 48 h Citric Acid 1% aq. 133 oral disease patients Citric Acid 2.5% aq. 49 atopic; 56 non-atopic patients Citric Acid not stated (most likely 100%) 702 contact dermatitis patients Sodium Citrate 10% aq. 49 atopic; 56 non-atopic patients Δ erythema:a, ~0.3; B, ~0.7 TEWL: A, ~4 g/m 2 /h (P 0.5); B, ~1.25 g/m 2 /h Δ s.c. hydration: A, ~ -1; B, ~ -1.9 Δ erythema: A, ~0.7 (p 0.5); B, ~0.45 TEWL: A, ~11 g/m 2 /h (p 0.5); B, 8 g/m 2 /h (p 0.5) Δ s.c. hydration: A, ~ -9.5(p 0.5); B, ~ -8 Δ erythema: A, ~2.7 (p 0.5); B, ~2.25 (p 0.5) TEWL: A, 14 g/m 2 /h; B, ~7.9 g/m 2 /h, diff. btwn. A&B (p 0.5) Δ s.c. hydration: A, ~ -7.9 (p 0.5); B, ~ -7.7 (p 0.5) 48 h patch test, occlusive no positive reactions 20 min occlusive application no immediate (non-immunologic contact urticaria) reactions Finn chambers were applied the back using Scanpore tape; 48 h Sodium Citrate no reactions 20 min occlusive application no immediate (non-immunologic contact urticaria) reactions Triethyl Citrate Triethyl Citrate 20% in pet. 22 subjects 48- closed patch test not irritating SENSITIZATION Citric Acid Citric Acid 4% in a cuticle cream Citric Acid 2.5% aq. 91 patients w/chronic urticaria or angioedema Triethyl Citrate 4.8% in a blush Distributed for Comment Only -- Do Not Quote or Cite 56 subjects HRIPT; semi-occlusive patches applied 3x/wk for 3 wks; a challenge patch was applied after 2 wks skin prick test Triethyl Citrate 106 subjects HRIPT; 0.2 g applied to a ¾ x ¾ occlusive patch and then moistened; applied 3x/wk for 3 wks; a challenge patch was applied after 2 wks not an irritant or a sensitizer positive results in 3 patients; 1 of the positive reactors also reacted to benzoic and propionic acids not a dermal irritant or a sensitizer CIR Panel Book Page 49

55 Table 10. Dermal irritation and sensitization Test Article Concentration Test Pop. Procedure Results Reference Triethyl Citrate Triethyl Citrate Triethyl Citrate Triethyl Citrate Triethyl Citrate Triethyl Citrate concentration range tested not specified (vehicle alcohol 39C) concentration ranged tested not specified (vehicle alcohol 39C) concentration range tested not specified (vehicle pet.) concentration range tested not specified (vehicle alcohol, SDA 39C) concentration range tested not specified (vehicle pet.) concentration range tested not specified (vehicle pet.) 41 subjects 5 males 36 females 41 subjects 10 males 31 females 45 subjects 10 males 35 females HRIPT; 0.5 ml applied to a Webril patch affixed to an elastic bandage; 9 24-h patches were applied during induction; challenge patches were applied to the test site and an untested site HRIPT; as above HRIPT; as above, except that 0.4 ml was applied 26 subjects modified maximization study: induction: 5 alternate 48-h occlusive patches applied to the back or forearm, with 2.5% SLS pre-treatment; challenge: 48-h semi-occlusive patch, with 2.5% SLS pretreatment not a primary irritant or sensitizer; no effects observed with 15% not a primary irritant or sensitizer; no effects observed with 33.33% not a primary irritant or sensitizer; no effects observed with 33.33% not a sensitizer according to the Kligman scale; irritant effects with 15% at induction ranged from mild erythema to erythema and edema with vesiculation and/ or ulceration; rxns at challenge included minimal to well-defined erythema; no sensitization at 15% 25 subjects as above 1 subject was not patched during challenge due to rxns to substances during induction; rxns at induction included minimal erythema to erythema and edema; rxns at challenge included minimal to well-defined erythema; not a sensitizer according to the Kligman scale; no effects at 33.33% 22 subjects maximization test: induction: 5 alternate 48-h occlusive patches applied to the forearm, with 5% aq. pretreatment with the 1 st patch only; challenge: 48-h semiocclusive patch, with 5% SLS pretreatment (occlusive) Triethyl Citrate 100% 59 subjects HRIPT; 0.4 ml, 20 x 20 mm Webril pad applied with a 40 x 40 mm adhesive square; 9 induction patches Tristearyl Citrate Tristearyl Citrate 25% in olive oil; heated until soluble Distributed for Comment Only -- Do Not Quote or Cite 110 subjects HRIPT; 0.2 ml applied to a 1sq. in. pad of a semi-occlusive patch; induction patches applied 3x/wk for 3 wks; a challenge patch was applied after 2 wks not effects observed with 20% not an irritant or a sensitizer not a primary irritant or sensitizer CIR Panel Book Page 50

56 Table 10. Dermal irritation and sensitization Test Article Concentration Test Pop. Procedure Results Reference Triisostearyl Citrate Triisostearyl Citrate Triisostearyl Citrate Trioctyldodecyl Citrate Laureth-7 Citrate 15.5% in a lip gloss 110 subjects HRIPT; 0.2 g applied to a 1sq. in. pad of a semi-occlusive patch; induction patches applied 3x/wk for 3 wks; a challenge patch was applied after 2 wks neat 114 subjects HRIPT; 150 µl applied to a 2 cm 2 absorbent pad of an occlusive patch; induction patches applied 4x/wk for 3 wks; 4 challenge applications were made on a previously untreated site Trioctyldodecyl Citrate neat 105 subjects HRIPT; 150 µl applied to a 2 cm 2 absorbent pad under a 4 cm 2 occlusive covering; induction patches applied 4x/wk for 3 wks; 4 challenge applications were made on a previously untreated site Laureth-7 Citrate 10%, with ph adjusted to 5.5 (vehicle not given) Distributed for Comment Only -- Do Not Quote or Cite 100 subjects HRIPT;.2 ml applied to a 3/4 sq. in. pad of an occlusive patch; induction patches applied 3x/wk for 3 wks; a challenge patch was applied after 2 wks not an irritant or a sensitizer not an irritant or a sensitizer not an irritant or a sensitizer not an irritant or a sensitizer Abbreviations: DBS dodecylbenzenesulfonate; FCA Freund s complete adjuvant; GPMT guinea pig maximization test; HRIPT human repeated insult patch test; LLNA local lymph node assay; pet petrolatum; PII- primary irritation index; SLS sodium lauryl sulfate; TEWL transepidermal water loss CIR Panel Book Page 51

57 Table 11. Ocular irritation studies Test Article Concentration/Dose Animals/Gp Method Results Reference ALTERNATIVE STUDIES Citric Acid Citric Acid 2% in NaCl --- luminescent bacteria toxicity moderate/ severe ocular 102 test (Microtox test) irritant; EC 50 =14 mg/l Citric Acid undiluted --- EYTEX assay severe/extreme irritant; 103 EDE>51 Triisostearyl Citrate Triisostearyl Citrate 10% in corn oil --- MatTek EpiOcular in vitro toxicity assay ; 100 µl Laureth-7 Citrate Laureth-7 Citrate 5% active µl; HET-CAM test; reactive-time method NON-HUMAN STUDIES Citric Acid Citric Acid (hydrate) 5.0% (0.26 M); ph NZW rabbits. Citric Acid 10 and 30% aq. 3 NZW rabbits modified Draize study; test material was placed directly on central portion of cornea; eyes rinsed in 1 gp 0.1 ml; Draize eye irritation study Citric Acid not given rabbits acute eye irritation/corrosion study Triethyl Citrate 15 and 33.3% in alcohol SDA 39C 3 NZW rabbits Triethyl Citrate 33.3% in pet 3 NZW rabbits Distributed for Comment Only -- Do Not Quote or Cite Triethyl Citrate 0.1 ml; Draize eye irritation study 0.1 ml; Draize eye irritation study non-irritating; ET 50 >256 min slightly irritating; irritation value of 0.00 no corneal opacity in rinsed or unrinsed eyes; conjunctivitis in all animals through day 7 (details not given) 10%: PII = 9.3; minimally irritating 30%: PII = 16.0; mildly to moderately irritating avg. scores (24-72 h): cornea=2.8; iris = 0.0; conjunctiva = 1.7 both concentrations: conjunctival irritation and corneal involvement which did not clear by day 7 conjunctival irritation and corneal involvement cleared on day 7 Trioctyldodecyl Citrate Trioctyldodecyl Citrate neat 6 rabbits 0.1 ml; Draize eye irritation study non-irritating; MMTS = 0.00 Abbreviations: EC 50 concentration causing a 50% reduction in light; EDE EYTEX/Draize equivalent; ET 50 - % viability 50%; MMTS-maximum mean total score; NZW New Zealand white CIR Panel Book Page 52

58 REFERENCES 1. Andersen FA. Final report on the safety assessment of glycolic acid, ammonium, calcium potassium, and sodium glycolates, methyl, ethyl, propyl, and butyl glycolates, and lactic acid, ammonium, calcium, potassium, sodium and TEA-lactates, methyl, ethyl, isopropyl, and butyl lactates, and lauryl, myristyl, and cetyl lactates. Int J Toxicol. 1998;17:(Suppl 1): Food and Drug Administration (FDA). Guidance: Labeling for Cosmetics Containing Alpha Hydroxy Acids. Guidance for Industry, Labeling for Topically Applied Cosmetic Products Containing Alpha Hydroxy Acids as an Ingredient Date Accessed Anastassiadis, S., Morgunov, I. G., Kamzolova, S. V., and Finogenova, T. V. Citric acid production patent review. Recent Pat Biotechnol. 2008;2:(2): Merck & Co., Inc. The Merck Index Date Accessed Berovic, M. and Legisa, M. Citric acid production. Biotechnol Annu Rev. 2007;13: de Guertechin, Luis Oldenhove. Surfactants: Classification. Chapter: 71. Barel, Andre O., Paye, Marc, and Maibach, Howard I.In: Handbook of Cosmetic Science and Technology. 3rd ed. Informa Healthcare; 2009: Gottschalck T.E. and Bailey, J. E. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA.Updated Feb Personal Care Products Council. Concentration of use on additional citrate ingredients Unpublisehd data submitted by the Council on march 15, (1 p). 10. Personal Care Products Council. Updated concentration of use by FDA product category: Citric acid and its salts and esters Unpublished data submitted by the Council of Jan 28, (9 pp). 11. Personal Care Products Council. Concentration of use by FDA product category: Citric acid and its salts and esters Unpublished data submitted by the Council on Jan 6, (9 pp). 12. European Commission. European Commission Health and Consumers Cosmetic Cosing database Date Accessed European Commission. European Commission Health and Consumers Cosmetic Cosing [Cosmetics Directive (v.1)] - Annex III/ Date Accessed Food and Drug Administration (FDA). Code of Federal Regulations Title 21, Chapter 1, Subchapter B, Part 184. Direct food substances affirmed as generally recognized as safe Date Accessed Food and Drug Administration (FDA). Code of Federal Regulations, Title Date Accessed Life Sciences Research Office. Evaluation of the health aspects of citric acid, sodium citrate, potassium citrate, calcium citrate, ammonium citrate, triethyl citrate, isopropyl citrate, and stearyl citrate as food ingredients Prepared for the Bureau of Foods of the Food and Drug Administration, Contract No. FDA Organisation for Economic Co-operation and Development (OECD). SIDS Initial Assessment Report - Citric Acid (CAS No ). =United Nations Environment Programme (UNEP) Chemicals: Date Accessed Hamm LL. Renal handling of citrate. Kidney International. 1990;38: CIR Panel Book Page 53

59 19. Tracor-Jitco, Inc. Scientific Literature Reviews on Generally Recognized as Safe (GRAS) food ingredients. Citric acid NTIS No. PB Fouda HG. Safety assessment of citroflex plasticizers in vitro by serum, liver and intestinal enzymes Unpublished data submitted by the Cosmetic, Toiletry,and Fragrance Association on Dec 4, (31 pp). 21. Slanina P, Falkeborn Y, Frech W, and Cedergren A. Aluminum concentrations int he brain and bone of rats fed citric acid, aluminum citrate or aluminum hydroxide. Fd Chem Toxic. 1984;22:(5): Vittori D, Nesse A, Pérez G, and Garbossa G. Morphologic and functional alterations of erythroid cells induced by long-term ingestion of aluminum. J Inorg Biochem. 1999;76: Madhavi, D. L. and Salunkhe, D. K. Toxicological aspects of food antioxidants. Madhavi, D. L., E, and D.K.SALUNKHE.In: Food science and technology (New york),71. NEW YORK,NEW YORK,USA; BASEL,SWITZERLAND: MARCEL DEKKER,INC.; 1996: Nicolau G, Dahlin DC, Kohlbrenner M, Chan PS, Ronsberg MA, Saunders TK, Yacobi A, and Cervoni P. Skin metabolism and transdermal absorption of viprostol, a synthetic PGE 2 analog, in the rat: Effect of vehicle. Skin Pharmacol. 1989;2: Deuel, H. J., Jr., Greenberg, S. M., Calbert, C. E., Baker, R., and Fisher, H. R. Toxicological studies on isopropyl and stearyl citrates. Food Res. 1951;16:(3): Finkelstein, M. and Gold, H. Toxicology of the citric acid esters: tributyl citrate, acetyl tributyl citrate, triethyl citrate, and acetyl triethyl citrate. Toxicol Appl Pharmacol. 1959;1:(3): Meyers DB, Autian J, and Guess WL. Toxicity of plastics used in medical practice. II. Toxicity of citric acid esters used as plasticizers. J Pharm Sci. 1964;53:(7): Gomez M, Domingo JL, and LLobet JM. Developmental toxicity evaluation of oral aluminum in rats: Influence of citrate. Neurotoxicol Teratol. 1991;13: Bechter, R. and Brouillard, J. F. The effects of different chemical forms of a test compound on embryotoxicity, distribution and metabolism in vitro. Toxicol In Vitro. 1988;2:(3): Brown-Woodman PDC, Post EJ, Chow PYW, and White IG. Effects of malonic, maleic, citric, and caffeic acids onthe motility of human sperm and penetration of cervical mucus. Int J Fertil. 1985;30:(3): Bala S and Grover IS. Antimutagenicity of some citrus fruits in Salmonella typhimurium. Mutat Res. 1989;222:(141): National Toxicology Program. Salmonella study overview with aluminum citrate. Study A &cas%5fno=31142%2d56%2d0&activetab=detail Date Accessed Ghadishah D and Gorchynski J. Airway compromise after routine alpha-hydroxy facial peel administration. Journal of Emergency Medicine. 2002;22:(4): Smith WP. Comparative effectiveness of a-hydroxy acids on skin properties. Int J Cosmet Sci. 1996;18:(2): Smith WP. Hydroxy acids and skin aging. Cosmetics & Toiletries. 1994;109: Green BA and Wildnauer RH. Effect of 10%, 20%, and 25% alpha-hydroxyacid (citric acid) formulations on skin morphology Cited in RIFM (2011) RIFM data synopsis on citric acid. 37. Bernstein, E. F., Underhill, C. B., Lakkakorpi, J., Ditre, C. M., Uitto, J., Yu, R. J., and van Scott, E. Citric acid increases viable epidermal thickness and glycosaminoglycan content of sun-damaged skin. Dermatol Surg. 1997;23:(8): Ditre CM, Griffin TD, Murphy GF, Sueki H, Telegan B, Johnson WC, Yu RJ, and van Scott EJ. Effects of -hydroxy acids on photoaged skin: A pilot clinical, histologic, and ultrastructural study. J Am Acad Dermatol. 1996;34: Berardesca E. Alpha hydroxy acids. Handb.Cosmet.Sci.Technol CAPLUS AN 2001:697206(Conference; General Review). 39 CIR Panel Book Page 54

60 40. Akeson, M. A. and Munns, D. N. Lipid bilayer permeation by neutral aluminum citrate and by three alpha-hydroxy carboxylic acids. Biochim Biophys Acta. 1989;984:(2): Laude EA, Higgins KS, and Morice AH. A comparative study of the effects of citric acid, capasicin and resiniferatoxin on the cough challenge in guinea-pig and man. Pulmonayr Pharmacology. 1993;6: Barros MJ, Zammattio SJ, and Rees PJ. Importance of inspiratoyr flow rate in the cough response to citric acid inhalation in normal subjects. Clinical Science. 1990;78: Allott C, Evan DP, and Marshall PW. A model ofr irritant-induced bronchoconstriction in the spontaneously breathing guineapig. Br J Pharmac. 1980;71: Archer Daniels Midland Co. Citric acid anhydrous, USP/FCC Unpublished data submitted by the Council on Nov. 19, (1 p). 45. ACD/Labs. Advanced Chemistry Development (ACD/Labs) Software (11.02):As cited in Chemical Abstracts Services Registry. Date Accessed American Chemistry Council. U.S. High Production Volume (HPV) Chemical Challenge Program. Robust summaries for acetic acid and salts category Date Accessed Research Institute for Fragrance Materials (RIFM). RIFM data synopsis on citric acid Unpublished document submitted by RIFM on March 21, (39 pp). 48. Archer Daniels Midland Co. Solubility fo citric acid in water Unpublished data submitted by the Council on Nov. 19, (1 p). 49. Archer Daniels Midland Co. ph of citric acid-sodium citrate solutions. Unpublished data submitted byt the Council on Nov. 19, (1 p). 50. Food and Drug Administration (FDA). Code of Federal Regulations, 21CFR Calcium citrate Date Accessed European Commission - European Chemicals Bureau. IUCLID Dataset - Sodium Dihydrogen Citrate Date Accessed Archer Daniels Midland Co. Potassium citrate, USP/FCC Unpublished data submitted by the Council on Nov. 19, (1 p). 53. National Institute for Occupational Safety and Health. International Chemical Safety Cards: trisodium citrate anhydrous Date Accessed European Commission - European Chemicals Bureau. IUCLID Dataset - Trisodium Citrate Date Accessed Research Institute for Fragrance Materials (RIFM). RIFM data synopsis on sodium citrate Unpublished document submitted by RIFM on March 21, (11pp). 56. Gooding CM, Vahlteich HW, and Neal RH. Citric acid esters (US ): 57. Research Institute for Fragrance Materials (RIFM). RIFM data synopsis on triethyl citrate Unpublished document submitted by RIFM on March 21, (33 pp). 58. National Institute for Occupational Safety and Health. International Chemical Safety Cards: Triethyl Citrate Date Accessed Morflex, Inc. Citroflex Citric Acid Esters. Technical Bulletin Unpublished data submitted by the Cosmetic, Toiletry,and Fragrance Association on Dec 4, (15 pp). 60. Salit NI and Sadykov AS. Esters of citric and malic acid. Zhurnal Obshchei Khimii. 1963;33:(8): CIR Panel Book Page 55

61 61. Personal Care Products Council. Information on trisostearyl citrate and triictyldodecyl citrate Unpublished data submitted by the Council on Jan 4, (1 p). 62. Product Investigations, Inc. Determination of the irritating and sensitizing propensities of EX-1028 (triisostearyl citrate) on human skin. Report: PII No Unpublished data submitted by the Council on Jan 4, (12 pp). 63. Cognis. Data profile on Plantapon LC 7 (laureth-7 citrate) Unpublished data submitted to the Council on Feb 22, (3 pp). 64. Food and Drug Administration (FDA). Code of Federal Regulations, 21CFR Ferric citrate Date Accessed Food and Drug Administration (FDA). Code of Federal Regulations, 21CFR Manganese citrate Date Accessed Archer Daniels Midland Co. Process stages: potassium citrate, USP/FCC Unpublished data submitted by the Council on Nov. 19, (1 p). 67. Food and Drug Administration (FDA). Code of Federal Regulations, 21CFR Ammonium citrate, dibasic Date Accessed Environmental Protection Agency. EPA R.E.D. Facts: Citric Acid NTIS No. PB Amidon GE, Peck, GE, Block LH, Moreton RC, Katdare A, Lafaver R, and Sheehan C. Proposed new USP general information chapter, excipient performance. Pharmacopeial Forum. 2007;33:(6): Opdyke DLJ. Monographs on fragrance raw materials. Triethyl citrate. Food Cosmet Toxicol. 1979;17:(4): Lubrizol. G-66 Guerbet Ester (trioctyldodecyl citrate) toxicology studies. TOX Tox summary data submitted by the Council on Jan 4, (2 pp). 72. Gruber, C. M., Jr. and Halbeisen, W. A. A study on the comparative toxic effects of citric acid and its sodium salts. J Pharmacol Exp Ther. 1948;94:(1): Al-Ani FY and Al-Lami SK. Absence of mutagenic activity of acidity regulators in the Ames Salmonella/microsome test. Mutat Res. 1988;206: Ishidate M, Jr, Sofuni T, Yoshikawa K, Hayashi M, Nohmi T, Sawada M, and Matsuoka A. Primary mutagenicity screening of food additives currently used in Japan. Fd Chem Toxicol. 1984;(8): Litton Bionetics, Inc. Mutagenic evaluation of compound FDA 71-54, citric acid NTIS No. PB Hayes S, Gordon A, Sadowski I, and Hayes C. RK bacterial test for independently measuring chemical toxicity and mutagenicity: Short-term forward selection assay. Mutat Res. 1984;130: Hartwig, A. and Schlepegrell, R. Induction of oxidative DNA damage by ferric iron in mammalian cells. Carcinogenesis. 1995;162:(12): Litton Bionetics, Inc. Mutagenic evaluation of compound. FDA , Potassium Citrate, NF. FCC Granular NTIS #PB Litton Bionetics, Inc. Mutagenic evaluation of compound FDA , Sodium Citrate, USP, FCC hydrous, granular NTIS No. PB Litton Bionetics, Inc. Mutagenic evaluation of compound FDA , Triethyl Citrate, FCC NTIS No. PB Wolfreys AM and Basketter DA. Mutagens and sensitizers - An unequal relationship? J Toxicol Cutaneous Ocul Toxicol. 2004;23:(3): CIR Panel Book Page 56

62 82. NOTOX B.V. Evaluation of the mutagenic activity of EX-1028 (triisostearyl citrate) in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). NOTOX Project No Unpublished data submitted by the Council on Jan 4, (26 pp). 83. RCC. Salmonella typhimurium reverse mutation assay with C-SAT (laureth-7 citrate). RCC - CCR study no Unpublished data submitted to the Council on Feb 22, (16 pp). 84. F. Hoffmann-LaRoche Ltd. Unpublished report, dermal irritation test, occlusive patch Unpublished data cited in OECD Kowalski RL and Hartnagel RE. Unpublished report; acute dermal and ocular irritation/corrosion study in rabbits Unpublished data cited in OECD Hill Top Biolabs, Inc. D.O.T. corrosivity potential study in rabbits of: citric acid solution, 60% Unpublished data cited in American Chemistry Council (2001). 87. Sousa, S. M., Bramante, C. M., and Taga, E. M. Biocompatibility of EDTA, EGTA and citric acid. Braz.Dent.J. 2005;16:(1): Unilever Limited. Sensitization potential of Citroflex A2, Citroflex A4,a nd Citroflex 2 (triethyl citrate) tested in guinea pigs Unpublished data submitted by the Cosmetic, Toiletry,and Fragrance Association on May 12, (31 pp). 89. Laden K. Studies on irritancy and stinging potential. J Soc Cosmet Chem. 1973;24: Schliemann-Willers S, Fuchs S, Kleesz P, Grieshaber R, and Elsner P. Fruit acids do not enhance sodium lauryl sulphate-induced cumulative irritant contact dermatitis in vivo. Acta Derm Venereol. 2005;85: Spoo J, Wigger-Alberti W, Berndt U, Fischer T, and Elsner P. Skin cleansers: Three test protocols for the assessment of irritancy ranking. Acta Dermato-Venereol. 2002;82:(1): Torgerson RR, Davis MDP, Bruce AJ, Farmer SA, and Rogers RS. Contact allergy in oral disease. J Am Acad Dermatol. 2007;57: Lahti, A. Nonimmunologic contact urticaria. Acta Dermato.-Venereol Suppl. 1980;60:(91): Reider N, Issa A, Hawranek T, Schuster C, Aberer W, Kofler H, Fritsch P, and Hausen BM. Absence of contact sensitization to Aloe veraa (L.) Burm. f. Contact Dermatitis. 2005;53: Clinical Research Laboratories Inc Repeated insult patch test of a cuticle cream containing 4% Citric Acid. CRL Study Number: CRL Unpublished data submitted by Personal Care Products Council. 96. Malanin G and KAlimo K. The results of skin testing with food additives and the effect of an elimination diet in chronic ad recurrent urticaria and recurrent angioedema. Clinical and Experimental Allergy. 1989;19: Consumer Product Testing Co Repeated insult patch test of a powder blush containing 4.8% Triethyl Citrate. Experiment Reference Number: C Unpublished data submitted by Personal Care Products Council. 98. Hill Top Research. Repeated insult patch test on Citroflex 2 liquid (triethyl citrate), Citroflex A-2 liquid, and Citroflex A-4 liquid Unpublished data submitted by the Cosmetic, Toiletry,and Fragrance Association on Dec 4, (3 pp). 99. Consumer Product Testing Co. Repeated insult patch test on test material C-SAT (tristearyl citrate). Experiment Ref. No. C Unpublished data submitted by the Council on Dec 10, (14 pp) Consumer Product Testing Co Repeated insult patch test of a lip gloss containing 15.5% Triisostearyl Citrate. Experiment Reference Number: C Unpublished data submitted by Personal Care Products Council Consumer Product Testing Co. Repeated insult patch test with C-SAT (laureth-7 citrate). Experiment Ref. No. C Unpublished data submitted to the Council on Feb 22, (13 pp) Bulich AA, Tung K-K, and Scheibner G. The luminescent bacteria toxicity test: Its potential as an in vitro alternative. J Biolumin Chemilumin. 1990;5: CIR Panel Book Page 57

63 103. Gordon VC. Utilization of biomacromolecular in vitro assay systems in the prediction of in vivo toxic responses. Lens and Eye Toxicity Research. 1992;9:(3&4): Consumer Product Testing Co. The MatTek corporation EpiOcular TM tissue model in vitro toxicity testing system. Experiment Ref. No. V Unpublished data submitted to the Council on Jan 4, (5 pp) Henkel KGaA. Final report on the in vitro HET-CAM [HCl] - reaction time method - with BHL-Fo-115 "H" (laureth-7 citrate) tested as 5% active substance Unpublished data submitted to the Council on Feb 22, (5 pp) Murphy JC, Ostenberg RE, Seabaugh VM, and Bierbower GW. Ocular irritancy responses to various phs of acids and bases with and without irrigation. Toxicology. 1982;23:(4): F. Hoffmann-LaRoche Ltd. Unpublished report; Draize ocular irritation study in rabbits. Unpublished data cited in OECD CIR Panel Book Page 58

64 Data

65 COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal CareProducts Council Committed to Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CW Expert Panel DATE: November 19, 2010 SUBJECT: Unpublished Information on Citric Acid and Potassium Citrate Archer Daniels Midland Co Citric Acid Anhydrous, USP/FCC. Archer Daniels Midland Co Material safety data sheet: Citric Acid Anhydrous. Archer Daniels Midland Co ph of Citric Acid-Sodium Citrate solutions. Archer Daniels Midland Co Solubility of Citric Acid in water. Archer Daniels Midland Co Potassium Citrate, USP/FCC. Archer Daniels Midland Co Material safety data sheet: Potassium Citrate granular. Archer Daniels Midland Co Process stages: Potassium Citrate, USPIFCC th Street, N.W, Suite 300 Washington, D.C (fax) CIR Panel Book Page 59

66 .. 19, aojo Distributed for Comment Only -- Do Not Quote or Cite Citric Acid Anhydrous, USPI FCC , , Acidulant ArcherDaniels Midland Co. Technical Services Phone: Fax: Description Citric acid anhydrous is widely used in the food, beverage, and pharmaceutical industries to impart a clean, refreshing tartness. Its prime use is as an acidulant, but it is also used as sequestrant ofmetal ions to give protection from the development of offflavors and off-odors in certain foodstuffs. A major industrial use of citric acid is in the manufacture of ecologically compatible detergents. It is also used in chemical cleaning, concrete admixtures, plasticizers, and a range of other applications. General Characteristics Formula Molecular Weight Solubility (g/ 100 ml at 25 C) In Water 162 In Alcohol 59.1 Taste Tart Standard Sncci Appearance Odor Identification Clarity of Solution Color of Solution Assay (anhydrous basis) Water Residue on Ignition Limit ofoxalic Acid Sulfate Heavy Metals (as lead) Lead Readily Carbonizable Substances Tridodecylamine Oxalate H3C6H507 Free-flowing, colorless, translucent crystals or as a white granular to fine crystalline powder. None Meets USP/ FCC Meets USP Meets USP 99.5 to 100.5% Maximum 0.5% Maximum 0.05% Maximum 0.036% Maximum 0.015% Maximum 5.0 ppm Maximum 0.5 ppm Not darker than matching fluid K. Not Applicable. ADM does not use solvent extraction process. Passes Test Ingredients Citric Acid Storae & Shelf Life Storage below 75 F and 55% relative humidity in tightly sealed container is recommended. This product is very stable and has been given a 3 year best by date. It is recommended to retest after 3 years. Granulations USS Sieves (microns) Granular On No. 16 (1 180 j.t), maximum 2% Through No.50 (300j.i), maximum 10% Fine Granular On No. 30(600j.t), maximum 3% Through No. 100 (150ii), maximum 5% Powder On No. 60 (250!,), Maximum 2% Through No. 200 (75ji), minimum 50% Packagine & Packaging Code Granular 50 lb. bags OH 25 kg bags O-2P 250 lb. drums S 1000 kg totes R Fine Granular 50 lb. bags OH 25 kg bags P 250 lb. drums S 1000 kg totes R Powder 200 lb. drums S Regulatory Status This Food Additive complies with all the compendial requirements of the U.S. Pharmacopeia, Food Chemical Codex, Code offederal Regulations, European Pharmacopoeia, British Pharmacopoeia, Japanese Pharmacopeia, and WHO.! F.A.O. Food Additive Specification. CAS Number: CA-00l For customers around the world, ADM draws on its resources its people, products, and market perspective to he p them meet today s consumer demands and envision tomorrow s needs. ADM The information contained herein is correct as of the date ofthis docnment to the host of oar knowledge. Any rccomn,endatinns or suggestions are made mcithotmt guarantee or rcprcsentntion as In eesalts and are subject to change svilhout notice. We suggcsm roil cvalaate aoy recommendations and suggestions indcpcodentlv. WE DtSCLAIM ANY AND ALL WARRANTIES. \VFIETttER EXPRESS OR IMPLIED, AND SPECIFICALLY DiSCLAIM THE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE. AND NON-1NPPJNGEMENT. Oar responsibility for claims arising from any claim for broach of wnernnty, negligence, or otherwise shall not inelade canseqnentiol, special, or incidental dansagcs, and is limited to the psirchase price of ntatcrial psrclmased from us. None of the statemnenlt made here shnll be construed as a graut, either express or implied, of nny license auder any patent held by Archer Daniels Midland Company or nther parlict. Customers ore responsible for obtaining any hermes oe other rights that nay he oecessary to make, smse, or sell products corstairming Archer Daniels Midland Company ingredients. CIR Panel Book Page 60 f-fl specialtyproducts@adm.com

67 ADM Material Safety Data Sheet r 1. PRODUCT AND COMPANY IDENTIFICATION Product Name: Citric Acid Nihydrous Contact Manufacturer: Archer Daniels Midland Company Product Code: , , Faries Parkway Decatur, IL 62526, USA Synonyms: Telephone Number: Acido 2-Hydroxy-1,2,3-Propanetricarboxyfic. Aciletten. Acido beta HydroxytricarballAic. Citrate. Citretten. Citronensaeure. NSC EmergencyTelephone Number: Acido 2-hydroxypropane-1,2,3-tricarbox1ic. E 330. Chemtrec Use of the Substance I Preparation: Food additi Revision Date 22-Apr HAZARDS IDENTIFICATION Emergency Overview Irritating to eyes, Irritating to skin, Corrosie to metals (as aqueous solution). Appearance White Colorless Physical State Solid Powder Odor Odorless Potential Health Effects Principle Routes of Exposure Acute Effects Eyes Skin Inhalation Ingestion Chronic Effects Aggravated Medical Conditions Potential Environmental Effects Toxicological information Eye contact, Skin contact, Inhalation, Ingestion. Contactwith eyes maycause irritation. Aid contact with skin. Prolonged skin contact may cause skin irritation. May cause irritation of respiratorytract. Ingestion maycause irritation to mucous membranes. Avid repeated exposure. No information ailable.. See Section 12 for additional ecological information. See Section 11 for additional toxicological information. NorTh America Page 1/7 CIR Panel Book Page 61

68 Revision Date 22-Apr-2009 Distributed for Comment Only -- Do Not Quote or Cite , , Citric Acid Anhydrous 3. COMPOSITION/INFORMATION ON INGREDIENTS Chemical Family Acids The following component(s) in this product are considered hazardous under applicable OSHA (USA), WHMIS (Canada), andlor NOM-002-SCT-2003 (Mexico) regulations Chemical Name CAS-No Weight % North American Hazard Indicator Citric acid yes 4. FIRSTAID MEASURES General Advice Eye Contact Skin Contact Inhalation Ingestion Notes to Physician Protection of First-aiders j Flammable Properties Suitable Extinguishing Media Unsuitable Extinguishing Media If symptoms persist, call a physician. Show this safetydata sheetto the doctor in attendance. Immediatelyflush with plentyofwater. After initial flushing, remove anycontact lenses and continue flushing for at least 15 minutes. Keep eyes wide open while rinsing. If symptoms persist, call a physician. Wash off immediatelywith soap and plentyof water removing all contaminated clothes and shoes. Move to fresh air. Clean mouth with water and afterwards drink plentyof water. Treat symptomatically. Use personal protective equipment. 5. FIRE-FIGHTING MEASURES Fine dust dispersed in air may ignite. Dry chemical. Carbon dioxide (CC 2). Water spray. Foam. Use extinguishing measures that are appropriate to local circumstances and the surrounding environment. No information available. Hazardous Combustion Products Explosion Data Sensitivity to mechanical impact Sensitivity to static discharge Specific Hazards Arising from the Chemical Carbon monoxide (CC), Carbon dioxide (CC 2),Thermal decomposition can lead to release of irritating gases and va pours. No No None known Protective Equipment and Precautions for Firefighters As in anyfire, wear self-contained breathing apparatus pressure-demand, MSHNNIOSH (approd or equivalent) and full protective gear. NorthAmerica Page 217 CIR Panel Book Page 62

69 Revision Date 22-Apr-2009 Distributed for Comment Only -- Do Not Quote or Cite , , Citric Acid Anhydrous NFPA Health 1 Flammability 1 Stability and Reactivity 0 Physical hazard - 6.ACCIDENTAL RELEASE MEASURES Personal Precautions Use personal protective equipment. Environmental Precautions Methods for Clean-up Prevent further leakage or spillage if safe to do so. Prevent product from entering drains. Pick up and transfer to properly labelled containers. Avoid dust formation. Keep in suitable, closed containers for disposal. 7.HANDLINGANDSTORAGE Handling Storage Wear personal protective equipment. Aveid contact with skin, eyes and clothing. Handle in accordance with good industrial hiene and safetypractice. Do not breathe vapours/dust. Use only in area provided with appropriate exhaust ventilation. Aveid dust formation in confined areas. Fine dustdispersed in air may ignite. Keep containers tightlyclosed in a cool, well-ventilated place. Keep in properly labelled containers. 8. EXPOSURE CONTROLS I PERSONAL PROTECTION Exposure Limits This product is not known to contain any hazardous materials with occupational exposure limits established bythe region specific regulatory bodies. Engineering Measures Personal Protective Equipment Eyelface Protection Skin and Body Protection Respiratory Protection General Hygiene Considerations Ensure adequate ventilation, especially in confined areas. Tightlyfitting safety goggles. Long sleeved clothing, Boots, lmperaous gloves. Breathing apparatus with filter. When using, do not eat, drink or smoke. Regular cleaning of equipment, work area and clothing. North America Page 3/7 CIR Panel Book Page 63

70 Revision Date 22-Apr-2009 Distributed for Comment Only -- Do Not Quote or Cite , , Citric Acid Anhydrous 9. PHYSICAL AND CHEMICAL PROPERTIES Appearance White Colorless Physical State Solid Powder Odor Odorless Odor Threshold No information available Flash Point 345 C I 653 F Autoignition Temperature 1010 C I F Boiling point No information available Melting/Freezing Point 153 C/307 F Flammability Limits in Air No information available Explosion Limits No information available ph 2.1 Vapor Pressure No information available Water Solubility Soluble Specific Gravity No information available Evaporation Rate No information available Vapor Density No information available Density STABILITY AND REACTIVITY Chemical Stability Conditions to Avoid Incompatible Materials Hazardous Decomposition Products Possibility of Hazardous Reactions Stable under normal conditions. Incompatible products. Arnines. Heavy metals. Strong oxiding agents. Strong bases. No information available. Hazardous polym erization does not occur. 11. TOXICOLOGICAL INFORMATION Acute Toxicity Product Information LD5O Oral: 3000(rat) mg/kg LD5O Dermal: No information available LC5O Inhalation: No information available Toxicology data for the components Chemical Name Weight % LD5O Oral Otric acid n/kg Rat LD5O Dermal LC5O Inhalation Chronic Effects Carcinogenicity There are no known carcinogenic chemicals in this product. OSHA: (Occupational Safety & Health Administration) Not Listed ACG1H: (American Conference of Governmental Industrial Hygienists) Not Listed NTP: (National Toxicity Program) Not Listed Mexico: (Official Mexican Norm NOM-010-STPS-1 999) Not Listed IARC: (International Agency for Research on Cancer) Not Listed NorthAmerica Page 4/7 CIR Panel Book Page 64

71 Revision Date 22-Apr , , Citric Acid An hydrous Subchronic Toxicity Corros ivity Neurological Effects Reproductive Effects Teratogenicity No information available. Corrosive to metals (as aqueous solution). No information available. No information available. No information available. Irritation Sensitization Mutagenic Effects Developmental Effects Target Organ Effects No information available. No information available. No information available. No information available. No information available. 12. ECOLOGICAL INFORMATION Ecotoxicity Environmental properties. Chemical Name Acute Fish Toxicity Oanhnia (Water flea) Fresh Water Algae Citric acid 96 Hr LC50 Leporris rrecrochirus: 72 Hr EC5O Daphnia rnagna: rmil [static rmil Chemical Name Weight% log Pow Citric acid Persistence/Degradability Readily biodegradable. Bioaccumulation/Accumulation No information available.... Mobility Soluble DISPOSAL CONSIDERATIONS Waste Disposal Methods Contaminated Packaging Hazardous waste. Dispose of in compliance with the laws and regulations pertaining to this product in sour jurisdiction. Emptycontainers should be taken for local recling, recoryor waste disposal. Domestic transport regulations (US DOT Not regulated 14. TRANSPORT INFORMATION Domestic transport regulations (Canada) TDG Not regulated Domestic transport regulations (Mexico) MDC Not regulated North America Page 5/7 CIR Panel Book Page 65

72 New Australian Domestic Distributed for Comment Only -- Do Not Quote or Cite Revision Date 22-Apr , , Citric Acid Anhydrous International transport regulations ICAO Not regulated IATA Not regulated IMDG IMO Not regulated International Inventories 15. REGULATORY INFORMATION Legend TSCA-. Todc Substances Control Act, Section 8(b) Inventory (USA). DSL - Substance List (Canada). NDSL - Non Domestic Substances List (Canada). EINECS- European InntoryofEdsting Commercial Chemical Substances (EU). ELINCS European List of Notified Chemical Substances (EU). AJCS - Inventory of Chemical Substances (Australia). ENCS - Edsting and New Chemical Substances (Japan). CHINA- Chinese InventoryofEdsting Chemical Substances (China). PICCS - Inentory of Chemicals and Chemical Substances (Philippines). KECL - Korean Edsting and Evaluated Chemical Substances (Korea). NZLoC - Zealand Inventory of Chemicals (New Zealand) USA Federal Regulations Ozone Depleting Substances: No Class I or Class II material is known to be used in the manufacture of, or contained, in this product. SARA 313 Section 313 of Title Ill of the Superfund Amendments and Reauthorization Act of 1986 (SARA). This product is not known to contain any chemicals which are subject to the reporting requirements of the Act or regulations contained in 40 CFR 372. SARA 311/312 Hazardous Categorization Acute Health Hazard Chronic Health Hazard Fire Hazard Sudden Release of Pressure Hazard Reactive Hazard Yes No No No No Clean Air Act, Section 112 Hazardous Air Pollutants (HAPs) (see 40 CFR 61) This product is not known to contain any HAPs. State Regulations California Proposition 65 Proposition 65 chemicals are not expected to be found in this product abo those naturally present in their agricultural source. Proposition 65 exempts naturallyoccurring listed chemicals from an obligation to label. State Right-to-Know No known components subject to Right-To-Know legislation in the following States: Chemical Name Weight % Illinois Massachusetts New Jersey Pennsylvania Rhode Island Citric acid No Nb No No No North America Page 617 CIR Panel Book Page 66

73 Revision Date 22-Apr-2009 Distributed for Comment Only -- Do Not Quote or Cite , , Citric Acid Anhydrous Canada WHMIS Product Classification Class E: Corrosive Material. WHMIS Ingredient Disclosure List IDL Component Information L hemical Name Citric acid I Weight % WHMIS IDL Threshold limits Listed 1 % (NPRI) Canadian National Pollutant Release Inventory No known component is listed on NPRI. This product has been classified in accordance with the hazard criteria of the Controlled Products Regulations (CPR) and the SOS contains all the information required by the CPR. Mexico Mexico - Grade Slight risk, Grade OTHER INFORMATION Prepared By Preparation Date Revision Date Revision Summary Specialty Food Ingredients 22-Apr Apr-2009 Implementation into software system. 1 Disclaimer The information provided on this (M)SDS is correct to the best of our knowledge, information and belief at the date of its publication. The information given is designed only as a guide for safe handling, use, processing, storage, transportation, disposal and release and is not to be considered as a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other material or in any process, unless specified in the text. End of (M)SDS North America Page 7/7 CIR Panel Book Page 67

74 - A ic _ejuo r 1oI ( I 4ADM Distributed for Comment Only -- Do Not Quote or Cite ph OF CITRIC ACID- SODIUM CITRATE SOLUTIONS* ThCHNICAL SERVICES 4666 FARIES PARKWAY DECATUR, IL PHONE: 217/ FAX: 217/ CITRIC ACID % S D 1.5 I 2 U M 4 : C 6 I 7 T 8 R 10 A 12 T 14 E % *The figures in this table apply only to water solutions. In foods the ph may be different. The concentrations are expressed as percent by weight citric acid anhydrous - sodium citrate dihydrate. I-he intcrmation contained herein is correct an of the date of this document to the beet of our knowledge. Any recemmendetiono or suggestions are made usthour guarantee or representation as to results and are subject to change without notice. We suggest you evaluate any reccmmendatioris and suggestions ondependestly. WE DISCLAIM ANY AND ALL WARRANTIES, WHETHER EXPRESS OR IMPLIED, AND SPECIFICALLY DISCLAIM THE IMPLIED WARRANTIES OF MEPCHANTASTLITY, FITNESS FOR A PARTICULAR PURPOSE, AND NON lnpp.tngement. Our responsibility far claims arising from any claim fur breech of aerrnnty, negligence, or otherwise shall sot incluae cossecuestia I, s,oecial, or incidental damages, and is limited to the purchase price op meter! 51 purchased Eros us. NOne of the statementu made here shall he construed as a grant, either eupress or implied, of any license under any patert held by Archer Daniels Mudlend Comoasy or other parties. Customers are responsible for obtaining any locenues or other rights that may be necessary to make, Jan, or sell products 000taiaing Archer Daniels MIdland Coepany logredients. CA Division of Archer Daniels Midland Company - CIR Panel Book Page 68

75 .,,.. i9o Distributed for Comment Only -- Do Not Quote or Cite ADM SOLUBILITYOF CITRIC ACID IN WATER , 430Q w TEMPERATURE DEGREES C. The information contained herein is correct as of the date of this document to the best of our knowledge. Any recommendations or suggestions are made without guarantee or representation as to results and are subject to change without notice. We suggest you evaluate any recommendations and suggestions independently. WE D1SCLAP ANY AND ALL WARRANTIES, WHETHER EXPRESS OR IMPLIED, AND SPECIFICALLY DISCLAIM THE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE. AND NON-INFRINGEMENT. Our responsibility for claims arising from any claim for breach of warranty, negligence, or otherwise shall not include consequential, special, or incidental damages, and is limited to the purchase price of material purchased from us. None of the statements made here shall be construed as a grant. either express or implied, of any license under any patent held by Archer Daniels Midland Company or other parties. Customers are responsible for obtaining any licenses or other rights that may be necessary to make, use, or sell products containing Archer Daniels Midland Company ingredients. CA-035-OOl 107 ARCHER DANIELS MIDLAND COMPANY BOX 1470 DECATUR, ILLINOIS TEL: 800/ or 217/ FAX: 217/ CIR Panel Book Page 69

76 37.2,... - pjoj 19 go?0 Distributed for Comment Only -- Do Not Quote or Cite Potassium Citrate, USP/ FCC Acidulant Archer Daniels Midland Co. Technical Services Phone: Fax: Description Potassium citrate is a highly soluble monohydrate salt, derived from citric acid and available as a crystalline granular material. Potassium citrate may be used as a partial or complete replacement for sodium citrate when reduced sodium or sodium free products are desired. Federal Standards of Identity for processed cheese allow the use of potassium citrate as an emulsifying salt. The substitution of potassium salt for sodium citrate will reduce the sodium content by 720 mg per 100 g product at 3% salt level. Potassium citrate will reduce sodium content in beverages, gelatin desserts, confections, jams and jellies. Potassium citrate may also be used as a source of potassium ion either as a nutritional supplement or to maximize gelation of kappa-carrageenan gels in applications such as surimi analogues. General Characteristics Formula K5C6HO;H,O Molecular Weight Potassium Content % LossonDrying 3 6% ph (5 g/ 100 ml at 25 C) Solubility (g/ lou ml at 25 C) Water 190 Alcohol Insoluble Ether Insoluble Standard Specifications Appearance White coarse powder, essentially free of foreign matter Odor None Identification Meets USP/ FCC Assay (dried basis) 99.0 to % Loss on Drying % Alkalinity Meets USP/ FCC Tartrate Meets USP Heavy Metals (as lead) Maximum 10.0 ppm Organic Volatile Impurities Meets USP Lead Maximum 2ppm Ingredients Potassium Citrate Storage This product is very hygroscopic and must be protected, during storage, from exposure to high temperatures and relative humidity. Storage below 75 F and 55% relative humidity in tightly sealed container is recommended. However, it is recommended to use the product within three years. Potassium citrate readily deliquesces in moist air. Adherence to the above storage conditions is highly recommended. Inspect product after six months of storage. Other than possible caking or altered water content, no effect ofproduct is anticipated from longer storage periods. It is suggested that all products be used on a First-Tn First-Out basis. G ranu lati o ns USS Sieves (microns) Granular On No. 16 (1180p), maximum 1% Through No. l00(150p), maximum 10% Packaging & Packaging Code Granular 50 lb. bags OH 200 lb drum kg tote R Regulatory Statns This Food Additive complies with all the compendial requirements of the U.S. Pharmacopeia, Food Chemical Codex, Code of Federal Regulations, European Pharmacopoeia, British Pharmacopoeia, Japanese Pharmacopeia, and WHO.! F.A.O. Food Additive Specification. CAS Number: CA t4 For customers around the world, ADM draws on its resources its people, products, and market perspective to help them meet today s consumer demands and envision tomorrow s needs. 4ADM wwwadrn corn J/ specialtyproducts@adrn corn I, --. The information containcd herein in correct an of the elate of this rioe,,ment tee the bent of our keoseleeige. Any rreommenelalions or snggestions re ntaele snithout getarantee or representation as to retolts and are seebjeet to change witho,,t notice. We n ggent yos evaluate smy recommendations and suggestions indepcoelently. WE DISCLAIM ANY AND ALL WARRANTIES. WHETHER EXPRESS OR tmplied, ANO SPECIFICALLY 0ISCLAIM TIlE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, AND NON-1NFRINOEMENT. O,,r eenponsihiltity foe claims arising froto asp clam, for breach of trorronly, negligence. or otherwise shall not include comequential, speeiol, or incidental damages, and in lintoed to the pterehane price of material porehosed froto on. None of the statements made here shall he ennnlroed ona grant. eiil,er enpresn or iotpleed. of any license ender any potent held by Aeeher Daniels Midland Cotopany or other portien. Contotners ore responsible for obtoining any licenses or otlter righto that otoy he tteeensory to,ttohe, ese, oe nell produce containing Archer Dattteln Elediand Cotopony ingredients. CIR Panel Book Page 70

77 4ADM Material Safety Data Sheet :ion Date 02-Mar-2009 Revision Date 01-May PRODUCT AND COMPANY IDENTIFICATION Product Name: Product Code: Potassium Citrate Granular Synonyms: 1,2,3-Propanetricarbolic acid, 2-hydroxy-, potassium salt, h1rate (1:3:1). CAS / EINECS Use of the Substance I Preparation: Food additve Contact Manufacturer: Ncher Daniels Midland Company 4666 Faries Parkway Decatur, IL 62526, USA Telephone Number: Emergency Telephone Number: Chemtrec HAZARDS IDENTIFICATION Emergency Overview The product contains no substances which at their gi en concentration, are considered to be hazardous to health. Appearance Colorless to White Physical State Powder / Crystalline Odor Odorless Granules Potential Health Effects Principle Routes of Exposure Acute Effects Eyes Skin Inhalation Ingestion Chronic Effects Main Symptoms Aggravated Medical Conditions Potential Environmental Effects Toxicological information Eye contact, Skin contact, Inhalation, Ingestion. May cause slight irritation Maycause slight skin irritation May cause irritation of respiratorytract Maybe harmful if swallowed. Repeated contact may cause allergic reactions in very susceptible persons Repeated orprolonged exposure maycause irritation of eyes and skin. Shortness of breath. No information available. See Section 12 for additional ecological information. See Section 11 for additional todcological information. North America Page 117 CIR Panel Book Page 71

78 Revision Date 01-May Potassium Citrate Granular 3. COMPOSITION/INFORMATION ON INGREDIENTS Chemical Family Esters Formula K3C6H507@H Non-hazardous Components Chemical Name CAS-No Weight % North American Hazard Indicator Tripotassium citrate mon ohj rate FIRSTAID MEASURES Eye Contact Skin Contact Inhalation Ingestion Notes to Physician I. 5. Rinse thoroughlywith plenty of water, also under the eiids Wash off with warm water and soap. Move to fresh air Clean mouth with water and afterwards drink plentyof water Treatsyrnptomatically FIRE-FIGHTING MEASURES.. flammable Properties Fine dustdispersed in air may ignite. i Suitable Extinguishing Media Unsuitable Extinguishing Media Hazardous Combustion Products Explosion Data Sensitivity to mechanical impact Sensitivity to static discharge Specific Hazards Arising from the Chemical Water. Carbon diodde (C0 2). Foam. Drychemical. Use extinguishing measures that are appropriate to local circumstances and the surrounding environment. Dry powder. No information available. Carbon monodde (CO), Carbon diodde (C02). No information available. No information available. Combustible material. Protective Equipment and Precautions for Firefighters As in anyfire, wear self-contained breathing apparatus pressure-demand, MSH,NNIOSH (approved or equivalent) and full protective gear. NFPA Health 0 flammability 1 Stability and Reactivity 0 Physical hazard - North America Page 217 CIR Panel Book Page 72

79 Revision Date O1-May Potassium Citrate Granular 6. ACCIDENTAL RELEASE MEASURES Personal Precautions Environmental Precautions Methods for Containment Methods for Clean-up Ensure adequate ventilation Prevent further leakage or spillage if safe to do so. Keep in suitable, closed containers for disposal. Shovel or sweep up. Avoid dustformation. After cleaning, flush awaytraces with water. 7.HANDLINGANDSTORAGE Handling Storage Ensure adequate ventilation. Aveid dustformation in confined areas. Fine dustdispersed in air may ignite. Keep in a dry, cool and well-ventilated place. 8. EXPOSURE CONTROLS I PERSONAL PROTECTION Exposure Limits This product is not known to contain any hazardous materials with occupational exposure limits established by the region specific regulatory bodies. Engineering Measures Protective Equipment Eyelface Protection Skin and Body Protection Respiratory Protection General Hygiene Considerations Ensure adequate ventilation, especially in confined areas Safety glasses with side-shields. Long sleeved clothing, lmperous gloves. Res pirator with a dust filter. Handle in accordance with good industrial hygiene and safety practice 9. PHYSICAL AND CHEMICAL PROPERTIES Appearance Colorless to White Physical State Powder / Crystalline Granules Odor Odorless Odor Threshold No information available Hash Point Not applicable Autoignition Temperature No information available Boiling point Not applicable MeltinglFreezing Point Decomposes before melting Hammability Limits in Air No information available Explosion Limits No information available ph approx. 8.0 Vapor Pressure No information available Solubility Insoluble Acohol Water Solubility (>50%) Specific Gravity No information available Evaporation Rate No information available Vapor Density No information available NorthAmerica Page 317 CIR Panel Book Page 73

80 Revision Date O1-May Potassium Citrate Granular 10. STABILITY AND REACTIVITY Chemical Stability Conditions to Avoid Incompatible Materials Hazardous Decomposition Products Possibilityof Hazardous Reactions Stable under normal conditions No information available. No information available. No information available. None under normal processing 11. TOXICOLOGICAL INFORMATION Acute Toxicity Product Information LD5O Oral: No information available LD5O Dermal: No information available LC5O Inhalation: No information available Toxicology data for the components No information available Chronic Effects Carcinogenicity There are no known carcinogenic chemicals in this product. OSHA: (Occupational Safety & Health Administration) Not Listed ACGIH: (American Conference of Governmental Industrial Hygienists) Not Listed NTP: (National Toxicity Program) Not Listed Mexico: (Official Mexican Norm NOM-Ol 0-STPS-1 999) Not Listed IARC: (International Agency for Research on Cancer) Not Listed Subchronic Toxicity Corros ivity Neurological Effects Reproductive Effects Teratogenicity No information available. No information available. No information available. No information available. No information available. Irritation Sensitization Mutagenic Effects Developmental Effects Target Organ Effects No information available. No information available. No information available. No information available. No information available. North America Page 417 CIR Panel Book Page 74

81 Revision Date O1-May Potassium Citrate Granular 12. ECOLOGICAL INFORMATION Ecotoxicity Contains no substances known to be hazardous to the environment. Contains no substances known to be notdegradable in waste water treatment plants. PersistencelDegradability Bioaccumulationl Accumulation Mobility Readily biodegradable. No information available. Partly soluble in water. 13. DISPOSAL CONSIDERATIONS Waste Disposal Methods Contaminated Packaging Dispose of in compliance with the laws and regulations pertaining to this product in ur jurisdiction. Can be landfilled or incinerated, when in compliance with local regulations. Empty containers should be taken for local recvling, recoryor waste disposal Domestic transport regulations (US DOT Not regulated 14.TRANSPORT INFORMATION Domestic transport regulations (Canada) TDG Not regulated Domestic transport regulations (Mexico) MEX Not regulated International transport regulations ICAO Not regulated IATA Not regulated IMDGIMO Not regulated 15. REGULATORY INFORMATION International Inventories The components of this product are reported in the following inntories: Chemical TSCA DSL NDSL EINECS ELINCS AICS ENCS CHINA PICCS KECL NZLoC Name Tripotassium Yes Yes No Yes No Yes Yes Yes Yes Yes Yes citrate KE rronohydrate NorthAmerica Page 5/7 CIR Panel Book Page 75

82 New Distributed for Comment Only -- Do Not Quote or Cite Revision Date O1-May Potassium Citrate Granular Legend TSCA- Toxic Substances Control Aut, Section 8(b) lnventory(usa). DSL - Domestic Substance List (Canada). NDSL - Non Domestic Substances List (Canada). EINECS - European lnntoryof Existing Commercial Chemical Substances (EU). ELINCS - European List of Notified Chemical Substances (EU).?JCS-Australian Inventoryof Chemical Substances (Australia). ENCS E)dsting and New Chemical Substances (Japan). CHINA- Chinese lnventoryof Existing Chemical Substances (China). PICCS - ln ntory of Chemicals and Chemical Substances (Philippines). KECL - Korean Existing and Evaluated Chemical Substances (Korea). NZLoC - Zealand Inventory of Chemicals (New Zealand) USA Federal Regulations Ozone Depleting Substances: No Class I or Class II material is known to be used in the manufacture of, or contained, in this product. SARA 313 Section 313 of Title Ill of the Superfund Amendments and Reauthorization Autof 1986 (SARA). This product is not known to contain any chemicals which are subject to the reporting requirements of the Aut or regulations contained in 40 CFR 372. SARA 311/312 Hazardous Categorization Acute Health Hazard Chronic Health Hazard Fire Hazard Sudden Release of Pressure Hazard Reactive Hazard No No No No No Clean Air Act, Section 112 Hazardous Air Pollutants (HAPs) (see 40 CFR 61) This product is not known to contain any HAPs. State Regulations California Proposition 65 Proposition 65 chemicals are not expected to be found in this product abo those naturally present in their agricultural source. Proposition 65 exempts naturallyoccurring listed chemicals from an obligation to label. State Right-to-Know No known components subject to Right-To-Know legislation in the following States: Chemical Name Weight % Illinois Massachusetts New Jersey Pennsylvania Rhode Island Tripotassium citrate 100 No No No No No monohd rate Canada WHMIS Product Classification Exempt: Food Product per Hazardous Products Azt Part II Controlled Products Section 12. WHMIS Ingredient Disclosure List IDL No known component is listed on the WHMIS ingredients disclosure list. (NPRI) Canadian National Pollutant Release Inventory No known component is listed on NPRI. This product has been classified in accordance with the hazard criteria of the Controlled Products Regulations (CPR) and the SDS contains all the information required by the CPR. Mexico Mexico - Grade No information available. Non hamerica Page 6/7 CIR Panel Book Page 76

83 Revision Date O1-May Potassium Citrate Granular 16 OTHER IN FORMATION Prepared By Preparation Date Revision Date Specialty Food Ingredients 02-Mar-2009 Ol-May-2009 Revision Summary This data sheet contains changes from the previous rsion in section(s) 1 3, 15 Disclaimer The information provided on this (M)SDS is correct to the best of our knowledge, information and belief at the date of its publication. The information given is designed only as a guide for safe handling, use, processing, storage, transportation, disposal and release and is not to be considered as a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other material or in any process, unless specified in the text. End of(m)sds NorthAmerica Page 7/7 CIR Panel Book Page 77

84 yjo). Distributed for Comment Only -- Do Not Quote or Cite 4ADM Process Stages Potassium Citrate, USP/FCC citric acid solution B B potassium hydroxide potassium citrate solution B crystallization II drying B sifting and packing The mformation contained herein is correct as of the date of this document to the best of our knowledge. Any recommendations or suggestions are made without guarantee or representation as to results and are subject to change without notice. We suggest you evaluate any recommendations and suggestions independently. WE DISCLAIM ANY AND ALL WARRANTIES, WI-1ETIIER EXPRESS OR IMPLIED. AND SPECIFICALLY DISCLAIM TUE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE. AND NON-INFRINGEMENT. Our responsibility br claims arising from any claim for breach of warranty, negligence, or otherwise shall not include consequential, special, or incidental damages, and is limited to the purchase price of material purchased from us. None of the statements made here shall be construed as a grant, either express or implied, of any license under any patent held by Archer Daniels Midland Company or other parties. Customers are responsible for obtaining any licenses or other rights that may be necessary to make, use, or sell products containing Archer Daniels Midland Company ingredients. CA ARCHER DANIELS MIDLAND COMPANY BOX 1470 DECATUR, ILLINOIS TEL: 800! or FAX: 217/ COM CIR Panel Book Page 78

85 COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) t 7 A - i FROM: John Bailey, Ph.D..i,L \ I L Industry Liaison to the CIR Expert Panel DATE: December 1, 2010 SUBJECT: HRIPT on Tnstearyl Citrate The attached HRIPT was completed on a material that contained approximately 90% Tnstearyl Citrate with 10-15% stearyl alcohol and 1-5% mono and diesters of citric acid (see the last page of the report). Consumer Product Testing Co Repeated insult patch test of C-SAT (Tnstearyl Citrate). Experiment Reference Number: C th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 79

86 Consumer Product Testing Co. EST 1975 C O1ooyéo-L FINAL REPORT CLIENT: ATTENTION: Cognis Deutschland GmbH & Co. KG. Henkelstr. 67 D Duesseldorf, Germany Mr. Peter Wierich TEST: Repeated Insult Patch Test ProtocolNo.: 1.01 TEST MATERIAL: C-SAT / ç i EXPERIMENT REFERENCE NUMBER: C Richard R. Eisenberg, M.D. Board Certified Dermatologist Robert W. Shanahan, Ph.D. Principal Investigator y F&nk, R.N. Study Director This report is submitted for the exclusive use of the person, partnership, or corporation to whom it is addressed, arid neither the report nor the name of these Laboratories nor any member of its staff, may be used in connection with the advertising or sale of any product or process without written authorization. 70 New Dutch Lane Fairfield, New Jersey (973) Fax (973) CIR Panel Book Page 80

87 Consumer Product Testing Co. EST 1975 QUALITY ASSURANCE UMT STATEMENT Study No.: C Ol The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical laboratory studies. These studies have been performed with adherence to ICH Guideline E6 for Good Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to standard operating procedures and applicable protocols. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study on the date(s) listed below. The findings of these inspections have been reported to management and the Study Director. All materials and data pertinent to this study will be stored in the Archive Facility at 70 New Dutch Lane, Fairfield, New Jersey, 07004, unless specified otherwise, in writing by the Sponsor. Date(s) of inspection: April 17, 2002 April 22, 2002 April 26, 2002 May 1, 2002 May 8, 2002 May 29, 2002 June 4, 2002 June 7, 2002 Senior personnel involved: OnChi Cheung, B.S. Kristin Filak, B.S. Quality Assurance Supervisor Quality Assurance Associate Kadileen Alworth, BA. Director of Quality Assurance The representative signature of the Quality Assurance Unit signifies that this study has been perfonued in accordance with standard operating procedures and study protocol as well as government regulations regarding such procedures and protocols. 70 New Duich Lane Fairfield. New Jersey ) Fax (973) Clinical Toxicology Analytical Chemistry Microbiology CIR Panel Book Page 81

88 Cognis Deutschland GmbH & Co. KG. C Page 3 Objective: To determine by repetitive epidermal contact the potential of a test material to induce primary or cumulative irritation andior allergic contact sensitization. Participants: One hundred fourteen (114) qualified subjects, male and female, ranging in age from 17 to 79 years, were selected for this evaluation. One hundred ten (110) subjects completed this study. The remaining subjects discontinued their participation for various reasons, none of which were related to the application of the test material. Inclusion Criteria: a. Male and female subjects, age l6 and over. b. Absence of any visible skin disease which might be confused with a skin reaction from the test material. c. Prohibition of use of topical or systemic steroids and/or antihistamines for at least seven days prior to study initiation. d. Completion of a Medical History form and the understanding and signing of an Informed Consent form. e. Considered reliable and capable of following directions. Exclusion Criteria: a. Ill health. b. Under a doctor s care or taking medication(s) which could influence the outcome of the study. c. Females who are pregnant or nursing. d. A history of adverse reactions to cosmetics or other personal care products. Test Material: C-SAT Study Schedule: Panel # Initiation Date Completion Date April 17, 2002 May23, April 22, 2002 May31, 2002 awith parental or guardian consent CIR Panel Book Page 82

89 Cognis Deutschland GmbH & Co. KG. C Ol Page 4 Methodology: Prior to the initiation of this study, the test material was prepared as a 25% dilution, using olive oil, which was heated until completely soluble. The upper back between the scapulae served as the treatment area. Approximately 0.2 ml of the test material, or an amount sufficient to cover the contact surface, was applied to the 1 x 1 absorbent pad portion of a clear adhesive dressing*. This was then applied to the appropriate treatment site to form a semi-occluded patch. Induction Phase: Patches were applied three (3) times per week (e.g., Monday, Wednesday, and Friday) for a total of nine (9) applications. The site was marked to ensure the continuity of patch application. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent Induction patches at home, twenty-four hours after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assigned test day, one (1) makeup day was permitted. This day was added to the Induction period. With the exception of the first supervised Induction Patch reading, if any test site exhibited a moderate (2-level) reaction during the Induction Phase, application was moved to an adjacent area. Applications are discontinued for the remainder of this test phase, if a moderate (2-level) reaction was observed on this new test site. Applications would also be discontinued if marked (3- level) or severe (4-level) reactivity was noted. Rest periods consisted of twenty-four hours following each Tuesday and Thursday removal, and forty-eight hours following each Saturday removal. Cha1lene Phase: Approximately two (2) weeks after the final Induction patch application, a Challenge patch was applied to a virgin test site adjacent to the original Induction patch site, following the same procedure described for Induction. The patch was removed and the site scored at the clinic twenty-four and seventy-two hours post-application. *Manufactured by TruMed Technologies, Inc., Burnsville, MN CIR Panel Book Page 83

90 Cognis Deutschland GmbH & Co. KG. C O1 Page 5 Evaluation Key: 0 = No visible skin reaction + = Barely perceptible or spotty erythema = Mild erythema covering most of the test site 2 = Moderate erythema, possible presence of mild edema 3 = Marked erythema, possible edema 4 Severe erythema, possible edema, vesiculation, bullae and/or ulceration. Results: The results of each participant are appended (Table 1). Observations remained negative throughout the test interval. Summary: Under the conditions of this study, test material, C-SAT , did not indicate a potential for dermal irritation or allergic contact sensitization. CIR Panel Book Page 84

91 Cognis Deutschland GmbH & Co. KG. C Page 6 Table 1 Panel # Individual Results C-SAT Site Virgin Challenge Subject Induction Phase Number 24*hr hr 72 hr * = Supervised removal of 1e Induction and Challenge Patch CIR Panel Book Page 85

92 Cognis Deutschland GmbH & Co. KG. C Page 7 Table I (continued) Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72 hr DID NOT COMPLETE STUDY * = Supervised removal of 1 Induction and Challenge Patch CIR Panel Book Page 86

93 Cognis Deutschland GmbH & Co. KG. C Page 8 Table I (continued) Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72 hr DID NOT COMPLETE STUDY DNC o 0 o * = Supervised removal of 1 Induction and Challenge Patch DNC = Did not complete study CIR Panel Book Page 87

94 Cognis Deutschland GmbH & Co. KG. C Page 9 Table 1 (continued) Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72 hr * = Supervised removal of 1 Induction and Challenge Patch CIR Panel Book Page 88

95 Cognis Deutschland GmbH & Co. KG. C Page 10 Table 2 Panel # Subject Data Subject Number Initials Age Sex 1 AS 75 F 2 RS 51 F 3 JT 67 F 4 AT 71 M 5 HK 73 F 6 CG 58 F 7 LV 69 F 8 AP 62 F 9 AK 62 F 10 FC 77 F 11 JC 76 M 12 DZ 57 F 13 RM 55 M 14 HV 70 F 15 GV 75 M 16 SO 30 F 17 CY 29 F 18 TL 62 M 19 SD 32 F 20 DH 42 F 21 MW 70 F 22 DR 20 M 23 ST 78 F 24 MV 30 F 25 KB 43 F 26 KG 37 F 27 JI 44 F 28 PF 42 F CIR Panel Book Page 89

96 Cognis Deutschland GmbH & Co. KG. C Page 11 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 29 LF 40 F 30 PH 46 F 31 DZ 23 F 32 SL 38 F 33 MM 55 M 34 JR 75 M 35 BM 60 F 36 DC 44 F 37 LM 44 F 38 EC 17 F 39 HG 31 F 40 DC 67 F 41 CG 21 F 42 AD 65 M 43 VD 70 F 44 LB 74 M 45 AE 41 F 46 DF 68 F 47 SC 53 F 48 SO 19 F 49 AO 49 F 50 BP 59 F 51 TM 39 F 52 SN 70 M 53 DL 30 F 54 CS 74 F 55 CA 57 F 56 PD 26 M CIR Panel Book Page 90

97 Cognis Deutsohiand GmbH & Co. KG. C Page 12 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 1 MA 25 F 2 PD 67 F 3 MC 57 F 4 MM 60 F 5 MH 32 F 6 EF 74 M 7 MC 66 F 8 MB 72 F 9 RS 24 F 10 SS 39 F 11 CA 43 F 12 TL 34 F 13 JM 79 F 14 FT 46 M 15 MT 40 F 16 NG 41 F 17 DK 43 F 18 FC 78 F 19 RT 62 M 20 DW 55 F 21 MB 18 F 22 AB 45 F 23 AO 25 F 24 JO 24 M 25 CC 23 F 26 AT 74 M 27 ET 73 F 28 EH 52 F 29 PR 54 M CIR Panel Book Page 91

98 Cognis Deutschland GmbH & Co. KG. C Page 13 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 30 LM 43 F 31 ES 65 M 32 SM 31 F 33 CO 58 F 34 GA 45 F 35 AS 64 F 36 GR 49 F 37 PL 56 F 38 BM 62 F 39 NM 54 F 40 AS 64 F 41 DP 45 F 42 MB 43 F 43 ED 70 F 44 TR 23 F 45 AL 42 F 46 AR 36 F 47 AP 31 M 48 MM 47 F 49 RG 54 F 50 GG 66 M 51 JP 37 F 52 VB 49 F 53 EA 73 F 54 MD 79 F 55 JS 63 F 56 IV 72 F 57 AD 74 F 58 RB 56 F CIR Panel Book Page 92

99 Substanz-Verwaltung CRT aktueller User CN=Peter Wierich/OU=Cognis/O=H ENKEL Is C-SAT-Nr Chemische Tristearyl citrate, Trioctadecyl citrate Bezeichnung frivial Name Cutina KE 3602 \S-Gehalt [%] ca. 90 LOsungsmittel (Neben-) % Stearylalkohol, Komponenten I - 5 % Mono/cu ester der Citronensãure Verunreinigungen Identifizierung der Prufsubstanz Herstelldatum Verfallsdatum Batch RIS S s CAS loslich iri/suspendierbar ri Farbe Aggregatzustand bei RT ph-wert fltichtig bei RT 01/02 51 Z nativen Olen, Olivenöl, ErdnullOl weir bis beige Feststoff Lagerung Auftraggeber CRT-Chemistry, Claus Nieendick, Tel 2333 ProduktbetreuerKleber Gefahrenhinweise (z. B. geftig/reizend/tzend/entzqndlich!explosiv) Weitere Iriformationen zur PrQfsubstanz CIR Panel Book Page 93

100 Personal Carei Products Council Committed to Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Dir ector - COSMETIC INGREDIENT REVIEW (CW) John Bailey, Ph.D. Industry Liaison to the Cifi Expert Panel DATE: January 4, 2011 SUBJECT: Information on Triisostearyl Citrate and Trioctyldodecyl Citrate A supplier has provided the following information concerning the chemical and physical properties and method of manufacture of Triisostearyl Citrate and Trioctyldodecyl Citrate. Triisostearyl Citrate Chemical and physical properties: Product is supplied as >90% Triisostearyl Citrate Molecular weight: approximately 944 Da It contains no antioxidants or preservatives Impurities: residual isostearyl alcohol (<10%) and citric acid (<0.5%) Method of Manufacture: Isostearyl alcohol and citric acid undergo a proprietary esterification process. No heavy metal catalysts are used in this process. Trioctyldodecyl Citrate Chemical and physical properties: Product supplied is supplied as 100% Trioctyldodecyl Citrate Molecular weight: approximately 1032 Da Impurities: residual octyldodecyl alcohol (<5%) and citric acid Method of Manufacture: Octyldodecyl alcohol and citric acid undergo a proprietary esterification process. No heavy metal catalysts are used in this process th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 94

101 COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal Care Products Counci Committed To Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CR Expert Panel DATE: January 4, 2011 SUBJECT: Summary of Safety Studies on Trioctyldodecyl Citrate Lubrizol G-66 Guerbet ester toxicology studies (INCI: Tnoctyldodecyl Citrate) th Street, N.W., Suite 3O0 Washington, D.C (fax) CIR Panel Book Page 95

102 Lutrizol nove-øri Consumer Specialties TOXICOLOGY & MICROBIOLOGY STUDIES TOX-Ol 7 Edition: January 12, 2005 Previous Edition: April, 2001 G-66 Guerbet Ester Toxicology Studies CTFA I INCI Name: Trioctyldodecyl Citrate Distributed for Comment Only -- Do Not Quote or Cite Acute Oral Toxicity The acute oral toxicity of G-66 Guerbet Ester was studied in rats according to FHSLA, 16 CF Five male and five female rats were administered a single dose of 5,000 mg/kg via gavage. The animals were housed individually and observed for mortality and clinical signs of toxicity for 14 days following exposure. No evidence of toxicity was observed. The acute oral LD 50 was determined to be greater than 5,000 mg/kg. Eye Irritation The potential for G-66 Guerbet Ester to cause eye irritation was evaluated according to FHSLA, 16 CFR A dose of 0.1 ml of the test material was administered to eyes of six rabbits. The eyes were evaluated according to the method of Draize et al. 1 The Draize scores were then classified according to the method of Kay and Calandra. 2 The eyes were evaluated at 24, 48, and 72 hours following exposure. The Maximum Mean Total Score was determined to be Based on these results the test material was considered to be non-irritating. 1 Draize et al., (1944) J. Pharmacol. Exp. Ther. 83: Kay and Calandra, (1962) J. Soc. Cos. Chem. 13: Skin Irritation The skin irritation potential of G-66 Guerbet Ester was evaluated according to FHSLA, 16 CFR A dose of 0.5 ml of the test material was applied to the intact and abraded skin of six rabbits and then covered by occlusive patches. The skin was evaluated at 24 and 72 hours. The Primary Irritation Index was determined to be Based on these results the test material was not considered to be a primary skin irritant. Skin Sensitization The irritation and sensitization potential of the test material was evaluated using the human repeat insult patch test. The test material (150 p1) was applied to a 2 cm x 2 cm absorbent pad centered on the adhesive-coated surface of a 4 cm x 4 cm water-impermeable plastic film that then was positioned on the skin of 107 numan volunteers. 3 For the induction phase u series of 12 applications, four consecutiv applications per week for three weeks, was conducted with ec,ch panelist at the designated site. The patches were removed and the contact sites were examined 24 hours after each application. Following a two week rest period, the challenge phase was conducted using 4 applications of the test material and evaluations on a naive site of each volunteer. A final evaluation of the sites was provided the following week. No skin irritation or sensitization responses were observed with the 107 volunteers participated in the induction phase; 105 completed the challenge phase Lubrizol Advanced Materials, Inc. /9911 Brecksville Road, Clevetand, Ohio ! TEL: or The information contained herein is believed to be equipment used commercially in processing these Materials, lncs direct control. THE SELLER MAKES NO reliable, but no representations, guarantees or materials, no warranties or guarantees are made as to WARRANTIE. EXPRESS OR IMPLIED, INCLUDING, warranties of any kind are made as to its accuracy, the suitability of the products for the application BUT NOT LIMITED TO, THE IMPLIED WARRANTIE. suitability for particular applications or the results to be disclosed. Full-scale testing and end product OF MERCI-çsNTABILITY AND FiTNESS FOR A obtained therefrom. The information is based on performance are the responsibility of the user. Lubrizol PARTICULAI PURPOSE. Nothing contained herein is laboratory work with small-scale equipment and does Advanced Materials, Inc. shall not be liable for and the to be considfred as permission, recommendation, nor not necessarily indicate end product performance. customer assumes all risk and liability of any use or as an induc4ment to practice any patented invention Because of the variations in methods, conditions and handling of any material beyond Lubrizot Advanced without permision of the patent owner. For further information, please visit Lubrizol Advanced Materials, Inc. is a wholly owned subsidiary of The Lubrizol Corporation * Trademark owned by The Lubrizol Corporation Copyright 2007? The Lubrizol Corporation CIR Panel Book Page 96

103 2 test material during the course of the study. The investigators concluded that the results do not contraindicate skin contact with the test material under equal or less stringent conditions.. The skin sensitization potential of a number of samples of the test material was evaluated in the mouse using the Local Lymph Node Assay based on the guidelines described in OECD, Section 4, Health Effects, No. 429, Paris Cedex, April, 2002, EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft), June 2001 and US EPA OPPTS , March Groups of five mice were treated with the test material a concentrations of 0%, 10%, 50%, and 100% w/v in acetone/olive oil (4:1 v!v) (25 p1/ear) by daily application to the dorsal surface of each ear for three consecutive, days. Five days following the first topical appliction, all mice were injected with 0.25 ml of phosphate buffered saline containing 3H-methyl thymidine via tail vein giving a total dose of 20 pci to each mouse. A single cell suspension of pooled lymph node cells was prepared by mechanical disaggregation through stainless steel gauze (125pm diameter). The cells were Washed and centrifuged, precipitated, and re-centrifuged at 40 C, and then were measured for 3HTdr incorporation. Alpha hexylcinnanhic aldehyde in acetone/olive ol (4:1 v/v) as Sc/u, 10%, and 25% solutions were used as the pohtive control. Untreated controls also were incluled. Slight erythema was noted ma few animals n the two highe dose groups. The majority of lymph nodes we normal; enlarged nodes were observed in one aimal in th vehicle control and the 50% group and in two animals in the 100% group. Th stimulation index (SI) for the test substance concentrations tested were found to range from 1.1 to 3.1. Because the SI values for several of the test substance concentrations were above the criteria for a positive response (test/control ratio > 3), the test substance was determined to cause a sensitization response under the oonditions of this test. The EC3 value ws caicuated to be 95.8%. Under the current prc)osed classification scheme the neat test material may be considered a mild sensitizer. Furthermonz based on these results it was conluded that the neat test material should be labeled as: may cause sensitization by skin contact (R43). CIR Panel Book Page 97

104 COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal CareProducts Council Memorandum Committed to Safety, ua ity nnovat ion TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel DATE: January 4, 2011 SUBJECT: Safety Studies of Triisostearyl Citrate Consumer Product Testing Co The MatTek Corporation EpiOcular tissue model in vitro toxicity testing system: Triisostearyl Citrate. Experiment Reference No.: V Product Investigations, Inc Determination of the irritating and sensitizing propensities of EX 1028 (Triisostearyl Citrate) on human skin. Report: P11 No NOTOX B.V Evaluation of the mutagenic activity of EX-1028 (Triisostearyl Citrate) in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). NOTOX Project th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 98

105 /it//27,7 EST Consumer Product Testing Co. FINAL REPORT CLIENT: ATTENTION: Jane Woo TEST: The MatTek Corporation EpiOcular Tissue Model In Vitro Toxicity Testing System TEST ARTICLE: r-, EXPERIMENT REFERENCE NO.: V Scott Krupa Quality Assurance Supervisor teven Nitka Vice President Laboratory Director This report is submitted for the exclusive use of the person, pertnersnip, or corporation to whom it is addressed, end neither the report nor the nsme of these Laboratories nor any member of its staff, may be used in connection with the advertising or sale of any product or process without written authorization 70 New Dutch Lane Fairfield, New Jersey (973) FaX (973) CIR Panel Book Page 99

106 EST Consumer Product Testing Co. QUALITY ASSURANCE UNIT STATEMENT Study No.: V The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of nonclinical laboratory studies. These studies have been performed under Good Laboratory Practice principles (including government regulations to the extent applicable) and in accordance with standard operating procedures and applicable standard protocols. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study on the date(s) listed below. The findings of these inspections may have been reported to management and the Study Director. Date of data inspection: July 22, 2004 Professional personnel involved: Steven Nitka, B.S. Vice President Lillian Deniza, B.S. Melissa Pandorf, B.S. Scott Krupa Laboratory Director (Study Director) Laboratory Supervisor Technician Quality Assurance Supervisor The representative signature of the Quality Assurance Unit on the front page signifies that this study has been performed in accordance with standard operating procedures and applicable study protocols. 70 Nev Dutch Lane Fairfield, New Jersey (973) Toxicology Clinical Analytical Chemistry Microbiology Fax (973) CIR Panel Book Page 100

107 Objective: To evaluate the test article for irritancy potential utilizing the MatTek Corporation EpiOcular in vitro toxicity testing system. Introduction: MatTek s patented EpiOcular corneal Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidennal cells, which are cultured on specially prepared cell culture inserts using serum free medium, differentiate to form a multilayered structure which closely parallels the corneal epithelium... This system.. provides a predictive, morphologically relevant in vitro means to assess ocular irritancy. 1 EpiOcular, when used with the recommended cell metabolism assay, can quickly provide toxicological profiles. The procedure utilizes a water-soluble, yellow, tetrazolium salt (MTT {3- {4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide}), which is reduced by succinate dehydrogenase in the mitochondria of viable cells to a purple, insoluble fonnazan derivative. Substances which damage this mitochondrial enzyme inhibit the reduction of the tetrazolium salt. The amount of MTT reduced by a culture is therefore proportional to the number of viable cells. Test Article: SCHERCEMOL TISC, Lot# Method: After the appropriate tissue preparation, 100 microliters of the test article (at 10% in corn oil) and the negative control (corn oil) were added to the Millicells containing the EpiOcular samples. The six (6) well plates containing the dosed EpiOcular samples were then incubated at 37 C, five (5)% carbon dioxide and 90% humidity. 1MatTek Corporation, 200 Homer Avenue, Ashland, Massachusetts CIR Panel Book Page 101

108 Method (continued): After the appropriate exposure period, each insert was individually removed from its plate and rinsed with phosphate buffered saline (PBS) to remove any residual material. Each was then rinsed a second and third time. Following the 3 rinses, each Millicell was submerged in 5 milliliters of assay media for 10 minutes, at room temperature. This final soak removed any residual, absorbed article. After the 10 minutes, excess liquid was shaken off and each EpiOcular tissue was placed into 300 microliters of MTT solution. The EpiOcular samples were then returned to the incubator. After the three (3) hour MIT exposure each insert was removed and gently rinsed with PBS to remove any residual MIT solution. Excess PBS was shaken from each of the inserts, which were then blotted on the bottom on paper towels. The inserts were then each placed into one (1) well of a 24 well extraction plate. Each insert was then immersed in two (2) milliliters of extraction, at room temperature, overnight. After the extraction procedure, the liquid within each insert was decanted back into the well from which it was taken. The remaining extractant solution was then agitated and a 200 microliter aliquot of each extract was removed for evaluation. A Dynatech MR 4000 Automatic Microplate Reader was used to determine the absorbance of each extract at 570mn. With the absorbance of a negative control (distilled water) defined as 100%, the percent absorbencies of the test and reference articles were determined. The percentages listed below directly correlate with the cell metabolism in the EpiOcular samples. Results: Article Percent Percent (% & Exposure) System Viabifity Inhibition.T- C-,1( (10% - 20 mm.) EpiOcular (lo%-lhr.) EpiOcular (10% - 4 hr.) EpiOcular CIR Panel Book Page 102

109 25 Distributed for Comment Only -- Do Not Quote or Cite When possible, using a semi-log scale, the percent viabilities for the articles were plotted on the linear y axis versus the dosing time on the log x axis. By interpolation, the time at which the percent viability would he 50% was determined (ET-50). As a general guideline (provided by MatTek) the following equation can be used to estimate the rabbit Draize eye score: Draize = O1.7/(ET-5O) 0.5 Based on the literature (Kay, J.H. and Calandra, J.C., Interpretation of eye irritation tests, I Soc. Cosmetic Chem., 13, (1962)), the ocular irritancy estimated potential has been categorized by MatTek into the following groups, based on the Draize score: Draize Score Irritancy Classification Example EpiOcular ET-50 (mlii) 0-15 Non-irritating, Minimal PEG-75 Lanolin, Tween 20 > Mild 3% Sodium Dodecyl Sulfate < Moderate 5% Triton X-l00 < Severe, Extreme 5% Benzalkonium Chloride <3.45 Discussion: JPc-// CIfc1J Under the conditions of this test, test article, at 10% in corn oil, elicited in vitro results, which place its ET-50 at greater than 256 minutes. Therefore, its estimated Draize ocular irritation scores is 0 with non-irritating irritancy classification. Conclusion: (/: 1J Under the conditions of this test, the results indicate that, at 10% in corn oil, has a non-irritating irritancy classification. CIR Panel Book Page 103

110 PRODUCT INVESTIGATIONS, INC. pi 1 REPORT: PH N East Tenth Avenue Conshohocken, PA fax DFTERMINATION OF THE IRRITATING AND SENSITIZING PROPENSITIES OF EX-1O28oNHuMANSKP 5o PREPARED FOR 29 May 2007 RcpDrt PJ CIR Panel Book Page 104

111 TABLE OF CONTENTS ABSTRACT PageT COMPLIANCE WITH GooD QUALITY ASSURANCE STANDARDS 1.00 OBJECTIVES Page SPONSOR 3.00 AUTHORIZATION 4.00 FEATURES OF THE METHOD 5.00 STUDY PRODUCT 6.00 GooD CLINICAL PRACTICES 7.00 SITE OF STUDY 8.00 DATES OF STUDY 9.00 SELECTION OF SUBJECTS Page 2.01 Recruiting.02 Informed Consent.03 Determination of Eligibility.04 Panel Information SITE INFORMATION Page PATCHING DEVICES DATA ACQUISITION SCHEDULE OF PROCEDURES Page STANDARD INSTRUCTIONS BEFORE INTERRUPTIONS IN THE REGIMEN REGIMEN: Week#1 Page6 Week #2 Week #3 Week #4 Week #5 Page 7 Week #6, # TABULATION OF CYCLES COMPLETED SUMMARY OF RESULTS SIGNIFICANCE OF THE RESPONSES Page CLINICAL RELEVANCE CONCLUSIONS Report PIt Moy 2007 CIR Panel Book Page 105

112 ABSTRACT A sample identified as EX-1028 was received by Product Investigations on 22 March, The sample was submitted by for a patch test to determine the sample s skin-irritating and sensitizing potentials. To accomplish this, Product Investigations, using a procedure based on that described in PROTOCOL ISM 059.NOV, initiated a study on more than one-hundred qualified volunteers. The regimen called for four twenty-four hour applications of the sample conducted seriatim during each of Weeks Nos. 1, 2, 3, and 7 on assigned skin sites on the right upper arm of each subject. Examinations of the contacted skin and grading of its condition were conducted within moments after devices containing the sample were removed. Weeks 1, 2, 3, and 4 formed the Initial or Induction Phase of the regimen. Data were acquired on one-hundred-andnineteen subjects during this phase. No clinically significant adverse effects were detected on any of the subjects during this phase. The Challenge or Diagnostic Phase of the regimen was conducted during Week #5. Data were acquired on one hundred-and-fourteen subjects during this phase. No clinically significant adverse effects were detected on any of the subjects during this phase. On the basis of the above-cited observations, EX-1028 was found to be devoid of clinically significant skinirritating and skin-sensitizing propensities that can be detected under the conditions of this patch test procedure. The investigator concluded that the data do not contraindicate usages of EX-1028 entailing conditions of contact commensurate with or less stringent than those that prevailed during the course of this study. COMPLiANCE WITH GooD OUALITY ASSURANCE STANDARDS In my review of the data I have found no discrepancies between the information presented in this report and the thcords that were kept during the conduct of this study. All information and raw data relating to this study will be retained in the study file and will he archived on the premises of Product Investigations, Inc. for a period of not less than seven (7) years. Date PageT Report P Moy 2007 CIR Panel Book Page 106

113 1.00 OBJECTIVES:. Determination of the Irritating and Sensitizing Propensities of EX-1028 on Human Skin.01 To identify and characterize the skin-damaging propensities that EX-1028 induced to exercise under the conditions of this intensified patch test procedure..02 To adjudge whether the exercise ofsuch propensities under the patch test conditions contraindicates the kind of skin contact that would be occasioned durinc the annronriate use of the product SPONSOR: Project Director: Karen Jordan Health, Toxicology & Product Safety 3.00 AUTHORIZATION: Letter from Karen Jordan dated 6 March FEATURES OF THE METHOD:.01 An intensified version of the Repeated Insult Patch Test regimen was conducted under double blind conditions on a panel consisting of more than one-hundred subjects at the outset..02 The regimen schedule entailed four 24-hour applications of the test sample conducted seriatim during each ofweeks one, two, and three, and four 24-hour applications conducted seriatim on a naive site during the challenge week..03 The contacted skin was examined after each application to assess and grade the elicited effect..04 During the initial phase the responses that were in evidence after removal of the sample were used to determine whether applications were to be continued on the same site, switched to a new site, or terminated STUDY PRODUCT: Type of Product: Cosmetic Ingredient Sponsor Identification: EX-1028 INCI Tnisostearyl Citrate CAS No.: Date of Receipt: 22 March, 2007 Description: Clear viscous oil Form used in study: As supplied PINt GOOD CLINICAL PRACTICES: The study was conducted in compliance with the standards of good clinical practices generally applicable for the protection of the privileges and well-being uf individuals who participate in patch test procedures SITE OF STUDY: Product Investigations, Inc. 142 North Ninth Street, Suite 16 Modesto, CA STAFF: Mecical Director: Dir. Derm. Services:. Dermatologist: Technician: Quality Assurance. Moms V. Shelanski, MDCM Joseph E. Nicholson III Clinton E. Prescott, MD Lisa Cortez Samuel J. Charles III 8.00 DATES OF STUDY: Started: 9 April, 2007 Completed: 14 May, 2007 Report PH May 2007 Page 1 CIR Panel Book Page 107

114 9.00 SELECTION OF SIJBJECTS:.01 REcRUITING: Individuals interested in participating were recruited from local areas by telephone, flyers, and direct contact..02 INFORMED CONSENT: An interested party had to obtain a reasonable understanding of the contents of an infonned consent document containing the following information and sign it willingly before she/he was engaged as a subject. a. The number of subjects that were to be enrolled in the study; b. The intended use of the product; c. The reasons for testing the product; d. The regimen for the test procedures; e. The different adverse effects that have been known to occur in patch test participants; f. The different ways that participation may be detriniental to a subject s health or quality of life; g. That not all detrimental effects could be foreseen and made known at the time the informed consent was presented for the prospective subject s signature; h. The commitments which a subject was asked to make to ensure that meaningful data would be generated; i. The rights endowed on a subject for her/his protection; j. The avenues of recourse available to a subject who believes that she/he has been misused; and k. The considerations a subject was entitled to receive and the conditions for receiving them..03 DETERMINATION OF ELIGIBILITY: An individual s eligibility was determined by checking her/his medical history and the answers given in response to specific questions in the informed consent document against the criteria listed below. a. Inclusion Criteria: Satisfaction of all the following items was obligatory for enrolment: I) The candidate was at least eighteen years old, and 2) agreed to comply fully with the scheduled study regimen, and 3) expressed awareness that a participant would incur risks that would affect her/his well-being, and 4) denied that the stipend had induced her/him to volunteer against her/his better judgement, and 5) had assured the interviewer that she/he had read the informed consent fonn and had no questions about the informed consent s contents that had not been answered to her/his satisfaction, and 6) had signed the consent form willingly and without reservation. b. Exclusion Criteria: Any of the following items was cause for rejection: i The candidate had an illness that contraindicated participation; or 2) -a condition that rendered the skin unsuitable-for use in this study; or 3) was using dosages of medications that could alter the skin s tolerance; or 4) had a documented history of intolerance to the category of products submitted for study; or 5) was a female who was pregnant or was breast feeding an infant..04 PANEL INFORMATION: a. Dedication: The subjects in Panel No were engaged concomitantly in the studies ofproducts submitted by sponsors other than. b. Demographics: SEX Number Age Ranue Female Male Repart P May 2007 Page 2 CIR Panel Book Page 108

115 10.00 SITE INFORMATION:.01 The test material was assigned the four sites on Band #6 on the left side of the back of each subject, to wit: Los INS I MS I L&] a. L6, was designated the first site that was to be exposed to the test material. b. M6, the site immediately lateral to L6, was designated as the first alternate site for use during the initial phase. c. N6, the site immediately lateral to M6, was designated as the second alternate site for use during the initial phase. d. 06, was the site designated to receive the challenge applications..02 IDENTIFICATION OF A CONTACT SITE: PATCHING DEVICES:.01 TYPE OF DEVICE:.02 PREPARATION OF A PATCHING DEVICE: I I I I I The skin around the site in current use was freshly marked at each visit. The marks made it possible for the technicians to locate the site for grading, etc., in the absence of the device or other distinguishing features. Occlusive patching devices were used to convey the material to the skin and to maintain it on its assigned site on each subject. These devices consisted of a 2 cm x 2 cm absorbent pad centered on the adhesive-coated surface of a 4 cm x 4 cm water-impermeable plastic film. The webril pad of a patching device was infused with 150 ui of the test material..03 APPLYING A PATCHING DEVICE: a. Each device was positioned on its designated site on a subject with the moistened webril pad in contact with the skin. b. Firm pressure was applied to the backing of the device to effect intimate contact of the material with the skin and to bond the adhesive of the flanges fast to the surrounding skin..04 REMOVING A PATCHING DEVICE AND ITS CONTENTS: When a subject returned to the clinic, a technician peeled the device and material off as gently as was feasible to prepare the subject for examination DATA ACOUISITION:.01 GRADING PROCEDURE: a. When a subject arrived, the technician examined the subject s back to ascertain whether the patch was present and on its proper site. b. The technician removed the patching device and examined the skin. 1) If no adverse effect was detected, the technician entered 0 in the space provided in the subject s record. 2) If an adverse effect was detected, the technician entered a grade indicating her assessment of the response s intensity into the subject s record. c. The subject was sent into the patching room with a note informing the patching technician of the grade assigned to the perceived effect. The grade was to be used by the patching technician to determine if the product was to be reapplied on the site in current use, applied on the next unused alternate site, or not applied altogether. d. The patching technician examined the contact site to ascertain independently whether the site should or should not be used again. If she disagreed with the first technician s assessment, the application was held in abeyance until the issue could be resolved with the help of the supervisor andlor the investigator. e. The supervisor or the investigator was called in when a disagreement had to be resolved but also to validate the technicians assessment I) When she assigns a grade of 2 or higher to a response, and 2) When she assigns a grade that is two or more points lower than the previous day s grade. Repoll J I My 2007 Page 3 CIR Panel Book Page 109

116 .02 CRITERIA FOR GRADING THE INTENSITY OF A RESPONSE: Grades were assigned in accordance with the following criteria to designate the intensity of the effects elicited on the skin by the material. STAGES OF VISIBLE CHANGE CLINICAL GRADE INFLAMMATION SIGNIFICANCE ABSENT, or sub-clinical None Indicates the absence of a 0 changes that are not gross perceptible on the skin inflammation-eliciting surface propensity. VASCULAR DILATATION Redness, faint; may Indicates a very weak to weak I not involve all of inflammation-eliciting contact area propensity. Redness, faint to Indicates a mild to moderate 2 moderate, inflammation-eliciting all of contact area propensity. involved Redness, intense, all Indicates a strong 3 of contact area inflammation-eliciting involved propensity, probably irritation. VASCULAR LEAKAGE WITH Redness, plus edema Indicates a strong 4 INFILTRATION andlor INDURATION.. and/or papules. mflammation-eliciting propensity, possibly sensitization. Redness, plus 5 vesicles, blisters or bullae Redness plus 6.03 CRITERIA FOR CHANGING A CONTACT SITE: a. - Applications are continued on the initial contact sire unless a response rated 3 or higher is elicited. When that happens, applications are discontinued on the affected site. b. If a response of 3 or higher is elicited on or before Monday of Week applications are stopped and not resumed for the remainder of the study if it is deemed to be a recall response, i.e., one indicative of a sensitized status that antedates the study. - applications are resumed on an alternate site on Monday of Week 2 if it is not deemed to be a recall response. c. If a response of 3 or higher is detected on Tuesday, Wednesday, Thursday or Friday of Week 2, applications are resumed on an alternate site on Monday of Week 3. d. If a response of 3 or higher is detected on Monday of Week 3, applications are switched immediately to an alternate site. e. If a response of 3 or higher is detected on or after Tuesday of Week 3, applications are discontinued and not resumed for the remainder of the Initial Phase. f. Applications are made for four consecutive days on the challenge site unless a response of 3 or higher is elicited. When that happens, applications are terminated. Rcpart P May 2007 Page 4 CIR Panel Book Page 110

117 13.00 SCHEDULE OF PROCEDURES:.01 APPLIcTIoNs: a. Initial/Induction Phase: Week #1: Week #2: Week #3: Week #4: Monday, Tuesday, Wednesday, Thursday. Monday, Tuesday, Wednesday, Thursday. Monday, Tuesday, Wednesday, Thursday. Hiatus, Make-up b. Challenge/Diagnostic Phase: Week #5: Monday, Tuesday, Wednesday, Thursday..02 EXAMINATIONS: a. lilitial/induction Phase: Week #1: Week #2: Week #3: Week#4: Monday (Baseline), Tuesday, Wednesday, Thursday, Friday. Monday, Tuesday, Wednesday, Thursday, Friday. Monday, Tuesday, Wednesday, Thursday, Friday. Monday, as needed. b. Challenge/Diagnostic Phase: Week #5: Week #6: Monday (Baseline), Tuesday, Wednesday, Thursday, Friday. Monday, etc. as needed STANDARD INSTRUCTIONS BEFORE INTERRUPTIONS IN TIlE REGIMEN:.01 WEEKENDS: Before being dismissed for the weekends during Weeks 1, 2, and 3, subjects were given instructions to notify the investigator without delay if the skin showed any of the following changes: j a substantial increase in the intensity of an already-elicited response, i) the spread of a response beyond the area of contact, or ii) the outbreak of a rash on a hitherto unaffected site..02 INTERMEDIATE PHASE: The same instructions were also given to each subject before she/he was dismissed for the hiatus in procedure before the challenge phase. Report P May 2007 Page 5 CIR Panel Book Page 111

118 WEEK Distributed for Comment Only -- Do Not Quote or Cite REGIMEN:.01 INITIAL PHASE - #1 of the INITIAL PHASE: MONDAY: 1) When a subject presented hersewhimself at the clinic, the skin on which the assigned contact sites were to be situated was ascertained to be in fit condition for receipt of the product before applications were begun. 2) A freshly-prepared patching device containing the test material was applied on its assigned initial contact site. 3) The skin around the device was marked. 4) The subject was dismissed with instructions to return on Wednesday. TUESDAY, WEDNESDAY, THURSDAY: I) When a subject returned, the marks identifying the location of the contact site were reinforced, 2) The device and test material were removed by a tecimician. 3) The contact site was examined; skin status was graded; the grade was recorded. 4) A freshly-prepared patching device was applied on the site in current use. 5) The subject was dismissed with instructions to return on the following day. FRIDAY: 1) When a subject returned, the marks identifying the location of the contact site were reinforced. 2) The device and test material were removed by a technician. 3) The contact site was examined; skin status was graded; the grade was recorded. 4) A freshly-prepared patching device was applied on the site in current use. 4) The subject was instructed to remove the patch at home on Saturday, to note the condition of the site, and to return on the following Monday. SATURDAY AND SUNDAY: No in clinic procedures were scheduled on these days..02 WEEKS #2 and #3 of the INITIAL PHASE: MONDAY: 1) When a subject returned, the marks identifying the location of the contact site were reinforced. 2) The site was examined; skin status was graded; the grade was recorded. 3) A freshly-prepared patching device was applied on an appropriate site. 4) The subject was dismissed with instructions to return on Tuesday. TUESDAY, WEDNESDAY, AND THURSDAY: 1) When a subject returned, the marks identifying the location of the contact site were reinforced. 2) The device and test material were removed by a technician. 3) The contact site was examined; skin status was graded; the grade was recorded. 4) A freshly-prepared patching device was applied on the site in current use. 5) The subject was dismissed with instructions to return on the following day. FRIDAY: 1) When a subject returned, the marks identifying the location of the contact site were reinforced. 2) The device and test material were removed by a technician. 3) The contact site was examined; skin status was graded; the grade was recorded. 4) The subject was given the standard instructions, and dismissed until the following Monday. SATURDAY and SUNDAY: No procedures were scheduled on these days..02 INTERMEDIATE PHASE WEEK 4: Make-up phase as needed MONDAY: 1) When a subject returned, the marks identifying the location of the site in current use were reinforced. 2) The contact site was examined; site status was graded; the grade was recorded. 3) All subjects dismissed until Monday of the Challenge week. Report P CIR Panel Book Page May 2007 Page 6

119 .03 CHALLENGE PHASE WEEK #5: MONDAY 1) When a subject returned, all previously used contact sites were examined. 2) If no persistent or delayed response that might have necessitated postponing the challenge applications was present, a freshly-prepared device was applied on a naive contact site. 3) The skin around the device was marked. 5) The subject was dismissed with instructions to return on the following day. TUESDAY, WEDNESDAY, TIJURSDAY: 1) When a subject returned, the marks identifying the location of the site were reinforced. 2) The device and material were removed by a technician. 3) The contact site was examined; skin status was graded; the grade was recorded. a) Absent a response a3, a freshly-prepared patching device was applied on the same site. b) If a response 3 was present, applications were stopped. 4) The subject was dismissed with instructions to return on the following day. FRIDAY: 1) When a subject returned, the marks identifying the location of the site were reinforced. 2) The device and material were removed by a technician. 3) The contact site was examined; skin status was graded; the grade was recorded. 4) If a reaction was present, the subject was dismissed with instructions to return on the following Monday. 5) Absent a need for treatment or continued observation, the subject was discharged with instructions to notify the investigator without delay should a response reappear on a previously involved site or break out on a hitherto unaffected one..04 FOLLOW-UP PHASE WEEK #6: WEEK #7: After the exit examination that was scheduled for the beginning of Week 6 was conducted, the subjects were given the balanoe of that week and all of Week 9 as a period to communicate any information that they believed was relevant to the effects oftheir exposure to the material as well as to express a need for treatment ofpersistent or newly-occurring responses. Deviations in Procedure: None were necessary. Report I ll CIR Panel Book Page May 2007 Page 7

120 3 120 Distributed for Comment Only -- Do Not Quote or Cite TABULATION OF CYCLES COMPLETED: Table Ia. INITIAL (INDUCTION) PHASE - (WEEKS 1,2,3 AND 4) NUMBER OF NO DATA ACQUIRED DATA ACQUIRED AECs F REQUIRED DROP OUTS EXCUSED EXCUSED NON-COMPLIANT I COMPLIANT 10 I I Subject I 0 subjects I 0 subjects I 4 subjects I 115 subjecls Table Tb. - CHALLENGE (DIAGNOSTIC) PHASE - (WEEKS) NUMBER OF No DATA ACQUIRED DATA ACQuIRED ARCs REQUIRED Drop outs Excused Excused Non-compliant Compliant 2 ) 6 subjects 0 subjects 0 subjects I 0 subjects I 114 subjects SUMMARY OF RESULTS: Table II: MAXIMUM ASSIGNED GIoDEs PER lnl)ivmual PARTICIPANT (MAGPIPs) GRADE TYPE OF RESPONSE INDUCTION CHALLENGE 0 NOVISIBLECI-IANGE II9SUBJECTS II4SUBJE-CIS I FAINT REDNESS, UNDEFINED BORDER MODERATE REDNESS, DEFINED BORDER 0 0 INTENSE REDNESS_ 0 0 REDNESS+OEFINITEEDEMAand/0rPAPULES 0 0 NUMBER OF RESPONDERS 0 SUBJECTS 0 SUBJECTS NUMBEROFSUBJECTSPATCHED NUEEER OF SJEJtCTS PROVODINC DAT,?!9 U 104 NUMBER PROVIDING NO DATA I 6 Table LII: WEEKLY incidence OF RESPONSES WEEK# WEEK #2 WEEK #3 WEEK #4 WEEK #5 #6 GRADE M I \V-F M T NV-F M 1 NV-F M T W-E M T V/-F M I B B B B B B B_ TOTAL Report P CIR Panel Book Page May 2007 Page 8

121 18.00 SIGNIFICANCE OF THE RESPONSES:.01 INITIAL/INDUCTION PHASE: No responses were noted on any of the 119 subjects who participated in all or part of the induction phase of the study. The absence of responses characterize the product as one which is devoid of clinically significant sldn irritating propensities..02 CHALLENGE PHASE: No responses were noted on any of the 114 subfrcts who participated in all or part of the challenge phase of the study. The absence of responses characterize the product as one which is devoid of clinically significant skinsensitizing propensities..03 FOLLOW-UP PHASE: The investigator received no communications from any of the subjects that provided a basis for altering his decision concerning the potential hazard presented by the product..04 GLOBAL: Under the conditions prevailing in this patch test study, the product was found to be incapable of eliciting clinically significant skin damage on any of the more that one hundred individuals concerning whom data were acquired CLiNICAL RELEVANCE:.01 The inability of the material to elicit substantial and persistent adverse changes suffices to characterize the product as one devoid of skin-damaging propensities that can be detected under the regimen used in this study. This characterization can be expressed with a high level of confidence because the regimen has proven itself to be one that affords products ample time and opportunity to exercise any latent propensities for eliciting gross skin changes indicative of irritation and/or sensitization..02 If the real-life use of this material entails non-occlusive contact or occlusive contact of less than twenty-four hours in duration, one may anticipate with a high level of confidence that the risk of skin damage that will be associated with the use of this material will be lower than that which can be extrapolated from the data generated in this study CONCLUSIONS: The data do not contraindicate exposure of the skin to the product represented by the sample identified as EX-1028 for usages entailing repeated applications under conditions commensurate with or less stringent than those that prevailed during the course of this study. PRODUCT INVESTIGATIONS, INC.? k34z7 Date of Clinical Services Report I IO May 2007 CIR Panel Book Page 115 Page 9

122 7ij /p 37cf REPORT Study Title EVALUATION OF THE MUTAGENIC ACTIVITY OF EX IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND THE ESCHERICH!A COLI REVERSE MUTATION ASSAY (WITH INDEPENDENT REPEAT),, / c. I- -C4 Author CM. Verspeek-Rip Study completion date 23 April 2007 Test Facility NOTOX B.V Hambakenwetering DO s-hertogenbosch The Netherlands Laboratory Project Identification NOTOX Project NOTOX Substance /A - Pagelof26 - CIR Panel Book Page 116

123 EX NOTOX Project CONTENTS 1. CONTENTS 2 2. STATEMENT OF GLP COMPLIANCE 3 3. QUALITY ASSURANCE STATEMENT 4 4. SUMMARY 5 5. INTRODUCTION Preface Aims of the study Guidelines Storage and retention of records and materials 7 6. MATERIALS AND METHODS Testsubstance , Test substance information Study specific test substance information Test substance preparation Reference substances Negative control Positive controls Test system Cell culture Metabolic activation system Preparation of S9-fraction Preparation of S9-mix Study design Dose range finding test Mutation assay Colony counting Electronic data capture Interpretation Acceptability of the assay Data evaluation and statistical procedures List of deviations List of protocol deviations List of standard operating procedures deviations RESULTS , Doserangeflndingtest Mutation assay DISCUSSIONANDCONCLUSlON REFERENCES 13 TABLES Table I Table 2 Experiment 1. Mutagenic response of EX in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay 14 Experiment 2: Mutagenic response of EX in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay 15 APPENDICES APPENDIX I SUPPORTING MATERIALS AND METHOD 16 APPENDIXII DETAILEDTABLES 17 - Page 2 - CIR Panel Book Page 117

124 EX-1028 NOTOX Project STATEMENT OF GLP COMPLIANCE NOTOX B.V., s-hertogenbosch, The Netherlands The study described in this report has been correctly reported and was conducted in compliance with: The Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice Guidelines (1997), Which essentially conform to: F The United States Food and Drug Administration Good Laboratory Practice Regulations. The United States Environmental Protection Agency Good Laboratory Practice Regulations. The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test substance information unless determined by NOTOX. Analysis of stability, homogeneity and concentration of the test substance under test conditions was not performed as part of this study. NOTOXB.V. G.M. Verspeek-Rip Study Director Ing. E.J. van de Waart, M.Sc. Head of In Vitro & Environmental Toxicology LEZ R... Date: rj Date -Page3- CIR Panel Book Page 118

125 EX NOTOX Project QUALITY ASSURANCE STATEMENT NOTOX By. s-hertogenbosch, The Netherlands This report was inspected by the NOTOX Quality Assurance Unit to confirm that the methods and results accurately and completely reflect the raw data. The dates of Quality Assurance inspections are given below. During the on-site process inspections procedures applicable to this type of study were inspected. The reporting date is the date of reporting to the Study Director. The QAU report was then forwarded to the Test Facility Management. Start End Type of.. Reporting PhaselP rocess Inspection Inspection inspections date date date Study Protocol 12-Mar Mar Mar-07 Report 1 3-Apr Apr Apr-07 Process Genetic and In Vitro Toxicology 09-Jan Jan Jan-07 Test substance handling Exposure Observations/Measurements Specimen handling Head of Quality Assurance C J. Mitchell B Sc -Page 4- CIR Panel Book Page 119

126 EX NOTOX Project SUMMARY Evaluation of the mutagenic activity of EX-1028 in the Salmonella typhimurium reverse mutation assay and the Escherichia coil reverse mutation assay (with independent repeat). EX was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1 535, TA1537, TA98 and TAIOO) and in the Escherichia coil reverse mutation assay with a tryptophan-requiring strain of Escherichia coil (WP 2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and l-naphthoflavone). The study procedures described in this report were based on the most recent OECD and EEC guidelines. Batch CM of EX-1028 was a clear colourless very viscous liquid with a purity of 100%. The test substance was dissolved in ethanol. In the dose range finding test, EX-1028 was tested up to concentrations of 5000 pg/plate in the absence and presence of S9-mix in the strains TA1 00 and WP 2uvrA. EX precipitated on the plates at dose levels of 1000 pg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, EX-1028 was tested in the first mutation assay at a concentration range of 10 to 1000 pg/plate in the absence and presence of 5% (vlv) 89-mix in tester strains TA1 535, TA1 537 and TA98. In an independent repeat of the assay with additional parameters, EX was tested at the same concentration range as the first assay in the absence and presence of 10% (vfv) 89-mix in tester strains TA TA TA98. TAIOD and WP 2uvrA. EX-1028 precipitated on the plates at the top dose of 1000 pg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. EX-1028 did not induce a significant dose-related increase in the number of revertant (His) colonies in each of the four tester strains (TA1 535, TA1 537, TA98 and TA1 00) and in the number of revertant (Trp) colonies in tester strain WP 2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. in this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that EX-1028 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay. - Page 5 - CIR Panel Book Page 120

127 EX-1028 NOTOX Project INTRODUCTION 5.1. Preface Sponsor Study Monitor Test Facility Study Director Technical Coordinator NOTOX By. Hambakenwetering DD s-hertogenbosch The Netherlands CM. Verspeek-Rip Ing. A. Tdink Study Plan Start 23 March 2007 Completion 05 April Aims of the study The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coil bacterial strain resulting in a tryptophan-independent strain. Backq round of the test system The Salmonella typhimurium reverse mutation assay and the Escherichia coil reverse mutation assay have been shown to be rapid arid adequate indicators for the mutagenic activity of a wide range of chemical compounds. The assay was conducted in the absence and presence of a metabolizing system (S9-mix). The Salmonella typhimurium strains used in this study were TAI 535, TA1 537, TA98 and TA100. The Escherichia coil strain used was WP 2uvrA. The strains TA1 537 and TA98 are capable of detecting frameshift mutagens, strains TM535, TA100 and WP 2uvrA are capable of detecting base-pair substitution mutagens (Ref. 1, 2, 3, 4 and 5) Guidelines The study procedures described in this report were based on the following guidelines; - Organisation - European for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: Genetic Toxicology: Bacterial Reverse Mutation Test (Adopted July 21, 1997). Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.13/14: Mutagenicity: Reverse Mutation Test using bacteria. EEC Publication Commission Directive (Published June 8, 2000). - Page 6 - CIR Panel Book Page 121

128 EX NOTOX Project Storage and retention of records and materials Records and materials pertaining to the study including protocol, raw data, specimens and the final report are retained in the NOTOX archives for a period of at least 10 years after finalization of the report. After this period, the sponsor will be contacted to determine whether raw data and specimens should be returned to them, retained or destroyed on their behalf. NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years after finalization of the report. After this period the sample will be destroyed. 6. MATERIALS AND METHODS 6.1. Test substance Test substance information Identification EX Structure -C 18H Molecular weight (theoretical) CAS Number Description Clear colourless very viscous liquid (determined at NOTOX) Batch CM (taken from label) Purity 100% Test substance storage At room temperature in the dark Stability under storage conditions Stable Expiry date 02 March 2008 (allocated by NOTOX, 1 year after receipt of the test substance) Study specific test substance information Stability in vehicle Solubility in vehicle Ethanol: Not indicated Ethanol: Not indicated Test substance preparation The test substance was dissolved in ethanol (Extra Pure, Merck, Darmstadt. Germany). Test substance concentrations were used within 3 hours after preparation 6.2. Reference substances Negative control The vehicle of the test article, being ethanol. - Page 7 - CIR Panel Book Page 122

129 EX NOTOX Project Positive controls Without metabolic activation (-S9-mix ): Strain Chemical Concentration/plate Solvent TA1 535 sodium azide (SA) 5 pg Saline (Sigma, Zwijndrecht, The Netherlands) TAI aminoacridine (9AC) 60 pg Milli-Q water (Acros Organics, Geel, Belgium) TA98 2-nitrofluorene (NF) (Merck) 10 pg DMSO TAIOO methylmethanesulfonate (MMS) (Sigma) 650 pg DMSO WP 2uvrA 4-nitroquinoline N-oxide (4-NQO) (Sigma) 10 pg DMSO With metabolic activation (+S9-mix): The positive control substance used for all tester strains was 2-aminoanthracene (2AA) (Sigma). The following doses were used: Strain Concentration/plate Amount of S9-mix Solvent TA pg 5 and 10% DMSO TA pg 5% DMSO TA pg 10% DMSO TA98 1 pg 5 and 10% DMSO TA100 1 pg 5% DMSO TA pg 10% DMSO WP 2uvrA 10 pg 5 and 10% DM80 Solvents for reference substances Saline physiological saline (B. Braun, Melsungen AG, Germany) DMSO = dimethyl sulfoxide of spectroscopic quality (Merck) Milli-Q water (Millipore Corp., Bedford, MA., USA) 6.3. Test system Test system Rationale Source Salmonella typhimurium bacteria and Escherichia coil bacteria Recommended test system in international guidelines (e.g. OECD and EEC). Salmonella typhimurium strains: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. TA98 received on , used batch: TA TAI 535 received on , used batch: TA TA1537 received on used batch: TA Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames>: TA100 received on , used batch: TA Escherichia coil strain: Prof. Dr. BA. Bridges, University of Sussex, Brighton, U.K. WP 2uvrA received on , used batch: EC The characteristics of the different Salmonella typhimurium strains were as follows: Strain Histidine mutation Mutation type TAI 537 hisc3076 Frameshift TA98 hisd3052lrfactor* Frameshift TAI 535 hisg46 Base-pair substitutions TA100 hisg46/rfactor* Base-pair substitutions *: R-factor = plasmid pkmio1 (increases error-prone DNA repair) - Page 8 - CIR Panel Book Page 123

130 EX NOTOX Project Each tester strain contained the following additional mutations: deep rough (defective iipopolysaccharide celicoat) gj : mutation in the galactose metabolism chi mutation in nitrate reductase bio : defective biotin synthesis loss of the excision repair system (deletion of the ultraviolet-repair B gene) The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TAIOO), UV-sensitivity and the number of spontaneous revertants. The Escherichia coil WP 2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants. jr r Stock cultures of the five strains were stored in liquid nitrogen (-1 96 C). 64. Cell culture Preparation of bacterial cultures Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD. Hampshire, England) and incubated in a shaking incubator (37 C, 150 spm), until the cultures reached an optical density of 1,0 ± 0.1 at 700 nm (10 cells/mi). Freshly grown cultures of each strain were used for a test. Agar plates Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (B. Braun, Melsungen, Germany). The agar plates for the test with the Salmonella fyph/murium strains also contained 12.5 pg/plate biotin (Merck) and 15 pg/plate histidine (Merck) and the agar plates for the test with the Escherichia coil strain contained 15 pg/plate tryptophan (Acros Organics). Milli-Q water containing 0.6% (wlv) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 mm at 121 ± 3 C. Environmental conditions All incubations were carried out in the dark at C (protocolied range 37.0 ± 1.0 C). Temporary deviations of maximally 1 hour (in the range of C) occurred due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity. 65. Metabolic activation system Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany Preparation of S9-fraction The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were orally dosed for three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and - Page 9 - CIR Panel Book Page 124

131 EX NOTOX Project B-naphthoflavone (100 mg/kg body weight) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0CC) sterile 0.1 M sodium phosphate buffer (ph 7.4) containing 0.1 mm 2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 mm at 9000 g. The supernatant (S9) was transferred into sterile ampoules, which were stored in liquid nitrogen (-19&C) for a maximum of 1 year. Na Before use, all S9-batches were characterized with the metabolic activation requiring positive control; benzo[ajpyrene (Sigma) in tester strain TA98 at the concentration of 5 pg/plate. r Preparation of S9-mix S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per 10 ml: 3D mg NADP (Randox) and 15.2 mg glucose-s-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer ph 7.4; 1 ml 0.08 M MgCI solution. The above solution was filter (0.22 pm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (vlv) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) SD-fraction) to complete the SD-mix in the second experiment. The S9-batch used was no Study design Dose range finding test 2 solution; 1 ml 0.33 M KCI Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP 2uvrA, both with and without S9-mix. Eight concentrations. 3, 10, 33, 100, , 3330 and 5000 pg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of EX used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility Mutation assay At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was molten and heated to 45CC. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10 cells/mi) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml SD-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 C for 48 h. After this period revertant colonies (histidine independent (His Escherichia coil) were counted. 4) for Salmonella typhimurium bacteria and tryptophan independent (Trp) for - Page 10- CIR Panel Book Page 125

132 59-mix S9-mix S9-mix S9-mix S9-mix S9-mix 59-mix S9-mix S9-mix Distributed for Comment Only -- Do Not Quote or Cite EX-1028 NOTOX Project Colony counting The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these could be counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope Electronic data capture Observations/measurements in the study were recorded electronically using the following programme: REES version 1.5 (REES scientific, Trenton, NJ, USA): Environmental monitoring Interpretation Acceptability of the assay A Salmonella typhimurium reverse mutation assay and/or Escherichia coil reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. Strain Minimum value Maximum value Mean ± 3 x S.D. TA ± 14 S9-mix ± 13 TA ± 8 +S9-mix ± 9 TA ± 19 +S9-mix ± 21 TA100 - WP 2uvrA ± mix ± ± 17 +S9-mix ± 17 b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean. Strain Minimum value Maximum value Mean ± 3 x S.D TA1535 S9-mix ± 907 S9-mix ± 235 TA ± S9-mix ± 405 TA ± mix ± 932 TA100 - WP 2vvrA ± S9-mix ± ± S9-mix ± 381 c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate. - Page 11- CIR Panel Book Page 126

133 EX-1028 NOTOX Project Data evaluation and statistical procedures No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TAI 535, TA1 537, TA98 orwp 2uvrA is not greater than three (3) times the concurrent control. b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1 535, TA1 537, TA98 orwp 2uvrA is greater than three (3) times the concurrent control. b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision List of deviations List of protocol deviations There were no deviations from the protocol List of standard operating procedures deviations Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study. 7. RESULTS 7.1. Dose range finding test EX was tested in the tester strains TA100 and WP 2uvrA with concentrations of 3. 10, , 1000, 3330 and 5000 pg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test (Table 1). The individual data are presented in Appendix II. Precipitate Precipitation of EX-1028 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 pg/plate and upwards. Toxicity To determine the toxicity of EX-1 028, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated in Appendix I. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. - Page 12 - CIR Panel Book Page 127

134 EX NOTOX Project Mutagenicity In the dose range finding test, no increase in the number of revertants was observed upon treatment with EX-1028 under all conditions tested Mutation assay Based on the results of the dose range finding test, EX-1028 was tested up to concentrations of 1000 hg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was per-formed with the strains TA1 535, TA1 537 and TA98 and the second mutation experiment was performed with the strains TA1535, TM 537, TA98, TA100 and WP 2uvrA. The results are shown in Table I and 2, the individual data are presented in Appendix II. Precipitate Precipitation of EX-1028 on the plates was observed at the start and end of the incubation period at the concentration of 1000 pg/plate. ToJcjt In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested ri all tester strains in the absence and presence of S9-mix. Mutagenicity In both mutation assays, no increase in the number of revertants was observed upon treatment with EX under all conditions tested. 8. DISCUSSION AND CONCLUSION All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that EX-1028 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay. 9. REFERENCES 1 Leonardo, J.M.. Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coil K12 343\ 113 and derived strains for microbial mutagenicity assays. Mutation Res., 130, Ames, B.N., McCann, 3. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, Erratum, 1983, Mutation Res., ii Green, M.H.L. and Muriel, W.J., 1976, Mutagen testing using Trp reversion in Escherichia col Mutation Res., 38, Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coil: partial purification and some properties. J. Biol. Chem., 218, Page 13 - CIR Panel Book Page 128

135 EX NOTOX Project Table I Experiment I: Mutagenic response of EX-I 028 in the Salmonella typhimurium reverse mutation assay and in the Escherlchla coil reverse mutation assay Day of performance: TAIOO and WP 2uvrA: 23 March 2007 TA1535, TA1537 and TA98: 30 March 2007 Dose (iglplate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coil strain TA1535 TA1537 TA98 TA100 WP 2uvrA F Without S9-mix Ix,sit±ve ctro] ± ± ± ± ± 19 1vit cmtxol 16 ± 4 8 ± 4 21 ± ± 7 20 ± ± 3 21± ± 3 8± 1 28± 3 68± 11 21± ± 6 8± 3 29± 3 70± 3 22± ± 6 9± 3 26± 4 89± 2 30± ± 4 5± 2 22± 1 83± 9 28± SE 16 ± 2 6 ± 2 33 ± 5 80 ± 5 30 ± SP 84± 4 21± 2 500D 96± 8 22± 6 With S9-mix 1 ptiw cxixtrol 268 ± ± ± ± ± 20 1vit cxxitrol 17 ± 4 7 ± 4 31 ± 2 80 ± 8 25 ± ± 9 20± ± 3 6± 3 33± 5 65± 10 28± ± 4 8± 4 29± 7 63± 7 25± ± 2 11 ± 2 34 ± 5 85 ± ± ± 3 8± 2 30± 7 86± 10 22± ± 6 7± 1 35± 10 79± 3 26± ? 82± 9 19± ? 67±11 17± 2 Solvent control: 0.1 ml ethanol 1 The S9-mix contained 5% (v/v) S9 fraction SP Slight Precipitate - Page 14- CIR Panel Book Page 129

136 EX NOTOX Project Table 2 Experiment 2: Mutagenic response of EX in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay Day of performance: 03 April 2007 Dose (pg/plate) Mean number of revertant colon ies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coil strain TA1535 TA1537 TA98 TA100 WP 2uvrA Without S9-mix positive cxntzx ± ± ± ± ± 17 so1t oatrt1 16 ± 2 12 ± 5 16 ± 3 77 ± 6 15 ± ± 3 9± 3 21± 4 84± 4 17± ± 4 8± 1 20± 6 82± 5 12± ± 5 9± 3 13± 3 84± 7 14± ± 2 7 ± 2 16 ± 5 80 ± 3 12 ± 5 i000sp 15± 5 11± 1 16± 3 75± 4 16± 5 With S9-mix 1 p,sitive citro1 213 ± ± ± ± ± 17 so1vxt itzo1 14 ± 6 12 ± 5 30 ± 4 71 ± 6 11 ± ± 8 4± 0 27± 6 83± 8 10± ± 2 8± 1 23± 1 76± 1 12± ± 3 8± 4 19± 4 84± 4 10± ± 3 5± 3 20± 3 82± 3 15± ± 2 8± 2 22± 9 77± 9 14± 3 Solvent control: 0.1 ml ethanol 1 The S9-mix contained 10% (VIV) SO fraction sr Slight Precipitate Page 15 - CIR Panel Book Page 130

137 EX-1028 NOTOX Project APPENDIX! SUPPORTING MATERIALS AND METHOD Bacterial background lawn evaluation The condition of the bacterial background lawn is evaluated (if indicated), both macroscopically and microscopically by using a dissecting microscope (results are normal unless indicated in tables). Definition Normal Slightly reduced Moderately reduced Extremely reduced Absent Characteristics Distinguished by a healthy microcolony lawn. Distinguished by a slight thinning of the microcolony lawn. Distinguished by a moderate thinning of the microcolony lawn. Distinguished by an extreme thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the solvent control plate. Distinguished by a complete lack of any microcolony background lawn. Ii- Precipitation evaluation Evidence of test article precipitate on the plates is recorded by addition of the following precipitation definition. Definition Characteristics Slight Precipitate Moderate Precipitate Heavy Precipitate Distinguished by noticeable precipitate on the plate. However, the precpitate does not influence automated counting of the plate. Distinguished by a marked amount of precipitate on the plate. requiring the plate to be hand counted. Distinguished by a large amount of precipitate on the plate, making the required hand count difficult. Evaluation of the reduction in the number of revertants The reduction in the number of revertant colonies compared to number of revertants in the solvent control is evaluated as follows: A reduction of 21-40%: slight reduction. A reduction of 41-60%: moderate reduction. A reduction of 61-99%: extreme reduction. If the size of the microcolonies was increased to small colonies due to an extremely reduced background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on the plates the reduction is evaluated as a complete lack of revertants. However, any mean plate count equal to the minimal value of the historical control data range should be considered not toxic. - Page 16 - CIR Panel Book Page 131

138 - Page Distributed for Comment Only -- Do Not Quote or Cite EX NOTOX Project APPENDIX II DETAILED TABLES lndividua) p ate counts; (following pages) Eziit 1 Strain Th1S35 witjr S9-MDC plate b N± J close (pg/plate) positive cxntrol ± 20 so1v-it ntro ± ± ± ± ± ± 2 WITH S9 MIX plate N± SD âdse (pg/plate) positive cxs-itrol ± 16 so1v-it odntrol ± ± ± ± ± SP ± 6 SP: Slight Precipitate 17 - CIR Panel Book Page 132

139 EX NOTOX Project APPENDiX II continued Eperit 1 Strain TA1537 WITHcX3T S9-+ID( p1at EN± (is (pg/plate) positive no ± 62 so1vit odntrol ± ± ± ± ± SP ± 2 WITH S9 MDC plate b?n± SD cbse (pg/plate) pdsitive cciol ± 10 o1vit cxniol ± ± ± ± ± SP ± 1 SP: Slight I recipitate - Page 18- CIR Panel Book Page 133

140 EX-l 028 NOTOX Project APPENDIX II continued Eriiit 1 Strain TA98 plate WITWXJT S9-IX NEAN± ±se (/p1ate) positive ntro1 solvit ODntrol ± 41 21± SP ± 3 29± 3 26± 4 22± 1 33± 5 plate WITH S9-MIX AN± SI) cbse (pg/plate) positive control so1vt control ± 29 31± SP ± 6 29± 7 34± 6 30± 7 35 ± 10 SF: Slight Precipitate - Page 19- CIR Panel Book Page 134

141 EX NOTOX Project APPENDIX II continued Eri2t1 Strain Th100 WITHYP s9-ndc plate E?iN± dose (/p1ate) ixsitive control ± 42 so1vit control ± ± ± ± ± ± SP ± SP ± SP ± 8 WITH S9-±IIX plate 1 2 3?E?N± J dose (pg/plate) positive control ± 31 so1vit control ± ± ± ± ± ± SP ± SP ± SP ± 11 5?: Slight Pripitate - Page 20 - CIR Panel Book Page 135

142 EX NOTOX Project APPENDIX II continued Eqe.riunt 1 Strain WITHTJ2 S9- plate dose (/p1ate) positive no ± 19 so1vt no ± ± ± ± ± ± SP ± SI ± SP ± 6 WITH S9-tX plate ME±.) dose (/plate) positive no ± 20 solvt oontrol ± ± ± ± ± ± SI ± SI ± SI ± 2 SI : Slight Pripitate - Page 21 - CIR Panel Book Page 136

143 - Page Distributed for Comment Only -- Do Not Quote or Cite EX NOTOX Project APPENDIX II continued Ezerit 2 Strain TA1535 plate wicur S9-c 1 2 3?yE?N± ±se (pg/plate) positive control solvit control ± 36 16± SP ± 3 13± 4 14± 5 11± 2 15± 5 plate WITH S9-MEX ElN± ±se (/plate) positive control olvit otrol ± 17 14± SI? ± 8 11± 2 15± 3 16± 3 13± 2 SI?: Slight Precipitate 22 - CIR Panel Book Page 137

144 - Page Distributed for Comment Only -- Do Not Quote or Cite EX NOTOX Project APPENDIX II continued Ecperiit 2 Strain Th1537 wrrcu S9-+ilDC plate l N± cse (ig/p1ate) positive ntrol ± 95 solvent ntrol ± ± ± ± ± SP ± 1 WI1 S9 NIX plate N± ) ckdse (/p1ate) positive cxntrol ± 10 solvent odntrol ± ± ± ± ± SP ± 2 SP: Slight Precipitate 23 - CIR Panel Book Page 138

145 EX-1028 NOTOX Project APPENDIX II continued Eeriit 2 Stxain TA98 WITHar S9-c plate len± dose (pg/plate) positive oritro ± 12 so1vit ntro ± ± ± ± ± ST ± 3 WITH S9-MtX plate N± Si) dose (pg/plate) positive odtitrol ± 8 so1vt odntrol ± ± ± ± ± ST ± 9 SP: Slight Precipitate - Page 24 - CIR Panel Book Page 139

146 EX-1028 NOTOX Project APPENDIX II continued Eeririt 2 Strain Th100 WITKUT S9 MIX plate NE± ddsg (pg/plate) positive control ± 33 so1vit control ± ± ± ± ± SP ± 4 WITH S9-D plate cidse (pg/plate) positive itro ± 70 so1vit control ± ± ± ± ± SP ± 9 SP: Slight Precipitate - Page 25 - CIR Panel Book Page 140

147 - Page Distributed for Comment Only -- Do Not Quote or Cite EX-1028 NOTOX Project APPENDIX II continued Eperinnt 2 Str.n WP 2UVIA WITHW1 S94ffX plate 1 2 3?N± SD cbse (pg/plate) positive control ± 17 solvit control ± ± ± ± ± SP ± 5 WITH S9-MIX plate EIN± SD ±se (pg/plate) positive control ± 17 soivt control ± ± ± ± ± SP ± 3 SP: Slight Pripitate 26 - CIR Panel Book Page 141

148 COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: DATE: SUBJECT: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel January 28, 2011 Updated Concentration of Use by FDA Product Category: Citric Acid and its Salts and Esters th Street, N.W., Suite 3OO Washington, D.C (fax) CIR Panel Book Page 142

149 Concentration of Use by FDA Product Category Citric Acid, Aluminum Citrate, Calcium Citrate, Copper Citrate, Dianimonium Citrate, Dilauryl Citrate, Disodium Cupric Citrate, Distearyl Citrate, Ethyl Citrates, Ferric Citrate, Isodecyl Citrate, Isopropyl Citrate, Magnesium Citrate, Manganese Citrate, Monosodium Citrate, Potassium Citrate, Propylene Glycol Citrate, Sodium Citrate, Stearyl Citrate, Tributyl Citrate, Tricaprylyl Citrate, Tri-C12-13 Alkyl Citrate, Tri-C1445 Alkyl Citrate, Triethyl Citrate, Triethyihexyl Citrate, Trihexyldecyl Citrate, Triisocetyl Citrate, Triisopropyl Citrate, Triisostearyl Citrate, Trilauryl Citrate, Trioctyldodecyl Citrate, Trioleyl Citrate, Tripropylene Glycol Citrate, Tristearyl Citrate and Zinc Citrate* Ingredient Product Category Concentration of Use Citric Acid Baby shampoo 0.2% Citric Acid Bath oils tablets and salts % Citric Acid Bubble baths % Citric Acid Other bath preparations 20% Citric Acid Eyebrow pencil % Citric Acid Eyeliner % Citric Acid Eye shadow % Citric Acid Eye lotion % Citric Acid Eye makeup remover % Citric Acid Mascara % Citric Acid Colognes and toilet waters % Citric Acid Perfumes % Citric Acid Powders (dusting and talcum) % Citric Acid Other fragrance preparations % Citric Acid Hair conditioners % Citric Acid Hair sprays (aerosol fixatives) 0.08% Citric Acid Hair straighteners 0.1% Citric Acid Permanent waves 0.1-3% Citric Acid Rinses (noncoloring) 2% Citric Acid Shampoos (noncoloring) % Citric Acid Tonics, dressings and other hair grooming aids % Citric Acid Other hair preparations (noncoloring) % Citric Acid Hair dyes and colors (all types requiring caution statement and % patch test) Page 1 of 8 CIR Panel Book Page 143

150 Citric Acid Hair tints 0.2% Citric Acid Hair rinses (coloring) % Citric Acid Other hair coloring preparations % Citric Acid Blushers (all types) % Citric Acid Face powders % Citric Acid Foundations % Citric Acid Leg and body paints 0.3% Citric Acid Lipstick % Citric Acid Makeup bases % Citric Acid Other makeup preparations 0.0 1% Citric Acid Basecoats and undercoats (manicuring preparations) % Citric Acid Cuticle softeners 4% Citric Acid Nail creams and lotions % Citric Acid Nail polish and enamel % Citric Acid Nail polish and enamel removers 0.1% Citric Acid Other manicuring preparations % Citric Acid Dentifrices (aerosol, pastes and powders) 0.2% Citric Acid Mouthwashes and breath fresheners (liquids and sprays) % Citric Acid Other oral hygiene products 0.1% Citric Acid Bath soaps and detergents 0.3-2% Citric Acid Deodorants (underarm) % Citric Acid Vaginal douches 0.3% Citric Acid Feminine hygiene deodorants 0.1% Citric Acid Other personal cleanliness products % Citric Acid Aftershave lotions % Citric Acid Shaving cream (aerosol, brushless and lather) % Citric Acid Skin cleansing (cold creams, cleansing lotions, liquids and pads) % Citric Acid Depilatories % Citric Acid Face and neck creams, lotions and powders % Citric Acid Body and hand creams, lotions and powders % Page 2 of 8 CIR Panel Book Page 144

151 Citric Acid Body and hand sprays % Citric Acid Foot powders and sprays 35% Citric Acid Moisturizing creams, lotions and powders % Citric Acid Moisturizing sprays 0.64% Citric Acid Night creams, lotions and powders % Citric Acid Paste masks (mud packs) % Citric Acid Skin fresheners % Citric Acid Other skin care preparations % Citric Acid Suntan gels, creams and liquids % Citric Acid Indoor tanning preparations % Citric Acid Other suntan preparations % Ethyl Citrates Bath soaps and detergents 1% Ethyl Citrates Skin cleansine (cold creams. cleansine lotions, liquids and oads 0.5% Ferric Citrate Bath soaps and detergents 0.5% Ferric Citrate Skin cleansine (cold creams. cleansine lotions, liquids and cads) 0.5% Magnesium Citrate Hair conditioners 0.5% Magnesium Citrate Shampoos (noncoloring) 0.5% Magnesium Citrate Tonics, dressings and other hair grooming aids 2% Maenesium Citrate Body and hand creams, lotions and nowders 0.0 1% Monosodium Citrate Baby lotions, oils powders and creams 5% Monosodium Citrate Bath oils, tablets and salts 5% Monosodium Citrate Other personal cleanliness products 2% Monosodium Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) 0.8% Monosodium Citrate Face and neck creams, lotions and powders 0.004% Monosodium Citrate Other skin care preparations 3 5% Potassium Citrate Colognes and toilet waters 0.06% Potassium Citrate Powders (dusting and talcum) 0.02% Potassium Citrate Hair sprays (aerosol fixatives) 0.07% Potassium Citrate Rinses (noncoloring) 0.002% Potassium Citrate Shampoos (noncoloring) 0.002% Page 3 of 8 CIR Panel Book Page 145

152 Potassium Citrate Dentifrices (aerosol, liquid, pastes and powders) 0.6% Potassium Citrate Bath soaps and detergents 0.002% Potassium Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) 0.5% Potassium Citrate Face and neck creams, lotions and powders 0.002% Potassium Citrate Body and hand creams, lotions and Dowders 0.06% Sodium Citrate Bath oils, tablets and salts 0.9% Sodium Citrate Eyeliner 0.07% Sodium Citrate Eye shadow % Sodium Citrate Eye lotion % Sodium Citrate Eye makeup remover 0.5-2% Sodium Citrate Mascara 0.2% Sodium Citrate Colognes and toilet waters 0.2% Sodium Citrate Perfumes 0.3% Sodium Citrate Hair conditioners % Sodium Citrate Hair sprays (aerosol fixatives) % Sodium Citrate Rinses (noncoloring) 0.2% Sodium Citrate Shampoos (noncoloring) 0.1-3% Sodium Citrate Tonics, dressings and other hair grooming aids % Sodium Citrate Other hair preparations (noncoloring) % Sodium Citrate Hair rinses (coloring) 0.1% Sodium Citrate Hair color sprays (aerosol) 0.1% Sodium Citrate Blushers (all types) 0.1% Sodium Citrate Face powders 0.03% Sodium Citrate Foundations % Sodium Citrate Lipstick % Sodium Citrate Makeup bases % Sodium Citrate Other makeup preparations 0.03% Sodium Citrate Basecoats and undercoats (manicuring preparations) 0.5% Sodium Citrate Nail creams and lotions 0.08% Sodium Citrate Nail polish and enamel removers 0.1% Page 4 of 8 CIR Panel Book Page 146

153 Sodium Citrate Mouthwashes and breath fresheners (liquids and sprays) % Sodium Citrate Bath soaps and detergents % Sodium Citrate Deodorants (underarm) % Sodium Citrate Other personal cleanliness products 0.04% Sodium Citrate Aftershave lotions % Sodium Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) % Sodium Citrate Face and neck creams, lotions and powders % Sodium Citrate Face and neck sprays 0.1% Sodium Citrate Body and hand creams, lotions and powders % Sodium Citrate Foot powders and sprays 0.1% Sodium Citrate Moisturizing creams, lotions and powders % Sodium Citrate Night creams, lotions and powders % Sodium Citrate Paste masks (mud packs) % Sodium Citrate Skin fresheners 0.6-2% Sodium Citrate Other skin care preparations % Sodium Citrate Suntan gels, creams and liquids % Sodium Citrate Indoor tanning preparations 0.2% Sodium Citrate Other suntan preparations % Stearyl Citrate Eyeliner 1% Stearyl Citrate Eye lotion 1% Stearyl Citrate Mascara 2% Stearyl Citrate Hair conditioners 1% Stearyl Citrate Foundations 0.3-2% Stearyl Citrate Lipstick 12% Stearyl Citrate Bath soaps and detergents % Stearyl Citrate Deodorants (underarm) 3% Stearyl Citrate Aftershave lotions 1% Stearyl Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) 0.8-2% Stearyl Citrate Depilatories 2% Stearyl Citrate Face and neck creams, lotions and powders 0.3-5% Page 5 of 8 CIR Panel Book Page 147

154 Stearyl Citrate Body and hand creams, lotions and powders 5% Stearyl Citrate Foot powders and sprays 3% Stearyl Citrate Night creams, lotions and powders 1% Stearyl Citrate Paste masks (mud packs) 2% Stearyl Citrate Other skin care preparations 4% Stearyl Citrate Suntan gels, creams and liquids 2% Stearvi Citrate Indoor tanning preparations 1% Tributyl Citrate Bubble baths % Tributyl Citrate Other fragrance preparations % Tributyl Citrate Basecoats and undercoats (manicuring preparations) % Tributyl Citrate Nail polish and enamel 9% Tributyl Citrate Nail polish and enamel removers 5% Tributyl Citrate Bath soaps and detergents 0.001% Tributyl Citrate Other personal cleanliness products % Tributyl Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) <0.05% Tributyl Citrate Body and hand creams, lotions and powders % Tributvl Citrate Other skin care preparations <0.05% Tri-C14-15 Alkyl Citrate Eye shadow 3% Tri-C14-15 Alkyl Citrate Makeup bases 5% Tri-C14-15 Alkvl Citrate Indoor tanninn orenarations 0.1-5% Tricaprylyl Citrate Eye shadow 3% Tricaprylyl Citrate Hair conditioners 0.8% Tricaprylyl Citrate Other hair preparations (noncoloring) 0.5% Tricaprylyl Citrate Foundations 9% Tricaprylyl Citrate Lipstick 14-19% Tricaprylyl Citrate Other makeup preparations 27% Tricaprylyl Citrate Face and neck creams, lotions and powders 0.3% Tricaprylyl Citrate Body and hand creams, lotions and powders 5% Tricaprylyl Citrate Other skin care preparations 4% Tricaprylyl Citrate Suntan eels, creams and linuids 5% Page 6 of 8 CIR Panel Book Page 148

155 Triethyl Citrate Other baby products 0.009% Triethyl Citrate Eye shadow 0.6% Triethyl Citrate Eye lotion 0.2% Triethyl Citrate Other fragrance preparations 2% Triethyl Citrate Hair conditioners 0.1% Triethyl Citrate Hair sprays (aerosol fixatives) 0.2-1% Triethyl Citrate Shampoos (noncoloring) 0.1% Triethyl Citrate Tonics, dressings and other hair grooming aids % Triethyl Citrate Other hair preparations (noncoloring) 5 0.2% Triethyl Citrate Hair color sprays (aerosol) 0.5% Triethyl Citrate Blushers (all types) 5% Triethyl Citrate Face powders 3% Triethyl Citrate Foundations 0.3% Triethyl Citrate Lipstick 0.3% Triethyl Citrate Deodorants (underarm) 2% Triethyl Citrate Other personal cleanliness products 0.2% Triethyl Citrate Skin cleansing (cold creams, cleansing lotions, liquids and pads) % Triethyl Citrate Face and neck creams, lotions and powders 6% Triethyl Citrate Body and hand creams, lotions and powders 0.004% Triethyl Citrate Body and hand surays 0.2% Triisocetyl Citrate Blushers (all types) 0.6% Triisocetyl Citrate Face powders 2% Triisocetyl Citrate Face and neck creams, lotions and jowders 3% Triisostearyl Citrate Foundations 0.3% Triisostearyl Citrate Lipstick 9-80% Triisostearyl Citrate Makeup fixatives 9% Triisostearyl Citrate Other skin care nrenarations 5% Trioctyldodecyl Citrate Eyeliner 5% Trioctyldodecyl Citrate Eye shadow 5-21% Trioctyldodecyl Citrate Eye lotion 11% Page 7 of 8 CIR Panel Book Page 149

156 Trioctyldodecyl Citrate Blushers (all types) 14% Trioctyldodecyl Citrate Face powders 1-3% Trioctyldodecyl Citrate Lipstick 1-19% Trioctyldodecyl Citrate Other makeup preparations 30% Trioctyldodecyl Citrate Face and neck creams, lotions and powders 2% Trioctyldodecyl Citrate Body and hand creams, lotions and powders 4% Trioctyldodecyl Citrate Foot powders and sprays 4% Trioctyldodecyl Citrate Suntan e1s, creams and liquids 14% Zinc Citrate Face powders 0.05% Zinc Citrate Foundations 0.05% Zinc Citrate Dentifrices (aerosol, liquid, pastes and powders) 2% Zinc Citrate Mouthwashes and breath fresheners (liquids and sprays) 0.3% *Ingredients included in the title of the table, but not found in the table were included in the concentration of use survey, but no uses were reported. 15% in a manicure soak that is diluted to 0.025% before use 210% in a professional peel 35% in a rinse-off product 2% and 7% in leave-on products 0.2% in a non-aerosol hair spray Information collected in 2010 Table prepared January 5, 2011 Updated January 28, 2011 (Citric Acid: added low concentration Other suntan preparations; Sodium Citrate: added low concentration Other suntan preparations; Tri-C14-15 Alkyl Citrate: added low concentration to Indoor tanning preparations) Page 8 of 8 CIR Panel Book Page 150

157 Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (Cifi) John Bailey, Ph.D. Industry Liaison to the CJR Expert Panel DATE: March 15, 2011 SUBJECT: Concentration of Use Additional Citrate Ingredients Laureth-6 Citrate, Laureth-7 Citrate, Disodium Laureth-7 Citrate, Dilaureth-7 Citrate, Sodium Dilaureth-7 Citrate, PEG-5 Tricapryl Citrate, PEG-5 Trilauryl Citrate, Trilaureth-9 Citrate, PEG-5 Trimyristyl Citrate and PEG-S Tristearyl Citrate were included in a concentration of use survey. No uses of these ingredients were reported th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 151

158 CCR COSMETIC Distributed for Comment Only -- Do Not Quote or Cite Personal Care iproducts Council Committed to Safety, Quality & Innovation Memorandum TO: FROM: DATE: SUBJECT: F. Alan Andersen, Ph.D. Director - INGREDffiNT REVifiW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel February 22, 2011 Unpublished Information on Laureth-7 Citrate Cognis Data profile Plantapon LC 7 (INCI name: Laureth-7 Citrate) (C-SAT and BHL-Fo H are identical) RCC Salmonella lyphimurium reverse mutation assay of C-SAT (Laureth-7 Citrate). RCC - Study Number Consumer Product Testing Co Repeated Insult Patch Test of C-SAT (Laureth-7 Citrate) (tested as a 10% dilution). Experiment Reference Number C Henkel KgaA In-vitro-test: NET-CAM [HCI] Reaction time method of BHL-Fo H (H is Laureth-7 Citrate) (tested at 5% active substance [AS]). Report No. R th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 152

159 COGNIS HYBR0S carechemicals Data Profile PLANTAPON LC 7 C 31T QOO)b CnC 1 Address Producer!Supplier Cognis GmbH Care Chemicals D Düsseldorf Tel.: Fax.: I 14 cjc -ci 4o L 4-h 1 CL-C, c General characterisation Chemical description Ester of ethoxylated Fatty Alcohol and Citric Acid Mol weight 677 g/mol Raw material basis Vegetable: Synthetic: (coconut/palm kernel oil, citric acid) (ethylene oxide) Labeling information lncl name(s) Laureth-7 Citrate (EU + CTFA) Composition hints for finished product label Active matter mm. 99,5 % Water max. 0,5 % Ingredient information Ingredient CASR-No. EINECS!ELINCS-No. Laureth-7 Citrate polymer Manufacturing procedure Esterification of fatty alcohol ethoxylate with citric acid. Cognis, PLANTAPON LC 7 Care Chemicals CIR Panel 1/3 Book Page 153 Rev. 2.1 js

160 Product properties Appearance Plantapon LC 7 is a clear, slightly yellow liquid. Example of use Plantapon LC 7 is a anionic citric acid-derived surfactant with an excellent dermatological and active care profile. When incorporated into personal wash formulations, such as body washes, shampoos, facial washes or baby cleansing products, Plantapon LC 7 provides moisturization and care to skin and hair. Being 100% active, it contains no water and can thus be used in non-aqueous products, such as bath and shower oils. Characteristic v&ues The specifications stated in the paragraphs Quality control data and Additional product descriptive data finally and conclusively describe the properties of the product. Quality control data (Data which is used for quality release and is certified for each batch.) Odor evaluation versus standard corresponds to the standard Visual appearance versus standard corresponds to the standard Active matter >= 99,5 % Cognis method (100 - water 0/ /0 Acid number ISO 660 Saponification value ISO 3657 C-chain distribution Cognis Method <C12 <=3,0% % % C % % >C18 <=1,0% Additional product descriptive data (Data which is proven statistically but not determined regularly.) phvalue(10%) 1,8-2,8 1S04316 Cognis, PLANTAPON LC 7 Care Chemicals Rev. 2.1 CIR Panel 2/3 Book Page 154

161 Stabilising additives I Auxiliaries (type and concentration) Preservatives not present Antioxidants approx % of a mixture of Tocopherol (and) Hydrogenated Palm Glycerides Citrate Solvents not present Others Citric acid max. 7 % Storage and transportation Shelf life 12 months Storage temperature Temperatures between + 20 C and 40 C Storage conditions In original sealed containers and protected from moisture. Disclaimer All products in the text marked with an are trademarks of the Cognis group. The information on product specifications provided herein is only binding to the extent confirmed by Cognis in a written Sales Agreement. COGNIS EXPRESSLY DISCLAIMS ANY RESPONSIBILITY FOR THE SUITABILITY OF THE PRODUCTS FOR ANY SPECIFIC OR PARTICULAR PURPOSES INTENDED BY THE USER Suggestions for the use and application of the products and guide formulations are given for information purposes only and without commitment. Such suggestions do not release Cognis customers from testing the products as to their suitability for the customers intended processes and purposes. Cognis does not assume any liability or risk involved in the use of its products as the conditions of use are beyond its control. The user of the products is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties. Cognis, PLANTAPON LC 7 Care Chemicals CIR Panel 3/3 Book Page 155 Rev. 2.1 Is

162 Ce JoO RCC - CCR STUDY NUMBER SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY WITH C-SAT LoJe CJqC REPORT STUDY COMPLETION DATE: May 09, 2003 CIR Panel Book Page 156

163 RCC-.CCR Study Number Report Page 2 of 16 C-SAT I CONTENTS 1 CONTENTS 2 2 PREFACE General Responsibilities Schedule Project Staff Signatures Guidelines Archiving 4 3 OBJECTIVE Aims of the Study Reasons for the Study 5 4 MATERIALS AND METHODS Test Item Controls Test System Mammalian Microsomal Fraction S9 Mix Dose Selection Experimental Performance Data Recording Acceptability of the Assay Evaluation of Results Biometry 11 5 DISCUSSION OF RESULTS 12 6 REFERENCES Distribution of the Report 13 7 ANNEXE: TABLES OF RESULTS 14 8 HISTORICAL CONTROL DATA 16 st2reoglp CIR Panel Book Page 157

164 RCC-CCR Study Number Report Page 3 of 16 C-SAT PREFACE 2.1 Genera Title: Salmonella typhimurium Reverse Mutation Assay with C-SAT Sponsor: Study Monitor: Test Facility: R C C CYTOTEST CELL RESEARCH GMBH In den Leppsteinswiesen 19 D Roldorf 22 Respon&bilities Study Director: Management: Dr. Albrecht Poth Dr. Wolfgang Völkner 2.3 Schedule Experimental Starting Date: April 03, 2003 Experimental Completion Date: April 08, 2003 Date of Final Report: May 09, 2003 st2reoglp CIR Panel Book Page 158

165 RCC-CCR Study Number Report Page 4 of 16 C-SAT Project Staff Signatures Study Director Dr. Albrecht Poth Date: May 09, 2003 Management Dr. Wolfgang Välkner C). U;LA Date: : May 09, Guidelines This study followed the procedures indicated by the following internationally accepted gui delines and recommendations: Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial Reverse Mutation Test, adopted July 21, Commission Directive 2000!321EC, L , Annexe 4D, dated May 19, Variation: The study was performed with the strains TA 98 and TA 100 only 2.6 Archiving RCC Cytotest Cell Research will archive the following data for 2 years: Raw data, copy of report, and a sample of the test item No raw data or matenal relating to the study will be discarded without the sponsor s prior consent. st2reoglp CIR Panel Book Page 159

166 RCC-CCR Study Number Report Page 5 of 16 C-SAT OBJECTIVE 3.1 Aims of the Study The experiments were performed to assess the potential of the test item to induce gene mutations by means of the Salmonella typhimurium reverse mutation assay. 3.2 Reasons for the Study The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker. The Salmonella typhimurium histidine (his) reversion system measures his- --> reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100) and frameshift (TA 98) mutations. According to the direct plate incorporation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted. To establish a dose response effect 8 dose levels with adequately spaced intervals were tested. The maximum dose level was 5000 pg/plate. To validate the test, reference mutagens are tested in parallel to the test item. his st2reoglp CIR Panel Book Page 160

167 RCC-CCR Study Number Report Page 6 of 16 C-SAT MATERIALS AND METHODS 4.1 Test Item Internal RCC-CCR Test Item Number: S The test item and the information concerning the test item were provided by the sponsor. Name: C-SAT Batch No.: Aggregate State at Room Temperature: Colour: Purity: Stability in Solvent: Storage: BHL-FO /Technikumsansatz liquid yellow 100 % active ingredient not indicated by the sponsor room temperature Expiration Date: August 13, 2004 On the day of the experiment, the test item C-SAT was dissolved in deionised water. The solvent was chosen because of its solubility properties. No precipitation of the test item occurred up to the highest investigated dose. st2reoglp CIR Panel Book Page 161

168 RCC-CCR Study Number Report Page 7 of 16 C-SAT Controls Negative Controls Concurrent untreated and solvent controls were performed Positive Control Substances Without metabolic activation Strains: TA 100 Name: sodium azide, NaN 3 Supplier: SERVA, D Heidelberg Catalogue No.: Purity: at least 99 % Dissolved in: water deionised Concentration: 10.Jg/plate Strains: TA98 Name: 4-nitro-o-phenylene-diamine, 4-NOPD Supplier: S IGMA, D Deisenhofen Catalogue No.: N 9504 Purity: > 99.9 % Dissolved in: Concentration: DMSO (purity >99 %, MERCK, D Darmstadt) 10 pg/plate in TA 98, 50 pg/plate in TA With metabolic activation Strains: TA 98, TA 100 Name: 2-aminoanthracene, 2-AA Supplier: SIGMA, D Deisenhofen Catalogue No.: A 1381 Purity: 97.5 % Dissolved in: DMSO (purity >99 %, MERCK, D Darmstadt) Concentration: 2.5 pg/plate The stability of the positive control substances in solution was unknown but a mutagenic response in the expected range is sufficient evidence of biological stability. st2reoglp CIR Panel Book Page 162

169 RCC-CCR Study Number Report Page 8 of 16 C-SAT Test System Characterisation of the Salmonella typhimurium Strains The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histiciine locus. Additionally due to the deep rough (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named uvrb-minus. In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pkm 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrb-mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid paq1 carrying the hisg428 mutation (ochre mutation in the hisg gene ) and a tetracycline resistance gene. In summary, the mutations of the TA strains used in this study can be described as follows: Salmonella typhimurium Strains Genotype Type of mutations indicated TA 98 his D 3052; rfa; uvrb;r-factor frame shift mutations TA 100 his G 46; rta; uvrb;r-factor base-pair substitutions Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled. The bacterial strain TA 100 was obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D Darmstadt) Storage The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D Darmstadt) in liquid nitrogen Precultures From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 p1 ampicillin (25 pg/mi) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D Darmstadt) 5 g NaCI (MERCK, D Darmstadt) The bacterial cultures were incubated in a shaking water bath for 4 hours at 370 C. st2reoglp CIR Panel Book Page 163

170 RCC-CCR Study Number Report Page 9 of 16 C-SAT Selective Agar The plates with the minimal agar were obtained from E. Merck, D Darmstadt Overlay Agar The overlay agar contains per litre: 6.0 g MERCK Agar Agar* 6.0 g NaCl* 10.5mg L-Histidine x HCI xh20* 12.2mg Biotin* * (MERCK, D Darmstadt) Sterilisations were performed at 121 C in an autoclave. 4.4 Mammalian Microsomal Fraction S9 Mix The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture S9 (Preparation by R C C - C C R) Phenobarbital/j3-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8-12 weeks old male Wistar Hanlbm rats, weight approx g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D Hamburg) and p-naphthoflavone p.o. (Aldrich, D Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquotes of the supematant are frozen and stored in ampoules at -80 C. Small numbers of the ampoules can be kept at -20 C for up to one week. The protein concentration in the S9 preparation was 35.1 mg/mi (lot no. R ). st2reoglp CIR Panel Book Page 164

171 RCC-CCR Study Number Report Page 10 of 16 C-SAT S9 Mix Before the experiment an appropriate quantity of S9 supernatarit was thawed and mixed with S9 co-factor solution. The amount of S9 supematant was 15% v/v in the cultures. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 2 8 mm MgCI 33mM KCI 5 mm Glucose-6-phosphate 5mM NADP in 100 mm sodium-ortho-phosphate-buffer, ph 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(4). 4.5 Dose Selection The maximum concentration was pg/plate. The concentration range covered two logarithmic decades. In this study eight adequately spaced concentrations were tested. The test item was tested at the following concentrations: 3; 10; 33; 100; 333; 1000; 2500; and 5000 pg/plate 4.6 Experimental Performance For each strain and dose level, including the controls three plates were used as a minimum. The following materials were mixed in a test tube and poured onto the selective agar plates: 100 p1 Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 p1 S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 p1 Bacteria suspension (cf. test system, pre-culture of the strains), 2000pl Overlay agar After solidification the plates were incubated upside down for at least 48 hours at 37 C in the dark. st2reoglp CIR Panel Book Page 165

172 RCC-CCR Study Number Report Page 11 of 16 C-SAT Data Recording The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D Karben). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). 4.8 Acceptability of the Assay The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria: - regular - the - the background growth in the negative and solvent control spontaneous reversion rates in the negative and solvent control are in the range of our historical data positive control substances should produce a significant increase in mutant colony frequencies 4.9 Evaluation of Results A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant Biometry A statistical analysis of the data is not required. st2reoglp CIR Panel Book Page 166

173 RCC-CCR Study Number Report Page 12 of 16 C-SAT DISCUSSION OF RESULTS The test item C-SAT was assessed for its potential to induce gene mutations ac cording to the plate incorporation test using Salmonella typhimurium strains TA 98 and TA 100. The assay was performed in with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations: 33, 100; 333; 1000; 2500; and 5000 pg/plate No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the two tester strains was observed following treatment with C-SAT at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct in crease in induced revertarit colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. st2reoglp CIR Panel Book Page 167

174 RCC-CCR Study Number Report Page 13 of 16 C-SAT REFERENCES 1. Ames, B.N., J. McCann, and E. Yamasaki (1977) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test In: B.J. Kilbey et al. (Eds.) Handbook of Mutagenicity Test Procedures Elsevier, Amsterdam, de Sen-es F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay Mutation Res. 64, Hollstein,M., J. McCann, F.A. Angelosanto and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens Mutation Res. 65, Maron, D.M., Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test Mutation Res. 113, Distribution of the Report Sponsor Study Director 2x (lx original, lx copy) lx (copy) st2reoglp CIR Panel Book Page 168

175 RCC-CCR Study Number Report Page 14 of 16 C-SAT ANNEXE: TABLES OF RESULTS Plate Incorporation Test Test item: C-SAT S9 mix from: Rat liver (Batch R ) Test strain: TA98 without S9 mix Concentration Plate Revertants / plate pg/plate mean s.d. factor* Negative Control Solvent Control Positive Control# with S9 mix Concentration Plate Revertants / plate pg/plate mean s.d. factor* Negative Control Solvent Control Positive Control * z revutants / conceuti. test jtn enhancement factor = rcvertants / solvent conijol # 4-nitro-o-phenylene-diamine 10 pg/plate 2-aminoanthracene 2.5 pg/plate st2reoglp CIR Panel Book Page 169

176 RCC-CCR Study Number Report Page 15 of 16 C-SAT Plate Incorporation Test Test item: C-SAT S9 mix from: Rat liver (Batch R ) Teststrain: TA 100 without S9 mix Concentration Plate Revertants / plate pg/plate mean s.d. factor* Negative Control Solvent Control Positive Control# with S9 mix Concentration Plate Revertants I plate pg/plate mean s.d. factor* Negative Control Solvent Control Positive Controls * z revtants / conc. test item enhanceinit ctor = revertants / so1vt conln)l sodium azide 10 pg/plate 2-aminoanthracene 2.5 pg/plate st2reoglp CIR Panel Book Page 170

177 RCC-CCR Study Number Report Page 16 of 16 C-SAT HISTORICAL CONTROL DATA Strain Range of mean value of revertants/plate (without metabolic activation) Negative control Solvent control Positive Control S. typhimurium TA S. typhimurium TA Range of mean value of revertants/plate (with metabolic activation) S.typhimuriumTA S. typhimurium TA These values represent our historical control data since st2reoglp CIR Panel Book Page 171

178 Consumer Product Testing Co. EST FINAL REPORT CLIENT: ATIENTTON: TEST: Repeated Insult Patch Test ProtocolNo.: 1.01 TEST MATERIAL: C-SAT EXPERIMENT REFERENCE NUMBER: C l Richard R. Eisenberg, MD. Board Certified Dermatolo st Michael Traudt, Director, Clinical Evaluations Robert W. S anahan, Ph.D. Prmcipal Investigator This report a submitted name t these Laboratories nor any memb5r ot for without written authorization. JfFf,nkR.N. the xclusjve use of the person, partnership, or corporation stall may be iced a ecutive Vice President, Clinical Evaluations in connection to with whom It addressed, and neither the report nor the the advertising or sate of any produr or p oeesa 70 New Dutch Lane Fairfield. New Jersey (973) Ii Fax 973) CIR Panel Book Page 172

179 EST 1975 Consumer Product Testing Co. OUALITY ASSURANCE UNIT STATEMENT Study No.: C The objective of the Quality Assurance Unit (QAU) is to monitor the laboratory studies. These studies have been performed with adherence Clinical Practice and requirements provided for in operating procedures and applicable protocols. The 21 CFR parts standard operating procedures and has inspected this study management and these inspections have been reported pertinent to to conduct and reporting of clinical Guideline accordance maintains copies of study protocols and and 50 QAU on the the at 70 this study will be stored in the Archive Facility Jersey, 07004, unless specified otherwise, in writing by the Sponsor. to ICH 56 in and E6 to for Good standard date(s) listed below. The findings of Study Director. All materials New Dutch Lane, and data Fairfield, New Date(s) of inspection: July July August 22, 23, , , 29, 2003 August25, August August Senior personnel involved: Richard Hettenbach, M.A. Senior Director of Regulatory Affairs & Quality Assurance Marie Terlizzese, M.S. Quality Assurance Associate The representative signature of the Quality Assurance Unit signifies that this study has been performed in accordance with standard operating procedures regarding such procedures and protocols. and study protocol as well as government regulations 70 New Dutch Lane Fairfield, New Jersey Clinical (973) Toxicology Analytical Chemistry Microbiology Fax (973) CIR Panel Book Page 173

180 Cognis Deutschland GmbH C O1 3 Page & Co. KG. Objective: To to determine by repetitive epidermal contact the potential of a test material induce primary or cumulative irritation and/or allergic contact sensitization. Participants: One hundred thirteen (113) qualified subjects, male age and female, ranging in from years, were selected for this evaluation. One hundred (100) subjects completed this 17 to 79 study. The remaining subjects discontinued the their participation for various reasons, none of which were related application of the test material. to Inclusion Criteria: a. b. c. d. e. Male and female subjects, age 16a and over. Absence of any visible skin disease which might be confused with reaction from the test material. Prohibition of use of topical or systemic steroids antihistamines study initiation. Completion of a Medical History form and the understanding for at least seven signing of an days prior to Informed Consent form. anchor Considered reliable and capable of following directions. a skin and Exclusion Criteria: a. Ill b. c. d. A health. Under a outcome of the doctor s care or taking medication(s) which could influence the study. Females who are pregnant or nursing. history of adverse reactions products. to cosmetics or other personal care Test Material: C-SAT Study Schedule: Panel # Proposed Actual Initiation Date Completion Date Completion Date July July 2, 9, August 8,2003 August 9,2003 August 21, 2003 August 21, 2003 awjth parental or guardian consent CIR Panel Book Page 174

181 Cognis Deutschland GmbH & Co. KG. C Page 4 Methodology: Prior to the initiation of this study, the test material was prepared as a 10% dilution with ph adjustment to 5.5. The upper back between the scapulae served as the treatment area. Approximately 0.2 ml of the test material, or an amount sufficient to cover the contact surface, was applied to the 3/4 x 3/4 absorbent pad portion of an adhesive dressing*. This was then applied to the appropriate treatment site to form an occluded patch. Induction Phase: Patches were applied three (3) times per week (e.g., Monday, Wednesday, and Friday) for a total of nine (9) applications. The site was marked to ensure the continuity of patch application. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent Induction patches at home, twenty-four hours after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assigned test day, one (1) makeup day was permitted. This day was added to the Induction period. It was noted that due to a holiday weekend which occurred during the Induction Phase, subjects who required a makeup day experienced a delay between applications. With the exception of the first supervised Induction Patch reading, if any test site exhibited a moderate (2-level) reaction during the Induction Phase, application was moved to an adjacent area. Applications are discontinued for the remainder of this test phase, if a moderate (2-level) reaction was observed on this new test site. Applications would also be discontinued if marked (3- level) or severe (4-level) reactivity was noted. Rest periods consisted of twenty-four hours following each Tuesday and Thursday removal, and forty-eight hours following each Saturday removal. Cha1lene Phase: Approximately two (2) weeks after the final Induction patch application, a Challenge patch was applied to a virgin test site adjacent to the original Induction patch site, following the same procedure described for Induction. The patch was removed and the site scored at the clinic twenty-four and seventy-two hours post-application. *Manufactured by TruMed Technologies, Inc., Bumsville, MN CIR Panel Book Page 175

182 Cognis Deutschland GmbH & Co. KG. C O 1 Page 5 Evaluation Key: 0 = No visible skin reaction + = Barely perceptible or spotty erythema 1 Mild erythema covering most of the test site 2 = Moderate erythema, possible presence of mild edema 3 = Marked erythema, possible edema 4 = Severe erythema, possible edema, vesiculation, bullae andjor ulceration Results: The results of each participant are appended (Table 1). Observations remained negative throughout the test interval. Summary: Under the conditions of this study, test material, C-SAT , did not indicate a potential for dermal irritation or allergic contact sensitization. CIR Panel Book Page 176

183 Cognis Deutschland GmbH & Co. KG. C Page 6 Table 1 Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72hr o 0 o o DID NOT COMPLETE STUDY DNC o o o * = Supervised removal of 1st Induction and Challenge Patch DNC = Did not complete study CIR Panel Book Page 177

184 Cognis Deutschland GmbH & Co. KG. C Page 7 Table I (continued) Panel # Individual Results C-SAT Virgin Challenge Subject - Induction Phase Site Number 24*hr *1w 721w DID NOT COMPLETE STUDY om o o 0 0 o o o o o DID NOT COMPLETE STUDY o o DID NOT COMPLETE STUDY o o om om 0 0 o o * = Supervised removal of 1st Induction and Challenge Patch m = Additional makeup day granted at the discretion of the clinic supervisor CIR Panel Book Page 178

185 Cognis Deutschland GmbH & Co. KG. C Page 8 Table 1 (continued) Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Number 24*hr Site 24*hr 72hr DNC DID NOT COMPLETE STUDY 13 DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY * = Supervised removal of l Induction and Challenge Patch DNC = Did not complete study CIR Panel Book Page 179

186 Cognis Deutschland GmbH & Co. KG. C l Page 9 Table I (continued) Panel # Individual Results C-SAT Virgin Challenge Subject Induction Phase Site Number 24*br *hr 72 hr DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY * = Supervised removal of 1st Induction and Challenge Patch CIR Panel Book Page 180

187 Cognis Deutschland GmbH & Co. KG. C Page 10 Table 2 Panel # Subject Data Subject Number Initials Age Sex I LV 70 F 2 CN 61 F 3 GC 48 F 4 KM 20 F 5 AT 30 M 6 DF 49 F 7 JM 70 F 8 LM 63 F 9 MC 45 M 10 MC 36 M 11 LII 69 F 12 PC 36 F 13 HU 39 F 14 EA 64 F 15 MZ 64 F 16 LC 75 M 17 DM 33 F 18 AM 71 F 19 JM 76 M 20 KM 55 F 21 RR 60 F 22 CL 32 F 23 DM 23 F 24 MS 55 F 25 DC 46 F 26 AB 75 F 27 EG 43 F 28 YG 18 F 29 JU 60 M CIR Panel Book Page 181

188 Cognis Deutschland GmbH & Co. KG. C l Page 11 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 30 FG 55 F 31 JC 42 M 32 WF 22 M 33 RB 50 F 34 LS 64 F 35 BP 67 F 36 AS 74 M 37 LL 64 F 38 JS 46 F 39 AS 17 F 40 FR 74 F 41 CC 66 F 42 WM 35 F 43 SC 43 F 44 AF 58 F 45 JL 52 F 46 BM 73 M 47 OL 42 F 48 NM 46 F 49 LC 44 F 50 PS 25 F 51 EK 54 M 52 SG 25 F 53 LS 19 F 54 MS 72 F 55 JS 22 F 56 WR 29 M CIR Panel Book Page 182

189 Cognis Deutschland GmbH & Co. KG. C l Page 12 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 1 PT 64 F 2 EM 53 F 3 AN 20 F 4 CV 29 F 5 SA 58 F 6 FC 61 M 7 AC 22 F 8 MA 61 F 9 LL 26 F 10 MR 35 F 11 TM 75 F 12 TS 25 M 13 DC 47 M 14 TP 58 M 15 CA 61 F 16 ES 29 F 17 EG 22 F 18 NL 46 F 19 NL 54 F 20 LC 39 F 21 AV 43 F 22 MK 41 F 23 JD 55 M 24 ES 43 F 25 MP 21 F 26 SO 59 F 27 EB 37 F 28 GD 47 F 29 VA 49 M CIR Panel Book Page 183

190 Cognis Deutschland GmbH & Co. KG. C Page 13 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 30 JV 58 F 31 OU 70 M 32 SM 55 M 33 VT 79 F 34 SV 58 F 35 RV 65 M 36 LR 60 M 37 FD 77 M 38 LD 53 F 39 IP 47 M 40 LS 34 F 41 CM 72 F 42 TL 63 M 43 RM 77 F 44 MV 65 F 45 SE 56 F 46 MM 67 F 47 RD 39 F 48 LO 59 F 49 JT 23 F 50 JC 27 F 51 AC 58 F 52 RS 63 F 53 DE 68 F 54 WE 75 M 55 EM 72 F 56 ML 54 F 57 Fl 72 M CIR Panel Book Page 184

191 Henkel KGaA Skin Biochemistry.myst.m 4 DNX Q91.i-W 44) OAA- A Final Report Report No. R In-vitro-test: RET-CAM CIj - Reaction-Time Method -,,BHL -Fo A to I = ( (4 ) As Or c g AUTOR: J. Kreutz Issue: January 2002 tirke KGA VTS- Skin Bloctomlstry D4Ol91 Dau&doif 1R ES TECt41OLo V T& Fax CIR Panel Book Page 185

192 ASS R R HCI 0201 Paee 2 of 3 Brief Report The aim of the test was the evaluation of the irritation potential of the test substances on the chorionallantoic membrane (CAM) of fertilized chicken eggs, in order to estimate the local irritation property of the substances after contact with mucous membranes or eyes. The transparent test substances were tested by the reaction-time method. The reaction-time period and reaction intensity of effects on the CAM like haemorrhage, lysis of the vessels and protein coagulation (intravascular and/or extravascular) were evaluated visually. The substances,,bhl -Fo A to I [5 % AS] were tested undiluted by adding approx. 300 gl to six eggs at least with well developed blood vessels. The amount of the added test substance covered 25 % of the CAM. In order to compare the results of in vitro with in vivo experiments and to minin,i7e effects due to biological variation of the charges of the eggs, an irritation index of the reference substance Texapon ASV 70 was calculated [5% active substance, ph approx. 7]. This concentration was classified as being moderately irritating to rabbit s eyes. Due to many years of experience in the validation of such in vitro studies, the following classification scheme of irritating properties was defined for Q-values: a ii EvaIOuio&WS.,, slightlyirritating.fl. > < 1.2 moderately irritating l.2--<2.0 irritating(r36) - up 2 severely irritating (P. 41) The required dilutions (percent of weight) of the reference substance and the test substances were made in bi-distilled water. The study was conducted in compliance with the regulations of Good- Laboratory- Practice (GL,P). The results of all test substances are shown in appendix 1 and the added diagram in appendix 2. CIR Panel Book Page 186

193 ASSRO2Oo44lLRo2oOO73.IICTO2Oj Page3of3 The test substances,,bhl -Fo F,(. and I (5 % AS) showed the lowest effects. They are predicted to be moderately irritating.,,bhl -Fo A (5 % AS) showed the highest effects on the CAM. It is predicted to be irritating. After the comparison of the in vitro and in vivo results and based on our experience we can suppose that the test substances do the same irritations alter the contact with the mucous membrane, particularly with the eye (L)raize-Test). Dt)sseldorf January 24, 2002 Dr.rer.K.R.Sc oder Dr.Pitte Study Director, Toxicology Product Manager CIR Panel Book Page 187

194 (n9 4. -YTh - Skin Biochemistry Appendix I \t_ In vitro test: HET-CAM - Reaction-Time Method (HCX) - Test substances Rez. No: 5%AS,,BHL SAT -Fo intcrnai Code-No. ph Irritation factor value [Q] Standard deviation Evaluation reference substance,,texapon ASV :1.00 ± 0.06 moderately irritating H I F G B ± 0.00 ± 0.00 ± ± C up i 0200X A ± ± ± ± 0.06 slightly irritating moderately irritating moderately irritating Classification scheme of the in vitro test: RET-CAM Q [Irritation valuej Reactjon-Thne Method Evaluation < 0.8 slightly irritating > moderately irritating irritating(r36) 2.0 severely irritating (R.41) -> 1.2-<2.0 Classification sch me of the in vitro test: RET-CAM - End-PolntAsse.cement Q [Irritation index] Evaluation 1-5 slightly irritating 6-12 moderately irritating irritating(r36) severely irritating (R41) ASS R , R , HCI 0201 CIR Panel Book Page 188

195 t.) D I U) C-) CIR Panel Book Page 189

196 Persona Care rnproducts Counci Memorandum Committed to Safety, ua ity Innovation TO: FROM: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR) John Bailey, Industry Liaison to the CIR Expert Panel DATE: June 21, 2011 SUBJECT: Clinical Data on Products Containing Citric Acid, Triethyl Citrate or Triisostearyl Citrate Clinical Research Laboratories, Inc Repeated insult patch test of a cuticle cream containing 4% Citric Acid. CRL Study Number: CRL Consumer Product Testing Co Repeated insult patch test of a powder blush containing 4.8% Triethyl Citrate. Experiment Reference Number: C Consumer Product Testing Co Repeated insult patch test of a lip gloss containing 15.5% Triisostearyl Citrate. Experiment Reference Number: C th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 190

197 Clinical Research Laboratories, Inc. cov Final Report Ri peated Insult Patch Test utic1e Cream I fo c CLIENT: ATTENTION: TEST MATERIAL: CRL STUDY NUMBER: Ms. Pamela Felice Senior Regulatory Affairs Associate Cuticle Cream I 023-8A CRL AUTHORIZED SIGNATURES: Executive Vice President/COO M.D. Diplomate American Board of Dermatology REPORT DATE; November 16, Hoes Lane Piscataay, NJ (732) FAX (732) CIR Panel Book Page 191

198 Clinical Research Laboratories. Inc. Good Clinical Practice QuaIity Assurance Audit Statement Clinical Study Number: CRLl Start Date: October 1, 2007 Completion Date: November 9, 2007 The clinical study listed above was conducted in accordance with Clinical Research LaboratorIes, Inc. Standard Operating Procedures, which incorporate the principles of Good Clinical Practice defined by applicable guidelines and regulations established by U.S. Regulatory Agencies. The conduct of the study was monitored for compliance, and the associated records, including source documents or raw data were reviewed for documentation practices arid accuracy by a Project Manager/Study Director and/or a Quality Assurance Representative Standard Quality Assurance audit procedures for this final report and study related documents were conducted, as indicated below. Signature of QA Aud Date //h/ CIR Panel Book Page 192

199 Clinical Reseach Laboratories, Inc. Fimz( Rrport Sru Nu,r.br: CRL PaciofiO FINAL REPORT REPEATED INSULT PATCH TEST PURPOSE The purpose of this study was to potential of a lest material. determine thc dermal irritation and sensitization INVESTIGATIVE SITE Clinical Research Laboratories, Inc. 371 Hoes Lane Piscataway, New Jersey )8l-l616 TEST MATERIAL The following test material was provided Clinical Research Laboratories, Inc. on September 27, 2007: was received by Test Materialj Test CondatI Yj Patch Fype jj Cuticle Cream A Test asrecei. ed Semiocc1usre* I The test material was coded with the following CRL identification number: CRLI STUDY DATES This study was initiated on October 1, 2007 and was completed on November 9, 2007 Srmi-occhcive Strip (TruMcd Techno]ogies Inc.. aurnsvill, Minncsota) CIR Panel Book Page 193

200 C, Study rnth r; crl1349o2 Page 4uflO Resear ch Laboratories, Inc. Fiil pr PANEL SELECTION Each subject was assigned a permanent CRL identification number. All subjects signed an Informed Consent Form in compliance with 21 CFR Part 50: Protection of Human Subjects and a HJPAA Authorization Form in compliance with 45 CFR Parts 160 and 164. All subjects completed a Subject Profile/Medical History Form provided by Clinical Research Laboratories, Inc. prior to the study (Subject Demographics Appendix I). Subjects who met the following criteria were impaneled: Male and female panelists between the ages of 18 and 70; Subjects who have completed a Panelist Profile/Medical History; - Subjects who arc in general good health as determined by a Panelist Profile/Medical History; Subjects who do not exhibit any skin diseases that might be confused with a skin reaction from the test material; a Subjects wilting to sign an Informed Consent Form in conformance with 21 CFR Pan 50: Protection of Human Subjects ; a Subjects who have completed a HWAA Authorization Form in conformance with 45 CFR Parts 160 and 164; a Females who are not pregnant or lactating; Subjects who demonstrate dependability and intelligence in following directions; a Subjects who are not currently using any systemic or topical corticosteroids, anti inflammatory drugs or antihistarnines; Subjects who do not exhibit skin disorder, sunburn, scars, excessive tattoos, etc. in the test area. TEST METRO!) Prior to the application of the patch, the test area was wiped with 70% isopropyl alcohol and allowed to dry. The test material, which was prepared as described in the Test Material section of the report, was applied to the upper back (between the scapulae) and was allowed to remain in direct skin contact for a period of 24 hours. CIR Panel Book Page 194

201 fihal Xtjwrt i SLwfly Ntr: L3$9U-3 d1iji1da1.5 f10 Research Laboratories, Inc. TEST METHOD (Continued) Patches ere applied to the same site on Monday, Wednesday and Friday for a total of 9 applications during the Induction Period This schedule ma have been modified to anow for missed visits or holidays If a subject was unable to report on an assiied test date, the test material was applied on 2 consecutive days during the Tnduction Phase and/or a makeup day was added at the end of the Induction Phase The sites were graded by a CRL technician for dermal imtation 24 hours after removal of the patches by the subjects on Tuesday and Thursday and 48 hours after removal of the patches on Saturday, unless the patching schedule was altered as described above. The sites were graded according to the following scoring system: Dermal Scoring Scale 0 No visible skin reaction ± Barely perceptible erythema 1+ Mild erythema 2+ Well defined erythema 3+ Erythema and edema 4+ Erythema and edema with vesiculation If a 2+ reaction or greater occurred, the test material was applied to an adjacent virgin site. If a 2+ reaction or greater occurred on the new site, the subject was not patched again during the Induction Phase but was challenged on the appropriate day of the study. At the discretion of the Study Director, patch sites with scores less than a 2+ may have been changed. Following approximately a 2-week rest period, the challenge patches were applied to pi eviously untreated test sites on the back After 24 hours, the patches were removed by a CRL technician and the test sites were evaluated for dermal reactions. The test sites were reevaluated at 48 and 72 hours Subjects exhibiting reactions during the Challenge Phase of the study may have been asked to return for a 96-hour reading CIR Panel Book Page 195

202 clinical Research Laboratories, Inc. Fin1 Repori Stud :nhei: CRL1349O72 Jizge 6ofIO RESULTS This study was initiated with 58 subjects. Two subjects discontinued study participation for reasons unrelated to the test material. A total of 56 subjects completed the study. Individual dermal scores recorded during the induction and Challenge Phases appear in Table I. CONCLUSION Based on the test population of 56 subjects and under the conditions of this study, the test material identified as Cuticle Cream I 023-8A did not demonstrate a potential for eliciting dermal irritation or sensitization. RETENTION Test materials and all original forms of this study will be retained by Clinical Research Laboratories, Inc. as specified in CRL Standard Operating Procedures 30.6 and 30.6C, unless designated otherwise by the Sponsor. CIR Panel Book Page 196

203 IIIIllJJI Clinical Research Laboratories, Inc. Fi*tal Repart Srudy Number: crl1349q7-2 Page 7oflO TABLE I Summary of Dermal Scares - - suj,jeci InduttioneeL- -. challenge Scores ii : 22 DJSCONTrNUEI) 22R S [o o 0 o o o 23 DISGONTLNUED a [ o o [ o o 23k () Due to early discontinuation these numbers were reassigned CIR Panel Book Page 197

204 Distributed for Comment Only -- Do Not Quote or Cite Clinical Research Iaboratories, Inc. FbwI Rprf &iidy Numben CRL1349O2 10 TABLE I (Continued) Summary of Dermal Scores Teija1: Cuticle Cream 1O238A Subject Jduction Sc!!!. 48 Numberi 234{ I F haflenge Scores J72 fl[ CIR Panel Book Page 198

205 . Distributed for Comment Only -- Do Not Quote or Cite Clinical Research Labortodes. Inc. Fhux! Report [ reste1 TABLE I (Continued) Summary of Dermal Scores jcutii1ecitarn1o2-.8a -1 -, Subject.1FndueUon Scores :4,I1euge Scores ZE 1 2 CIR Panel Book Page 199

206 ,J 4 l 7 V :i WY. j J t 3 00 J C t) SO C L U k) 00 C) V 3 4 i -) 3 0 ts) SO J tc) SC) i 3 ts) C) 0 s ] C C) M t i 0 0 S3 S) 0 L 0-3 SO 4 0-5C 4 D,J 35 V Distributed for Comment Only -- Do Not Quote or Cite I :±::1.I - tfl 3 00 Os SJ 3 W t 0 SC 00 Os :- -4 t? I ) L-3 1.) C) * V J ) V ) Lti cti OS CS (- Os L W 4 i JJ Ln!J ( (-M 0 u- ) ) Cs V 00 LM L ) 00 () Os Ui OS ;: CIR Panel Book Page 200

207 ET H75 Consumer Product Testing Co. FINAL REPORT CLIENT: ATTENTION: Pamela Felice Senior Regu atory Affairs Associate TEST: Repeated Insult Patch Test Protocol NoV: L0I TEST MATERiAL: Cc-h 7-cf y C,i,.tr EXPERIMENT REFERENCE NUMBER: Powder :f. ) q4 -i 1e. C Richard R. Liscnherg. Mi). Board Certified Dermatologist j? Robert W. Shanahan, Phi). Principal Investigator RN. Study Director Ins reporl s submtfed for th e usve use of the person, r,armershp or co oraron Ip whom b re adsod, and nedhar the repori nor the nrme of these Laboratohes nor any rnprnlpr of rs stafi ma be used to c0000chvn wrth the aovorflsnq or saic 01 any pro0ue or process wrihout written authprrzation 70 Ntv Dutch Lui it Fih Iir hf, r v Jersey ICt t4 4 )71I 80H w )73J CIR Panel Book Page 201

208 Consumer Product Testing Co. ES1 197 QUALITY ASSURANCE UNIT STATEMENT Study No.: C The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical laboraton studies. These studies have been perfonned with adherence to 10-1 Guideline E6 for Good Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to standard operating procedures and applicable protoco1. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study on the date(s) listed below. The findings of these inspections have been reported to manatement and the Study Director. All materials and data pertinent to this study will be stored in the Archive Facility at 70 New Dutch Lane, Fairfield. New Jersey , unless specified otherwise, in writing by the Sponsor. Date(s) of inspection: October 14, 2002 October October 29, 2002 November December 3, 2002 December 4, 2002 December Januaiw Senior personnel involved: Laura A. Artiles, M.A, Manager, Quality Assurance Marie Terlizzese. M.S. Quality Assurance Associate The representative signature of the Quality Assurance Unit signifies that this study has been performed in accordance with standard operating procedures and study protocol as well as zovernment regulations regarding such procedures and protocols. rw Dutch Lane 1-aIr)ekl. Ncv Jcrsc i973) 8t Lax (973) SI (iinical %.l (jiici1 (1 Ius(rv r1i(.r,)! i_\ CIR Panel Book Page 202

209 C X12 Page 3 Objective: To determine by repetitive epidermal contact the potential of a test material to induce primary or cumulative irritation andior allergic contact sensitization. Participants One hundred telve (112) qualified subjects male and female ranging in age from 19 to 79 years, were selected for this evaluation. One hundred six (106) cubjects completed this ctud The remainmg cuhect discontinued their participation for various reasons, none of which were related to the application of the test material. Jnclusion Criteria: a. Male and female subjects. age 16E and over. b. Absence of any visible skin disease which might he confused with a skin reaction from the test material. C. Prohibition of use of topical or systemic steroids andior antihistamines for at least seven days prier to study initiation. d. Completion of a Medical History form and the understanding and signing of an Informed Consent form. e. Considered reliable and capable of following directions. Exclusion Criteria: a. Dl health. b. Under a doctor s care or taking medication(s) which could influence the outcome of the study. c. Females who are pregnant or nursing. d. A history of adverse reactions to cosmetics or other personal care products. Test Material: Powder A Proposed Actual Study Schedule: Panel # Initiation Date CompIeon Date Ietion Date October 14, 2002 November 21, 2002 November 22, October 16, 2002 November 21, 2002 December With parental or guardian consent CIR Panel Book Page 203

210 C Page 4 Methodology: The upper back between the scapulae served as the treatment area. Approximately O2 g of the test material, or an amount sufficient to cover the contact surface, was applied to the 3/4fl x 3/4V absorbent pad portion of an adhesive dressing*, This pad was moistened with several drops of water to ensure adherence of the test material. This was then applied to the appropriate treatment site to form an occluded patch. Induction Phase: Patches were applied three (3) times per week (eg, Monday, Wednesday. and Friday) for a total of nine (9) applications. The site was marked to ensure the continuity of patch application. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent Induction patches at home, twenty-four hours after application. The evaluation of this site was made again just prior to re-application. If a participant as unable to report for an assigned test day one (1) makeup day was permitted. This day was added to the Induction period. With the exception of the first supervised Induction Patch reading. if any test site exhibited a moderate (2-level) reaction during the induction Phase. application was moved to an adjacent area. Applications are discontinued for the remainder of this test phase if a moderate (2-level) reaction was observed on this new test site. Applications would also be discontinued if marked (3- level) or severe (4-level) reactivity was noted. Rest periods consisted of twenty-four hours following each Tuesday and Thursday removal, and fortyeight hours following each Saturday removal. Challenge Phase: Approximately to (2) weeks after the final Induction patch application a Challenge patch was applied to a virgin test site adjacent to the original Induction patch site, following the same procedure described for Induction. The patch was removed and the site scored at the clinic twenty-four and seventy-two hours post-application. *Manufacmred by TruMed Technologies Inc Burnsville MN CIR Panel Book Page 204

211 C Page 5 Evaluation Key: 0 = No visible skin reaction + = Barely perceptible or spotty erthema 1 = Mild erythema covering most of the test site 2 = Moderate eiythema, possible presence of mild edema 3 = Marked erythema, possible edema 4 = Severe erythema, possible edema, vesiculation, bullae and/or ulceration Results: The results of each participant are appended (Table 1). Observations remained negative throughout the test interval. Summary: Under the conditions of this study. test material, Powder 1004-ISA. did not indicate a potential for derrnal irritation or allergic contact sensitization. CIR Panel Book Page 205

212 C Page 6 Table I Panel # Individual Results Powder A Subject * - Induction Phase Numbcr 24hr I \ irgin Challenge Site 24*hr 72 hr ] ( o o om C) o :o o o o (1 0 Cl o o o o o o o o 0 24* Supervised removal of] Induction and Challenge Patch in Addmonal makeup da granted at the dlclrction of the chine upenior CIR Panel Book Page 206

213 C Page 7 Table I (continued) Panel # Individual Results Powder A Virgin Challenge Subject -lnductionphase Site Number hr *hr 72 hr o o o o o o DID NOT COMPLETE STUDY * = Supervlsud removal of 1 induction and Challenge Patch CIR Panel Book Page 207

214 Induction Distributed for Comment Only -- Do Not Quote or Cite C Page 8 Table I (continued) Panel # individual Results Powder 1004-ISA - \ irgin CLaaice Subject Phase-- Number 24*br Site 24*hr 72 hr DIDNOTCOMPLETESTUDY DID NOT COMPLETE STUDY * Supervised removal of] Induction and Cbaflenge Patch CIR Panel Book Page 208

215 CO2-0969O2 Page 9 Table I (continued) Panel Individual Results Powder 1004-ISA Vircin Challenge Subject Lnduction Phase Site Number 24*hr *hr 72hr o o DID NOT o o 0 0 -D]D NOT COMPLETE STUDY -DID NOT COMPLETE STUDY COMPLETE STIJDY o o = Supervised removal of i induction and Challenge Patch CIR Panel Book Page 209

216 C Pag 10 Table 2 Panel # Subject Data Subject Number Initials Age Sex FP 63 F 2 NL 53 F 3 MO 67 F 4 TO 67 M 5 HM 77 M 6 JM 66 F 7 HR 54 M 8 HK 73 F 9 DF 48 F 10 WP 73 M 11 SW 47 F 12 LD 52 F 13 IV 48 F 14 RP 72 F 15 U) 27 F 16 RD 26 F 17 DB 58 M 18 MB 78 F 19 FR 63 F 20 BA 31 F 21 YC 37 F 22 MP 50 M 23 VE 42 F 24 DC 46 F 25 BG 48 F 26 DC 61 F 27 SV 38 F 28 DB 24 F 29 it 30 F CIR Panel Book Page 210

217 C Page 11 Table 2 (continued) Pane] Subject Data Subject Number Initials Age Sex 30 SC 54 F 31 BM 38 F 32 DG 42 F 33 EG 25 F 34 ES 52 M 35 SA 26 F 36 JC 64 M 37 JP 35 M 38 ST 79 F 39 JP 66 M 40 JG 38 M 41 LA 72 F 42 MG 31 M 43 DP 45 F 44 SL 36 F 45 LG 41 F 46 AG 56 M 47 Al 46 F 48 OC 23 M 49 1M 19 F 50 JV 50 F 51 ES 71 M 52 AS 70 F 53 DO 77 F 54 FF 49 M 55 ER 75 F 56 PS 55 M CIR Panel Book Page 211

218 C02-O96902 Page 12 Table 2 (continued) Panel O3 ject Data Subleot i.umher Initials Age Sex I SB 37 F 2 CF 61 F 3 LV 70 F 4 PF 55 F 5 PT 66 M 6 WT 63 F 7 MS 58 F 8 LG 45 F 9 DG 37 F 10 LA 79 F 11 MF 49 F 12 Al 54 F 13 RM 56 M 14 DQ 37 F 15 GN 75 F 16 FE 31 F 17 EB 67 M 18 AD 66 M 19 VD 71 F 20 LC 32 F 21 JS 69 M 22 PH 25 F 23 NM 40 M 24 MP 57 F 25 BB 57 M 26 ES 28 F 27 LK 62 F 28 LP 37 F 29 NM 31 M CIR Panel Book Page 212

219 C020969M2 Page 13 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 30 DC 44 p 31 CT 31 F 32 AD 40 F 33 ED 64 F 34 LC MD 33 F 36 JE 39 F 37 in 52 F 38 PP 52 M 39 NS 23 F 40 RF 34 F 41 GH 38 M 42 SD 51 M 43 AS 64 F 44 AB 39 F 45 MB 48 F 46 JR 30 F 47 HB 62 M 48 KB 46 F 49 MB AW TW RM 41 F 53 NJ 47 F 54 JB 66 F 55 VG 41 F 56 AC 33 F CIR Panel Book Page 213

220 Distributed for Comment Only -- Do Not Quote or Cite LESt 75, Consumer Product Testing Co. FINAL REPORT CLIENT: ATTENTION: Heather Brandell Licensed Cosmetologist TEST: TEST MATERIAL Repeated Insult Patch Test ProtocolNo.: 1.01 Lip Gloss EXPERIMENT REFERENCE NUMBER: C IsJ-I Ci4 Reviewed by: Richard R. Eisenberg, M.f). Medical Director Board Certified Dermatologist Approved by: J! FnI, RN. jecutive Vice President, Clinical Evaluations Report Date: This report Is submitted for the exch.isive use of he parson. partnership, or corporation 10 whom it is addressed, arid neither the report nor the name of these Laboratories nor any member ol its stall, may be used in connec:ion with the advertising or sate ol any product or process without written authorization. 70 New Dutch Lane Fairfield, Now Jersey (973) Fax (973) CIR Panel Book Page 214

221 EST H175 Consumer Product Testing Co. QUALITY ASSURANCE UNIT STATEMENT Study NoV: C09-099L01 The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical laboratory studies. These studies have been performed with adherence to the applicable ICH Guideline E6 for Good Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to standard operating procedures and applicable protocols. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study. All data pertinent to this study Will be stored in the Consumer Product Testing Company archive, unless specified otherwise, in writing by the Sponsor. Quality Assurance personnel involved: Quality Assurance te The representative signature of the Quality Assurance Unit signifies that this study has been performed in accordance with standard operating procedures and study protocol as well as government regulations regarding such procedures and protocols. 70 New Dutch Lane Fairfield, New Jersey O7O (973) Fax (973) Clinical Toxicology Analytical Chemistry Microbiology CIR Panel Book Page 215

222 C Page 3 Objective: To determine by repetitive epidermal contact the potential of a test material to induce primary or cumulative irritation and/or allergic contact sensitization. Participants: One hundred fifteen (115) qualified subjects, male and female, ranging in age from 17 to 78 years, were selected for this evaluation. One hundred ten (110) subjects completed this study. The remaining subjects discontinued their participation for various reasons, none of which were related to the application of the test material. Inclusion Criteria: a. Male and female subjects, age 6a and over. b. Absence of any visible skin disease which might be confused with a skin reaction from the test material. c. Prohibition of use of topical or systemic steroids and/or antihistamines for at least seven days prior to study initiation. d. Completion of a Medical History form and the understanding and signing of an Informed Consent form. e. Considered reliable and capable of following directions. Exclusion Criteria: a. Ill health. b. Under a doctor s care or taking medication(s) which could influence the outcome of the study. c. Females who are pregnant or nursing, d. A history of adverse reactions to cosmetics or other personal care products. Test Material: Lip Gloss - Study Schedule: Panij Initiation Date Completion Date March 2, 2009 April 9, March 4, 2009 April 9, 2009 awith parental or guardian consent CIR Panel Book Page 216

223 C Mi Page 4 Methodology: The upper back between the scapulae served as the treatment area. Approximately 0.2 g of the test material, or an amount sufficient to cover the contact surface, was applied to the I x 1 absorbent pad portion of a clear adhesive dressing. This was then applied to the appropriate treatment site to form a semi-occlusive patch. Induction Phase: Patches were applied three (3) times per week (e.g., Monday, Wednesday, and Friday for a total of nine (9) applications. The site was marked to ensure the continuity of patch application. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent Induction patches at home, twenty-four hours after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assigned test day, one (1) makeup day was permitted. This day was added to the Induction period. With the exception of the first supervised Induction Patch reading, if any test site exhibited a moderate (2-level) reaction during the Induction Phase, application was moved to an adjacent area. Applications were discontinued for the remainder of this test phase, if a moderate (2-level) reaction was observed on this new test site. Applications would also be discontinued if marked (3-level) or severe (4-level) reactivity was noted. Rest periods consisted of twenty-four hours following each Tuesday and Thursday removal, and forty-eight hours following each Saturday removal. Challenge Phase: Approximately, two (2) weeks after the final Induction patch application, a Challenge patch was applied to a virgin test site adjacent to the original Induction patch site, following the same procedure described for Induction. The patch was removed and the site scored at the clinic twenty-four and seventy-two hours post-application. CIR Panel Book Page 217

224 C Page 5 Methodology (continued) Evaluation Critena (EErythema and additional Dermal Siuelae 0 = No visible skin reaction E = Edema 0.5 / + = Barely perceptible 1) Dryness I = Mild S = Staining 2 Moderate P Papules 3 Marked V = Vesieles 4 Severe B = Bullac U = Ulceration Sp = Spreading Erythema was scored numerically according to this key. If present, additional Dermal Sequeiae were indicated by the appropriate letter code and a numerical value for severity. Results: The results of each participant are appended (Table 1). Qbservations remained negative throughout the test interval. Subject demographics arc presented in Table 2. Summary: Under the conditions of this study, test material, Lip Gloss - did not indicate a potential for dermal irritation or allergic contact sensitization. CIR Panel Book Page 218

225 C i Page 6 Table I Panel # Individual Results Lip Gloss Virgin Ch& lenge Subject - Induction Phase Site Number 24*hL *hr 72 hr ii 0 0 0, o * Supervised removal of l Induction and Challenge Patch CIR Panel Book Page 219

226 C Page 7 Table I (continued) Panel # Individual Results Lip Gloss - Virgin Challenge Subject lnduetionphase Site Number 24*hr *hr 72 hr * o o o o o o o o O o o o O o o o o o DID NOT COMPLETE STUDY o o * Supervised remova of 1st Induction and Challenge Patch - Subject not present for supervised removal. CIR Panel Book Page 220

227 C Page 8 rable I (continued) Panel # individual Results Lip Gloss - Virgin Challenge Subject Indu&ion Phase Site Number 24*1w *hr 72 hr a? $ 29 o a 0 0 o o () m o a o o o 0 o o o o o o a o O 0 0 o 0 o o 0 o a DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY---- o C) 0 a * Supervised removal of 1 Induction and Challenge Patch m Additional makeup day granted at the discretion of the clinic supervisor CIR Panel Book Page 221

228 C Page 9 Table I (continued) Panel # Individual Results Lip Gloss- Virgin Challenge Subject lnduction Phase Site Number 24*1w *hr 72 hr Urn & !D NOT COMPLETE STUDY () DID NOT COMPLETh STUDY * Supervised removal of 1 Induction and Challenge Patch m Additional makeup day granted at the discretion of the clinic supervisor CIR Panel Book Page 222

229 C I Page 10 Table 2 Panel # Subject Data Subject Number Initials Age Sex BD RF Cv RC JP lb FB AF GL Os Jv DH AA FA CR GV CL TS RR in PF Sv MM AW BD BC 113 RM KG F F F M F F M F F M F F F M M M F F F F F F F F F M M F CIR Panel Book Page 223

230 C l01 Page 11 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 30 MJ 52 M 3l KG 52 F 32 1K 42 F 33 MK 74 F 34 PM 61 F 35 SC 48 F 36 AP 40 F 37 CD 77 F 38 MD 41 F 39 RB 63 F 40 LE 38 F 41 LM 50 F 42 PR 31 M 43 LR 38 F 44 AR 32 P 45 LC 56 F 46 AC 73 M 47 CC 42 F 48 LL 37 F 49 MK 54 M 50 TM 54 p 51 BT 51 F 52 ZL 31 F 53 MB 57 F 54 M}I 39 F 55 ill 27 F 56 RK 40 F CIR Panel Book Page 224

231 C Page 12 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex I KP 20 M 2 ZF 18 M 3 MD 65 F 4 RD 43 M 5 HB 45 F 6 MP 70 F 7 3M 52 F 8 JL 50 F 9 RD 55 F 10 JF 50 F 11 MM 22 F 12 EW 54 p. 13 DB 25 F 14 JP 42 M 15 AZ 43 F 16 AP 37 F 17 DR 17 F 18 TB 42 F 19 BP 41 M 20 EG 18 M 21 SR 48 F 22 RS 41 M 23 ZR 44 F 24 JO 25 F 25 OS 45 M 26 RS 67 F 27 JM 59 F 28 MC 47 F 29 AH 56 M CIR Panel Book Page 225

232 C Page 13 Table 2 (continued) Panel # Subject Data ect Number Initials Age Sex 30 PF 49 F 31 BA 31 M 32 IS 19 M 33 DB 32 M 34 NM 64 F 35 AM 55 F 36 YB 56 F 37 JB 58 F 38 SR 62 F 39 RG 27 F 40 PN 28 F 41 JC 42 M 42 RM 32 F 43 FC 49 M 44 OC 41 F 45 IQ 68 F 46 AE 52 F 47 FE 19 M 48 HE 50 M 49 GO 18 M 50 MB 40 F 51 UC 37 M 52 CS 20 F 53 ST 21 M 54 CV 51 F 55 SC 58 F 56 LR 51 F 57 SV 32 F 58 DD 30 F 59 DC 55 F CIR Panel Book Page 226

233 China Europe Japan South America USA Other World Bands Kg Sodium or NR 0 or NR <1 0 or NR 10 to or NR 10 to 100 citrate Bands Sodium <1 <1 <1 <1 <1 <1 <1 Tonnes citrate Bands Kg Citric acid >1000 > to >1000 > or NR > Bands Citric acid to to 100 <1 1 to 10 1 to 10 <1 10 to 100 Tonnes Bands Kg Triethyl >1000 >1000 >1000 >1000 > to >1000 citrate 1000 Bands Triethyl to to 10 to to to <1 100 to Tonnes citrate Fragrance Consumption Data Above DATA IN THIS SYNOPSIS HAVE NOT BEEN PEER REVIEWED BY A SCIENTIFIC PANEL Citric acid Synonyms Citric acid Principal EINECS Citronensäure Trade 2-Hydroxy-1,2,3-propanetricarboxylic acid 1,2,3-Propanetricarboxylic acid, 2-hydroxy- CAS CAS Number RIFM ID FEMA EINECS Registration EINECS DSL TSCA Fragrance Structure-Activity Group: Carboxylic Acids/Branched Chain/Saturated Formula C 6 H 8 O 7 Molecular Weight SMILES Notation O=C(O)C(O)(CC(=O)O)CC(=O)O Generic Class Carboxylic Acids Description Colorless to white crystals, odorless, with a pleasant sour taste Physical Data Boiling Point >200 C Boiling Point (calculated) C FMA EPI Suite CIR Panel Book Page 227

234 Flash Point >200 F;CC FMA Henry's Law (calculated) 8.327e-018 Pa m 3 /mol EPI Suite Log K OW (calculated) EPI Suite Melting Point dec. FMA Melting Point (calculated) C EPI Suite Vapor Pressure <0.001 mm Hg 20C FMA Vapor Pressure (calculated) 5.64e-009 mm 25 C EPI Suite Water Solubility (calculated) 1e+006 mg/l EPI Suite Natural Occurrence Citric acid is reported to occur in nature. Product Flavor Consumption (in kg) 1995 EUROPE USA USA USA USA USA USA Average Usual Uses (in ppm) Average Maximum Mean Daily Consumption (gms) Updated Alcoholic Beverage Jul-88 Baked Goods Jul-88 Breakfast Cereals Jul-88 Cheese Jul-88 Chewing Gum Jul-88 Condiment Relish Jul-88 Confection Frosting Jul-88 Egg Products Jul-88 Fats Oils Jul-88 Fish Products Jul-88 Frozen Dairy Jul-88 Fruit Ices Jul-88 Fruit Juice Jul-88 CIR Panel Book Page 228

235 Gelatin Pudding Jul-88 Gravies Jul-88 Hard Candy Jul-88 Imitation Dairy Jul-88 Instant Coffee Tea Jul-88 Jam Jelly Jul-88 Meat Products Jul-88 Milk Products Jul-88 Non-alcoholic Beverage Jul-88 Nut Products Jul-88 Other Grain Jul-88 Poultry Jul-88 Processed Vegetables Jul-88 Reconstituted Vegetable Jul-88 Snack Foods Jul-88 Soft Candy Jul-88 Soups Jul-88 Sweet Sauce Jul-88 PADI 3.12 Food Products Containing Citric acid (in ppm) Product Code Lower Limit Upper Limit Rum category II (total volatiles ppm) 65-II 1 Status Citric acid was included by the Council of Europe in the list of substances granted A - may be used in foodstuffs ( COE No. 20), was approved by the FDA as GRAS Affirmed 12/12/94 ( 21 CFR ). Flavor and Extract Manufacturers' Association states: Generally Recognized as Safe as a flavor ingredient - GRAS 3. ( 2306) Hall,1965 Indicative Non-Exhaustive List states: Listed The Industrial Safety and Health Law (Japan) states: ISHL Number ( (2)- 1318) CIR Panel Book Page 229

236 Joint Expert Committee on Food Additives states: ADI not limited. ( 1973) United Nations Transport Classification Codes states: Not regulated R36 Irritating to eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Irritant FFIDS Volume I Updated 1-Nov-85 European Hazard Classification Labeling Irritation Data IR1 May be irritating to skin. Eye irritant. US OSHA Health Hazard Statements 1-Apr-89 Hazard Category SCI 3 EDI 2A Signal Word Global Harmonized System Hazard Statements Code Warning H316 Warning H319 Hazard Statement Causes mild skin irritation Causes serious eye irritation Precautionary Code P264 P280 P305/351/358 Precautionary Statement Wash.. (hands/face) thoroughly after handling Wear protective gloves/protective clothing/eye protection/face protection IF IN EYES: Rinse cautiously with water for several minutes. Remove CIR Panel Book Page 230

237 contact lenses if present and easy to do. Continue rinsing P332/313 If skin irritation occurs: Get medical advice/attention P337/313 If eye irritation persists: Get medical advice/attention Human Health Data Acute toxicity Route: oral. Species: rat. Ten animals per dose were tested. The rats were observed for mortality and/or systemic effects. A gross necropsy was carried out on all animals. No further information was provided. LD G/KG 5 g/kg lethal, 9 deaths. Toxic signs were flaccid and lethargy g/kg lethal, 3 deaths. Toxic signs were diarrhea, ptosis, slight lethargy and piloerection g/kg nonspecific effects, 0 deaths g/kg lethal, 4 deaths. Toxic signs were piloerection and lethargy. 3.8 g/kg calculated LD50, 95% limits= gm/kg. All necropsy findings routine. (RIFM,1977) [Moreno,1977] Route: skin. Species: rabbit. Ten animals were tested. The animals were observed for mortality and/or systemic effects. A gross necropsy was conducted on all animals. No further information provided. 5 g/kg nonspecific effects, 0 deaths. Acute dermal LD50>5 gm/kg. Necropsy findings routine. (RIFM,1977) [Moreno,1977] Route: gavage. Species: rabbit. Rabbits (Belgian Hare & Flemish Giant breeds) were gavaged with the test chemicals and observed for at least 24 hrs. Vehicle not reported. 7 g/kg lethal, Estimated initial minimum Fatal dose. (Weiss,1923) Route: intravenous. Species: mouse. Animals (no., sex, & strain unspecified) were various dosage a rate of 0.01 ml/sec; w/ soln's of test cpd. Clinical signs and necropsy wre conducted. LD50 was calculated according to the method of Litchfield & Wilcoxon. [No further experimental detail given]. 200 mg/kg lethal, 6/13 animals died. 203 mg/kg calculated LD50, respiratory tract, immediate convulsive deaths noted. Thought to be due to acute acidosis. Hemorrhagic lungs present. 225 mg/kg lethal, 10/13 animals died. 250 mg/kg lethal, 3/3 animals died. 175 mg/kg lethal, 2/13 animals died. (Horn,1957) Route: oral. Species: mouse. Six four wk old male ICR-JCL animals/dose grp were adm. test cpd. Behavior & mortality were observed for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon mg/kg clinical signs, gastrointestinal tract, calculated LD50, motor ataxia 50 min. Mydriasis, decrease in resp. rate & heart beat observed. Hemorrhage of gastric mucosa noted. Respiratory failure. Confidence interval (Yokotani,1971) Route: oral. Species: rat. Six five wk old male SD-JCL animals /dose grp were adm. test cpd. Behavior & mortality were observed for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon mg/kg clinical signs, gastrointestinal tract, calculated LD50, motor ataxia 50 min. Mydriasis, decrease in resp. rate & heart beat observed. Hemorrhage of gastric mucosa noted. Respiratory failure. Confidence interval (Yokotani,1971) Route: intraperitoneal. Species: rat. Six five wk old male SD-JCL animals/dose grp were adm. test cpd. Behavior & mortality were observed for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon. 725 mg/kg clinical signs, calculated LD50, CIR Panel Book Page 231

238 liver, stretching, slow crawling, tremor, slight opisthotonus & slow avoidance rxn noted. Surface of spleen & liver covered w/ a thin whitish membrane & slight hypertrophy of liver. Confidence intervals (Yokotani,1971) Route: intraperitoneal. Species: mouse. Six four wk old male ICR-JCL animals/dose grp were adm. test cpd. Behavior & mortality were oberved for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon. 940 mg/kg clinical signs, calculated LD50, liver, stretching, slow crawling, tremor, slight opisthotonus & slow avoidance rxn noted. Surface of spleen & liver covered w/ a thin whitish membrane & slight hypertrophy of liver. Confidence interval (Yokotani,1971) Route: subcutaneous. Species: mouse. Six four wk old male ICR-JCL animals/dose grp were adm. test cpd. Behavior & mortality were observed for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon mg/kg clinical signs, calculated LD50, mydriasis & decrease in rate of respiration noted. Death occurred w/in 15 min to 6 days by respiratory failure or emaciation. Confidence interval was (Yokotani,1971) Route: subcutaneous. Species: rat. Six five wk old male SD-JCL animals/dose grp were adm. test cpd. Behavior & mortality were observed for 7 days after adm. of test cpd & LD50s were calculated by the method of Litchfield & Wilcoxon mg/kg clinical signs, calculated LD50, mydriasis & decrease in rate of respiration noted. Death occurred w/in 15 min to 6 days by respiratory failure or emaciation. Confidence interval was (Yokotani,1971) Route: inhalation. Species: guinea pig. Female albino, outbred, English short-hair animals were exposed for 30 min to test cpd. A microphone was attached to port of the exposure chamber and animal respiration was monitored. Coughing episodes were defined as to duration and the number of such episodes was recorded. Animals were exposed To test cpd twice. A 7-day rest period was observed between exposures mg/m3 positive effects, total number of coughs recorded was mg/m3 positive effects, total number of coughs recorded was (Zelenak,1982) Route: unreported. Species: dog. A dog weighing 4.25 kg was fed dog biscuits and water for several weeks, then fasted for 36 hours. Following the fast period, the animal was administered a mixture containing the test material, water and hydrochloric acid. A second dose was administered the next day. No additional data was provided. 0.2 % no effects, The animal was entirely unaffected. (Lucas,1909) Irritation Route: skin. Species: human 18+ yrs. Following previous work on tandem irritation, 20 healthy volunteers were exposed twice daily for 4 days to the organic fruit acids citric acid, maleic acid and lactic acid, either alone or in tandem application with sodium lauryl sulfate (SLS) 0.5%, a model surfactant, in a repetitive irritation test. Irritant reactions were assessed by using clinical scoring and non-invasive bioengineering methods (choromanmetry and evaporimetry). Interaction, twice daily application of either malic or citric acid alone did not induce a significant barrier disruption, whereas exposure to malic acid and SLS, citric acid and SLS, or lactic acid and SLS, respectively, caused marked barrier disturbance. However, the latter irritant effect was smaller compared to that obtained by combined exposure to SLS and H2O. No enhancement of SLS-induced irritation by combined exposure to the above mentioned fruit acids, was observed. (Schlierman-Willers,2003) The EYTEX method was used as a predictor of ocular irritancy potential. The doses tested were 30, 50 and 100 ul or 30, 50 and 100 mg The EYTEX assay system is a macromolecular-based CIR Panel Book Page 232

239 test method. Alterations induced by test material in the confirmation and hydration of an ordered macromolecular matrix are quantitated. The major active component of the matrix is an oligomeric protein. The predictive value of this test is 88%. Irritant effects, severe to extreme. (Gordon,1992) Route: skin. Species: human 18+ yrs. Dansyl chloride was mulled into petrolatum 5% & applied under occlusive patch (2 cm x 3 cm) to test sites (usually volar forearm) for a period of 24 hrs. After patch removal, staining was verified w/ a Quartz mineral lamp. Product was applied to test sites twice a day; removal of the Fluorescent stain was monitored daily. Exfoliation, measured as the number of skin cells shed during treatment with test cpd was also assessed. 5 % positive effects, cell renewal values for phs 3, 5, & 7 were 18, 14 & 8, resp. (Smith,1994) Route: skin. Species: human 18+ yrs. Skin irritation was evaluated clinically, by subjective assessment of stinging (1-5 scale) in the nasal fold area, and w/ the Minola Chroma meter. With the Meter, changes in a measure of skin redness were recorded after product use on the cheek area of the face. 5 % positive effects, irritation values for phs 3, 5 & 7 were 2.3, 2.1 and 1.1 resp. (Smith,1994) Route: in vitro. Species: chicken. The chorioallantoic membrane vascular assay was conducted using at least eight 10- or 14-day-old fertilized white Leghorn eggs/dose. The vehicle was water. Signs of vascular change were evaluated 30 minutes after treatment The RC50 is that dose which results in 50% of the eggs showing a positive response. An RC50 of under 1% indicates no irritation. 18% citric acid. No effects, the RC50 was 0.2% for the 10-day-old eggs and 1.3% for the 14-day-old eggs. (Bagley,1994) Route: in vitro. Species: rabbit. The agar diffusion cytolysis assay was conducted using Staatens Scruminstitute rabbit cornea fibroblast cells. Doses were 1 to 30 mg or ul As an a screen for ocular irritation this method has demonstrated sensitivity of 100%, specificity of 93% and an overall predictive value of 96%. Irritant effects. (Reboulet,1994) Route: surface of eye. Species: rabbit. A modified Draize test was conducted using at least 12 New Zealand albino rabbits. A 0.1 ml aliquot of test material was instilled in the right eye of each rabbit. The untreated left eye served as a control. The treated eye of half of the rabbits in each group was washed 30 secs after instillation The test mateial was placed directly on the central portion of the cornea. The reactions were assessed 1 h and 1, 2, 3 and 7 days after application. Fluorescein stain was used. This study was designed to evaluate the effect of ph on eye irritation. The vehicle was likely water. 5 % eye effects, irritant effects, conjunctivitis was seen. (Murphy,1982) Route: oral. Species: human 18+ yrs. A man, aged 37 years, an Army veteran, was first seen in the Outpatient Allergy Clinic of the Veterans Adm. in May, 1953, complaining of nasal blockage, asthma, & headaches. These symptoms had begun at the age of 5, following an attack of whooping cough, and had been recurring since then. Little Improvement was noted either after a tonsillectomy and adenoidectomy at the age of 7 or after a submucous resection done at age 19. Irritant effects, oral canker sores were produced upon application of citric acid to the mouth or upon ingestion of substances containing citric acid. (Tuft,1956) Route: skin. Species: rabbit. 10 rabbits were observed during an associated dermal LD50 study. Irritation was evaluated on day 1 No further information provided. 5 g/kg irritant effects, redness mild in 3, moderate in 4 and severe in 2. Edema mild in 8 and moderate in 2. (RIFM,1977) [Moreno,1977] Route: skin. Species: human 18+ yrs. In a pre-test for a human maximization study, a 48 hour CIR Panel Book Page 233

240 closed patch test was conducted with test material in petrolatum on the backs of 30 healthy male inmate volunteers % no effects. (RIFM,1977) [Epstein,1977] Route: skin. Species: human 18+ yrs. Non-immunologic contact urticaria was evaluated in 105 male and female atopic and non-atopic dermatitis patients. Material applied for 20 minutes using a Finn chamber and Scanpor applied to the upper back. Reactions evaluated immediately after chamber removal The number of positive reactions in the results is the total for those with redness and edema and those with redness only. Vehicle was water. 2.5 % no effects. (Lahti,1980) Route: in vitro. Species: bacteria. Mouse fibroblasts were plated either on glass coverslips or on surface of a porous membrane contained w/in a plastic cup. Cells were incubated until confluent. On day of assay - cells were placed face down inside a silicon microphysiometer flow chamber. After an equilibration period, baseline metabolic rate of fibroblasts was determined. The cells were then exposed to increasing conc. of test material for approx. 500 sec/dose. After each exposure test material was washed away and metabolic rate was measured. This cycle was repeated until production of acid metabolites ceased. in water or soyabean oil. 18 %, scores in two laboratories performing assay were 1.94 & 2.75, resp as compared to 27 obtained from an in vivo assay. (Bagley,1992) Route: in vitro. Species: bacteria. Stock soln's/suspensions of test material (1%) were prepared in distilled water & incubated overnight. After centrifuging to remove insoluble material, serial dilutions were prepared in Microtox diluent (2% NaCl) & cooled in wells of Microtox analyser. The freeze-dried bacteria were reconstituted & 5 C throughout experiment. Control values of light emission from the bacteria were determined. Bacteria were then incubated w/ test cpd & light emission was remeasured after 5 & 15 min. The ratio of light lost to light remaining was calculated. in water or soyabean oil. 18 %, scores in two laboratories performing assay were 1.92 & 2.06, resp. as compared to 27.0 obtained from an in vivo assay. (Bagley,1992) Route: in vitro. Species: mouse. Mouse fibroblast 3T3 cells were cultured and then plated & incubated overnight before being treated w/ test substances. After a 24 h period of contact between cells & test cpd or media, the latter were replaced by 200 ul neutral red sol'n (50 ug/ml), which was left in situ for 3 h. After this time the plates were rinsed w/ warmed phosphate buffered saline, destained & absorbance of ea. well measured in a microplate reader. in water or soyabean oil. 18 %, score derived from this assay was 4.0 as compared to 27.0 obtained from an in vivo assay. (Bagley,1992) Route: in vitro. Species: hamster. Chinese hamster ovary cells were used. The cells were added to each well of 24 well a conc. of 2 X 10(5) cells in 2 ml medium. After a 24 h incubation period the cells were rinsed & incubated for a further 24 h before being treated w/ test substance. After 2 h of contact w/ the test substance the cells were rinsed & incubated for a further 24 h in fresh medium. After 1 h, stain was removed & cells were air-dried and destained. Eluted solution was 540 nm in a spectrophotometer. in water or soyabean oil. 18 %, score obtained with this assay was 3.7 vs. 27. obtained from an in vivo assay. (Bagley,1992) Route: surface of eye. Species: rabbit. A Draize rabbit eye test was conducted with 2 New Zealand White albino rabbits. An aliquot of 100 ul neat material was intilled in the eye with washing 24 hours later. The eyes were examined 1, 24, 48, 72 hrs and 7 d after instillation. Ultrasonic pachymetry was used to measure corneal thickness. 100 µl eye effects, irritant effects, a fairly good correlation was seen between changes in corneal thickness and clinical eye observations. (Martins,1992) CIR Panel Book Page 234

241 Route: in vitro. Species: human 18+ yrs. Cellular viability, mitochondrial function and histological and ultrastrutural changes were evaluated after the incubation of test material with Testskin Testskin is a human living skin equivalent consisting of differentiated human keratinocytes grown on a human fibroblast and bovine collagen matrix coated filter support. Irritant effects, changes were noted with doses of mg with a 3 hr incubation or 25 mg with an 18 hr incubation. (Laska,1992) Sensitization Route: skin. Species: human 18+ yrs. A multicentre patch tesing study was conducted with a total of 702 male and female patients to determine the prevalence of contact sensitization with Aloe vera. A specially designed questionnaire was used to interview patients for the use o faloe vera and other Liliaceae, their reasong for use and location of application, adverse reactions, occupation, hobbies and atopy. Patch testing was performed at the Departments of Dermatology in Innsbruck, Salzburg, Graz, the Allergy Outpatient Clinic in Hall in Tirol from September 2003 to February All study subjects were patients who had visited the centers for suspected allergic contact dermatitis and were therefore being tested for other substances as well. In most cases, at least the European standard series (from Hermal, Germany) was performed. The test material was applied to Finn Chambers that were fixed to the subjects' upper back with Scanpore tape. The patches were removed after 48 hours and read 15 minutes and 24 hours later. An exception was at the Buxtehude center where 176/702 patients were tested. Patches for patients at Buxtehude were removed after 24 hours of exposure. Positive reactions were graded from + to +++ as recommended by the International Contact Dermatitis Research Group Pure Aloe gel (concentrated 10-fold), oil (from the leaves macerated in soja oil) and Aloe pulvis (from the whole plant) were tested. The oil was stabilized with vitamin E (tocopherol acetate) and the gel with citric acid, both substances were tested. Sorbic acid, which is frequently contained in commercial Aloe products was also tested. Prior to testing, the extracts were applied on 20 volunteer staff members; none of them experienced an irritative or allergic reaction. Subjects: 702 Unspecified Sex Summary 0/702 patients exhibited positive patch test reactions with the test material. No effects, no concentration provided. 0/702 patients exhibited positive patch test reactions with the test material. (Reider,2005) Route: skin. Species: human 18+ yrs. A 35 year old male baker who had apparently been cured of recurrent cold urticaria and mucous membrane hypersensitivity to cold of 4 years duration by avoidance of foods containing acetic acid was tested. Skin tests with 1.0% test material were conducted. Skin tests were conducted with 1.0% test material followed by application of an ice cube over the skin test area for 15 minutes. Ice cube tests were performed concomitantly over untested skin. Simlar testing with 1.0% test material followed by application of an ice cube over the test site on normal control subjects was also conducted No further details were provided. Subjects: 1 Male Summary Direct skin testing with 1.0% test material was negative. When an ice cube was immediately placed over the test site for 15 minutes, no effects were observed. Ice cube tests performed concomitantly over untested skin were negative. Simlar testing with 1.0% test material plus an ice cube over the test site in normal control subjects gave negative reactions. 1.0 % no effects, direct skin testing with 1.0% test material was negative. When an ice cube was immediately placed over the test site for 15 minutes, no effects were observed. Ice cube tests performed concomitantly over untested skin were negative. Similar testing with 1.0% test material plus an ice cube over the test site in normal control subjects gave negative reactions. (Wiseman,1956) CIR Panel Book Page 235

242 Route: in vitro. Species: rat. The effect of food additives, including test material on immediate allergic reactions using an assay system which measures the release of B-hexosaminidase (determined by colorimetry) from cultured rat basophilic leukemia cells (RBL-2H3) as an index of chemical mediator release which occurs during an immediate allergic response. The cells were stimulated with the reaction mixture prepared by adding a complex of biotinylated mouse anti- DNP IgE antibody (3 ug/ml), avidin (1 ug/ml) as the stimulator, and 10 ul of a test material solution to the releasing medium and diluting the mixture to a total quantity of 1 ml. Test material was dissolved in ultra-pure water, hydrochloric acid solution, methanol or dimethyl sulfoxide to a concentration of 100 mm. Final concentration of test material in the mixture was 1 mm. Cells were incubated with the mixture for 1 hour at 37 C, centrifuged, and the B- hexosaminidase activity measured in the supernatant (extracellular fluid) and lysed cell fraction (intracellular fluid) using p-nitrophenyl-2-acetamide-2-deoxy-b-d-glucopyranoside as substrate. The resulting p-nitrophenol was measured at an OD of 405 nm and the percent release of B- hexosaminidase was calculated. Promotion of B-hexosaminidase release is triggered by cell injury. Vehicle was water. No effects. (Tanaka,1991) Route: skin. Species: human 18+ yrs. Skin prick and skin scratch tests were conducted on subjects with chronic or recurrent urticaria, or recurrent angioedema, in addition to 247 control subjects. Food additives were tested at concentrations of 1-15% in water, petrolatum or 70% ethyl alcohol. Histamine chloride and physiological saline were used as the positive and negative controls, respectively. The tests were read at 15 minutes, and only weals as large or larger than that of histamine were considered positive. From 91 initial urticaria subjects, 23 were found to be "skin test positive", and the results on these 23 subjects were reported. The food coloring and food flavorings, plus tocopherol were evaluated by the skin scratch test, due to the difference in vehicles used. The food preservatives were tested by the skin prick test. Vehicle was water. Tested by the skin prick method. 2.5 % sensitization effects, Positive skin test reactions were produced in 3/23 subjects. (Malanin,1989) Route: skin. Species: human 18+ yrs. Skin-prick and skin-scratch tests were conducted on subjects with chronic or recurrent urticaria, or recurrent angioedema, in addition to 247 control subjects. Food additives were tested at concentrations of 1-15% in water, petrolatum or 70% ethyl alcohol. Histamine chloride and physiological saline were used as the positive and negative controls, respectively. The tests were read at 15 minutes, and only weals as large or larger than that of histamine were considered positive. From 91 initial uriticaria subjects, 23 were found to be "skin test positive", and 11 of these 23 positive subjects were retested 2 months to 3.5 years later. The results on these 11 subjects were reported. Vehicle was water. Tested by the skin prick method. 2.5 % no effects, No positive skin test reactions were produced. (Malanin,1989) Route: oral. Species: human 18+ yrs. Skin prick and skin scratch tests were conducted on 91 subjects with chronic or recurrent urticaria, or recurrent angioedema, in addition to 247 control subjects. Food additives were tested at concentrations of 1-15% in water, petrolatum or 70% ethyl alcohol. Histamine chloride and physiological saline were used as the positive and negative controls, respectively. The tests were read at 15 minutes, and only weals as large or larger than that of histamine were considered positive. From the 91 initial subjects, 23 were found to be "skin test positive". Of these 23 positive subjects, 10 were tested by the oral provocation method with the food additive(s) that caused the positive skin test reactions. The subjects were symptomfree from 2 weeks - 3 years (median of 1 month) before the oral provocation test. The food additives were administered in capsules at doses of mg alone or as solutions in water at doses of mg. The capsules or solutions were given at hourly intervals. On the 1st day, CIR Panel Book Page 236

243 the subjects were given only a placebo. At the same time of the oral provocation tests, the skin prick and skin scratch tests were repeated. Refer to MF 39675, sub-ref 3 for results of the repeated skin tests (skin prick and scratch tests). Administered as capsules. 1 subjects were challenged. 100 mg no effects, No positive reactions were produced. 200 mg no effects, No positive reactions were produced. (Malanin,1989) Route: skin. Species: human 18+ yrs. Epicutaneous patch tests were conducted on a 18-year-old male who currently had eczema on the insides of the fingers and palms of his hands, as a result of his occupational activity which consisted of mixing different non-alcoholic, aromatized and sugared drinks. The subject had a history of allergic eczema from 8 weeks old to 6 years old. The test material in water was applied to the patient's upper back, and were reactions were read at 24 and 48 hours. A standard series of substances was also tested, but the doses and vehicle used for these substances were not provided. No further details were provided. An "odorous substance series" consisting of several odor and aromatic substances was compiled, but the substances were tested as mixtures, not individually % no effects. (Schultheiss,1957) Route: skin. Species: human 18+ yrs. A 30-year-old woman had undergone sclerotherapy for varicose veins, following which she had developed hyperpigmentation. She was prescribed a cream containing 10% urea and Kojicol Plus (hydroquinone 2%, lactic acid 4%, hazel 74%, castor oil 5%, citric acid 1%, cellulose 1%, propylene glycol 10% and kojic acid 3%). After applying this cream for 4 months, she noted no improvement and so Angiogel was added (a mixture of melilotus, alpha bisabolol, Ginkgo biloba extract and ascorbic acid). A few weeks later, she presented with an eczematous eruption on and around the hyperpigmented areas. No effects. (Serra-Baldrich,1998) Route: skin. Species: human 18+ yrs. Five healthy men (age years) completed this 30 day study. Four test sites (2 cm x 2 cm) were selected and randomly rotated on the ventral forearms. Three cream formulations containing increasing amounts of citric acid (10, 20, and 25%) were tested along with an untreated control (UT). The test creams (0.2 ml) were applied in the following manner: 3 times weekly under occlusion (Duhring chambers) for 1 week, 3 times weekly with semi-occlusive patches for 2 weeks, and once daily without occlusion for one week. This product application regimen was utilized to minimize the development of irritation under conditions of occlusion. At endpoint, a 3 mm punch biopsy was obtained from each test site. The skin specimens were analyzed microscopically with the aid of image analysis to determine the effect of treatment on viable epidermal thickiness (VET), stratum corneum thickness (SCT), glycosaminoglycan (GAG) content using Hales strain and number of Langerhans cells. 10 % positive effects, a dose-responsive increase in VET was observed at this dose level and above. 20 % positive effects, a substantial increase in Langerhans cells was observed with the 20 and 25% formulations, corresponding with visual irritation on these test sites. GAG concentrations were markedly increased on the 20 and 25% treated sites in comparison to UT and the 10% strengths. 25 % positive effects. (Green,2000) Route: skin. Species: woman. Tests were conducted on a 22-year-old college student following the occurence of contact uritcaria of the face from shampoo. The vehicle was water. 1 % no effects. (Rietschel,1978) Route: skin. Species: human 18+ yrs. Patch tests were conducted to determine the prevalence of contact allergy to 85 test materials in patients with oral disease. In this retrospective study, 620 patients who underwent patch testing to allergens in an oral antigen screening series were identified from a clinical database. Allergen patch testing was performed between May 2, 2000 CIR Panel Book Page 237

244 and April 30, 2004 at Mayo Clinic (Rochester, Minn. and Scotsdale, Ariz.) 331 patients (268 females and 63 males) with burning mouth syndrome, lichenoid tissue reaction, chelitis, stomatitis, gingivitis, orofacial granulomatosis, perioral dermatitis or recurrent aphthous stomatitis were retained for this study. Age of the subjects ranged from years old with a mean age of 58 years old. Patch testing was conducted using Finn Chambers on Scanpor tape. Test materials were applied to the skin of the back, torso, extremities or a combination of the above and left in place for 48 hours. Readings were obtained at 48 and 96 hours. Patch test reactions were evaluated using criteria similar to the North American Contact Dermatitis Group criteria, negative reaction, macular erythema, weak reaction (nonvesicular erythema, infiltration, and possible papules), strong reaction (edematous or vesicualr lesions), extreme reaction (spreading, bullous, and ulcerative lesions) or irritant reaction. For this study, weak, strong or extreme reactions were considered positive results. For each patient, the treating physician judged each positive patch test result to be relevant or irrelevant on the basis of the patient's history and clinical examination findings. Vehicles not provided for the majority of test materials. Subjects: 63 Male 268 Female Study Length: 48 hours Summary Of the 289 patients tested with 1% test material aqueous, no subjects exhibited positive reactions. 1.0 % no effects, Of the 289 patients tested with 1% test material aqueous, no subjects exhibited positive reactions. (Torgerson,2007) Route: skin. Species: human 18+ yrs. Eighty patients (between 17 & 70 yrs) w/ hand eczema varying in duration from several wks to over 20 yrs were tested. Test cpds were tested either by the scratch-chamber method (SCT) or w/ Finn chambers. Rxns using scratch chambers were evaluated according to ICDRG criteria. Both immediate and Delayed reactions were assessed. A follow-up study was performed 1-8 yrs after the hospital investigation. in petrolatum. 2.5 % no effects. (Niinimaki,1987) Route: skin. Species: human 18+ yrs. A 78-yr old atopic man w/ sulphonamide allergy and chronic venous insufficiency had been treated for over 40 yr w/ many systemic and topic medications. Since the early years of treatment a dermatitis had developed in the summer, mainly involving sun-exposed areas. The rash faded 4-6 wks later, after changing his medication. Patch testing w/ a variety of standard series (GEIDC, cosmetic, Scandinavian) was performed. Twentytwo volunteers were also tested. (No further experimental details given). in aqueous solution. 1 % no effects, results in both subject and 22 healthy volunteers were negative. (Aguirre,1993) Route: skin. Species: woman. Patch tests using Finn chambers on Scanpor tape were conducted on a 60-year-old female patient with recurrent eczema. Reactions were noted 24 hour after removal of the 48 hour patch Vehicles and doses not listed for the negative test materials. No effects. (Bojs,1987) Route: skin. Species: man. A 50 yr old man with a history of psoriasis & a tendancy toward erythrodermia had an acute dermatitis 1 and 1/2 yrs after instituting regular treatment with Alphosyl cream. He was subsequently patch tested with Alphosyl as is & diluted with 50% pet., the ingredients of Alphosyl cream and the ICDRG, series. 10% aq. No effects. (Liden,1975) Route: skin. Species: human 18+ yrs. A maximization test was carried out with test material in petrolatum on 30 healthy male inmate volunteers. Application was under occlusion to the same site on the forearms of all subjects for five alternate-day 48 hour periods. Patch sites were pretreated for 24 hours with 5% aqueous sodium lauryl sulfate (SLS) under occlusion for the inital patch only. Following a ten to fourteen day rest period, challenge patches were applied under occlusion to fresh sites for 48 hours. Challenge applications were preceded by 30 minute CIR Panel Book Page 238

245 applications of 5% aqueous SLS under occlusion on the left side whereas the test material was applied without SLS treatment on the right side. Additional SLS controls were placed on the left and petrolatum on the right. (Kligman,1966) % no effects. (RIFM,1977) [Epstein,1977] Pharmacokinetics Route: food. Species: rat. Groups of ten female animals, 7 months old, were administered experimental diets containing the test material. Seven days later, the animals were administered an intraperitoneal injection of [32-P]-labeled phosphate, and the urine and feces were collected in two periods of 7 days. The specific activities of urine and feces were determined, as well as their ratio giving the percentage of endogenous phosphorus in the total fecal phosphorus excretion. No further details were provided. Subjects: 20 Female 1.2 % no effects. (Bonting,1956) Route: food. Species: rat. The retention of phosphorus in bone was evaluated. Groups of 11 male rats, 13 months old, were administered experimental diets containing the test material. The tibia of 1 hind leg from the animals was amputated 24 hours after an intraperitoneal injection of [32-P]-labeled phosphate (to serve as an internal reference standard). After six weeks, the animals were sacrificed and the other tibia was obtained. The specific activities in both tibiae were determined, and their ratio giving the retention of phosphorus by the bone was reported. No further details were provided. Subjects: 22 Male 1.2 % no effects, No significant difference in the average phosphorous retention over a 6-week period was reported. (Bonting,1956) Pharmacokinetics and metabolism studies Route: food. Species: rat. Groups of 8 female rats were administered either the basal diet (controls) or the experimental diet (treated) since weaning, and the metabolic profiles of each group were compared over five weeks. Calcium, phosphorus, and nitrogen balances were determined as the differences between the intake of these elements and their excretion in urine and feces (expressed as mg/day). In addition, the fixed base excretions, and the acidity, ph, and volume of urine were assessed. No further information was provided. Subjects: 16 Female 1.2 % no effects, No influence on the phosphorus balance or excretion was exhibited. No significant change in the calcium balance and excretion was produced, and urinary calcium output was not increased. No effect on ammonia or urea excretion was produced. No effect on the urinary fixed base excretion was produced. No change on the titratable acidity, ph, or volume of urine was produced. (Bonting,1956) Species: domestic animals. The presence of the test material in the kidney, liver and brain of untreated, healthy sheep, horses cows and pigs was evaluated. Citric acid was identified as a normal component of the kidneys of sheep, horses, cows and pigs, the brain of sheep and pigs and the liver of cows. (Halpern,1959) Route: food. Species: rat. A total of 24 female rats, 8 months old, were treated with the control diet for 6 weeks. During the next 8 weeks, 8 animals were continued on the control diet, and 8 other animals were administered an experimental diet while the remaining 8 animals were administered another experimental diet. Following the treatment with the test diets, another control period for 6 weeks was conducted in which all the animals were treated with the control diet. Calcium, phosphorus, and nitrogen balances were determined as the differences between the intake of these elements and their excretion in urine and feces (expressed as mg/day). In addition, the fixed base excretions, and the acidity, ph, and volume of urine were assessed. No further information was provided. Subjects: 16 Female Study Length: 8 weeks 1.2 % no effects, No influence on the phosphorus balance or excretion was exhibited. No significant CIR Panel Book Page 239

246 change in the calcium balance and excretion was produced, and urinary calcium output was not increased. No effect on ammonia or urea excretion was produced. No effect on the urinary fixed base excretion was produced. No changes in the titratable acidity, ph of urine, or volume of urine were produced. No increased urinary citric acid excretion was produced. (Bonting,1956) Species: mouse. The level of test material in homogenates prepared from male dd mice was studied. Normal, pantothenic acid deficient and vitamin B1 deficient mice were used. The homogenate was prepared 60 minutes after the abdominal injection of fructose. The level of citric acid in the homogenate of normal mice averaged mg%. The average level for pantothenic acid deficient mice was mg% and for vitamen B1 deficient mice was mg%. (Kawano,1959) Route: intravenous. Species: rabbit. The occurence of the "citric acid cycle" in the mammalian body was studied using rabbits. Various materials were infused into an ear vein. Urine was collected for analysis Test materials were generally sodium salts of acids. Two-thirds of the acid equivalents were neutralized with NaOH. Citric acid was seen in the urine of rabbits after the iv infusion of succinate, fumarate, malate, oxaloacetate or malonate. (Krebs,1938) Route: subcutaneous. Species: rat. The occurence of the "citric acid cycle" in the mammalian body was studied using rats. Various materials were administered. Urine was collected for analysis Test materials were generally sodium salts of acids. Two-thirds of the acid equivalents were neutralized with NaOH. Citric acid was seen in the urine of rats after the subcutaneous administration of malate, oxaloacetate or malonate. (Krebs,1938) Pharmacology Route: oral. Species: rabbit. Rabbits were used which had been resting for at least five days between any two experiments. The animals were fed mixed green and dry fodder and were sometimes used while fasting; three rabbits only were fed with dextrose or with calf liver respectively. The substances to be tested were administered either by mouth or subcutaneously. For the test, blood was withdrawn from the ear blood vessels. Before each feeding or injection, an analysis as always performed: the first estimation was usually performed one hour or half an hour later, in some experiments only a quarter of an hour after administration of the substance. The estimation itself did not take more than 3 to 4 min and was only done after the apparatus had been tested for absence of ammonia in control estimation. The same control estimation was performed after finishing the main estimations. Positive effects, feeding of malic, citric and betahydroxybutyric acids in some cases lead to a sometimes very marked increase of ammonia nitrogen, in other cases no change occurred. The cause of these differences could not be ascertained. (Wantoch,1929) Interaction Route: in vitro. Species: Salmonella typhimurium. Sudachi, yuzu, unshu, hassaku, iyokan, lemon, grapefruit, navel orange, lime were purchased at local markets in Japan. Sudachi, lemon and yuzu were fractionated into juice and flavedo (the outer colored portion of peel). For the other citrus fruits, only the juices were used. The citrus samples were prepared aseptically by using sterile glassware and flame sterilization. Well-washed citrus fruits were cut in half and hand-squeezed to obtain their juice samples. The flavedo portions were grated. One gram of each grated sample was ground with water in a mortar with a pestle and was adjusted to a volume of 10 ml. (Higashimoto,1998) Route: in vitro. Species: Salmonella typhimurium. Chemicals were dissolved or suspended in sterilized water. Pectin was used as a suspension in dimethyl sulfoxide. Soy sauce was used after CIR Panel Book Page 240

247 filtration through a sterilized membrane filter (pore size, 0.45 um). Aqueous solutions of 1 ml containing mg of MTCCA (or mg of tyramine or 0.05 ml of soy sauce), ml of ethanol and ml of citrus juice sample or citrus flavedo sample were mixed in brown tubes with 1 ml of 0.1 M sodium nitrite and adjusted to ph 3.0 with 6 N HCl with monitoring by a ph meter equipped with a small electrode. The reaction mixtures were incubated at 37 C for 60 min in the dark. The nitrosation was stopped by addition of 1 ml of 0.1 M ammonium sulfamate to decompose the residual nitrite. The effect of each citrus sample was examined at three or six doses. All the nitrite treatments were performed in triplicate. The nitrite-treated sample was immediately used for the mutation assay. (Higashimoto,1998) Route: in vitro. Species: Salmonella typhimurium. The mutation test with Salmonella typhimurium strain TA100 was conducted in duplicate by preincubation procedure in the absence of S9 mix. A mixture containing 0.1 ml of a nitrite-treated sample, 0.5 ml of buffer solution (ph 7.4) and 0.1 ml of an overnight culture of strain TA100 was preincubated at 37 C for 20 min, mixed with 2 ml of soft agar and plated on agar plates. The His+ revertant colonies were counted after incubation at 37 C for 48 h. The mutation was assayed with brown tubes under a yellow lamp in the dark. The numbers of spontaneous revertants (106) were subtracted in all the data on mutagenicity. A positive control, 2(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, 10 ng) induced 418 His+ revertants from strain TA100 in the absence of S9 mix. 2.5 mg/ml positive effects, Relative mutagenicity was 71%. [Relative mutagenicity is defined as the mutagenicity compared to 100% mutagenicity of nitrite-treated MTCCA in the presence of ethanol.]. 0.5 mg/ml positive effects, Relative mutagenicity was 93%. [Relative mutagenicity is defined as the mutagenicity compared to 100% mutagenicity of nitrite-treated MTCCA in the presence of ethanol.]. (Higashimoto,1998) Route: unreported. Species: dog. A dog weighing 3.5 kg was fasted for 24 hours, after which it was administered the test material. Twenty-four hours after the first dose, the dog was administered a second dose of the test material with citric acid. No additional data was provided. 0.2 % clinical signs, gastrointestinal tract, kidney, liver, lethal, Within an hour of the administration of 4 grams sodium benzoate + citric acid, the animal became very weak, breathing was labored and respirations were reduced to 9 per minute. Tonic and clonic convulsions began 1 hour and 15 minutes after the dose was given, and the animal died 2 hours and 20 minutes after the dose was given. At necropsy, pronounced congestion of the kidneys, stomach and intestines was found with some ulceration. The liver and lungs exhibited evidence of infarcts. (Lucas,1909) Route: unreported. Species: dog. A dog weighing 3.5 kg, was fed dog biscuits and water for several weeks, then fasted for 36 hours. Following the fast period, the animal was administered a mixture containing sodium benzoate and citric acid. A second dose was administered the next day. No additional data was provided. 0.2 % gastrointestinal tract, kidney, liver, musculoskeletal, After receiving the first dose of 35 grams sodium benzoate + citric acid, the animal became quite uneasy, and at the end of an hour showed great muscular weakness and tremor. Following the second dose, the animal was more prostrated than on the previous day and showed general stiffness of the muscles. At the end of 6 hours, the animal was sacrificed and a necropsy was conducted. Ulceration in the stomach and intestines was observed, with marked congestion in the intestines. The liver and lungs exhibited considerable congestion with evidence of infarcts. The kidneys were cyanotic with a congested cortex and a pale and anemic medulla. (Lucas,1909) Route: food. Species: rat. Groups of 15 F344 males were simultaneously given carcinogen [0.025% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or 0.021% N-ethyl-N-(4- CIR Panel Book Page 241

248 hydroxybutyl)nitrosamine (EHBN)] in drinking H2O and test chemical supplemented or control diet for 20 wk. Body weights, food & H2O intake. Osmolality & ph, determined in urine at wk 10 & 14. Urinary metabolites of BBN or EHBN determined in 24 hr urine samples at wk 20. Liver & kidneys weighed. Liver & kidneys & semiserial sections of urinary bladder examined histologically % body weight changes, interaction, liver, lower final BW & higher liver wt in rats given BBN & EHBN; lower urine ph in rats given BBN; no differences in induction of preneoplastic lesions of the bladder, papillary or nodular hyperplasia. (Inoue,1988) Route: skin. Species: mouse. The ability of the test material to inhibit UVR-induced ornithine decarboxylase (ODC) activity was evaluated using female albino hairless Skh:HR-1 mice. A single application of test material was given 2 hr prior to irradiation with UVA & UVB at 1.2 J/cm2. Epidermal ODC activity was measured. Vehicle was PG:H2O (1:3). 5 % interaction, the induction of epidermal ODC activity by UV irradiation was increased by the application of citric acid. (Hillebrand,1993) Route: skin. Species: mouse. The effect of the test material on chronic UVB induced skin tumors was evaluated using groups of 8-15 female albino hairless Skh:HR-1 mice. Test material was applied 2 hr prior to each UVB exposure of 30 mj/cm2. Applications & irradiation were conducted 3x/wk. Tumor yield & incidence were evaluated. Vehicle was PG:H2O (1:3). 5 % no effects. (Hillebrand,1993) Route: oral. Species: mouse. Test cpds were tested against seizures in mice induced by ipr inj of semicarbazide HCl (168 mg/kg) neutralized to ph mice/grp were pretreated orally w/ test cpd immediately prior to semicarbazide inj & sometimes 20 to 30 min after inj. of semicarbazide. sodium citrate. 6.3 g/kg interaction, sign. incr. in time to initial convulsions noted (+30%). (Jenney,1958) Route: oral. Species: rat. Female Sprague-Dawley animals (6/grp) were treated w/ aluminum in combination w/test cpd in drinking water 5x/wk for 5 wks. Control grps received no test cpd in drinking water. Weight gain, fluid & food intake were recorded daily or twice a week, whereas urine was collected daily. After 5 wks - animals were sacrificed & brain, femur, kidneys, liver & spleen were removed. Aluminum conc. of these organs was determined by atomic absorp. spectrophotometry. 62 mg/kg interaction, sig. incr. in aluminum levels in liver, spleen, brain & bone were noted. Dose = 62 mg/kg/day. (Domingo,1991) Route: gavage. Species: rat. The effect of the test material on blood alcohol levels after simultaneous adminstration of test material and ethanol was evaluated using male Sprague- Dawley rats. Test material was given in ethanol mmole/kg interaction, the maximum blood ethanol level declined and the time needed to reach this maximum increased. (Mochizuki,1987) Route: gavage. Species: rat. Three groups of pregnant Sprague-Dawley animals were given daily doses of test substance concurrently w/ aluminum hydroxide or gd Control received distilled water. All animals were observed daily for body wt. gain, food consumption, & clinical signs of toxicity. Animals were sacrificed on d 20 and maternal body, liver, kidney, brain & uterine wts were recorded. All females were evaluated for the number of ovarian corpora lut, and p status of uterine implantation sites. Fetuses were weighed, sexed and examined for external, internal, and skeletal abnormalities. Reproductive effects, fetal body weight was sig. decr. in aluminum hydroxide and citric acid group. Skeletal variations were significantly increased. (Gomez,1991) Route: inhalation. Species: human 18+ yrs. Eight normal & eight asthmatic subjects were selected. All were non-smokers & had not had respiratory infections in the eight wks preceeding CIR Panel Book Page 242

249 the study. Subj. were asked to breathe through a mouthpiece and a Fleish pneumotachygraph. Nebulized test cpd were either vented to the atmosphere or Directed to the mouthpiece via a side tube. A mercury strain gauge was loosely attached round subjects neck so that movement of the thyroid cartilage could be detected and cough recorded. Airway flow, coughing movements and valve opening were recorded on a Medeler fibreoptic recorder. 32 % no effects, no significant difference in cough response between normal & asthmatic subj. noted. 16 % no effects, no significant difference in cough response between normal & asthmatic subj. noted. 8 % no effects, no significant difference in cough response between normal & asthmatic subj. noted. (Pounsford,1985) Subchronic toxicity Route: oral. Species: dog. Three or four animals were given test cpd in a gelatin capsule for days. Parameters evaluated included clinical signs of toxicity, body wt., hematology, urinalysis, gross & microscopic pathology. No effects. (Krop,1945) Route: oral. Species: rat. Male SD-JCL animals were divided into 4 grps consisting of 10 animals each and adm. test cpd for 6 wks. Parameters evaluated were body wt., daily food intake, behavior abnormality & urinalysis. On the last feeding day, bromosulfothalein (BSP) measurements were made. After fasting for 24 hrs, Blood sampled from the abdominal artery of the animals slightly anesthetized by ether inhalation was subjected to morphological & biochemical examinations. Animals were sacrificed, gross & microscopic analyses were performed. No effects. (Yokotani,1971) Route: unclassified. Species: rat. Male and female Sprague-Dawley rats received test material during the fetal stage and for 200 days after birth. Test material was added to the maternal diet and later to the pup diets. Effects on growth and physiological characteristics were evaluated. The calcium intake was restricted The study was initiated with 12 pregnant rats each receiving reduced calcium. Half of these received citric acid. The number of pups was 63 with and 64 without citric acid. 5 % body weight changes, lethal, musculo-skeletal, reduced body weight, bone weight, packed cell volume and haemoglobin in males only. (Wright,1976) Route: food. Species: guinea pig. Groups of 10 young male guinea pigs received test diet for 24 days. Effects on growth and metabolism were studied The actual dose may have been higher than indicated due to the presence of citric acid in the basal diet. 3 % blood effects, reduced packed cell volume. (Wright,1976) Route: food. Species: guinea pig. Groups of 10 older male guinea pigs received test diet for 60 days. Effects on growth and metabolism were studied The actual dose may have been higher than indicated due to the presence of citric acid in the basal diet. 5 % blood effects, reduced packed cell volume. 1 % blood effects, reduced packed cell volume. (Wright,1976) Route: food. Species: rat. Groups of 10 male rats received test diet for 60 days with and without a calcium-restricted diet. Effects on growth and physiological characteristics were studied The rats given adequate calcium were older than the calcium-restricted rats. 4 % body weight changes, blood effects, Citric acid produed decreased body weight and packed cell volume with but not without calcium restriction. (Control groups receiving the high and low doses of calcium without citric acid were included). (Wright,1976) Route: oral. Species: rabbit. Sixty male albino, New Zealand animals, were divided into 4 grps of 15 animals ea. and fed diets containing test cpd for 150 days. Control grp received ground Rockland Rabbit Diet. Parameters evaluated included food intake, body wt., hematology, urinalysis, gross & microscopic pathology. No effects. (Packman,1963) CIR Panel Book Page 243

250 Chronic toxicity Route: food. Species: rat. Groups of Wistar rats (26/sex) were fed bread containing 50 times the normal concentration of chlorine dioxide, propyl gallate and BHA, polyoxyethylene (8) monostearate, or sodium propionate as 75% of the diet for 1 year. Body weights and food intake. Three/sex/group killed at 13 & 26 wk & tissues, preserved. Remaining rats killed after 52 wk. Heart, liver, spleen & kidneys weighed. Gross & microscopic pathology. (Antioxidants were added to lard as a commerical prep containing 20% BHA, 6% propyl gallate, 4% citric acid & 70% propylene glycol [PG]). 4% with 20% BHA, 6% propyl gallate & 70% PG mg/kg no effects mg/kg no effects. (Graham,1954) Route: food. Species: rat. Groups of 15 Wistar males were fed diets containing 50 times the normal concentration of chlorine dioxide, propyl gallate & BHA, polyoxyethylene (8) monostearate, or sodium propionate for 32 wk. Body weights & food intake. Two/grp killed after 16 wk for histopath & hemoglobin exams. Remaining, rats killed after 32 wk. Organ weights & histopath. (Antioxidants were added to lard as a commercial prep containing 20% BHA, 6% propyl gallate, 4% citric acid & 70% propylene glycol [PG]). 4% with 20% BHA, 6% propyl gallate & 70% PG. No effects. (Graham,1955) Route: food. Species: rat. Twenty male & 10 female albino Carworth Farm animals/dose level were placed on either basal laboratory diet or basal diet containing test cpd. Parameters evaluated were body wt., food consumption, clinical signs of toxicity, gross & microscopic pathology. 3 % body weight changes, male body wt. was sign. below controls. 5 % body weight changes, male body wt. was sign. below controls. (Horn,1957) Carcinogenesis and mutagenesis studies Route: in vitro. Species: Salmonella typhimurium. Ames test using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94, TA98 and for some samples TA2637 with and without S-9 liver microsome fraction. Each sample was tested at 6 doses; only maximum dose reported. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). 99.9% purity solvent was phosphate buffer. 5 mg no effects. (Ishidate,1984) Route: in vitro. Species: Salmonella typhimurium. In test tubes, an overnight nutrient broth culture of Salmonella typhimurium strains TA98, TA100, TA1535 or TA1537, and the test material in an unspecified vehicle were added to 2 ml of molten top agar maintained at 45oC. The test tube contents were mixed and then poured on minimal glucose agar plates. The plates were allowed to harden for a few minutes, and then incubated at 37oC for 48 hours. The numbers of revertants per plate were counted and the results recorded.if the test material was non-toxic, it was tested over a dose range of 10, 100 and 500 ug or 10, 100 and 1000 ug (or higher for TA100). If the test material was inhibitory, it was tested up to the maximum allowable concentration. The assay was conducted in the absence of S9 metabolic activation. A threefold or greater increase in the spontaneous mutation frequency was used as the criterion for a positive mutagenic response. The material was considered non-mutagenic if there was no dose-response. The specific protocol was from McCann, 1975 (Location 4023), which then cited the protocol from Ames,1975 (Location 3466). This paper discusses results that were previously presented in Location The results for materials that were not presented in Location 4023 are presented here. The non-mutagenic materials reported were tested in the absence and presence of S9 activation. (Ames,1975) No effects, Non mutagenic with all 4 strains, with and without S9. With TA100, the number of revertants per umol was <0.01. (McCann,1976) CIR Panel Book Page 244

251 Route: in vitro. Species: Salmonella typhimurium. Mutagenicity was evaluated by the Ames assay using the standard plate incorporation assay in the presence and absence of metabolic activation prepared from rat livers plus co-factors (S9 mix). A 0.1-ml aliquot of the test material dissolved in distilled water was mixed in capped culture tubes with 0.5 ml of S9 mix and 0.1 ml of a fresh overnight culture of Salmonella typhimurium strains TA97, TA98, TA100, and TA104. A 2-ml aliquot of top agar was added to the tube, and the tube contents were poured on a minimal glucose agar plate. The plates were incubated at 37oC for 48 hours, and the number of revertant colonies was counted. The positive control was 10 ug/plate of 2-aminoanthracene, and the experiment was conducted in triplicate. (Maron,1983) Anhydrous citric acid was tested µg no effects, Unit was ug/plate. No mutagenic activity was detected in the absence or presence of S µg no effects, Unit was ug/plate. No mutagenic activity was detected in the absence or presence of S µg no effects, Unit was ug/plate. No mutagenic activity was detected in the absence or presence of S9. (Al-Ani,1988) Route: in vitro. Species: hamster. Chromosomal aberration test without metabolic activation in a Chinese hamster fibroblast cell line (CHL) using multiple harvest times (24 and 48 hours after the initiation of treatment). The assay was conducted at 3 different doses but only the maximum dose was reported. The cells were exposed for a total of 24 or 48 hours; colcemid was added two hours prior to cell harvesting. Preparations were processed wtih Giemsa and 100 well spread metaphases were analyzed. Untreated cells and solvent treated cells served as negative controls. The results were considered negative if the incidence of aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When doseresponse relationships were not found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of positive samples, the D20 (dose in mg/ml at which structural aberrations (including gaps) were detected in 20% of metaphases observed) was determined. Additionally, the TR value (indicates frequency of cells with exchange-type aberrations per unit dose (mg/ml)) was calculated. A high TR value exists for chemicals that show carcinogenic potential in animals. 99.9% purity solvent was physiological saline. 1 mg/ml no effects. (Ishidate,1984) Route: in vitro. Species: Salmonella typhimurium. The antimutagenic effects of citric acid and ascorbic acid were evaluated separately and in a 1:1 mixture. Activity against N-nitro-ophenylenediamine was tested in strain TA97a and against sodium azide in strain TA100. The Ames test procedure was followed. Interaction, a dose-dependent antimutagenic effect was seen at ug/plate. A synergistic effect with ascorbic acid was noted. (Bala,1989) Developmental and reproduction studies Route: food. Species: rat. Male and female rats, 32 weeks old, were administered experimental diets containing the test material for 29 weeks and mated. Eleven weeks later, these same animals were mated again. A group of control animals were administered the basal diet alone. The weight of the mothers, number of living and stillborn per litter, the average birth weight of the living offspring, and the number of young remaining at weaning were evaluated. No further information was provided. Study Length: 26 weeks 1.2 % no effects, No detrimental effect on reproduction was reported after the first or second mating. (Bonting,1956) Cytotoxicity Route: in vitro. Species: human 18+ yrs. Cytotoxicity assay using Eagle's KB strain of human carcinoma cells. Test samples were dissolved in sterile H2O, dimethylformamide or ETOH. Agents with ID50<10 ug/ml were reassayed at 4-5 concentrations, 2 replicates/concentration. CIR Panel Book Page 245

252 ID50>1000 ug/ml. (Smith,1959) Route: in vitro. Species: human 18+ yrs. The cultured corneal endothelial cell model system was used. Cell viability was measured via the 51Cr release assay Vehicle not provided. ED50 values of and M were obtained in duplicate tests. Known irritant potentcy in vivo - moderate to severe irritant. (Douglas,1983) Route: in vitro. Species: human 18+ yrs. This study provides a new cell culture system for determining cytotoxicity. Hep G2 cells (derived from a human hepatoma) were maintained at 37 C in a 7% CO2 atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U penicillin G/ml and 100 ug streptomycin/ml (complete medium). The cells were plated with 1 ml complete medium in each well (2 cm2 growth surface) of a 24-well tissue culture test plate. After 24 hours the medium was removed from the wells. Four control wells were refilled with 1 ml fresh complete medium. The other wells received the test materials, in five different concentrations, freshly prepared in complete medium, using four wells per concentration. The test materials were dissolved directly in complete culture medium. The highest concentration was chosen on the basis of a preliminary range-finding test. The assays were performed on logarithmically growing cells. After treatment for 24 hours at 37 C, the supernatant was discarded, the cells were washed three times with 1 ml Hank's balanced salt solution, and the cells were lysed. This solution was further used for protein measurement using bovine serum albumin as a standard. The results are presented as percentages of control cultures, which typically contained ug protein/well. The relative toxicity of the test materials was determined by use of the PI(50), which is the concentration of test material required to induce a 50% inhibition in cell protein content of the culture. Reproducibility was assessed by conducting 3 independent assays of specific test materials to determine PI(50)'s. The PI(50) value of test material in culture with Hep G2 cells was 20 mm. (Dierickx,1989) Miscellaneous Species: human 18+ yrs. The test material was present in urine obtained from a normal subject. Citric acid was present in urine obtained from a normal subject. (Osteux,1954) Route: in vitro. Species: Salmonella typhimurium. The inhibitory effect of several aromatic compounds and acidic compounds used alone or in combination were tested against Salmonella enterica subsp. enterica serovar Typhimurium ATCC To determine effective combinations of antimicrobials, two experimental designs were used. The first one with only aromatic compounds and the second one with both acidic and aromatic compounds. This protocol describes the experimental design with both acidic and aromatic compounds. The bacteria were grown in BHI broth in absence or presence of antimicrobials. Acids were added in the medium before autoclaving while aromatic compounds were added after sterilization from the ethanol stock solutions adjusting the final ethanol concentration in the medium at 0.2% v/v. For the determination of effective combinations, the concentrations of each antimicrobial in the medium for a given culture were set by the experimental design. The medium was inoculated at 1% v/v (approximately 10(6) cells/ml) with a standardized inoculum obtained after three subcultures in BHI broth. Growth was carried out with the Bioscreen Apparatus. It follows multiple growth curves in 100 wells microplates by an automatic measurement of the optical density (OD). Each well contained 200 ul of culture. The growth was carried out at 37 C with a mild and discontinuous agitation for 10 seconds every 3 minutes. The OD at 600 nm was measured every 30 minutes up to 72 hours. Growth analysis was the growth percentage at 12 hours of culture. The experimental design was as follows: A 2(7)-1 regular fractional factorial CIR Panel Book Page 246

253 design of resolution (7 (half-complete design of 7 compounds at 2 levels). It allows the estimation of the main effects, two-factors and three-factors interactions (Box and Hunter, 1961) The seven compounds (citric acid, acetic acid, lactic acid and polyphosphoric acid, thymol, citral and carvacrol) were used at two levels: "absence" for the low level and (IC5)/2 for the high level. The experimental designs were constructed and analysed by the softwares PLANOR (Kobilinski, 1997) and ANALYS which are specifically adapted to regular fractional designs. In the analysis of the "mixed" design, the determination of active effects was made by an internal graphical comparison, the Daniel graphic (Daniel, 1959). In both factorial designs used to determine efficient combinations of antimicrobials, the levels for the different compounds were chosen such that total growth inhibition after 12 hours of incubation was avoided. Data was analyzed in terms of growth percentage and growth delay. Results are provided as the "main effect" of each compound, the "two-factors interaction" or "three-factors interaction" for interaction of two or three compounds. These factorial effects and the general mean are necessary to calculate the mean result on growth percentage or growth delay. The factorial effect is added to the general mean when the compound is at its high level and subtracted when it is at its low level. When the factorial effect is negative, it means that the variable (growth percentage or growth delay) will be lower when the compound is at its high level comparatively to low level. The effect of each combination of three compounds was then calculated when the three compounds are all either at their high level or at their low level Note: Pyropolyphosphoric acid was also tested but is not a RIFM/FEMA material. Study Length: 12 hours Summary Citric acid has the highest "main effect" on the growth percentage (-19.16%). For both growth percentage and growth delay, all acidic combinations were approximately of equal activity. High level (IC5)/2 concentration = 5 mm; Low level (absence) = 0. Citric acid has the highest "main effect" on the growth percentage (-19.16%). When citric acid is present at its high level (IC5)/2 (5.0 mm), the mean result on growth percentage is = 37.4%, while it is = 75.8% when citric acid is absent. (Nazer,2005) Route: in vitro. Species: Salmonella typhimurium. The inhibitory effect of several aromatic compounds and acidic compounds were tested individually against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311, prior to being tested in combination with other compounds. The Minimum Inhibitory Concentrations (MIC) at 72 hours were determined. Three separated experiments were conducted for each range of concentrations. Five wells were inoculated in parallel for each experiment. The MIC of an antimicrobial is defined at its lowest concentraiton which leads to no increase of the optical density (OD) during 72 hours of incubation. The inhibitory concentration x (ICx) of an antimicrobial is defined as the concentration that leads to x% of reduction of the growth percentage at 12 hours Concentrations of test material tested not provided. Study Length: 72 hours Summary The MIC of the test material was 40 mm. The MIC of the test material was 40 mm. (Nazer,2005) Route: in vitro. Species: fungi. The onset of inhibitory action of test cpd(s) upon cell multiplication of the blue-green alga, Microcystis aeruginosa and the green alga, Scenedesmus quadricauda was determined and the test cpd concentration causing the onset of cell multiplication inhibition in ea. case (toxicity threshold = TT) for both test organisms established. On this basis the ratio between toxicity threshold of ea. tested substance for the blue-green alga (TTmi) and the toxicity threshold of the corresponding substance for the green alga (TTsc) was calculated by setting TTmi = 1. TTmi = 80 mg/l; TTsc = 640 mg/l. (Bringmann,1978) Route: in vitro. Species: fungi. Test material was tested for antifungal properties on Penicillium digitatum, Penicillium italicum, Penicillium verrucosum and Fusarium oxysporum. Fungi were CIR Panel Book Page 247

254 inoculated into media that contained test material and incubated for 8-12 days at 25 C. 10 mg/ml positive effects, 100% inhibition of colony formation was noted when combined with nootkatone at 1000, 500, and 250 ug/ml. 5 mg/ml positive effects, only slight colony formation (1 mm diameter) was noted when combined with nootkatone at 1000, 500, or 250 ug/ml. 20 mg/ml positive effects, 100% inhibition of colony formation was noted when combined with nootkatone at 1000, 500, or 250 ug/ml. (Morozumi,1989) Route: skin. Species: human 18+ yrs. Eleven males & three female patients who had ichthyosiform dermatitis of several types participated. Their ages ranged from 1-22 yrs. Diagnosis of type of disease was established by a study of genetic pattern & by clinical, histologic, & ophthalmologic exam. Test materials that were dissolved in either water, ethanol, a hydrophilic ointment or plain petrolatum. The patient or parent was instructed to apply each test material to its appropriate test site twice daily for 14 days. Observations were made daily or weekly. Biopsies of treated & untreated sites were performed. 5 % positive effects, disappearance of scale from lesions & restoration to normal looking skin was observed upon treatment of lamellar ichthyosis. (VanScott,1974) Route: oral. Species: rabbit. Histochemically detectable changes in the adrenal cortex of female rabbits after adm. of test cpd were investigated. [No further experimental detail given, article in Polish, summary in English]. Positive effects, presence of vacuoles in the cellular plasma, contents of lipids in the adrenocortical layers, increase of the individual layer and of the whole adrenal wt, the presence of degenerative changes in adrenal cortex noted. (Jonek,1961) Route: inhalation. Species: human 18+ yrs. Sensitivity to test cpd was assessed by determining the lowest concentration of inhaled test cpd required to produce involuntary coughing in 23 healthy white males, ages Approx. 1 wk later, changes in cough, pain on deep inspiration, FVC, FEV1, and SRaw were measured immediately following a One hour exposure to 0.4 ppm ozone during heavy, intermittent exercise. [Data presented in Abstract form only]. Respiratory tract, individual sensitivity to citric acid ranged from 0.5 to 32.0 mg/l. Sign. association between sensitivity to citric acid & degree of ozone-induced cough. (Schreiber,1986) Route: oral. Species: human 18+ yrs. Twenty fasting subj came to laboratory at 8:00 AM.Following a gastric washout w/250 ml tap water, 750 ml of sol'n of acid were given down a tube into stomach in about 75 sec. Either conc. of acid or type of acid used was varied. Only one meal was given to ea. subj. in any one day. After an Interval of min, constant for ea. subj., gastric contents were recovered. Ea. litre of meal contained about 30 ml phenol red. Conc. of phenol red in gastric contents recovered at end of fixed period was used to calculate the volume of original meal recovered. A values ranged from (A = volume of meal calculated to be recoverd at zero conc. of acid). B values ranged from (B = ml. increase in vol of meal recovered per m-equiv. increase of acids/litre of meal). (Hunt,1969) Route: unclassified. Species: rat. Recordings were obtained from the lingual nerve of 6 adult male rats weighing g (Sprague-Dawley, CrL:CD(SD)BR). Rats were housed in transparent plastic cages, a maximum of two animals per cage, in a temperature-controlled colony room on a h light-dark cycle with lights on at 0500 h. All animals had free access to Purine Rat Chow 5001 and deionized-distilled water ad libitum. All preparations began approximately 4 h into the light phase. Rats were anesthetized with urethane (1.5 mg/g body weight). The trachea was cannulated and a small suture was attached to the ventral surface to the tongue After placement in a nontraumatic head holder, the right lingual branch of the trigeminal nerve was exposed using a mandibular approach. A small nerve strand was dissected from the CIR Panel Book Page 248

255 nerve trunk and placed on a nichrome wire electrode. The animal was grounded via the headholder. The action potentials were stored on a video recorder tape. The tongue was slightly extended from the oral cavity and held in place by fixing the ventral tongue suture to the preparation table. Stimuli were presented to the anterior portion of the tongue. Two stimulus bottles and a water rinse bottle were connected by polyethylene tubing and luer-lock adapters to separate input ports on the stimulus mixing platform. From a delivery faucet, the solution flowed through a Peltier heat exchange device before being presented to the anterior portion of the tongue. (Pittman,1998) Route: unclassified. Species: rat. Chemical stimuli consisted of NaCl (150 mm), glucose (150 mm), quinine-hcl (3 mm), and citric acid (0.3 mm). The effect of temperature adaptation was assessed by presenting the four stimuli in two series. Throughout the experiment, distilled deionized water at the appropriate temperature condition continuously flowed over the tongue to maintain the adaptation temperature. Baseline neural activity was recorded during the water rinse for at least 20 sec preceeding each stimulus presentation. The stimuli were presented for 10 s and were followed by a water rinse of at least 90 sec. 0.3 millimolar positive effects, 29% of thermally sensitive lingual neurons were responsive to citric acid at 35 C and 35% were responsive to citric acid at 25 C. (Pittman,1998) Route: skin. Species: woman. A 56-year-old female was presented to an emergency room complaining of severe facial pain and breathing difficulties, 4 hours after a 10% citric acid facial peel was administered on to her face. The facial peel had been applied for 4 hours after which it was washed off just before the patient was presented in the emergency room. At the time of her presentation, the patient was alert, rapidly breathing (tachypnea) and in severe pain. Initial blood pressure = 162/88 mm Hg, heart rate = 123 beats per minute, respiratory rate = 28 breaths per minute, temperature = 36.6oC, and oxygen saturation = 100%. Initial physical finding were first and second degree burns to the face and anterior neck with blisters (bullae), normal phonation and clear lungs with no evidence of stridor (vibrating sounds signifying obstruction of the respiratory passages). It was noted that there were other compounds present in the facial peel, but these were not specified. 10 % skin effects, Treatment consisted of fluid hydration, IV narcotics, oxygen supplementation, continuous cardiac and pulse/oxygen monitoring, chest radiography and burn consultation. However, the patient became more tachypneic with enlarging bullous lesion over the anterior neck, stridor and abnormal phonation. Laryngoscopy revealed supraglottic edema of the oropharynx without vocal cord involvement. Pulse/oxygen readings remained normal but the patient was prophylacticly intubated for airway protection secondary to increasing airy edema in the oropharynx. The patient was admitted to the Burn ICU and the intubation was removed the next day. The patient had permanent facial and neck scars without any airway pathology. (Ghadishah,2002) The chelating effect of the test material was determined. The test material in methanol was mixed with 3.7 ml of methanol and 0.1 ml of 2 mm FeCl3(4H2O), and the reaction was allowed to proceed for 30 seconds. Next, 0.1 ml of 5 mm ferrozine was added and the solution was mixed and left to stand for 10 minutes at room temperature. The absorbance of the mixture was measured with a spectrophotometer at 562 nm, and the lower the absorbance, the higher the chelating power. Tested as the positive control. 20 mg/ml positive effects, The chelating effect was 25.2%.positive effects, The chelating effect on ferrous ion increased steadily up to 20 mg/ml. (Mau,2003) A model for studying specific effects. CIR Panel Book Page 249

256 Serra,2003. A classification model using genotoxicity data from an invitro chromosomal aberration assay with Chinese hamster lung cells is presented. Allergenicity other than sensitization (SEN) or photosensitization (PSEN). Fuglsang,1994. An open oral challenge of a solution of food additives in carbonated water was conducted on children. Analytical methodology. Can also refer to physical properties such as boiling temperature, flash point, etc. Lees,1957. Organic acids recovered in bone are reported. Bose,1962. Some organic acids present in the raw juice, clarified juice and molasses of sugar cane are identified by analytical procedures. Bakowski,1964. Yang,1983. Turemis,2003. The aromatic compounds of 5 blackberry varieties using Immersion Solid Phase Micro Extraction (Im-SPME) were determined. Article in foreign language; no English translation available. Villard,1927. No English abstract or translation available. Villard,1927a. No English abtract or translation available. Villard,1927b. No English abstract or translation available. Villard,1927c. No English abstract or translation available. Baraud,1957. Buchloh,1958. The paper is in German; only an English abstract is available. Carbonic acid components of apple juice were mentioned. Geistlich,1970. Biological tests other than classical toxicology testing and pharmacological tests. Bickerman,1954. The usefulness of the test material for the artificial stimulation of the cough reflex for the study of antitussive agents was evaluated Nieth,1966. The consumption of lactate, pyruvate, citrate and glucose by the human kidney was determined. Carcinogenicity studies and/or effects. Gaworski,1999. The flavor ingredients were tested in combination; not individually. Citations. No original data; the authors cite others work. Jarvinen,1991. Rodgman,2002. Flavorants and other tobacco components were previously examined individually for their effect on cigarette mainstream smoke composition. Computational methodology including use of computers in toxicology or safety evaluation. Also includes articles on databases. Rosenkranz,1990a. The mutagenicity of various substances including physiological substances, was predicted using the CASE system. Rosenkranz,1990b. The carcinogenicity of various substances including physiological substances, was predicted by the CASE system. CIR Panel Book Page 250

257 Editorial comments, including letters to editors, authors responses, etc. Gangolli,1992. Metabolism or non-ecological (not bacteria or fungi) biotransformation studies. See also pharmacokinetics (PKIN). Krusius,1940. The effect of various diets on the excretion of certain acids is discussed. Marshall,1949. Haynes,1965. Labeled carbons from CO2 was incubated with pyruvate and were later found in synthesized citrate, maleate and fumerate. Atfield,1972. The handling of pyruvate, citrate, succinate and alpha-ketoglutarate by the guinea pig small intestine was studied. Methods for determining priorities. Johnson,1984. Mixture. Gaworski,1998. Mixtures were tested not individual ingredients Roemer,2000. The flavor components of cigarettes are listed. Smith,2004. Compounds used by the pharmaceutical industry to enhance the percutaneous penetration of drugs were identified. Baker,2004. The potential chemical changes and biological activity of smoke from cigarettes with added ingredients was investigated. The ingredients of the experimental cigarettes were listed. Occurrence such as physiological (PHYSIOL) occurrence. Also non-animal natural occurrence. See (NAT) for natural occurrence in animals. Miller,1949. The tricarboxylic acid cycle was studied in dogs and man Piper,1967. The content of lactic, citric and pyruvic acids in the gastric juice of normal subjects and patients with peptic ulcers and gastric carcinomas was investigated. Papers that are a review on a general subject, not a specific material. TREV papers may contain one or more RIFM materials. TREV papers do not contain original data. Sawada,1957. A pharmacological and clinical review on the use of several acids. Henson,1959. Wieland,1968. Chalmers,1971. Gardner,1972. Alarie,1973. Fisher,1982. Bronaugh,1984. FASEB,1984. Dziezak,1986. Dziezak,1986a. Ishidate,1988. DeGroot,1991. CIR Panel Book Page 251

258 Hurley,1993. Taylor,1993. Additives and ingredients most likely to be used in seafood products are listed, along with their typical functions. Sainio,1995. Christian,1996. Swirsky-Gold,1999. Pharmacokinetics. See also metabolism (METAB). Saeki,1956. Test material was the sodium salt Predictability of a test. See also model (MODEL), relevance (RELEV). Pavan,2006. The predictive capability of the proposed model for the SIDS data set was compared with five well known literature QSAR models for acute fish toxicity to Pimephales promelas. 408 heterogeneous chemicals were selected for this study. Processed flavors. Shinohara,1986. The formation of furan compounds by the reaction of amino acids with sugars in the presence of acids was studied Reports on patients such as patch testing. Fisher,1975. Patch test concentrations and vehicles are discussed DeGroot,1994. Synonyms, patch test concentrations and vehicles with references, in addition to comments for each chemical are listed. Davis,2008. Patch-test reaction rates among patients tested on day 5 and on day 7 or later were shown. Reproductive effects. See also teratogenicity (TERAT) and fetal (FETAL) effects. Material is given to mother and father before or during gestation to see how it affects the ability to reproduce viable offspring. Toxic effects may be noted Berking,1991. Sodium salts of the acids were tested Review articles. Papers that are reviews on a material or a class of materials. A review paper may be on one or more RIFM materials. Review papers do not contain original data. FASEB,1977. Katz,1994. Berry,2001. A review that encompasses the chemistry, function properties and food applications of several acidulants. Blackburn,2005. The list of chemicals was used to identify chemicals for retrieval of toxicity data and identification of a set of NOEL values. Skin or respiratory irritation. Schaper,1993. Following a review of available literature, a database with 295 chemicals and mixtures was developed for airborne materials that have been evaluated for sensory irritation, using the ASTM mouse bioassay or some variation of it. Swanson,1995. The use of the Tissue Equivalent Assay (TEA) and Bovine Corneal Opacity and Permeability (BCOP) assay compared to the Draize assay were evaluated using industrial and household cleaning formulations. The components of the formulations that contribute to irritancy were reported. CIR Panel Book Page 252

259 Kartono,2006. The results of tandem irritation studies were reviewed and the mechanisms that led to the responses were investigated. Structure activity relationships such as studies of a group of aldehydes or other similar structures. Jurs,1979. Haas,1982. Ability to stimulate penetration of Schistosoma mansoni cercariae into agar was evaluated Rosenkranz,1990. Rosenkranz,1990c. Williams,1993. Barratt,1995. Barratt,1995a. Barratt,1995b. Barratt,1996. Purdy,1996. Walker,2003. The Integrated 4-Phase model was used to predict estrogen receptor binding affinities for various chemicals. Studies on cosmetic products or directly related thereto. Maibach,1980. Studies on flavor and flavor components other than processed flavors (PF). Sanders,1966. Gatfield,1988. Test methodology (new or altered classical), testing procedures, guidelines, etc. If applicable, see also biological tests (BIOTS). Schormuller,1960. A method to detect organic acids in biological material was presented. Tordoff,2002. The sensitivity of the 2-bottle choice test was evaluated. Environmental Data Acute toxicity Route: unclassified. Species/Media : invertebrate. The ability of the test material to interfere with reattachment of zebra mussels was studied. A 48 hr exposure was conducted under static conditions. Mussels were acclimated to the test conditions over a 4 day period before testing and received no food during this time. Well water was used as test water. Temperature, disssolved oxygen, and ph were measured daily. The water temperature was controlled at 17 C. The lighting cycle was controlled to give 16 hours light and 8 hours darkness. For each test concentration, a 3.8 liter glass jar containing 2.5 liter of well water (and appropriate test concentration) was used. At time of testing 15 mussels/per replicate were removed from the PVC substrates they were previously allowed to attach to and placed in a glass petri dish preconditioned in an aquarium containing well water for 5 days prior. The petri dish and mussels was placed onto the bottom of an exposure chamber and mussels were turned on their left valve CIR Panel Book Page 253

260 side. The ability to right themselves and reattach was evaluated after a 48 hour exposure period. Mussels that were not upright and attached were transferred to a separate 3.8 liter glass jar containing 2.5 liter well water, placed on their side and observed for a 48 hour treatment free postexposure period. Reattachment and mortality were evaluated at the end of the 48 hour period. The EC50 and LC50 were calculated using Probit analysis. 50 mg/l no effects. (Cope,1997) Route: unclassified. Three model organisms, Pseudomonas putida (bacteria), Scendesmus quadricauda (green algae) and Entosiphon sulcatum (protozoa), were used to assess the potential toxic action of water pollutants on water micro-organisms. The cell multiplication inhibition test was applied using pure cultures of single-cell model organisms. Culture media and conditions for preparation of stock cultures, preliminary cultures and test cultures are standardized. Inoculum, cell multiplication and its inhibition are determined quantitatively. The analogous test methods permitted comparison of the damaging action of a pollutant between organisms. In each instance, the multiplication of the cells is inhibited by dissolved toxic water ingredients. After a certain period the increase in the number of cells in a test culture free from toxic influence and with a fixed standardized offer of nutrients will exceed that observed in test culture containing dissolved toxic substances and kept under identical conditions. The concentration of the bacterial and algal suspensions is measured turbidimetrically and expressed by the extinction of the primary light of the monochromatic radiation. The number of protozoa is determined by means of a cell counter. The concentration at which the inhibitory action of a pollutant starts for bacteria and alga is present in the dilution series of a pollutant having an extinction value at the end of the test period that is >/=3% below the mean value of extinction for non-toxic dilutions of the test culture. The pollutant concentrations at which the onset of inhibitory action was observed for protozoa was obtained by mathematical evaluation of counts. The toxicity threshold (TT) of each test materials is determined for the bacteria (TTps), algae (TTsc) and protozoa (TTen). Lowest Effect Concentration reported, TTps is >10,000 mg/l; TTsc is 640 mg/l; TTen is 485 mg/l. (Bringmann,1980) Inhalation Route: Freshwater. Species/Media : fish. The LC0, LC50 and LC100 of various test materials were calculated using Golden Ofre fish. Some of the test materials were tested under comparable conditions in two different laboratories, and the different results were reported. No further information was reported. Article in German; The information was obtained from an English abstract and result tables. 760 mg/l calculated LC mg/l calculated LC mg/l lethal, LC mg/l lethal, LC mg/l no effects, LC mg/l no effects, LC0. (Juhnke,1978) Cytotoxicity Route: in vitro. Species/Media : fish. The neutral red uptake inhibition assay was conducted using cell cultures obtained from the fathead minnow. The vehicle was medium. The contact time was 2 hours The NI50 is that concentration of the test material which induces a 50% inhibition in neutral red uptake mg/l positive effects, NI millimolar positive effects, NI50. (Brandao,1992) Route: in vitro. Species/Media : fathead minnow. Cells were derived from tissue posterior to the anus of fathead minnows and cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, 100-units/ml penicillin and 0.1-mg/ml streptomycin (complete medium). Cells were seeded in the wells of titer plates (15,000 CIR Panel Book Page 254

261 cells/well) in 200 ul of 0.4 mm L-buthionine-S,R-sulfoximine (BSO) in complete medium for 24 hrs at 34º Celsius. BSO reduced the endogenous glutathione (GSH) to 22% of control cells without affecting growth. BSO is a specific inhibitor of gamma-glutamyl-cysteine synthetase and was added to increase the sensitivity of the assay by reducing the protective effect of GSH against the test materials. After the 24-hr incubation with BSO, the medium was removed and replaced with different concentrations of test materials in complete medium containing BSO and sodium dodecyl sulfate (as a surfactant). After a further 24-hr incubation the cells were rinsed and then incubated with Triton X-100 as a dispersing agent for one hour. 3-(-4-Carboxybenzoyl)- quinoline-2-carboxaldehyde and KCN was added to each well to react with protein amines to produce fluorescence measured at 530 nm emission with excitation at 485 nm using a Dynatech Fluorolite 1000 fluorescence microplate reader. Cytotoxicity was calculated as the concentration (mm) causing a 50% reduction of total protein content of the cells (EC50 or PI50) compared to controls. The study also produced data showing a wide variation in the effect of test materials on endogenous glutathione in the cells millimolar calculated EC50 value, Total protein content of cells reduced 50%. (Dierickx,1998) Environmental Route: unclassified. Sampling of gray wastewater was conducted at a residential building in Denmark, which contained 17 flats with 38 tenants. Gray wastewater is wastewater from households, business complexes, hotels, schools and some types of industries where no contributions from toilets, bidets or heavily polluted process water are included. The gray wastewater was produced in the bathrooms and originated from the showers and hand basins, with a daily production of approximately 750 liters. The water was collected on site and treated within the building with the purpose of being reused for toilet flushing. The sampling was conducted at the inlet to the gray wastewater treatment facility. The samples were taken in glass bottles and transported in the dark at 4oC to the laboratory, where the analysis was conducted immediately or the samples stored in the dark at 4oC until analysis. The xenobiotic organic compounds present in the gray wastewater were qualitatively determined by solid-phase extraction (SPE) with 4 different solid phases after which the extracts were analyzed by gas chromatography-mass spectroscopy (GC-MS). The internal standards, 4-fluorobenzoic acid and 4-chloro-aniline were added before extractions to all samples. The semi-quantitative detected concentrations of the xenobiotic organic compounds were reported, and for some materials, the quantitative concentration range was also reported. The detected concentration was 15 ug/l. (Eriksson,2003) Biodegradation. Gerike,1979. Ecotoxicity studies, tests or effects. Definition: Studies such as algal growth & inhibition, aquatic toxicity on various species (D. magna, shrimp, fish, mollusks, etc.). Includes studies on other species (earthworm, birds, plants, etc.) Bringmann,1979. Results were not specified by material in the English abstract of this paper Environmental studies, tests or effects. Definition: fate and effect studies that include biodegradation, bioaccumulation, biotransformation, bioconcentration, soil adsorption, soil migration, sediment and water studies, etc. Walker,1988. CIR Panel Book Page 255

262 References Aguirre A., Gardeazabal J., Izu R., Raton J.A. and Diaz-Perez J.L. (1993) Allergic contact dermatitis due to plant extracts in a multisensitized patient. Contact Dermatitis, 28(3), Al-Ani F.Y. and Al-Lami S.K. (1988) Absence of mutagenic activity of acidity regulators in the Ames Salmonella/microsome Test. Mutation Research-Genetic Toxicology, 206(4), Alarie Y. (1973) Sensory irritation by airborne chemicals. Critical Reviews in Toxicology, 2(3), Atfield J.L., Sanford P.A. and Smyth D.H. (1972) Transfer and metabolism of citric acid cycle intermediates by hamster small intestine. Journal of Physiology, 222(1), 63P-64P. Bagley D.M., Bruner L.H., DeSilva O., Cottin M., O'Brien K.A.F., Uttley M. and Walker A.P. (1992) An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo. Toxicology in Vitro, 6(4), Bagley D.M., Waters D. and Kong B.M. (1994) Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the Draize rabbit eye irritation test. Food and Chemical Toxicology, 32(12), Baker R.A., Massey ED and Smith G. (2004) An overview of the effects of tobacco ingredients on smoke chemistry and toxicity. Food and Chemical Toxicology, 42S, S53-S83. Bakowski J., Schanderl S.H. and Markakis P. (1964) Nonvolatile acids of green beans, Phaseolus vulgaris, cv. green crop x Romano. Michigan State University, Arg. Expt. Sta., Quart. Bull., 47(2), Bala S. and Grover I.S. (1989) Antimutagenicity of some citrus fruits in Salmonella typhimurium. Mutation Research-Genetic Toxicology, 222(3), Baraud J., Genevois L. and Redeuilh J. (1957) Organic acids and mineral salts in potatoes. Proces-verbaux soc. sci. phys. nat. Bordeaux, Barratt M.D. (1995) The role of structure-activity relationships and expert systems in alternative strategies for the determination of skin sensitisation, skin corrosivity and eye irritation. Alternatives to Laboratory Animals (ATLA), 23, Barratt M.D. (1995a) Quantitative structure activity relationships for skin corrosivity of organic acids, bases and phenols. Toxicology Letters, 75, Barratt M.D. (1995b) Quantitative structure activity relationships for skin corrosivity of organic acids, bases and phenols. Toxicology Letters, 75(1-3), Barratt M.D. (1996) Quantitative structure-activity relationships (QSARs) for skin corrosivity of organic acids, bases and phenols: Principal components and neural network analysis of extended datasets. Toxicology in Vitro, 10(1), Berking S. (1991) Effects of the anticonvulsant drug valproic acid and related substances on developmental processes in hydroids. Toxicology in Vitro, 5(2), Berry S.K. (2001) Role of acidulants in food industry. Journal of Food Science and Technology, 38(2), Bickerman H.A., Barach A.L., Itkin S. and Drimmer F. (1954) The experimental production of cough in human subjects induced by citric acid aerosols. Preliminary studies on the CIR Panel Book Page 256

263 evaluation of antitussive agents. American Journal of Medicine Sci., 228, Blackburn K., Stickney J.A., Carlson-Lynch H.L., McGinnis P.M., Chappell L. and Felter S.P. (2005) Application of the threshold of toxicological concern approach to ingredients in personal and household care products. Regulatory Toxicology and Pharmacology, 43(3), Bojs G., Nicklasson B. and Svensson A. (1987) Allergic contact dermatitis to propyl gallate. Contact Dermatitis, 17(5), Bonting S.L. and Jansen B.C.P. (1956) The effect of a prolonged intake of phosphoric acid and citric acid in rats. Voeding, 17, Bose S. and Datta A.S. (1962) Non-nitrogenous organic acids of sugarcane. Part I. A chromatographic analysis of non-nitrogenous organic acids present in raw juice, clarified juice and molasses. Sharkara, 5(2), Brandao J.C., Bohets H.H.L., VanDeVyver I.E. and Dierickx P.J. (1992) Correlation between the in vitro cytotoxicity to cultured fathead minnow fish cells and fish lethality data for 50 chemicals. Chemosphere, 25(4), Bringmann G. and Kuhn R. (1978) Testing of substances for their toxicity threshold: Model organisms Microcytis (Diplocytis) aeruginosa and Scenedesmus quadricauda. International Association Theoretical Applied Limnology, 21, Bringmann G. and Kuehn R. (1979) Comparison of toxic limiting concentrations of water contaminants toward bacteria, algae and protozoa in the cell-growth inhibition test. Gesundheitsingenieur Haustechnik Bauphysik Umwelttechnik, 100(8), Bringmann G. and Kuhn R. (1980) Comparison of the toxicity threholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test. Water Research, 14(3), Bronaugh R.L. and Maibach H.I. (1984) Safety Evaluation of Cosmetic Preservatives. In Cosmet. Sci Technol. Series, Cosmet Drug Preserv., Princ Pract. 1, Buchloh G. and Hane M. (1958) Determination of fumaric acid and some other organic acids in apple fruits. Gartenbauwissenschaft, 23, Chalmers L. (1971) Antioxidants: Research and development report. Soap, Perfumery and Cosmetics, January, 1971, Christian M.S. and Diener R.M. (1996) Soaps and detergents: Alternatives to animal eye irritation tests. Journal of the American College of Toxicology, 15(1), Cope W.G., Bartsch M.R. and Marking L.L. (1997) Efficacy of candidate chemicals for preventing attachment of zebra mussels (Dreissena polymorpha). Environmental Toxicology and Chemistry, 16(9), Davis M.D.P., Bhate K., Rohlinger A.L., Farmer S.A., Richardson D.M. and Weaver A.L. (2008) Delayed patch test reading after 5 days: The Mayo Clinic experience. Journal of the American Academy of Dermatology, 59(2), DeGroot A.C. (1991) Choosing preservatives: Dermatoallergenic considerations. Cosmetics and Toiletries, 106(2), DeGroot A.C. (1994) Patch Testing. Test Concentrations and Vehicles for 3700 Allergens. In: Test Concentrations and Vehicles for 3700 Allergens, Dierickx P.J. (1989) Cytotoxicity testing of 114 compounds by the determination of the protein CIR Panel Book Page 257

264 content in HEP G2 cell cultures. Toxicology In Vitro, 3(3), Dierickx P.J. (1998) Increased cytotoxic sensitivity of cultured FHM fish cells by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine. Chemosphere, 36(6), Domingo J.L., Gomez M., Llobet J.M. and Corbella J. (1991) Influence of some dietary constituents on aluminum absorption and retention in rats. Kidney International, 39, Douglas W.H.J. and Spilman S.D. (1983) In vitro ocular irritancy testing. In Product Safety Evaluation, Dziezak J.D. (1986) Preservatives: Antioxidants, the ultimate answer to oxidation. Food Technology, 40(9), Dziezak J.D. (1986a) Preservatives: Antimicrobial agents, a means toward product stability. Food Technology, 40(9), Eriksson E., Auffarth K., Eilersen A.-M., Henze M. and Ledin A. (2003) Household chemicals and personal care products as sources for xenobiotic organic compounds in grey wastewater. Water, 29(2), Federation of American Societies for Experimental Biology (1977) Evaluation of the health aspects of citric acid, sodium citrate, potassium citrate, calcium citrates, ammonium citrate, triethyl citrate, isopropyl citrate, and stearyl citrate as food ingredients. FDA ; NTIS PB ; FDA/BF-78/96. Federation of American Societies for Experimental B (1984) Interim scientific report on evaluation of the evidence of carcinogenicity and genotoxicity of drugs and cosmetic ingredients. Unpublished. Fisher A.A. (1975) Patch testing with perfume ingredients. Contact Dermatitis, 1, Fisher A.A. (1982) Dermatits of the hands from food additives. Cutis, 30(3), Fuglsang G., Madsen C., Halken S., Jorgensen M., Ostergaard P.A. and Osterballe O. (1994) Adverse reactions of food additives in children with atopic symptoms. Allergy, 49(1), Gangolli S.D. and Borzelleca [Editors] J.F. (1992) Foods for infants and young children. [Editorial] Food and Chemical Toxicology, 30(9), Gardner W.H. (1972) Acidulants in Food Processing. In: Handbook of Food Additives, Chapter 5, Gatfield I.L. (1988) Production of flavor and aroma compounds by biotechnology. Food Technology, Oct., & 169. Gaworski C.L., Dozier M.M., Heck J.D., Gerhart J.M., Rajendran N., David R.M., Brennecke L.H. and Morrissey R. (1998) Toxicologic evaluation of flavor ingredients added to cigarette tobacco: 13-week inhalation exposures in rats. Inhalation Toxicology, 10(4), Gaworski C.L., Heck J.D., Bennett M.B. and Wenk M.L. (1999) Toxicologic evaluation of flavor ingredients added to cigarette tobacco: Skin painting bioassay of cigarette smokle condensate in SENCAR mice. Toxicology, 139(1-2), Geistlich P. (1970) Aerosol foams for female hygiene. Patent, German, , 95/N , 2/3/69. CIR Panel Book Page 258

265 Gerike P. and Fischer W.K. (1979) A correlation study of biodegradability determinations with various chemicals in various tests. Ecotoxicology and Environmental Safety, 3(2), Ghadishah D. and Gorchynski J. (2002) Airway compromise after routine alpha-hydroxy facial peel administration. Journal of Emergency Medicine, 22(4), Gomez M., Domingo J.L. and Llobet J.M. (1991) Developmental toxicity evaluation of oral aluminum in rats: Influence of citrate. Neurotoxicology and Teratology, 13, Gordon V.C. (1992) Utilization of biomacromolecular in vitro assay systems in the prediction of in vivo toxic responses. Lens Eye Tox Res., 9(3-4), Graham W.D., Teed H. and Grice H.C. (1954) Chronic toxicity of bread additives to rats. Journal of Pharmacy and Pharmacology, 6(8), Graham W.D. and Grice H.C. (1955) Chronic toxicity of bread additives to rats. part II. Journal of Pharmacy and Pharmacology, 7(2), Green B.A. and Wildnauer R.H. (2000) Effect of 10%, 20% and 25% alpha-hydroxyacid (citric acid) formulations on skin morphology. 58th Annual Meeting of the American Academy of Dermatology, San Francisco, March Haas W. and Schmitt R. (1982) Characterization of chemical stimuli for the penetration of Schistosoma mansoni cercariae. 1. Effective substances, Host Specificity. Parasiten, 66, Halpern M.J., Relvas M.E.A. and Ferraz F.G.P. (1959) Comparative chromatographic study of the organic acids of the brain, liver and kidney of several animal species. Preliminary tests. Arquiv. Port. Bioquim., 4, Haynes Jr. R.C. (1965) The fixation of carbon dioxide by rat liver mitochondria and its relation to lguconeogenesis. The Journal of Biological Chemistry, 240(16), Henson E.V. (1959) Toxicology of the fatty acids. Journal of Occupational Medicine, June, Higashimoto M., Yamoto H., Kinouchi T. and Ohnishi Y. (1998) Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol. Mutation Research-Genetic Toxicology and Environmental Mutagenesis, 415(3), Hillebrand G.G. and Bissett D.L. (1993) Inhibition of acute ornithine decarboxylase induction can predict tumor prevention by topical photoprotective agents in the skin of hairless mice exposed to ultraviolet radiation. Journal of the Society of Cosmetic Chemists Japan, 44(3), Horn H.J., Holland E.G. and Hazleton L.W. (1957) Safety of adipic acid as compared with citric and tartaric acid. Journal of Agricultural and Food Chemistry, 5(10), Hunt J.N. and Knox M.T. (1969) The slowing of gastric emptying by nine acids. Journal of Physiology, 201, Hurley P.M., Chambers W.A., Green S., Gupta K.C., Hill R.N., Lambert L.A., Lee C.C., Lee J.K., Liu P.T., Lowther D.K., Roberts C.D., Seabaugh V.M., Springer J.A. and Wilcox N.L. (1993) Screening procedures for eye irritation. Food and Chemical Toxicology, 31(2), Inoue T., Imaida K., Suzuki E., Okada M. and Fukushima S. (1988) Combined effects of l- ascorbic acid, citric acid or their sodium salts on tumor induction by N-butyl-N-(4- CIR Panel Book Page 259

266 hydroxybutyl)nitrosamine or N-Ethyl-N-(4-hydroxybutyl) Cancer Letters, 40, Ishidate Jr. M., Sofuni T., Yoshikawa K., Hayashi M., Nohmi T., Sawada M. and Matsuoka A. (1984) Primary mutagenicity screening of food additives currently used in Japan. Food and Chemical Toxicology, 22(8), Ishidate Jr. M., Harnois M.C. and Sofuni T. (1988) A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures. Mutation Research-Reviews in Genetic Toxicology, 195(2), Jarvinen V.K., Rytomaa I.I. and Heinonen O.P. (1991) Risk factors in dental erosion. Journal of Dental Research, 70(6), Jenney E.H. and Pfeiffer C.C. (1958) The convulsant effect of hydrazides and the antidotal effect of anticonvulsants and metabolites. The Journal of Pharmacology and Experimental Therapeutics, 122(1), Johnson O.H., Casey S., Doeltz M.K., McCaleb K.E., Miller A.M., Papa P.A., Swett L.B., Valentini M.A. and Helmes C.T. (1984) A study of biocides for the selection of candidates for carcinogen bioassay. Journal of Environmental Science and Health, A19(1), Jonek J. (1961) The effect of glutamic acid and other acidifiers on the histochemical changes in the endocrine glands of female rabbits (the adrenals). Endokrynologia Polska, 12(3), Juhnke I. and Luedmann D. (1978) Results of the study of 200 chemical compounds on acute fish toxicity using the Golden Orfe test. Zeitschrift fur Wasser und Abwasser-Forschung, 11, Jurs P.C., Chou J.T. and Yuan M. (1979) Computer-assisted structure-activity studies of chemical carcinogens. A heterogenous data set. Journal of Medicinal Chemistry, 22(5), Kartono F. and Maibach H.I. (2006) Irritants in combination with a synergistic or additive effect on the skin response: An overview of tandem irritation studies. Contact Dermatitis, 54(6), Katz G.V. and Guest D. (1994) Aliphatic Carboxylic Acids. In Patty's Industrial Hygiene Toxicology, 4th Edit., Cpt. 36, Pt. E, Kawano T. (1959) On the relation of acetoin with panthothenic acid. Fukuoka Igaku Zasshi, 50, Krebs H.A., Salvin E. and Johnson W.A. (1938) The formation of citric acid and alphaketoglutaric acids in the mammalian body. Biochemical Journal, 32, Krop S., Gold H. and Paterno C.A. (1945) On the toxicity of hydroxyacetic acid after prolonged administration: Comparison with its sodium salt and citric and tartaric acids. Journal of the American Pharmaceutical Association, 24(1), Krusius F.-E. (1940) Animal experiments on the urinary excretion of pyruvic acid, alphaketoglutaric and citric acids as well as of some other substances associated with their metabolism. Acta physiol. scand., Suppl. 3, Lahti A. (1980) Non-immunologic contact urticaria. Acta Dermato-Venereologica,, Stockh., 60(91), Laska D.A., Poulsen R.G., Horn J.W., Meador V.P. and Hoover D.M. (1992) An evaluation of TESTSKIN: An alternative dermal irritation model. In Vitro Toxicology: Journal of Molec. CIR Panel Book Page 260

267 Cell. Toxicol., 5(4), Lees H. and Kuyper A.C. (1957) The organic acids of bones. Biochemical Journal, 225(2), Liden S. (1975) Alphosyl sensitivity and propyl gallate. Contact Dermatitis, 1(4), Lucas D.R. (1909) Some effects of sodium benzoate. Proceedings of the Society for Experimental Biology and Medicine, 6, Maibach H.I., Akerson J.M., Marzulli F.N., Wenninger J., Greif M., Hjorth N., Andersen K.E. and Wilkinson D.S. (1980) Test concentrations and vehicles for dermatological testing of cosmetic ingredients. Contact Dermatitis, 6, Malanin G. and Kalimo K. (1989) The results of skin testing with food additives and the effect of an elimination diet in chronic and recurrent urticaria and recurrent angioedema. Clinical and Experimental Allergy, 19(5), Marshall L.M., Orten J.M. and Smith A.H. (1949) The determination of fumaric acid in animal tissue by partition chromatography. Biochemical Journal, 179(3), Martins T., Pauluhn J. and Machemer L. (1992) Analysis of alternative methods for determining ocular irritation. Food and Chemical Toxicology, 30(12), Mau J.-L., Lai E.Y.C., Wang N-P., Chen C-C., Chang C-H. and Chyau C-C. (2003) Composition and antioxidant activity of the essential oil from Curcuma zedoaria. Food Chemistry, 82(4), McCann J. and Ames B.N. (1976) Detection of carcinogens as mutagens in the salmonella/microsome test: Assay of 300 chemicals: Discussion. Proceedings of the National Academy of Sciences of the United States of America, 73(3), Miller M., Bueding E., Strauch R.O., Owens J. and Woodward H. (1949) Pyruvate and citrate metabolism in man and animal. Proc. Amer. Diabetes Assoc., 9, Mochizuki S., Hata M., Takeuchi F. and Masai H. (1987) Effects of organic acids on blood ethanol concentrations in rats. Nutrition Reports International, 35(3), Morozumi S., Wauke T., Kudoh Y. and Hitokoto H. (1989) Antifungal effects of commercial foods and spices, and their components. Mycotoxins Phycotoxins, 7th Int. IUPAC Symposium, Aug , 1988, Murphy J.C., Osterberg R.E., Seabaugh V.M. and Bierbower G.W. (1982) Ocular irritancy responses to various phs of acids and bases with and without irrigation. Toxicology, 23(4), Nazer A.I., Kobilinsky A., Tholozan J.-L. and Dubois-Brissonnet F. (2005) Combinations of food antimicrobials at low levels to inhibit the growth of Salmonella sv. Typhimurium: A synergisic effect? Food Microbiology, 22(5), Nieth H. and Schollmeyer P. (1966) Substrate-utilization of the human kidney. Nature (London), 209(5029), Niinimaki A. (1987) Scratch-chamber tests in food handler dermatitis. Contact Dermatitis, 16(1), Osteux R. and Laturaze J. (1954) Paper chromatography of fixed organic acids found in urine. Compt. Rend., 239, Packman E.W., Abbott D.D. and Harrison W.E. (1963) Comparative subacute toxicity for CIR Panel Book Page 261

268 rabbits of citric, fumaric, and tartaric acids. Toxicology and Applied Pharmacology, 5(2), Pavan M., Netzeva T. and Worth A.P. (2006) Validation of a QSAR model for acute toxicity. SAR & QSAR in Environmental Research, 17(2), Piper D.W., Fenton B.H. and Goodman L.R. (1967) Lactic, pyruvic, citric and uric acid and urea content of human gastric juice. Gastroenterology, 53(1), Pittman D.W. and Contreras R.J. (1998) Responses of single lingual nerve fibers to thermal and chemical stimulation. Brain Research, 790(1-2), Pounsford J.C., Birch M.J. and Saunders K.B. (1985) Effect of bronchodilators on the cough response to inhaled citric acid in normal and asthmatic subjects. Thorax, 40, Purdy R. (1996) A mechanism-mediated model for carcinogenicity: Model content and prediction of the outcome of rodent carcinogenicity bioassays currently being conducted on 25 organic chemicals. Environmental Health Perspectives, 104(5), Reboulet J.T., Houchins J.O., Clair R.L.St., Hoffman W.P. and Laska D.A. (1994) The Agar diffusion cytolysis method: An alternative in vitro screen for the prediction of a severe ocular response. Toxicology Methods, 4(4), Reider N., Issa A., Hawranek T., Schuster C., Aberer W., Kofler H., Fritsch P. and Hausen B.M. (2005) Absence of contact sensitization to Aloe vera (L.) Burm. f. Contact Dermatitis, 53(6), Research Institute for Fragrance Materials, Inc, (1977). Report on human maximization studies. Report to RIFM. Unpublished report 1691 from Epstein W.L. July 00 Research Institute for Fragrance Materials, Inc, (1977). Acute toxicity study in rats, rabbits and guinea pigs. Report to RIFM. Unpublished report 1695 from Moreno O.M. August 22 Rietschel R.L. (1978) Contact urticaria from synthetic cassia oil and sorbic acid limited to the face. Contact Dermatitis, 4(6), Rodgman A. (2002) Some studies of the effects of additives on cigarette mainstream smoke properties. I. Flavorants. Beitraege zur Tabakforschung International, 20(2), [Contributions to Tobacco Research] Roemer E., Rustemeier K., Vanscheeuwijck P.M., Meisgen T.J., Veltel D.J., Haussmann H. and Carmines E.L. (2000) Effects of the addition of flavor ingredients to the tobacco on the chemical composition and biological activity of cigarette smoke. The Toxicologist, 54(1), 16. Rosenkranz H.S. and Klopman G. (1990) New structural concepts for predicting carcinogenicity in rodents: An artificial intelligence approach. Teratogenesis, Carcinogenesis and Mutagenesis, 10,(2) Rosenkranz H.S. and Klopman G. (1990a) The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: The National Toxicology Program Data Base. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 228(2), Rosenkranz H.S. and Klopman G. (1990b) Structural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 228(2), Rosenkranz H.S., Ennever F.K., Dimayuga M. and Klopman G. (1990c) Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal CIR Panel Book Page 262

269 aberrations in Chinese hamster ovary cells. Environmental and Molecular Mutagenesis, 16(3), Saeki Y. (1956) The action of sodium succinate on isolated cat heart, considered mainly in comparison with the corresponding action of other sodium compounds of organic acids. Fukuoka Igaku Zasshi, 47, Sainio E.-L. and Kanerva L. (1995) Contact allergens in toothpastes and a review of their hypersensitivity. Contact Dermatitis, 33(2), Sanders H.J. (1966) Food additives. Chemical and Engineering News, Oct Sawada T. and Mivaji K. (1957) The clinical application of organic acids of the tricarl oxylic acid cycle. Sogo Rinsho, 6, Schaper M. (1993) Development of a database for sensory irritants and its use in establishing occupational exposure limits. American Industrial Hygiene Association Journal (AIHAJ) (AIHAJ), 54(9), Schlierman-Willers S., Fuchs S., Kleesz P. and Elsner P. (2003) Fruit acids do not contribute to cumulative irritant contact dermatitis in a human tandem irritation model in vivo. Proceedings from the 14th Annual ACDS Meeting, March 20, San Francisco, California, page 51. Schormuller J. and Langner H. (1960) Quantitative determination of organic acids in foods. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung A, 113, Schreiber R.T., McDonnell W.F., Horstman D.H., Ives P.J., Abdul-Salaam S. and Bromberg P.A. (1986) The relationship of inhaled citric acid sensitivity and responsiveness to ozone. American Rev. resp. Dis., 132(4 pt.2), A216. Schultheiss E. (1957) Sensitivity to ionone and benzyl alcohol. Dermatologische Wochenschrift, 135, Serra J.R., Thompson E.D. and Jurs P.C. (2003) Development of binary classification of structural chromosome aberrations for a diverse set of organic compounds from molecular structure. Chemical Research in Toxicology, 16(2), Serra-Baldrich E., Tribo M.J. and Camarasa J.G. (1998) Allergic contact dermatitis from kojic acid. Contact Dermatitis, 39(2), Shinohara K., Kim E.-H. and Omura H. (1986) Furans as the Mutagens Formed by Amino- Carbonyl Reactions. In: Developments in Food Science, 13, Smith C.G., Lummis W.L. and Grady J.E. (1959) An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals, and solvents. Cancer Research, 19(8), Smith W.P. (1994) Hydroxy acids and skin aging. Cosmetics and Toiletries, 109(9), Smith C.J., Perfetti T.A., Garg R., Martin P. and Hansch C. (2004) Percutaneous penetration enhancers in cigarette mainstream smoke. Food and Chemical Toxicology, 42(1), Swanson J.E., Lake L.K., Donnelly T.A., Harbell J.W. and Huggins J. (1995) Prediction of ocular irritancy of full-strength cleaners and strippers by tissue equivalent and bovine corneal assays. Journal of Toxicology: Cutaneous and Ocular Toxicology, 14(3), Swirsky-Gold L., Manley N.B., Slone T.H. and Rohrbach L. (1999) Supplement to the carcinogenic potency database (CPDB): Results of animal bioassays published in the general literature in 1993 to 1994 and by the National Toxicology Program in CIR Panel Book Page 263

270 Environmental Health Perspectives, 107(Supplement 4), Tanaka Y., Takagaki Y. and Nishimune T. (1991) Effects of food additives on B- hexosaminidase release from rat basophilic leukemia cells (RBL-2H3). Eisei Kagaku, 37(5), Taylor S.L. and Nordlee J.A. (1993) Chemical additivies in seafood products. Clinical Reviews Allergy, 11(2), Tordoff M.G. and Bachmanov A.A. (2002) Influence of test duration on the sensitivity of the two-bottle choice test. Chemical Senses, 27(9), Torgerson R.R., Davis M.D.P., Bruce A.J., Farmer S.A. and Rogers III R.S. (2007) Contact allergy in oral disease. Journal of the American Academy of Dermatology, 57(2), Tuft L. and Ettelson L.N. (1956) Canker sores from allergy to weak organic acids (citric and acetic). Journal of Allergy, 27(6), Turemis N., Kafkas E., Kafkas S., Kurkcuoglu M. and Baser K.H.C. (2003) Determination of aroma compounds in blackberry by GC/MS analysis. Chemistry of Natural Compounds (Traslantion of Khimiya Prirodnykh Soedinenii), 39(2), VanScott E.J. and Yu R.J. (1974) Control of keratinization with alpha-hydroxy acids and related compounds. I. Topical treatment of ichthyotic disorders. Archives of Dermatology, 110(4), Villard H. (1927) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927a) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927b) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927c) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Walker J.D. (1988) Effects of chemicals on microorganisms. Journal Water Pollution Control Federation, 60(6), Walker J.D., Fang H., Perkins R. and Tong W. (2003) QSARs for endocrine disruption priority setting database 2: The integrated 4-phase model. QSAR Comb. Sci., 22(1), Wantoch H. (1929) Influence of various substances on the sugar and ammonia content of blood. Archives of exp. Path. Pharmak., 143, Weiss J.M., Downs C.R. and Corson H.P. (1923) Inactive malic acid as a food acidulent. Ind. Engng Chem., 15(6), Wieland O. (1968) Ketogenesis and its regulation. Adv. metab. Disorders, 3, Williams W., Riddle M., Copeland C., Andrews D. and Smialowicz R. (1993) Dermal exposure with 2-methoxyethanol (ME) is immunotoxic in F344 rats. The Toxicologist, 13(1), 323. Wiseman R.D. and Adler D.K. (1956) Acetic acid sensitivity as a cause of cold urticaria. Journal of Allergy, 27(1), Wright E. and Hughes R.E. (1976) Some effects of dietary citric acid in small animals. Food and Cosmetics Toxicology, 14(6), Yang S.-T. and Lee E.-H. (1983) Taste compounds of fresh-water fishes. 6. Taste compounds of Korean catfish meat. Bulletin Korean Fish. Soc., 16(3), Yokotani H., Usui T., Nakaguchi T., Kanabayashi T., Tanda M. and Aramaki Y. (1971) Acute and subacute toxicological studies of TAKEDA-citric acid in mice and rats. Journal Takeda CIR Panel Book Page 264

271 Research Laboratories, 30(1), Zelenak J.P., Alarie Y. and Weyel D.A. (1982) Assessment of the cough reflex caused by inhalation of sodium lauryl sulfate and citric acid aerosols. Fundamental and Applied Toxicology, 2(4), No data added since 4-Feb-11. DATA IN THIS SYNOPSIS HAVE NOT BEEN PEER REVIEWED BY A SCIENTIFIC PANEL Sodium citrate Synonyms Citratin Citrosodine 1,2,3-Propanetricarboxylic acid, 2-hydroxy-, trisodium salt CAS Sodium citrate Principal Trisodium citrate CAS Number RIFM ID FEMA EINECS Registration EINECS DSL TSCA Formula C 6 H 5 O 7 Na 3 Molecular Weight SMILES Notation [Na]OC(=O)CC(O)(C(=O)O[Na])CC(=O)O[Na] Generic Class Carboxylic Acids Physical Data Boiling Point (calculated) C EPI Suite Henry's Law (calculated) 8.43e-018 Pa m 3 /mol EPI Suite Log K OW (calculated) EPI Suite Melting Point (calculated) C EPI Suite Vapor Pressure (calculated) 2.09e-012 mm 25 C EPI Suite CIR Panel Book Page 265

272 Water Solubility (calculated) mg/l EPI Suite Natural Occurrence Sodium citrate is a functional material. Flavor Consumption (in kg) Product 1995 USA USA USA USA USA USA Average Usual Uses (in ppm) Average Maximum Mean Daily Consumption (gms) Updated Alcoholic Beverage Jul-88 Baked Goods Jul-88 Breakfast Cereals Jul-88 Cheese Jul-88 Fats Oils Jul-88 Frozen Dairy Jul-88 Fruit Ices Jul-88 Fruit Juice Jul-88 Gelatin Pudding Jul-88 Gravies Jul-88 Hard Candy Jul-88 Imitation Dairy Jul-88 Instant Coffee Tea Jul-88 Jam Jelly Jul-88 Meat Products Jul-88 Milk Products Jul-88 Non-alcoholic Beverage Jul-88 Other Grain Jul-88 Processed Vegetables Jul-88 Snack Foods Jul-88 Soft Candy Jul-88 CIR Panel Book Page 266

273 Soups Jul-88 Sweet Sauce Jul-88 PADI 1.56 Status Sodium citrate was approved by the FDA as GRAS Affirmed 12/12/94 ( 21 CFR ). Flavor and Extract Manufacturers' Association states: Generally Recognized as Safe as a flavor ingredient - GRAS 3. ( 3026) Hall,1965 EPA- High Production Volume Program states: Robust summary is available here Joint Expert Committee on Food Additives states: ADI not limited (see citric acid). ( 1973) Human Health Data Acute toxicity Route: unclassified. Species: chick embryo. As part of a Teratogenicity study, LD50 values were estimated from the regression line for one of the four conditions: injection via air cell or via the yolk sac at two time intervals (preincubation and 96 hour) for each route. All test materials were administered by a single injection in a volume of 100 ul or less, and solvent controls were treated with the same volume of the solvent only. The test material was dissolved or suspended in an appropriate vehicle. For each condition, at least 100 embryos per dose level were treated at a minimum of five dose levels. Eggs from Single-Comb White Leghorn chickens were used. Appropriate groups of vehicle controls and untreated controls were included. After treatment, all eggs were candled daily and nonviable embryos were removed. The percentage mortality at each dose level was calculated as the total number of nonviable embryos divided by the total number of treated eggs. Dose units are actually mg/egg. Vehicle was water.ld MG 2.06 mg calculated LD50, with air cell injection at 96 hours. (Verrett,1980) Route: intraperitoneal. Species: mouse. Ten male Swiss mice weighing grams were used per dose level. The mice were maintained on ad libtium diet of commercial chow and water. Ambient temperature was maintained between 21 and 23 C at all times and mice were exposed to a 12 hour light/dark cycle. The test materials were administered by a single intraperitoneal injection and survival recorded at the end of 14 days. The LD50 values were then calculated. Solution concentrations were adjusted so that 0.2 ml volume was administered /30 grams bodyweight. Solutions were prepared just prior to administration in 0.9% saline and administered at a ph of approximately 7.0; sodium bicarbonate was used to adjust solution ph when required. The LD50 values were calculated according to the standard method of Litchfield and Wilcoxon (1949). LD mg/kg bodyweightsummary The acute intraperitoneal LD50 of the test CIR Panel Book Page 267

274 material = 40.8 mg/kg bodyweight mg/kg calculated LD50, concentrations were mg/kg bodyweight. The acute intraperitoneal LD50 of the test material = 40.8 mg/kg bodyweight. (Ortega,1989) Route: in vitro. Species: human 18+ yrs. Blood from healthy volunteers was collected & platelet rich plasma was prepared and used for platelet aggregation study. The effect of test substance was studied by measuring the inhibition of norepinephrine induced platelet activation. 10 millimolar positive effects, inhibited platelet aggregation by inhibiting cyclo-oxygenasethromboxane pathway. (Williams,1989) Sensitization Route: skin. Species: human 18+ yrs. Non-immunologic contact urticaria was evaluated in 105 male and female atopic and non-atopic dermatitis patients. Material applied for 20 minutes using a Finn chamber and Scanpor applied to the upper back. Reactions evaluated immediately after chamber removal The number of positive reactions in the results is the total for those with redness and edema and those with redness only. Vehicle was water. 10 % no effects. (Lahti,1980) Route: skin. Species: human 18+ yrs. A 40 yr old woman had severe acute dermatitis of the face, neck, & upper trunk associated w/ the use of 2 proprietary sunscreens. She had used these previously with no trouble. After recovery, she was patch-tested to the standard GEIDC series, to the 3 sunscreens & their ingredients. [No further Experimental detail given]. in water. 1 % positive effects, a 3+ reaction was noted 4 days after testing w/ sunscreens A or B. [Scores for individual ingredients not provided]. (Camarasa,1986) Pharmacokinetics and metabolism studies Route: intravenous. Species: rabbit. The occurence of the "citric acid cycle" in the mammalian body was studied using rabbits. Various materials were infused into an ear vein. Urine was collected for analysis Test materials were generally sodium salts of acids. Two-thirds of the acid equivalents were neutralized with NaOH. The iv administration of disodium citrate and acid calcium citrate to rabbits led to the presence of citic, a-ketoglutaric and succinic acids in the urine. (Krebs,1938) Pharmacology Route: unreported. Species: rabbit. The test susbtance was injected in fasting (18 h) normal rabbits and the blood sugar level was studied at hourly intervals for a period of 3 hours. Positive effects, produced hypoglycemic response. (Brahmachari,1963) Route: subcutaneous. Species: rat. The ability of the test material to deplete adrenal ascorbic acid was studied using female Wistar rats. An aqueous vehicle was used. Sacrifice was 2 hours afer the single dose. 300 mg/kg no effects. (Cronheim,1952) Route: in vitro. Species: cat. The action of the test material on the pulsation of an isolated adult cat heart was studied Data from English abstract only. 100 millimolar positive effects, a reduction in the amplitude of movement, inducing arrhythmia in many cases, was seen with fatigued and normal hearts. (Saeki,1956) Interaction Route: oral. Species: mouse. One month old female Swiss-Webster mice (44) were fed purified diet containing the test substance & either 2 ug Aluminium (Al/g) diet or 1000 ug/g as AlCl3. After 5 or 7 weeks of feeding these diets, changes in behavior were assessed using the NIEHS neurobehavioral Test Battery (NBTB). 3.5 % neurological effects, Al accumulated in the nervous CIR Panel Book Page 268

275 system with simultaneous administration of the test substance which was associated with overt sign of neurotoxicity. (Oteiza,1992) Route: intraperitoneal. Species: mouse. The antidotal efficacy of the test material was initially tested using survival as a measure of effect. Therapeutic effectiveness (TEF) of the test material was assessed quantitatively by dividing the LD50 of the test material treated mice by the LD50 control. Twenty male Swiss mice weighing grams were used per group. The mice were maintained on ad libtium diet of commercial chow and water. Ambient temperature was maintained between 21 and 23 C at all times and mice were exposed to a 12 hour light/dark cycle. The first groups received the test material (1/4 of their respective intraperitoneal LD50 values) 10 minutes after the subcutaneous administration of different dosages of uranyl acetate dihydrate ( mg/kg). The second groups received 0.9% saline instead of the test material. Uranyl acetate solutions were prepared in 0.5% saline and administered at a ph of 7.0. The therapeutic indices for the test materials which showed the highest therapeutic effectiveness was subsequently determined. Summary The test material enhanced the survival rate of animals. The therapeutic effectiveness (TEF) = 2.0 Interaction, The test material enhanced the survival rate of animals. The therapeutic effectiveness (TEF) = 2.0. (Ortega,1989) Route: unreported. Species: rabbit. The test substance was injected simultaneously but separately with two unit of insulin (in normal saline) in different legs of the rabbits to study the in vivo effects on the insulin response. No effects. (Brahmachari,1963) Route: unreported. Species: rabbit. The test substance was mixed in vitro with two units of insulin, mixture was incubated at 37 C for 30 minutes and the clear solution was injected in one leg of the rabbit. The blood sugar level was then examined. Positive effects, decreased the potency of exogenous insulin. (Brahmachari,1963) Route: food. Species: rat. A tumor promotion study in male F344 rats with N-butyl-N-(4- hydroxybutyl)nitrosamine as the initiator. The test material diet was fed for 32 weeks after a 4 week treatment with the bladder tumor initiator. Observations included body weight and histology of the bladder. 5 % body weight changes, interaction, the promotion of preneoplastic and neoplastic lesions of the bladder was noted. (Fukushima,1986) Route: food. Species: rat. Groups of 15 F344 males were simultaneously given carcinogen [0.025% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or 0.021% N-ethyl-N-(4- hydroxybutyl)nitrosamine (EHBN)] in drinking H2O and test chemical supplemented or control diet for 20 wk. Body weights, food & H2O intake. Osmolality & ph, determined in urine at wk 10 & 14. Urinary metabolites of BBN or EHBN determined in 24 hr urine samples at wk 20. Liver & kidneys weighed. Liver & kidneys & semiserial sections of urinary bladder examined histologically. 6.5 % body weight changes, interaction, liver, tumorigenic, lower final BW, higher liver wt & higher urine ph in rats given BBN & EHBN; higher concentration of BBN metabolites than BBN controls; significantly greater incidence & no. of carcinomas per 10 cm basement membrane. (Inoue,1988) Route: oral. Species: rat. Various dietary supplements were evaluated for their growthstimulating effect on rats stunted by sodium benzoate. Male rats (115) were kept in individual cages and given a basal diet ad libitum. When the bodyweights of the rats were approximately grams, sodium benzoate was added to each 100 grams of the basal diet, which was given to 106 rats. Following 3 6 weeks on the sodium benzoate stunting diet, dietary supplements were individually incorporated into the diet containing sodium benzoate and fed to groups of 2-6 rats. After 2 weeks on the sodium benzoate + dietary supplements diet, animals that had been CIR Panel Book Page 269

276 administered the growth-stimulating dietary supplements were placed back on the diet containing sodium benzoate alone, and those that had been given ineffective dietary supplements, were placed on a diet containing sodium benzoate + glycine. The average weight change and food consumption of the treated animals was calculated. See sub. ref. 2 for the basal diet + sodium benzoate control group. 1.5 gm body weight changes, Sodium citrate was completely incapable of stimulating the growth of animals stunted by the sodium benzoate-containing basal diet. (White,1941) Route: intraperitoneal. Species: mouse. The effect of the test material on the 24 hour excretion and distribution of uranium was determined. Twenty male Swiss mice weighing grams were used per group. The mice were maintained on ad libtium diet of commercial chow and water. Ambient temperature was maintained between 21 and 23 C at all times and mice were exposed to a 12 hour light/dark cycle. Animals were kept in metabolic cages which permitted separate collection of urine and faeces. Mice received subcutaneous injections of uranyl acetate at a dose of 10 mg/kg and 10 minutes after either saline or the test material was administered intraperitoneally. The dose of the test material was approximately 1/4 of the LD50. Urine and faeces were collected from each group 24 hours after administration. The animals were then removed from the metabolic cages, anaesthetized with ether and 1 ml of blood was obtained by cardiac puncture. The mice were sacrificed and the following organs and tissues were removed: heart, brain, liver, kidneys and bone (femur). For the determination of uranium concentrations, 0.5 ml of urine, 200 mg of ground faeces or 300 mg of tissue were mixed with 1 ml of 65% HNO3 and heated under pressure at 190 degrees. The samples were then diluted up to 5 ml with deionized water, filtered and the uranium concentration measured in a computer-controlled sequential inductively coupled plasma spectrometer (Jobin Ybon JY 38 VHR). Data were compared by a one-way analysis of variance. When the analysis indicated a significant difference existed, the treatment groups were compared with controls by Duncan's new multiple range test. The level of significance was set a P<0.05. Summary No significant increase in urine or faecal excretion was observed after administration of the test material. No significant changes when compared to controls was observed on the tissue distribution of uranium after administration of the test material. No effects, No significant increase in urine or faecal excretion was observed after administration of the test material. No significant changes when compared to controls was observed on the tissue distribution of uranium after administration of the test material. (Ortega,1989) Route: intraperitoneal. Species: mouse. Determination of the ED50 and therapeutic index of the test material when given intraperitoneally 10 minutes after a single subcutaneous injection of uranyl acetate dihydrate (50.0 mg/kg). Ten male Swiss mice weighing grams were used per dose level. The mice were maintained on ad libtium diet of commercial chow and water. Ambient temperature was maintained between 21 and 23 C at all times and mice were exposed to a 12 hour light/dark cycle. Therapeutic indices were calculated by dividing the intraperitoneal LD50 of each test material by its ED50 (the amount of test material protecting 50% of the animals against the lethal effects of subcutaneous LD99 of uranyl acetate dihydrate: 50 mg/kg. Summary Antidotal efficacy of the test material was poorer than Tiron, gallic acid and DTPA. The ED50 of the test material = 390 mg/kg. The Therapeutic index (TI) = 2.4 Interaction, Antidotal efficacy of the test material was poorer than Tiron, gallic acid and DTPA. The ED50 of the test material = 390 mg/kg. The Therapeutic index (TI) = 2.4. (Ortega,1989) Subchronic toxicity CIR Panel Book Page 270

277 Route: food. Species: rat. 5 male F344 rats received the test diet for 16 weeks. Urine examinations were conducted at 4, 8 and 16 weeks. 5 % positive effects, increases in urinary ph, crytals and sodium ion content were noted. (Fukushima,1986) Route: oral. Species: rabbit. Sixty male albino, New Zealand animals, were divided into 4 grps of 15 animals ea. and fed diets containing test cpd for 150 days. Control grp received ground Rockland Rabbit Diet. Parameters evaluated included food intake, body wt., hematology, urinalysis, gross & microscopic pathology. No effects. (Packman,1963) Route: food. Species: rat. Groups of 10 male F344 rats received test diet for 10 weeks. Sacrifice was at the end of feeding. Observations were food & water intake, body weight, urinalysis, necropsy, bladder, forestomach, glandular stomach & kidney histology and bladder cell proliferation. 8.9 % body weight changes, organ weight changes, increased water intake, urinary ph & urine volume, decreased urine creatine, increased urine sodium & bladder weight, bladder hyperplasia, increased bladder labeling index, increased kidney weight & kidney calcification. (Cohen,1995a) Developmental and reproduction studies Route: unclassified. Species: chick embryo. Toxicity and teratogenicity were evaluated under four conditions: injection via air cell or via the yolk sac at two time intervals (preincubation and 96 hour) for each route. All test materials were administered by a single injection in a volume of 100 ul or less, and solvent controls were treated with the same volume of the solvent only. The test material was dissolved or suspended in an appropriate vehicle. For each condition, at least 100 embryos per dose level were treated at a minimum of five dose levels. Eggs from Single- Comb White Leghorn chickens were used. Appropriate groups of vehicle controls and untreated controls were included. After treatment, all eggs were candled daily and nonviable embryos were removed. Embryos and hatched chicks were examined grossly for any functional or structural abnormalities in development. Abnormalities were tabulated and compared with values for the vehicle controls to determine teratogenicity. At least five embryos and hatched chicks were randomly selected from the highest dose level group for examination of the viscera. Dose listed is highest of 5 tested and units are actually mg/egg. Mortality is reported in the associated LD50 study. Methodology Reference: McLaughlin and Verrett, 1964 (MF1137). Vehicle was water. 10 mg no effects. (Verrett,1980) Cytotoxicity Route: in vitro. Species: human 18+ yrs. This study provides a new cell culture system for determining cytotoxicity. Hep G2 cells (derived from a human hepatoma) were maintained at 37 C in a 7% CO2 atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U penicillin G/ml and 100 ug streptomycin/ml (complete medium). The cells were plated with 1 ml complete medium in each well (2 cm2 growth surface) of a 24-well tissue culture test plate. After 24 hours the medium was removed from the wells. Four control wells were refilled with 1 ml fresh complete medium. The other wells received the test materials, in five different concentrations, freshly prepared in complete medium, using four wells per concentration. The test materials were dissolved directly in complete culture medium. The highest concentration was chosen on the basis of a preliminary range-finding test. The assays were performed on logarithmically growing cells. After treatment for 24 hours at 37 C, the supernatant was discarded, the cells were washed three times with 1 ml Hank's balanced salt solution, and the cells were lysed. This solution was further used for protein measurement using bovine serum albumin as a standard. The results are presented as percentages of control cultures, CIR Panel Book Page 271

278 which typically contained ug protein/well. The relative toxicity of the test materials was determined by use of the PI(50), which is the concentration of test material required to induce a 50% inhibition in cell protein content of the culture. Reproducibility was assessed by conducting 3 independent assays of specific test materials to determine PI(50)'s. The PI(50) value of test material in culture with Hep G2 cells was 22 mm. (Dierickx,1989) Route: in vitro. Species: rat. Transformed rat bladder epithelial cell line AY-27 cells were seeded in a 35 mm tissue culture well and incubated for 24 hour with sodium citrate dissolved in the medium. After incubation, cells were observed under microscope, & then processed for cytotoxicity evalution. The effect of treatment on rat urothelial cells was evaluated by two parameter: percentage attachment & percent viability. 1.7 millimolar no effects, No effect on percent cell attachment or percent cell viability millimolar positive effects, Significant decrease in percent cell attachment and percent cell viability millimolar no effects, No effect on percent cell attachment or percent cell viability. (Garland,1989) Enzyme processes Route: in vitro. Species: rat. Kidney slices from hooded animals were cut using a Stadie-Riggs tissue slicer and were immediately placed on top of a dry Petri dish containing cracked ice. They were then blotted, weighed on a torsion balance & placed directly into chilled Warburg vessels. All incubations w/ [14C]test cpd were Made for 1 37 C in an atmosphere of O2 using Warburg apparatus. Oxidation of test cpds was measured by collection of [14C]CO2, conversion to BaCO2 and counting of radioactivity. Enzyme induction, an increase in the rate of [14C]CO2 production from ethylthio fatty acids was obtained. (Brown,1954) Article in foreign language; no English translation available. Villard,1927. No English abstract or translation available. Villard,1927a. No English abtract or translation available. Villard,1927b. No English abstract or translation available. Villard,1927c. No English abstract or translation available. Carcinogenicity studies and/or effects. Gaworski,1999. The flavor ingredients were tested in combination; not individually. Editorial comments, including letters to editors, authors responses, etc. Gangolli,1992. Mixture. Gaworski,1998. Mixtures were tested not individual ingredients Baker,2004. The potential chemical changes and biological activity of smoke from cigarettes with added ingredients was investigated. The ingredients of the experimental cigarettes were listed. Papers that are a review on a general subject, not a specific material. TREV papers may contain one or more RIFM materials. TREV papers do not contain original data. Ishidate,1988. Ito,1989. Anderson,1991. Monro,1993. Cohen,1995. CIR Panel Book Page 272

279 Review articles. Papers that are reviews on a material or a class of materials. A review paper may be on one or more RIFM materials. Review papers do not contain original data. FASEB,1977. Studies on cosmetic products or directly related thereto. Maibach,1980. Environmental Data Inhalation Route: multiple routes. Species/Media : Daphnia magna. 24 hour LC50 of test materials hazardous in water was determined. 24 hour old animals from a clone of Daphnia magna were used. At the same time, the LC0 and LC100 for each test material was determined. The test medium was tap water free from chlorine, saturated with oxygen, hardness 16 degrees (German), ph , temperature C Information obtained from English abstract and German tables. Article was in German. LC MG/L 3330 mg/l calculated LC50, LC0 = 625 mg/l; LC100 = 5000 mg/l. (Bringmann,1977) References Anderson R.L. (1991) Early indicators of bladder carcinogenesis produced by non-genotoxic agents. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 248(2), Baker R.A., Massey ED and Smith G. (2004) An overview of the effects of tobacco ingredients on smoke chemistry and toxicity. Food and Chemical Toxicology, 42S, S53-S83. Brahmachari H.D. and Sarma G.R. (1963) Effects of some intermediary metabolites on the `exogenous insulin response' of normal rabbits. Proc. Rajasthan-Acad. Sci., 10(1), Bringmann G. and Kuhn R. (1977) Results of the damaging effects of water pollutants on Daphnia magna. Zeitschrift Wasser Abwasser-Forschung, 10(5), Brown W.T. and Scholefield P.G. (1954) Studies of fatty acid oxidation. I. The oxidation of the alkylthio fatty acids. Biochemical Journal, 58, Camarasa J.G. and Baldrich E.S. (1986) Allergic contact dermatitis to sunscreens. Contact Dermatitis, 15(4), Cohen S.M. (1995) Cell proliferation in the bladder and implications for cancer risk assessment. Toxicology, 102(1-2), Cohen S.M., Cano M., Garland E.M., John M.St. and Arnold L.L. (1995a) Urinary and urothelial effects of sodium salts in male rats. Carcinogenesis, 16(2), Cronheim G., King Jr. J.S. and Hyder N. (1952) Effect of salicylic acid and similar compounds on the adrenal-pituitary system. Proceedings of the Society for Experimental Biology and Medicine, 80, Dierickx P.J. (1989) Cytotoxicity testing of 114 compounds by the determination of the protein content in HEP G2 cell cultures. Toxicology In Vitro, 3(3), Federation of American Societies for Experimental Biology (1977) Evaluation of the health CIR Panel Book Page 273

280 aspects of citric acid, sodium citrate, potassium citrate, calcium citrates, ammonium citrate, triethyl citrate, isopropyl citrate, and stearyl citrate as food ingredients. FDA ; NTIS PB ; FDA/BF-78/96. Fukushima S., Thamavit W., Kurata Y. and Ito N. (1986) Sodium citrate: A promoter of bladder carcinogenesis. Japanese Journal of Cancer Research, 77, 1-4. Gangolli S.D. and Borzelleca [Editors] J.F. (1992) Foods for infants and young children. [Editorial] Food and Chemical Toxicology, 30(9), Garland E.M., Parr J.M., Williamson D.S. and Cohen S.M. (1989) In vitro cytotoxicity of the sodium, potassium and calcium salts of saccharin, sodium ascorbate, sodium citrate and sodium chloride. Toxicology in Vitro, 3(3), Gaworski C.L., Dozier M.M., Heck J.D., Gerhart J.M., Rajendran N., David R.M., Brennecke L.H. and Morrissey R. (1998) Toxicologic evaluation of flavor ingredients added to cigarette tobacco: 13-week inhalation exposures in rats. Inhalation Toxicology, 10(4), Gaworski C.L., Heck J.D., Bennett M.B. and Wenk M.L. (1999) Toxicologic evaluation of flavor ingredients added to cigarette tobacco: Skin painting bioassay of cigarette smokle condensate in SENCAR mice. Toxicology, 139(1-2), Inoue T., Imaida K., Suzuki E., Okada M. and Fukushima S. (1988) Combined effects of l- ascorbic acid, citric acid or their sodium salts on tumor induction by N-butyl-N-(4- hydroxybutyl)nitrosamine or N-Ethyl-N-(4-hydroxybutyl) Cancer Letters, 40, Ishidate Jr. M., Harnois M.C. and Sofuni T. (1988) A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures. Mutation Research-Reviews in Genetic Toxicology, 195(2), Ito N. and Fukushima S. (1989) Promotion of urinary bladder carcinogenesis in experimental animals. Expl Path., 36(1), Krebs H.A., Salvin E. and Johnson W.A. (1938) The formation of citric acid and alphaketoglutaric acids in the mammalian body. Biochemical Journal, 32, Lahti A. (1980) Non-immunologic contact urticaria. Acta Dermato-Venereologica,, Stockh., 60(91), Maibach H.I., Akerson J.M., Marzulli F.N., Wenninger J., Greif M., Hjorth N., Andersen K.E. and Wilkinson D.S. (1980) Test concentrations and vehicles for dermatological testing of cosmetic ingredients. Contact Dermatitis, 6, Monro A. (1993) The paradoxical lack of interspecies correlation between plasma concentrations and chemical carcinogenicity. Regulatory Toxicology and Pharmacology, 18(1), Ortega A., Domingo J.L., Gomez M. and Corbella J. (1989) Treatment of experimental acute uranium poisoning by chelating agents. Pharmacology and Toxicology, 64(3), Oteiza P.I., Han B., Keen C.L. and Golub M.S. (1992) Aluminum accumulation and neurotoxicity after chronic exposure to aluminum and citrate. The Toxicologist, 12(1), 211. Packman E.W., Abbott D.D. and Harrison W.E. (1963) Comparative subacute toxicity for rabbits of citric, fumaric, and tartaric acids. Toxicology and Applied Pharmacology, 5(2), Saeki Y. (1956) The action of sodium succinate on isolated cat heart, considered mainly in comparison with the corresponding action of other sodium compounds of organic acids. CIR Panel Book Page 274

281 Fukuoka Igaku Zasshi, 47, Verrett M.J., Scott W.F., Reynaldo E.F., Alterman E.K. and Thomas C.A. (1980) Toxicity and teratogenicity of food additive chemicals in the developing chicken embryo. Toxicology and Applied Pharmacology, 56(2), Villard H. (1927) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927a) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927b) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), Villard H. (1927c) Les brulures chimiques de l'oeil. Archives Ophthalomologie, 44(1), White A. (1941) Growth-inhibition produced in rats by the oral administration of sodium benzoate. Effects of various dietary supplements. Yale Journal of Biology and Medicine, 13, Williams W.R., Pawlowicz A. and Davies B.H. (1989) Aspirin-like effects of selected food additives and industrial sensitizing agents. Clinical and Experimental Allergy, 19(5), No data added since 18-Mar-11 DATA IN THIS SYNOPSIS HAVE NOT BEEN PEER REVIEWED BY A SCIENTIFIC PANEL Triethyl citrate Synonyms Citric acid, triethyl ester Citroflex 2 Trade Ethyl citrate 2-Hydroxy-1,2,3-propanetricarboxylic acid, triethyl ester 1,2,3-Propanetricarboxylic acid, 2-hydroxy-, triethyl ester CAS Triethyl citrate Principal EINECS RIFM Triethyl 2-hydroxy-1,2,3-propanetricarboxylate Uniplex 80 Trade CAS Number RIFM ID FEMA EINECS Registration EINECS DSL TSCA CIR Panel Book Page 275

282 RIFM Monograph: 700 (Published 1979: FCT,v17,p389) Fragrance Structure-Activity Group: Esters/Dioic and Trioic/Tricarboxylic Acids Formula C 12 H 20 O 7 Molecular Weight SMILES Notation O=C(OCC)C(O)(CC(=O)OCC)CC(=O)OCC Generic Class Aliphatic Esters Description Practically colorless, oily, odorless, bitter liquid Physical Data Boiling Point 127 C at 1 mm Hg Finkelstein,1959 Boiling Point (calculated) C EPI Suite Flash Point 155 C Finkelstein,1959 Flash Point >200 F;CC FMA Henry's Law (calculated) 6.392e-010 Pa m 3 /mol EPI Suite Log K OW (calculated) 0.33 EPI Suite Log K OW (measured) (OECD 117) 1.3 at 35C Givaudan,1998daq Melting Point (calculated) C EPI Suite Specific Gravity 1.14 FMA Specific Gravity 25 C (25/25 C) Finkelstein,1959 Vapor Pressure (calculated) mm Hg 20C FMA Vapor Pressure (calculated) mm 25 C EPI Suite Water Solubility 6.5 g/ C Finkelstein,1959 Water Solubility (calculated) mg/l EPI Suite Preparation By esterification of ethyl alcohol with citric acid (Arctander,1969) Natural Occurrence Triethyl citrate is reported to occur in nature. Flavor Consumption (in kg) 1995 EUROPE USA USA USA USA USA 4540 Uses (in ppm) CIR Panel Book Page 276

283 Product Average Usual Average Maximum Mean Daily Consumption (gms) Updated Alcoholic Beverage Jul-88 Baked Goods Jul-88 Chewing Gum Jul-88 Frozen Dairy Jul-88 Gelatin Pudding Jul-88 Hard Candy Jul-88 Non-alcoholic Beverage Jul-88 Soft Candy Jul-88 PADI 0 Food Products Containing Triethyl citrate (in ppm) Product Code Lower Limit Upper Limit Cabbage (raw) 18-I 0 Status Triethyl citrate was approved by the FDA as GRAS affirmed 12/12/94 ( 21 CFR ). Flavor and Extract Manufacturers' Association states: Generally Recognized as Safe as a flavor ingredient - GRAS 3. ( 3083) Hall,1965 Indicative Non-Exhaustive List states: Listed The Industrial Safety and Health Law (Japan) states: ISHL Number ( (3)- 1320) Joint Expert Committee on Food Additives states: The JECFA Evaluation Summary is available here. Joint Expert Committee on Food Additives states: The Aliphatic Primary Alcohols, Aldehydes, Carboxylic Acids, Acetals and Esters Containing Additional Oxygenated Functional Groups Safety Evaluation is available here. Joint Expert Committee on Food Additives states: ADI 20 mg/kg. ( 1984) Joint Expert Committee on Food Additives states: The Joint FAO/WHO Expert Committee on Food Additives (JECFA) concluded that the substance CIR Panel Book Page 277

284 does not present a safety concern at current levels of intake when used as a flavouring agent. ( 629) United Nations Transport Classification Codes states: Not regulated FFIDS Volume IX-B Updated 12-Oct-04 Presented at the September 2002 HCWG meeting. European Hazard Classification Labeling NC Based on available data, classification and labeling was not considered necessary as per the EFFA Code of Practice. Hazard Category NC Signal Word Code Global Harmonized System Hazard Statements Hazard Statement H000 Not Classified Human Health Data Acute toxicity Route: unclassified. Species: chick embryo. As part of a Teratogenicity study, LD50 values were estimated from the regression line for one of the four conditions: injection via air cell or via the yolk sac at two time intervals (preincubation and 96 hour) for each route. All test materials were administered by a single injection in a volume of 100 ul or less, and solvent controls were treated with the same volume of the solvent only. The test material was dissolved or suspended in an appropriate vehicle. For each condition, at least 100 embryos per dose level were treated at a minimum of five dose levels. Eggs from Single-Comb White Leghorn chickens were used. Appropriate groups of vehicle controls and untreated controls were included. After treatment, all eggs were candled daily and nonviable embryos were removed. The percentage mortality at each dose level was calculated as the total number of nonviable embryos divided by the total number of treated eggs. Dose units are actually mg/egg. Vehicle was absolute ethanol.ld50 67 MG 67 mg calculated LD50, with air cell injection at 0 hours. (Verrett,1980) Route: skin. Species: rabbit. A dermal LD50 study was conducted in which a group of 4 rabbits received a single dermal application of the test material. The animals were observed for mortality CIR Panel Book Page 278

285 and/or systemic effects for 14 days No further details were provided LD50 5 G/KG > 5 g/kg calculated LD50, 0/4 deaths. Acute dermal LD50 greater than 5 g/kg. (RIFM,1975) [Levenstein,1975] Route: intraperitoneal. Species: mouse. Doses were chosen from a logarithmic scale and the median lethal dose (LD50) calculated by method of Bliss. All esters were administered suspended or dissolved in 3% acacia. Animals were of the Swiss albino strain weighing between 16 and 20 g. No necropsy was performed. No further details of experimental procedure were given. LD MG/KG 1750 mg/kg calculated LD50. (Meyers,1964) Route: gavage. Species: rat. An acute LD50 study was conducted on groups consisting of 1-20 Wistar rats. The animals were administered a single dose of test material in an unspecified vehicle by a stomach tube. All animals were observed for general appearance and behavior, and the time for the onset of the effects, recovery time and the time to death were determined. No further details were provided. 12 ml/kg lethal, 4/5 deaths. Time to effects' onset = 6.4 minutes; recovery time = 40 hours; and time to death = 26 hours ml/kg lethal, 3/4 deaths. Time to effects' onset = 7 minutes; recovery time = 36 hours; and time to death = 85 hours. 8 ml/kg lethal, 12/16 deaths. Time to effects' onset = 18.6 minutes; recovery time = 15.5 hours; and time to death = 6.3 hours. 10 ml/kg lethal, 4/5 deaths. Time to effects' onset = 2.4 minutes; recovery time = 16 hours; and time to death = 6.7 hours. 8.5 ml/kg lethal, 4/5 deaths. Time to effects' onset = 23 minutes and time to death = 1.8 hours. 14 ml/kg lethal, 1/1 death. Time to effects' onset = 3 minutes and time to death = 15 hours. 7.5 ml/kg lethal, 19/20 deaths. Time to effects' onset = 28.2 minutes; recovery time = 36 hours; and time to death = 8.4 hours.calculated LD50, Reported to be near 7 ml/kg. 15 ml/kg lethal, 1/1 death. Time to effects' onset = 3 minutes and time to death = 7.5 hours. 6 ml/kg lethal, 7/15 deaths. Time to effects' onset = 32 minutes; recovery time = 34.5 hours; and time to death = 11.3 hours. 5 ml/kg lethal, 3/10 deaths. Time to effects' onset = 88 minutes; recovery time = 36 hours; and time to death = 30.7 hours. 7 ml/kg lethal, 6/15 deaths. Time to effects' onset = 13.1 minutes; recovery time = 8.9 hours; and time to death = 13.6 hours. 13 ml/kg lethal, 5/5 deaths. Time to effects' onset = 6.6 minutes and time to death = 15.2 hours. (Finkelstein,1959) Route: gavage. Species: cat. An acute LD50 study was conducted on groups consisting of 1-20 cats. The animals were administered a single dose of test material by a stomach tube, after a 24- hour predose fast. All animals were observed for general appearance and behavior, and the time for the onset of the effects, recovery time and the time to death were determined. No further details were provided. LD MG/KG 3.5 mg/kg calculated LD50. 5 mg/kg lethal, 4/4 deaths. Time to effects' onset = 11.2 minutes and time to death = 4.1 hours. 2 mg/kg lethal, 1/5 deaths. Time to effects' onset = 14 minutes; recovery time = 14 hours; and time to death = 64 hours. 8 mg/kg lethal, 3/3 deaths. Time to effects' onset = 8 minutes and time to death = 3 hours. 6 mg/kg lethal, 1/1 death. Time to effects' onset = 4 minutes and time to death = 1.5 hours. 9 mg/kg lethal, 3/3 deaths. Time to effects' onset = 10.7 minutes and time to death = 3.5 hours. 3 mg/kg nonspecific effects, 0/3 deaths. Time to effects' onset = 16.2 minutes; and recovery time = 16.7 hours. 1 mg/kg nonspecific effects, 0/5 deaths. Time to effects' onset = 50 minutes; and recovery time = 14 hours. 7 mg/kg lethal, 2/2 deaths. Time to effects' onset = 6.5 minutes and time to death = 4.6 hours. 4 mg/kg lethal, 3/4 deaths. Time to effects' onset = 20.6 minutes; recovery time = 108 hours; and time to death = 31.2 hours. (Finkelstein,1959) Route: gavage. Species: cat. The effect of the test material on the heart and blood pressure was evaluated. One cat per dose was administered the test material by a stomach tube, and then the CIR Panel Book Page 279

286 animal was placed on an animal board to be prepared for reading of the blood pressure from the carotid artery. Electrocardiograms were used to record the effect on the heart. No further details were provided. 9 ml/kg blood pressure, Progressive lowering of the blood pressure to shock levels was exhibited. In addition, a progressive slowing of the heart from the rapid rates of about 200 or over in the control, to rates in the range about 150 a minute was exhibited. There was progressive lowering of the T-wave. There were no changes in the heart rhythm. 6 ml/kg blood pressure, Progressive lowering of the blood pressure to shock levels was exhibited. In addition, a progressive slowing of the heart from the rapid rates of about 200 or over in the control, to rates in the range about 150 a minute was exhibited. There was progressive lowering of the T-wave. There were no changes in the heart rhythm. (Finkelstein,1959) Route: intraperitoneal. Species: mouse. A 14-day toxicity test was performed in which animals weighing from gm were given daily doses which were approximately 1/5 of the acute median lethal dose. All chemicals to be tested were suspended in 3% acacia which was also used in control groups. Five groups of 20 mice each were selected a nd the weight, red blood cell count, white blood cell count, clotting time and hemoglobin level of ea individual were determined. Animals were weighed daily and at the end of the 2-wk period blood measurements were made. At the end of the study, slides were made of liver, lung, and kidney tissues. 350 mg/kg body weight changes, weight gain was significantly 7 da. (Meyers,1964) Route: intravenous. Species: rabbit. Blood pressures were recorded from the cannulated carotid artery with a mercury manometer. A tampour connected to the tracheal cannula simultaneoulsy recorded respiration. Test compounds were administered through the ear vein or through the cannulated jugular. Dose levels not specified. Cardiovascular effects, complete loss of blood pressure was noted when administered in toxic doses. (Meyers,1964) Route: intravenous. Species: cat. Blood pressures were recorded from the cannulated carotid artery with a mercury manometer. A tampour connected to the tracheal cannula simultaneoulsy recorded respiration. The esters were administered through the ear vein or through the cannulated jugular. Dose levels not specified. Cardiovascular effects, complete loss of blood pressure was noted when administered in toxic doses. (Meyers,1964) Route: unclassified. Species: rabbit. Test substances were tested on the isolated rabbit heart after the method of Langendorff (details of experimental procedures not given) in order to more clearly delineate the depressor effects noted in earlier experiments. Cardiovascular effects, a sharp decline in both rate & amplitude of heart rate was observed. (Meyers,1964) Route: intraperitoneal. Species: mouse. Saturated saline solutions of the test material were evaluated by intraperitoneal injection in mice. A 0.5 ml aliquot per 20 grams body weight was administered. No further details were provided. Positive effects, Severe depression and ataxia were observed. (Guess,1968) Route: in vitro. Species: mouse. Mouse fibroblasts (L-929) cells were allowed to form a monolayer in petri dishes, afterwhich the liquid nutrient media was aspirated off and replaced with 1% agar suspension in nutrient media. After gelation, the test material (undiluted or as an aqueous solution in normal saline) was placed onto a paper disk which was then placed on the agar overlay, and incubated for 24 hours. A zone of dead cells around the disk denoted a toxic test material. Positive effects, Toxicity occurred with the undiluted form but not with the saturated saline solution. (Guess,1968) Route: in vitro. Species: chick embryo. Ten-day chick embryo cells were allowed to form a monolayer in petri dishes, afterwhich the liquid nutrient media was aspirated off and replaced CIR Panel Book Page 280

287 with 1% agar suspension in nutrient media. After gelation, the test material (undiluted or as an aqueous solution in normal saline) was placed onto a paper disk which was then placed on the agar overlay, and incubated for 24 hours. A zone of dead cells around the disk denoted a toxic test material. Positive effects, toxicity occurred with undiluted form but not with saturated saline solution. (Guess,1968) Route: in vitro. Species: unknown. Saturated saline solutions of the test material were evaluated for hemolysis liability to a 2% suspension of washed red blood cells. Distilled water served as the 4+ hemolysis control. Positive effects, 4+ hemolysis. (Guess,1968) Route: gavage. Species: cat. The effect of the test material on neuromuscular conduction was evaluated during an associated study in which the effect of the test material on the heart and blood pressure was evaluated. One cat per dose was administered the test material by a stomach tube, and then the animal was placed on an animal board to be prepared for reading of the blood pressure from the carotid artery. Electrocardiograms were used to record the effect on the heart. When respiration ceased, the animals chests were opened and the phrenic nerves were stimulated by an electric shock. In addition, the sciatic nerved was also exposed and stimulated with a shock. No further details were provided. 6 ml/kg positive effects, With stimulation of the phrenic nerves, a sustained contraction of the diaphragm was produced, which was similar to that in normal animal that succumb to ether or hemorrhage. With stimulation of the sciatic nerves, a vigorous and sustained contraction of the muscles was produced, similar to that in untreated controls. This response to stimulation of the phrenic and sciatic nerve indicated that the test material did not interfere with neuromuscular transmission during advanced poisoning by the test material. 9 ml/kg positive effects, With stimulation of the phrenic nerves, a sustained contraction of the diaphragm was produced, which was similar to that in normal animal that succumb to ether or hemorrhage. With stimulation of the sciatic nerves, a vigorous and sustained contraction of the muscles was produced, similar to that in untreated controls. This response to stimulation of the phrenic and sciatic nerve indicated that the test material did not interfere with neuromuscular transmission during advanced poisoning by the test material. (Finkelstein,1959) Route: gavage. Species: cat. Two cats were fasted for 24 hours, and then administered a single dose of the test material by stomach tube. Blood counts (red blood count RBC, white cell count WBC, and hemoglobin), blood chemistry (blood sugar, blood NPN and blood creatinine), and urine tests were conducted for 2 months at intervals of 2 weeks. The 2 controls were not administered the test material. No further details were provided. 1 cat was treated. 5 ml/kg clinical signs, Severe poisoning was produced but the animals recovered. No effects on the blood count, hemoglobin, sugar, non-protein nitrogen or creatinine was exhibited. (Finkelstein,1959) Irritation Route: skin. Species: rabbit. A study was conducted to determine the degree of irritation of the test material may produce when applied to the clipped intact and abraded skin of the rabbit. Three healthy, normal, albino rabbits were used for this experiment. On the day prior to the experiment 10% of the total body area of the animals was carefully clipped free of hair. On the posterior of the clipped area several minor abrasions were made so as to penetrate the stratum corneum but not disturb the derma. A volume of 0.5 ml of the test material in alcohol SDA 39C was patched over the scarified area and 0.5 ml over the unscarified area. The 2 x 2 patch area was covered with Webril patches and the entire experimental area sealed with Blenderm Surgical Tape. The vehicle was tested separately in the same manner as the test material. The animals were immobilized in racks for 24 hours. At the end of the 24 hour contact period and again 48 CIR Panel Book Page 281

288 hours later the treated skin was evaluated according to the Draize method as described in "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics", published by the Association of Food and Drug Officials of the United States. The scale was as follows: no erythema=0, no edema=0; very slight erythema (slightly perceptible)=1, very slight edema=1; well-defined erythema=2, slight edema=2; moderate to severe erythema=3, moderate edema=3; severe erythema to slight eschar formation=4, severe edema=4. The vehicle alcohol SDA 39C in distilled water, tested separately in the same manner as the test material, produced no irritant effects. Subjects: 3 Unspecified Sex Study Length: 24 hours (Draize, JH;1959) Vehicle was alcohol SDA 39C.Summary Under the conditions of this study, the test material was not considered a primary skin irritant. 15 % no effects, No irritant effects were observed on the intact or abraded skin of rabbits at 24 and 72 hours after administration of test material in alcohol SDA 39C. The test material produced a primary skin irritation index of 0 and was not considered a primary skin irritant. (IFF,1977) Route: skin. Species: rabbit. A study was conducted to determine the degree of irritation of the test material may produce when applied to the clipped intact and abraded skin of the rabbit. Three healthy, normal, albino rabbits were used for this experiment. On the day prior to the experiment 10% of the total body area of the animals was carefully clipped free of hair. On the posterior of the clipped area several minor abrasions were made so as to penetrate the stratum corneum but not disturb the derma. A volume of 0.5 ml of the test material in petrolatum was patched over the scarified area and 0.5 ml over the unscarified area. The 2 x 2 patch area was covered with Webril patches and the entire experimental area sealed with Blenderm Surgical Tape. The vehicle was tested separately at 100% in the same manner as the test material. The animals were immobilized in racks for 24 hours. At the end of the 24 hour contact period and again 48 hours later the treated skin was evaluated according to the Draize method as described in "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics", published by the Association of Food and Drug Officials of the United States. The scale was as follows: no erythema=0, no edema=0; very slight erythema (slightly perceptible)=1, very slight edema=1; well-defined erythema=2, slight edema=2; moderate to severe erythema=3, moderate edema=3; severe erythema to slight eschar formation=4, severe edema=4. The vehicle petrolatum, tested separately in the same manner as the test material, produced no irritant effects. Subjects: 3 Unspecified Sex Study Length: 24 hours (Draize,JH;1959) Vehicle was petrolatum.summary Under the conditions of this study, the test material in petrolatum was not considered a primary skin irritant % no effects, No irritant effects were observed on the intact or abraded skin of rabbits at 24 and 72 hours after administration of test material in petrolatum. The test material produced a primary skin irritation index of 0 and was not considered a primary skin irritant. (IFF,1977a) Route: skin. Species: rabbit. A study was conducted to determine the degree of irritation of the test material may produce when applied to the clipped intact and abraded skin of the rabbit. Three healthy, normal, albino rabbits were used for this experiment. On the day prior to the experiment 10% of the total body area of the animals was carefully clipped free of hair. On the posterior of the clipped area several minor abrasions were made so as to penetrate the stratum corneum but not disturb the derma. A volume of 0.5 ml of the test material in alcohol SDA 39C was patched over the scarified area and 0.5 ml over the unscarified area. The 2 x 2 patch area was covered with Webril patches and the entire experimental area sealed with Blenderm Surgical Tape. The vehicle was tested separately in the same manner as the test material. The animals were immobilized in racks for 24 hours. At the end of the 24 hour contact period and again 48 CIR Panel Book Page 282

289 hours later the treated skin was evaluated according to the Draize method as described in "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics", published by the Association of Food and Drug Officials of the United States. The scale was as follows: no erythema=0, no edema=0; very slight erythema (slightly perceptible)=1, very slight edema=1; well-defined erythema=2, slight edema=2; moderate to severe erythema=3, moderate edema=3; severe erythema to slight eschar formation=4, severe edema=4. The vehicle alcohol SDA 39C in distilled water, tested separately in the same manner as the test material, produced no irritant effects. Subjects: 3 Unspecified Sex Study Length: 24 hours (Draize,JH;1959) Vehicle was alcohol SDA 39C.Summary Under the conditions of this study, the test material in alcohol SDA 39C was not considered a primary skin irritant % no effects, No irritant effects were observed on the intact or abraded skin of rabbits at 24 and 72 hours after administration of test material in alcohol SDA 39C. The test material produced a primary skin irritation index of 0 and was not considered a primary skin irritant. (IFF,1977b) Route: surface of eye. Species: rabbit. In an eye irritation study, a single concentration of test material in a vehicle of alcohol SDA 39C was applied to one eye of each of three New Zealand White rabbits. The test material was applied by pulling out the lower lid from the right eye and placing 0.1 ml in the sac. The untreated left eye of each animal served as its own control. The treated and control eyes were examined before application of the test material, every 24 hours after treatment for four days and then again on the seventh day. They were scored for corneal swelling, and corneal, conjunctival and iridial damage according to the Draize scale for scoring ocular lesions. Subjects: 3 Unspecified Sex (Draize,JH;1959) Vehicle was alcohol SDA 39C.Summary Under the conditions of this study, test material in alcohol SDA 39C produced a conjunctival irritation and corneal involvement which did not clear on the seventh day of observation. 15 % irritant effects, Under the conditions of this study, test material in alcohol SDA 39C instilled into the right eye of each of three rabbits produced a conjunctival irritation and corneal involvement which did not clear on the seventh day of observation. (IFF,1977c) Route: surface of eye. Species: rabbit. In an eye irritation study, a single concentration of test material in a vehicle of alcohol SDA 39C was applied to one eye of each of three New Zealand White rabbits. The test material was applied by pulling out the lower lid from the right eye and placing 0.1 ml in the sac. The untreated left eye of each animal served as its own control. The treated and control eyes were examined before application of the test material, every 24 hours after treatment for four days and then again on the seventh day. They were scored for corneal swelling, and corneal, conjunctival and iridial damage according to the Draize scale for scoring ocular lesions. Subjects: 3 Unspecified Sex (Draize,JH;1959) The vehicle was alcohol SDA 39C.Summary Under the conditions of this study, test material produced a conjunctival irritation with corneal involvement which did not clear on the seventh day % irritant effects, Under the conditions of this study, test material instilled into the right eye of each of three rabbits produced a conjunctival irritation with corneal involvement which did not clear on the seventh day. (IFF,1977d) Route: surface of eye. Species: rabbit. In an eye irritation study, a single concentration of test material in a vehicle of petrolatum was applied to one eye of each of three New Zealand White rabbits. The test material was applied by pulling out the lower lid from the right eye and placing 0.1 ml in the sac. The untreated left eye of each animal served as its own control. The vehicle was tested separately in the same manner as the test material. The treated and control eyes were examined before application of the test material, every 24 hours after treatment for four days and then again on the seventh day. They were scored for corneal swelling, and corneal, conjunctival CIR Panel Book Page 283

290 and iridial damage according to the Draize scale for scoring ocular lesions. The vehicle (petrolatum), tested separately in the same manner as the test material, produced no irritant effects. Subjects: 3 Unspecified Sex (Draize,JH;1959) Vehicle was petrolatum.summary Under the conditions of this study, the test material in petrolatum produced a conjunctival irritation which cleared on the seventh day of observation with corneal involvement also clearing on the seventh day % irritant effects, Under the conditions of this study, the test material in petrolatum instilled into the right eye of each of three rabbits, produced a conjunctival irritation which cleared on the seventh day of observation with corneal involvement also clearing on the seventh day. (IFF,1977e) Route: skin. Species: rabbit. Irritation was assessed during an acute dermal LD50 study, that was conducted on a group of 4 rabbits administered a single dermal application of test material. Animals were observed over a 14 day period No further details provided g/kg no effects. (RIFM,1975) [Levenstein,1975] Route: skin. Species: human 18+ yrs. The purpose of this study was to determine the contact sensitizing potential of the test article. Twenty seven healthy volunteers started the study and 25 completed. During the pre-test phase the material was tested on all subject to determine if pretreatment with sodium lauryl sulphate was required. For this, a patch with the test article was applied to the back for 48 hours under semi-occlusive conditions. The subjects were also tested with 2.5% sodium lauryl sulphate to determine the length of treatment required. For the induction phase of the study, the patch sites were pre-treated with 2.5% sodium lauryl sulphate as required for each subject, with time ranging from 5 to 36 hours. The test article was applied to the same site on the volar forearms or back of each subject for five alternate 48 hour periods under occlusive conditions. The challenge portion was conducted 10 to 14 days after application of the last induction patch. The challenge patch was applied to a new site on the back which had been pre-treated with 2.5% sodium lauryl sulphate for 1 hour. The patch was applied in a semiocclusive manner and remained in place for 48 hours. The skin reactions were read at patch removal and at 24 and 48 hours after. The reactions were scored according to the following scale; 0=no reaction 1+=slight erythema, 2+=well defined erythema, 3+=erythema and edema, 4+= erythema and edema with vesiculation and/or ulceration and ±=minimal erythema. Sensitization was assessed using the Kligman scale; 0=not sensitized, 1=mild sensitization (erythema and a little edema), 2=moderate sensitization (erythema with infiltration; raised, spreading beyond borders with or without vesiculation), 3=strong sensitization (large, vesiculobulous, vividly red, infiltrated plaques). The vehicle used was petrolatum. There was no reason given for the three discontinued subjects. One subject was not patched during the challenge phase as she reacted to other products on the panel and developed a generalized rash. Subjects: 25 Unspecified Sex Study Length: 21 days Regulatory Compliance GLP No Sample code HRE ( )-P.Summary Under the conditions of this study, skin irritation was observed % irritant effects, The reactions noted ranged from minimal erythema to erythema and edema across the 5 exposure days in the induction phase. Some reactions were noted during the challenge phase, ranging from minimal erythema to well defined erythema. (IFF,1977g) Route: skin. Species: human 18+ yrs. The irritation potential of the test article was assessed during a Modified Maximization Study. Twenty eight healthy volunteers started the study and 26 completed. During the pre-test phase the material was tested on all subject to determine if pretreatment with sodium lauryl sulphate was required. For this, a patch with the test article was applied to the back for 48 hours under semi-occlusive conditions. The subjects were also tested CIR Panel Book Page 284

291 with 2.5% sodium lauryl sulphate to determine the length of treatment required. For the induction phase of the study, the patch sites were pre-treated with 2.5% sodium lauryl sulphate as required for each subject, with time ranging from 5 to 36 hours. The test article was applied to the same site on the volar forearms or back of each subject for five alternate 48 hour periods under occlusive conditions. The challenge portion was conducted 10 to 14 days after application of the last induction patch. The challenge patch was applied to a new site on the back which had been pre-treated with 2.5% sodium lauryl sulphate for 1 hour. The patch was applied in a semiocclusive manner and remained in place for 48 hours. The skin reactions were read at patch removal and at 24 and 48 hours after. The reactions were scored according to the following scale; 0=no reaction 1+=slight erythema, 2+=well defined erythema, 3+=erythema and edema, 4+= erythema and edema with vesiculation and/or ulceration and ±=minimal erythema. Sensitization was assessed using the Kligman scale; 0=not sensitized, 1=mild sensitization (erythema and a little edema), 2=moderate sensitization (erythema with infiltration; raised, spreading beyond borders with or without vesiculation), 3=strong sensitization (large, vesiculobulous, vividly red, infiltrated plaques). The vehicle used was alcohol (SDA 39C). There was no reason given for the two discontinued subjects. Subjects: 26 Unspecified Sex Study Length: 21 days Regulatory Compliance GLP No Sample code HRE Summary Under the conditions of this study, irritation was observed. 15 % irritant effects, The reactions noted ranged from mild erythema to erythema and edema with vesiculation and/or ulceration across the 5 exposure days in the induction phase. Some reactions were noted during the challenge phase, ranging from minimal erythema to well defined erythema. (IFF,1977f) Route: skin. Species: human 18+ yrs. Irritation potential was assessed during an associated repeated insult patch test. A total of 45 subjects, 10 males and 35 females were placed on test, all subjects completed the study. Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.4 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the patch site, H=small petechial erosions and/or scabs. The vehicle was petrolatum. No details were given regarding the timing of the challenge patches. Subjects: 10 Male 35 Female Regulatory Compliance GLP No (Draize,1959) HRE ( )-P.Summary Under the conditions of the study, the test article is not a primary irritant % no effects, There was little or no CIR Panel Book Page 285

292 primary irritation noted. (IFF,1977h) Route: skin. Species: human 18+ yrs. Irritation potential was assessed during an associated repeated insult patch test. A total of 48 subjects were initially enrolled in the study, 41 subjects completed the study (5 males and 36 females). Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.5 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the patch site, H=small petechial erosions and/or scabs. The vehicle was alcohol 39C. The panellists did not complete the study for the following reasons; 3 could not meet the schedule, vacation interfered in one, one did not realize the number of visits required, one objected to the odour and one did not wish to continue. No details were given regarding the timing of the challenge patches. Subjects: 5 Male 36 Female Regulatory Compliance GLP No (Draize,1959) Brown liquid, HRE ( ).Summary Under the conditions of the study, the test article is not a primary irritant. 15 % no effects, There was little or no primary irritation noted. (IFF,1977i) Route: skin. Species: human 18+ yrs. Irritation potential was assessed during an associated repeated insult patch test. A total of 47 subjects were initially enrolled in the study, 41 subjects completed the study (10 males and 31 females). Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.5 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no CIR Panel Book Page 286

293 erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the patch site, H=small petechial erosions and/or scabs. The vehicle was alcohol 39C. The panellists did not complete the study for the following reasons; 2 objected to the odour, 1 was injured in a fall and bedridden, 1 left for vacation, 1 could not attend challenge as infant was hospitalized and one did not attend challenge and could not be reached. Subjects: 10 Male 31 Female Regulatory Compliance GLP No (Draize,1959) Brown liquid, HRE ( )-1.Summary Under the conditions of the study, the test article is not a primary irritant % no effects, There was little or no primary irritation noted. (IFF,1977j) Route: skin. Species: human 18+ yrs. In a pre-test for a human maximization study, a 48-hour closed patch test with the test material in petrolatum was conducted on the backs of 22 healthy volunteers. 20 % no effects. (RIFM,1975) [Epstein,1975] Route: skin. Species: human 18+ yrs. Irritation was assessed during an associated repeated insult patch test. Sixty-one male and female subjects were enrolled in the study. Fifty-nine subjects (51 females and 8 males) completed the study. Two subjects who dropped from the test did so for personal reasons unrelated to applications of the test materials. Subjects' ages ranged from 21 to 60 years old. 0.4 ml of the test material was applied to the test patch which was a 20 x 20 mm square of Webril (a thick, non-woven cotton fabric) affixed to a 40 x 40 mm adhesive square. The patches, one of each of the test materials, were applied to the upper arms of each subject on Monday, Wednesday and Friday for three consecutive weeks. The subjects were instructed to keep the patches dry and to remove them 24 hours after application. Duplicate challenge applications of each test material were made two weeks after the final induction applications, one set of patches to the original sites and one to adjacent virgin sites. The patch sites were scored just prior to the patch applications at the second through the ninths visits and on the tenth visit. The challenge application sites were scored at 48 and 96 hours after application. All readings were made under light supplied by a 100 watt incandescent blue bulb. Test material was applied as received. No concentration or vehicle was provided. No further information was provided. Study was conducted in Subjects: 8 Male 51 Female Study Length: 3 weeks (Draize, J. H., 1959) Lot No. N60051-H8110; Laboratory Code A.Summary Under the conditions of the study, the test material was non-irritating to the subjects tested. No effects, concentration not provided. Under the conditions of the study, the test material was nonirritating to the subjects tested. (CIR,1978) Route: intradermal. Species: guinea pig. A preliminary irritation test was conducted as part of an associated sensitization study in guinea pigs to determine the appropriate dose for the intradermal induction phase of of the main study. Four guinea pigs of the same sex and weighing grams were each injected intradermally on the shaved flanks with 0.1 ml aliquots of test material at concentrations of 0.05%, 0.1%, 0.25%, 0.5% and 1.0% in Dobs/saline. 24 hours later the reactiosn were examined for size (2 largest diameters), erythema and oedema and the concentration giving a definite irritatin reaction (~10 x 10 mm, pale pink with or without oedema) was selected as the intradermal injection induction concentration. Strain of guinea pigs not provided. No further information provided. Study conducted in Subjects: 4 Unspecified Sex Study Length: 24 hours (Magnusson and Kligman, 1969 and 1970) Specified as Citroflex 2 vehicle was 0.01% Dobs/Saline.Summary 2.5% test material in Dobs/Saline was CIR Panel Book Page 287

294 selected for the intradermal induction phase of the main sensitization study % irritant effects, skin effects, 2/4 animals exhibited faint pink skin reactions covering an area of 7x7 mm. 2/4 animals exhibited faint pink skin reactions covering an area of 7x8 mm in one animal and 10x11 mm in another animal. 0.1 % irritant effects, skin effects, 4/4 animals exhibited faint pink skin reactions covering an area of 7x8 mm in one animal, 8x8 mm in one animal, 8x9 mm in one animal and 11x11 mm in another animal % irritant effects, skin effects, 3/4 animals exhibited faint pink skin reactions covering an area of 7x7 mm in one animal, 8x9 mm in one animal and 9x9 mm in another animal. 1/4 animals exhibited faint pink skin reactions with oedema covering an area of 8x8 mm. 0.5 % irritant effects, skin effects, 4/4 animals exhibited faint pink skin reactions covering an area of 9x9 mm in one animal, 8x9 mm in one animal, 8x8 mm in one animal and 9x10 mm in another animal. 1.0 % irritant effects, skin effects, 2/4 animals exhibited faint pink skin reactions covering an area of 8x8 mm. 1/4 animals exhibited faint pink skin reactions covering an area of 8x9 mm. 1/4 animals exhibited faint pink reactions with oedema covering an area of 7x8 mm. (CIR,1976) Route: skin. Species: guinea pig. A preliminary irritation test was conducted as part of an associated sensitization study in guinea pigs to determine the appropriate dose for the topical neck induction and flank challenge in the main study. Four guinea pigs of the same sex and weighing grams were tested. 8 mm diameter filter paper patches in aluminium patch test cups were saturated with three different concentrations of the test material in absolute alcohol. The cups were applied to the shaved flanks of each of the guinea pigs and held in place by adhesive plaster (Poroplast) wound around the trunk. 24 hours later the patches were removed and the reactions sites examined 24 hours and 48 hours after removal of the patches. Reactions were scored for irritation on a scale from 0 to +++. A concentration giving definite irritation (~+) was selected for shoulder induction. The highest concentration which causes no irritation was selected for the flank challenge. Strain of guinea pigs not provided. No further information provided. Study conducted in Subjects: 4 Unspecified Sex Study Length: 24 hours (Magnusson and Kligman, 1969 and 1970) specified as Citroflex 2 vehicle was absolute alcohol.summary 100% test material was selected for the topical induction phase of the main sensitization study. 50% test material in absolute alcohol was selected for the challenge phase of the main sensitization study. 100 % irritant effects, skin effects, barely perceptible erythema was observed in 1/4 animals at the 24 hour reading. No skin reactions were observed at the 48 hour reading. 70 % no effects, no skin reactions were observed at the 24 and 48 hour readings in any of the 4 animals tested. 40 % no effects, no skin reactions were observed at the 24 and 48 hour readings in any of the 4 animals tested. (CIR,1976) Route: intradermal. Species: rabbit. A rabbit intradermal irritation test was conducted, in which saturated saline solutions of the test material was administered to the animals by intradermal injection. Ethanol (20%) served as the 4+ control. No further details were provided. No effects. (Guess,1968) Sensitization Route: skin. Species: human 18+ yrs. The purpose of this study was to determine the contact sensitizing potential of the test article. Twenty seven healthy volunteers started the study and 25 completed. During the pre-test phase the material was tested on all subject to determine if pretreatment with sodium lauryl sulphate was required. For this, a patch with the test article was applied to the back for 48 hours under semi-occlusive conditions. The subjects were also tested with 2.5% sodium lauryl sulphate to determine the length of treatment required. For the CIR Panel Book Page 288

295 induction phase of the study, the patch sites were pre-treated with 2.5% sodium lauryl sulphate as required for each subject, with time ranging from 5 to 36 hours. The test article was applied to the same site on the volar forearms or back of each subject for five alternate 48 hour periods under occlusive conditions. The challenge portion was conducted 10 to 14 days after application of the last induction patch. The challenge patch was applied to a new site on the back which had been pre-treated with 2.5% sodium lauryl sulphate for 1 hour. The patch was applied in a semiocclusive manner and remained in place for 48 hours. The skin reactions were read at patch removal and at 24 and 48 hours after. The reactions were scored according to the following scale; 0=no reaction 1+=slight erythema, 2+=well defined erythema, 3+=erythema and edema, 4+= erythema and edema with vesiculation and/or ulceration and ±=minimal erythema. Sensitization was assessed using the Kligman scale; 0=not sensitized, 1=mild sensitization (erythema and a little edema), 2=moderate sensitization (erythema with infiltration; raised, spreading beyond borders with or without vesiculation), 3=strong sensitization (large, vesiculobulous, vividly red, infiltrated plaques). The vehicle used was petrolatum. There was no reason given for the three discontinued subjects. One subject was not patched during the challenge phase as she reacted to other products on the panel and developed a generalized rash. Subjects: 25 Unspecified Sex Study Length: 21 days Regulatory Compliance GLP No Sample code HRE ( )-P.Summary Under the conditions of this study, there was no contact sensitization observed % no effects, Some reactions were noted during the challenge phase, ranging from slight erythema to well defined erythema. When the Kligman scale was used, there was no evidence of sensitization. (IFF,1977g) Route: skin. Species: human 18+ yrs. The purpose of this study was to determine the contact sensitizing potential of the test article. Twenty eight healthy volunteers started the study and 26 completed. During the pre-test phase the material was tested on all subject to determine if pretreatment with sodium lauryl sulphate was required. For this, a patch with the test article was applied to the back for 48 hours under semi-occlusive conditions. The subjects were also tested with 2.5% sodium lauryl sulphate to determine the length of treatment required. For the induction phase of the study, the patch sites were pre-treated with 2.5% sodium lauryl sulphate as required for each subject, with time ranging from 5 to 36 hours. The test article was applied to the same site on the volar forearms or back of each subject for five alternate 48 hour periods under occlusive conditions. The challenge portion was conducted 10 to 14 days after application of the last induction patch. The challenge patch was applied to a new site on the back which had been pre-treated with 2.5% sodium lauryl sulphate for 1 hour. The patch was applied in a semiocclusive manner and remained in place for 48 hours. The skin reactions were read at patch removal and at 24 and 48 hours after. The reactions were scored according to the following scale; 0=no reaction 1+=slight erythema, 2+=well defined erythema, 3+=erythema and edema, 4+= erythema and edema with vesiculation and/or ulceration and ±=minimal erythema. Sensitization was assessed using the Kligman scale; 0=not sensitized, 1=mild sensitization (erythema and a little edema), 2=moderate sensitization (erythema with infiltration; raised, spreading beyond borders with or without vesiculation), 3=strong sensitization (large, vesiculobulous, vividly red, infiltrated plaques). The vehicle used was alcohol (SDA 39C). There was no reason given for the two discontinued subjects. Subjects: 26 Unspecified Sex Study Length: 21 days Regulatory Compliance GLP No Sample code HRE Summary Under the conditions of this study, there was no contact sensitization observed. 15 % no effects, Some reactions were noted during the challenge phase, ranging from minimal erythema to well defined erythema. When the Kligman scale was used, there was no evidence of sensitization. (IFF,1977f) CIR Panel Book Page 289

296 Route: multiple routes. Species: guinea pig. A sensitization study was conducted in male and female guinea pigs according to Magnusson and Kligman, 1969 and Ten male and female animals were used for the test group along with 4 animals of the same sex for the treated control group and 4 animals of the same sex for the untreated control group. The guinea pigs weighed approximately 320 grams at the beginning of the study. For the induction phase, six 0.1 ml intradermal injections were made close together within a 2 x 4 cm area of the shoulder region as follows: 2 injections of 2.5% test material in 0.01% Dobs/Saline; 2 injections of 2.5% test material in 50% Complete Freunds Adjuvant (CFA) in saline; 2 injections of 50% CFA in saline. Seven days later, sensitization was boosted by placing a 2 x 4 cm filter paper patch saturated with 100% test maerial over the shoulder injection site. The patch was occluded with Blenderm and held in place by Poroplast and left in place for 48 hours. Fourteen days after application of the shoulder patch the guinea pigs were challenged on the flank by an occluded patch. For each animal an 8 mm diameter filter paper patch in a patch test cup was saturated with test material and the cup applied to the shaved flank and held in place by Poroplast wound around the trunk. 24 hours later the patch is removed and reaction site examined 24 hours and 48 hours after removal of the patch. Reactions were scored according to the following scale: 0 = no reaction; +/- = barely perceptible erythema; + = scattered, mild erythema (faint pink); ++ = moderate and diffuse erythema (pale pink); +++ = intense erythema (deep pink) and oedema. Reactions were considered positive if they were "+" or greater and there were no irritation reactions in the controls. Further challenges and cross-challenges were made on alternate flanks at weekly or greater intervals as required. Body weights were recorded weekly. The treated control group consisted of animals of the same sex and were treated exactly as for the test animals as described above, except the test material was omitted from the intradermal injection and covered patch induction procedures. These animals were challenged exactly as for the test animals. The untreated control group consisted of previously untreated animals of the same sex and were each challenged exactly as the test animals. Strain of guinea pigs not provided. No further information provided. Study conducted in Subjects: 10 Unspecified Sex (Magnusson and Kligman, 1969 and 1970) Specified as Citroflex 2 vehicle was 0.01% Dobs/Saline for the intradermal induction and absolute ethanol for challenge.summary Under the conditions of the study, the test material was considered a strong sensitizer. 50 % sensitization effects, 9/9 animals exhibited positive sensitization reactions at the 24 and 48 hour readings (one animal was killed prior to challenge because of a broken leg). Barely perceptible erythema was observed in 1 animal of the treated control group at the 24 hour reading only. No skin reactions were observed in untreated control group. A second challenge with the test material and cross-challenge with Citroflex A-2 and Citroflex A-4 was conducted. (CIR,1976) Route: skin. Species: human 18+ yrs. A 24 to 48 hour occluded patch test was conducted on patients. The test material concentration was % in a vehicle which was either a base cream or 99% ethanol (type of vehicle per test material was not specifically identified). Patch tests were always carried out with both perfumed and non-perfumed cream base at the same time. Patches consisted of a piece of 1 cm2 lint with a 2 cm2 cellophane disc placed on the lint and then covered with 4 cm2 plaster. The patches were applied to the back, the forearm and the inside of the upper arm for 24 to 48 hours. Reactions were evaluated 30 minutes after removal of patch. The skin reaction was graded as follows: - No visible reaction; +- Slight erythema; + Erythema; ++ Erythema and swelling or marked erythema. The period of study extended over 4 yrs and 3 months and patch testing was performed 1-3 times per month; September to May (summer months were excluded). The total number of subjects was 4737 CIR Panel Book Page 290

297 (2341 Japanese men and 2396 Japanese women aged 16-45, mostly 17-30, living in Tokyo and Osaka). Volume dose not provided. 99% purity. Positive effects, 2/177 exhibited erythema (+). (Takenaka,1986) Route: skin. Species: human 18+ yrs. A study was conducted to evaluate the irritative and contact sensitization properties of the test materials after repeated applications. Sixty-one male and female subjects were enrolled in the study. Fifty-nine subjects (51 females and 8 males) completed the study. Two subjects who dropped from the test did so for personal reasons unrelated to applications of the test materials. Subjects' ages ranged from 21 to 60 years old. 0.4 ml of the test material was applied to the test patch which was a 20 x 20 mm square of Webril (a thick, non-woven cotton fabric) affixed to a 40 x 40 mm adhesive square. The patches, one of each of the test materials, were applied to the upper arms of each subject on Monday, Wednesday and Friday for three consecutive weeks. The subjects were instructed to keep the patches dry and to remove them 24 hours after application. Duplicate challenge applications of each test material were made two weeks after the final induction applications, one set of patches to the original sites and one to adjacent virgin sites. The patch sites were scored just prior to the patch applications at the second through the ninths visits and on the tenth visit. The challenge application sites were scored at 48 and 96 hours after application. All readings were made under light supplied by a 100 watt incandescent blue bulb. Test material was applied as received. No concentration or vehicle was provided. No further information was provided. Study was conducted in Subjects: 8 Male 51 Female Study Length: 6 weeks (Draize, J. H., 1959) Lot No. N H8110; Laboratory Code A.Summary Under the conditions of the study, the test material showed no contact sensitization potential in any of the subjects tested. No effects, concentration not provided. Under the conditions of the study, the test material showed no contact sensitization potential in any of the subjects tested. (CIR,1978) Route: skin. Species: human 18+ yrs. The purpose of this study was to evaluate the potential of the test article to induce allergic sensitization in human following repeated exposure. A total of 47 subjects were initially enrolled in the study, 41 subjects completed the study (10 males and 31 females). Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.5 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the CIR Panel Book Page 291

298 patch site, H=small petechial erosions and/or scabs. The vehicle was alcohol 39C. The panellists did not complete the study for the following reasons; 2 objected to the odour, 1 was injured in a fall and bedridden, 1 left for vacation, 1 could not attend challenge as infant was hospitalized and one did not attend challenge and could not be reached. Subjects: 10 Male 31 Female Regulatory Compliance GLP No (Draize,1959) Brown liquid, HRE ( )-1.Summary Under the conditions of this study, the test article did not induce allergic contact dermatitis in human subjects % no effects, No subjects were sensitized. (IFF,1977j) Route: skin. Species: human 18+ yrs. The purpose of this study was to evaluate the potential of the test article to induce allergic sensitization in human following repeated exposure. A total of 48 subjects were initially enrolled in the study, 41 subjects completed the study (5 males and 36 females). Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.5 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the patch site, H=small petechial erosions and/or scabs. The vehicle was alcohol 39C. The panellists did not complete the study for the following reasons; 3 could not meet the schedule, vacation interfered in one, one did not realize the number of visits required, one objected to the odour and one did not wish to continue. No details were given regarding the timing of the challenge patches. Subjects: 5 Male 36 Female Regulatory Compliance GLP No (Draize,1959) Brown liquid, HRE ( ).Summary Under the conditions of this study, the test article did not induce allergic contact dermatitis in human subjects. 15 % no effects, No subjects were sensitized. (IFF,1977i) Route: skin. Species: human 18+ yrs. The purpose of this study was to evaluate the potential of the test article to induce allergic sensitization in human following repeated exposure. A total of 45 subjects, 10 males and 35 females were placed on test, all subjects completed the study. Subjects ranged in age from 16 to greater than 60. Any subject with a history of diabetes, psoriasis or any active dermatological conditions were excluded from the study. A Webril swatch was affixed to an elastic bandage and was prepared as a special order from Duke laboratories. The test article (0.4 ml) was applied to the swatch prior to application by means of a calibrated stopper. The patches were applied to the upper arms and this test article was one product in a panel of products tested per subject. The subjects were instructed to remove the patches after 24 CIR Panel Book Page 292

299 hours. The next patch was applied to the same location unless the reaction was of sufficient severity to preclude this. If possible, the patch was applied to an adjacent site or omitted. A total of 9 patches were applied during the induction phase. For the final challenge application, duplicate patches were applied, one to a virgin site and one on the original site. The scoring scale used for both the induction and challenge phases of the study was: 0=no evidence of irritation, 1=minimal erythema, barely perceptible, 2=definite erythema, readily visible, 3=erythema and papules, 4=definite edema and erythema, 4e=definite edema, no erythema present, 4Min=minimal edema and definite erythema, 4eMin=minimal edema, no erythema or minimal erythema present, 5=erythema, edema and papules, 6=vesicular eruption, 7=strong reaction spreading beyond test site, Min=used in conjunction with grades 1-3, 5-7 indicates reaction meets minimal requirements for grade assigned. Effects on superficial layers of the skin were also scored; A=slight glazed appearance, B=marked glazing, C=glazing with peeling and cracking, F=glazing with fissures, G=film of dried serous exudates covering all or portions of the patch site, H=small petechial erosions and/or scabs. The vehicle was petrolatum. No details were given regarding the timing of the challenge patches. Subjects: 10 Male 35 Female Regulatory Compliance GLP No (Draize,1959) HRE ( )-P.Summary Under the conditions of this study, the test article did not induce allergic contact dermatitis in human subjects % no effects, No subjects were sensitized. (IFF,1977h) Route: skin. Species: human 18+ yrs. A human maximization test was conducted on 22 healthy volunteers with the test material in petrolatum. Applications were made under occlusion to the same site on the volar forearms for five alternate-day 48-hour periods. The test sites were pretreated for 24 hours with 5% aqueous sodium lauryl sulfate (SLS) under occlusion for the first patch application only. Following a 10 to 14-day rest period, the challenge patches were applied to fresh sites on the back for 48 hours under occlusion. Prior to the challenge applications, 5% SLS was applied to the test site for 30 minutes under occlusion on the left side of the back, whereas the test materials were applied without SLS treatment on the right side. Additional SLS controls were placed on the left, while a fifth site challenged with petrolatum was placed on the right. The challenge sites were read at patch removal and 24 hours after patch removal. 20 % no effects. (RIFM,1975) [Epstein,1975] Cross sensitization Route: skin. Species: guinea pig. A second rechallenge and cross-challenge was conducted with the test material (Citroflex A-4) two weeks after the rechallenge to confirm the strong sensitization seen in the original challenge and rechallenge and to see if the related materials, Citroflex A-2 and Citroflex 2 cross-react with the test material Citroflex A-4. Patches were applied for 24 hours. Skin reaction sites were examined 24 and 48 hours after removal of the patches. Reactions were scored according to the following scale: 0 = no reaction; +/- = barely perceptible erythema; + = scattered, mild erythema (faint pink); ++ = moderate and diffuse erythema (pale pink); +++ = intense erythema (deep pink) and oedema. Strain of guinea pigs not provided. No further information provided. Study conducted in Subjects: 10 Unspecified Sex Study Length: 24 hours (Magnusson and Kligman, 1969 and 1970) Specified as Citroflex 2 vehicle was 0.01% Dobs/Saline for the intradermal induction and absolute ethanol for challenge.summary Citroflex 2 did not cause cross-reactions in animals treated with Citroflex A % skin effects, barely perceptible erythema was observed in 2/10 animals at the 24 hour reading. No skin reactions were observed at the 48 hour reading. One animal was sacrificed after the 24 hour reading because of an ulcerated neck-injection site. There was no evidence of crossreactions to Citroflex A-4. No skin reactions were observed in the control group. (CIR,1976) CIR Panel Book Page 293

300 Route: skin. Species: guinea pig. A rechallenge and cross-challenge was conducted with the test material (Citroflex A-2) two weeks after the original challenge to confirm the strong sensitization seen in the original challenge and to see if the related materials, Citroflex 2 and Citroflex A-4 cross-react with the test material Citroflex A-2. Patches were applied for 24 hours. Skin reaction sites were examined 24 and 48 hours after removal of the patches. Reactions were scored according to the following scale: 0 = no reaction; +/- = barely perceptible erythema; + = scattered, mild erythema (faint pink); ++ = moderate and diffuse erythema (pale pink); +++ = intense erythema (deep pink) and oedema. Strain of guinea pigs not provided. No further information provided. Study conducted in Subjects: 10 Unspecified Sex Study Length: 24 hours (Magnusson and Kligman, 1969 and 1970) Specified as Citroflex 2 vehicle was 0.01% Dobs/Saline for the intradermal induction and absolute ethanol for challenge.summary Under the conditions of the study, Citroflex 2 cross-reacts with Citroflex A % cross sensitization, 8/10 animals exhibited positive sensitization reactions at the 24 hour reading. 9/10 animals exhibited positive sensitization reactions at the 48 hour reading. No skin reactions were observed in the control group. The test material (Citroflex 2) cross-reacts with Citroflex A-2. (CIR,1976) Immunotoxic or immunological effects. Route: in vitro. Species: human 18+ yrs. The dilution effects of various test materials 1:20 on anti-a antibody, both immune and natural, were evaluated. No further details were provided. No effects, Both immune and natural. (Guess,1968) Interaction Route: skin. Species: rat. The effects of the test material as the vehicle on the dermal metabolism, transdermal absorption and antihypertensive activity of viprostol were studied in spontaneously hypertensive male rats. Materials were simultaneously applied to the skin for 24 hours. Interaction, Triethyl citrate decreased the antihypertensive effect, skin metabolism and skin penetration of viprostol. The rate of in vitro hydrolysis of viprosol in rat skin hemogenates was also reduced. (Nicolau,1989) Route: skin. Species: rabbit. The effects of the test material as a vehicle on the irritation produced by alpha-hexyl cinnamic acid were studied. A closed patch test was applied to the backs of rabbits. Data from English abstract and figures only (values estimated from figures). 50 % interaction, The primary irritation score for alpha-hexyl cinnamic acid was about 1.5 at 50% in diethyl phthalate, 1.6 at 50% in triethyl citrate, 1.9 at 50% in diisobutyl adipate, 2.5 at 50% in glyceryl-tri-2-ethyl-hexanoate, 3.2 for neat material, 3.7 at 50% in dipropylene glycol and 5.6 at 50% in ethanol. (Hyakutake,1985) Route: in vitro. Species: rabbit. The effects of various vehicles on the permeation of alphahexylcinnamic acid throught rabbit skin were studied. The Piled Membrane method was used with stratum corneum obtained from rabbit ears. The stratum corneum was swollen with water prior to use. Data from English abstract and figures only. 50 % interaction, The permeation of alpha-hexyl cinnamic acid was about the same with neat material, glyceryl-tri-2-ethyl-hexanate as the vehicle or dipropylene glycol as the vehicle. Ethanol as a vehicle increased permeation. Diethylphthalate decreased permeation. (Hyakutake,1985) Subchronic toxicity Route: food. Species: rat. The effect of the test material on the growth and blood count of rats was evaluated. Immature male and female Wistar rats, 21 days old, were allowed free access to food pellets and water. Various concentrations of the test material were thoroughly mixed with the food pellets, and the food was given to the animals for 6 weeks. The controls were given food CIR Panel Book Page 294

301 that was not treated with the test material. The animals were weighed weekly to evaluate the effect on nutrition and growth. A urine examination and complete blood count were made at the start, after 3 weeks, and again after 6 weeks of feeding. 0.5 % no effects, Daily consumption was 1 g/kg. No negative effect on the growth was produced. 1 % no effects, Daily consumption was 2 g/kg. No negative effect on the growth was produced. 2 % no effects, Daily consumption was 4 g/kg. No negative effect on the growth was produced. (Finkelstein,1959) Route: food. Species: rat. The effect of the test material on the growth and blood count of rats was evaluated. Immature male and female Wistar rats, 21 days old, were allowed free access to food pellets and water. Various concentrations of the test material were thoroughly mixed with the food pellets, and the food was given to the animals for 8 weeks. The controls were given food that was not treated with the test material. The animals were weighed weekly to evaluate the effect on nutrition and growth. In addition, a complete blood count (red blood count (RBC), white cell count (WBC), and differential) was made at the start, and again at 4 and 8 weeks, using selected animals from each group. At the end of the 8-week treatment period, treated and control rats were sacrificed and gross examination was conducted. A total of 63 histological sections were examined. 2 % no effects, Daily consumption was 4 g/kg. No negative effect on the growth or the RBC, WBC and differential counts were produced. No gross abnormalities in the thoracic and abdominal organs were observed, and the histological sections were indistinguishable from the controls. 0.5 % no effects, Daily consumption was 1 g/kg. No negative effect on the growth or the RBC, WBC and differential counts were produced. No gross abnormalities in the thoracic and abdominal organs were observed, and the histological sections were indistinguishable from the controls. 1 % no effects, Daily consumption was 2 g/kg. No negative effect on the growth or the RBC, WBC and differential counts were produced. No gross abnormalities in the thoracic and abdominal organs were observed, and the histological sections were indistinguishable from the controls. (Finkelstein,1959) Route: gavage. Species: cat. A subchronic study was conducted in which 6 cats were administered multiple doses of the test material by stomach tube for 2 months, while the 2 controls were not administered the test material. Observations and tests were made at intervals of 7 10 days, and included blood counts (red blood count RBC, white cell count WBC, and hemoglobin), blood chemistry (blood sugar, blood nitrogen and blood creatinine), and urine tests. In addition, electrocardiograms were taken at various intervals. Gross examinations of the thoracic and abdominal organs were made after the animals died or were sacrificed. No further details were provided. The ATEC dose tested was 7% of the reported LD50 (7 ml/kg) and the TEC dose tested was 7% of the reported LD50 (3.5 ml/kg) - see sub. ref #4 for the LD50 study. The text states that TEC was tested at 0.25 ml/kg, and the results table lists it as 0.3 ml/kg ml/kg clinical signs, No effect on body weight, blood chemistry, blood count and hemoglobin was observed. The electrocardiograms showed considerable fluctuations in the T-waves, but no difference between the treated and control animals was observed. Mild symptoms of poisoning appeared after the 4th or 5th doses such as weakness, ataxia and depression, but all animals survived the entire 8-week treatment period and recovered within 1 4 days after the doses were discontinued. (Finkelstein,1959) Route: gavage. Species: cat. To evaluate the cumulative effects of the test material over 8 weeks, 3 cats per group were administered the test material by stomach tube daily at approximately 50% of an LD50, while another group was administered the test material at approximately 25% of an LD50. No further details were provided. This study was a continuation of the study in sub-ref. #9 (test doses at 7% of the LD50). Also see sub-ref. #4 for the LD50 CIR Panel Book Page 295

302 study. 2 ml/kg lethal, after 8 daily doses. 1 ml/kg lethal, after 9 doses given at 4-hour intervals. (Finkelstein,1959) Developmental and reproduction studies Route: unclassified. Species: chick embryo. Toxicity and teratogenicity were evaluated under four conditions: injection via air cell or via the yolk sac at two time intervals (preincubation and 96 hour) for each route. All test materials were administered by a single injection in a volume of 100 ul or less, and solvent controls were treated with the same volume of the solvent only. The test material was dissolved or suspended in an appropriate vehicle. For each condition, at least 100 embryos per dose level were treated at a minimum of five dose levels. Eggs from Single- Comb White Leghorn chickens were used. Appropriate groups of vehicle controls and untreated controls were included. After treatment, all eggs were candled daily and nonviable embryos were removed. Embryos and hatched chicks were examined grossly for any functional or structural abnormalities in development. Abnormalities were tabulated and compared with values for the vehicle controls to determine teratogenicity. At least five embryos and hatched chicks were randomly selected from the highest dose level group for examination of the viscera. Dose listed is highest of 5 tested and units are actually mg/egg. Mortality is reported in the associated LD50 study. Methodology Reference: McLaughlin and Verrett, 1964 (MF1137). Vehicle was absolute ethanol. 10 mg no effects. (Verrett,1980) Neurotoxic or neurological effects (don' Route: unclassified. Species: rat. White animals were anesthetized with pentobarbital, and right anterior tibialis muscle freed and arranged for recording contractions. Shielded electrodes were placed central to a piece of cotton around the sciatic nerve. Stimulus was supplied through the electrodes by a stimulator set to deliver a current which would produce a submaximal response every 10 seconds After obtaining a normal response, 3 drops of test substance were placed on the cotton. Neurological effects, complete reversible sciatic block was observed. (Meyers,1964) Route: unclassified. Species: rat. Pentobarbital anesthetized white animals were set up so that the contralateral reflex could be recorded. After freeing the anterior tibialis muscle in the left leg and arranging it for recording contractions, shielded electrodes were placed around the sciatic nerve in the right leg. The stimulator was set to deliver a series of five impulses 10 sec apart when activated by a switch. After determining a normal submaximal contralateral reflex, each ester was administered through the jugular vein which had previously been cannulated. Doses not specified. Neurological effects, complete blockade of the reflex was observed. (Meyers,1964) Route: surface of eye. Species: rabbit. Three drops of a 5% suspension of test substance were placed in the conjunctival sac of the animal and corneal reflex was tested. Neurological effects, corneal reflex was temporarily abolished. (Meyers,1964) Neurotoxicity Route: intraperitoneal. Species: mouse. Test materials were administered to Swiss albino mice. Test materials were suspended or dissolved in 3% acacia. No further details were provided. Clinical signs, at doses slightly exceeding 400 mg/kg, a very rapid loss of righting reflex wtihout loss of consciousness was observed. Animals regained their posture within 15 minutes. Respiration rate was markedly increased and frequently clonic convulsions were observed. (Meyers,1964) Route: intraperitoneal. Species: rat. Wistar rats were used in this study. Test material was CIR Panel Book Page 296

303 administered suspended or dissolved in 3% acacia. No further information provided. Clinical signs, a rapid loss of righting reflex of short duration when the dose exceeded 400 mg/kg. (Meyers,1964) Route: intravenous. Species: rabbit. Test materials were administered suspended or dissolved in 3% acacia. No further information was provided. 100 mg/kg clinical signs, marked increases in motor activity and respiration were observed. (Meyers,1964) Route: unclassified. Species: frog. Test material was administered suspended or dissolved in 3% acacia into the ventral lymph sac of the frog. No further information provided mg/kg clinical signs, observations included a short period of clonic activity, followed by the complete abolishment of all reflex activity. (Meyers,1964) Cytotoxicity Route: in vitro. Species: unspecified mammal. The cytotoxicity of 29 plasticizers toward HeLa cells was tested by the Metabolic Inhibition Test supplemented by microscopy of cells after 24 hr incubation (MIT-24). The compounds were directly suspended in medium & incubated at 37 degrees C for 7 days. Cell viability determined after 24 hr by, microscopy. Another endpoint of viability was measured after 7 days by observing the color change of the phenol red of the medium. The "time-progressive acidity of substances" (hydrolysis) as well as a zone of hyperacid reaction at sub-inhibitory conc were also recorded at 7 days for some cmpds. 10 mg/ml caused partial inhibition at both 24 hr & 7 days with a zone of hyper-acid reaction at 7 days at 0.45 mg/ml in 1/2 cultures; 50 mg/ml caused total inhibition at both 24 hr & 7 days with timeprogressive acidity. (Ekwall,1982) Route: in vitro. Species: mouse. Mouse fibroblast cells were exposed to test material for 4 hours. The vehicle was growth medium. Phase-contrast time-lapse photography was used to evaluated morphological alterations. 7.9 millimolar positive effects, mitochodrial swelling and slowed growth were seen. 9 millimolar positive effects, mitochodrial swelling and slowed growth were seen. 48 millimolar lethal. 24 millimolar lethal. 12 millimolar lethal, plasma membrane contraction and cytoplasmic gelation were seen prior to mitochondrial swelling and lysis at this dose and above. (Jones,1968) Miscellaneous Route: food. Species: rat. Re-examined the nutritional value of 36 chemicals in rats & compared the ability to utilize these chemicals between rats & chicks (previously published data). Groups of 4 Wistar males were fed diets containing chemicals. BW taken every 6 days. Gross energy (kcal/g), available energy (kcal/g), availability (AE/GE) & palatability indices (%), digestibility coefficient(%) & evaporation loss (%)were calculated for each chemical in 7 trials. Figures in parentheses denote availability for chicks. Gross energy = 5.28 kcal/g; available energy -; AE/GE - (32); palatability index = 85. (Yoshida,1971) Analytical methodology. Can also refer to physical properties such as boiling temperature, flash point, etc. Stull,1947. Vapor pressures were collected for over 1200 organic compounds. Ramsey,1980. Hull,1984. Bouma,2002. Sixty two soft toys and childcare items were analyzed for plasticizers and other compounds. DINP and DEHP release was measured by the head over heels method. CIR Panel Book Page 297

304 Krebs,2003. Five air fresheners were analyzed for the constituent VOC's. Haggard,2006. The purpose of this study was to provide a reconnaissance of the occurrence of antibiotics, pharmaceuticals, and other organic chemicals of emerging concern in selected streams in northwestern and north-central Arkansas. The importance of this survey study was to provide an inventory of pharmaceuticals and other organic chemicals that may be found in these streams so that more detailed studies can be conducted on the transport and fate of these chemicals in aquatic environments. cals in aquatic environments. Article in foreign language; no English translation available. Guiot,1992. Biological tests other than classical toxicology testing and pharmacological tests. Golaz,1967. The mechanism of triethyl citrate toxicity toward in vitro mouse L-929 fibroblast cells was studied Kannikoski,1984. Comparison of test methodologies, models. Munro,2001. Comparison of ADI with NOELs from animal studies. Computational methodology including use of computers in toxicology or safety evaluation. Also includes articles on databases. Abraham,1995. Interaction of substances. See also promotion (PROMOTE), cocarcinogenicity (COCAR), cross reactivity (CROSS), enzyme induction (ENZ), quenching (QUENCH), protection (PROTECT), drugs (DRUG), synergism (SYN). Groten,2000. Mixture. Gaworski,1998. Mixtures were tested not individual ingredients Baker,2004. The potential chemical changes and biological activity of smoke from cigarettes with added ingredients was investigated. The ingredients of the experimental cigarettes were listed. Baker,2004a. The effects of flavoring and additives on smoke chemistry was investigated. The individual ingredients used in the experimental ingredient mixtures were reported. Papers that are a review on a general subject, not a specific material. TREV papers may contain one or more RIFM materials. TREV papers do not contain original data. Lanzet,1986. Reports on patients such as patch testing. DeGroot,1994. Synonyms, patch test concentrations and vehicles with references, in addition to comments for each chemical are listed. Review articles. Papers that are reviews on a material or a class of materials. A review paper may be on one or more RIFM materials. Review papers do not contain original data. FASEB,1977. Phillips,2005. A two phase study was designed to provide information concerning the character and concentration of ECs detected in wastewaters and the transport and fate of ECs through wastewater treatment plants and their impact on streams. The second phase focused on the CIR Panel Book Page 298

305 activated sludge preformed in wastewater plants. Blackburn,2005. The list of chemicals was used to identify chemicals for retrieval of toxicity data and identification of a set of NOEL values. Structure activity relationships such as studies of a group of aldehydes or other similar structures. Lipnick,1988. Cronin,1994. Rastogi,1998. Devillers,2000. Studies on cosmetic products or directly related thereto. Maibach,1980. Lukacs,1991. Test methodology (new or altered classical), testing procedures, guidelines, etc. If applicable, see also biological tests (BIOTS). ICCVAM,1999. This study provides a comparison of breakthrough times for acids as a function of concentration. Environmental Data Environmental Species/Media : sludge. A study was conducted to determine the ready and ultimate biodegradability of the test material using the sealed vessel test. The test was conducted in 160 ml vessels (hypovials) containing 100 ml mineral salts medium inoculated with secondary effluent and the respective test or reference substance. The inoculum used was 10% by volume of activated sludge plant secondary effluent, filtered through a Whatman filter paper (541) to remove coarse particulate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the ph to 6.5. Test concentration was nominal 10 mg/l organic carbon. Test temperature range was C. Multiple vessels were prepared per test material sealed with a butyl rubber septum and an aluminium crimp seal. The headspace in each vessel had a volume of 60 ml and when filled with air, contained approximately 6 times the mass of oxygen required for the complete oxidation of the test material. The sealed vessels were incubated at 20 C on a rotary shaker. At intervals during the 28 day test period a vessel was removed and concentration of carbon dioxide in the headspace gas determined. The seal is then broken and the concentration of inorganic carbon in the test medium was determined. Analysis of both the headspace gas and the liquid medium for CO2/DIC was performed on day numbers: 3, 8, 10, 14, 17, 21, 24 and 28 using the Ionics 555 Inorganic Carbon Analyser. The total inorganic carbon in the vessel was calculated and corrected by subtracting the inorganic carbon produced in the control. The control vessels were identical to the test vessels except for the omission of the test material. From a knowlege of the initial organic carbon concentration added as test substance, the extent of mineralisation was determined. A test substance was considered readily and ultimately biodegradable if the material exceeded 60% biodegradation within a 10 day window over 28 days Reference material not provided. Nominal carbon concentrations of the test materials were used based on the calculated CIR Panel Book Page 299

306 percentage carbon (from molecular formula) assuming 100% purity of the test material. Study Length: 28 days Regulatory Compliance OECD Guideline 301(B) GLP Yes R554/ Summary The test material failed the test and therefore cannot be classified as readily and ultimately biodegradable. 10 mg/l non-biodegradable, % biodegradation (nominal) of test material after 28 days (95% confidence limits) = 28.4 ( ). (Quest,1995) Species/Media : sludge. The Ready Biodegradability of the test material was determined by the Manometric Respirometry Test. Fresh activated sludge from a biological wastewater treatment plant treating predominantly domestic sewage was used. The sludge was collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day. The dry weight of suspended solids was determined by taking two 50 ml samples of the homogenized sludge, evaporating water on a steam bath, drying in an oven at C for two hours and weighing the residue. The toxicity of the test chemical for the inoculum was checked. A pair of flasks of the volumetric respirometer (SAPROMAT D 12) were filled with: mineral medium + test chemical (100 mg/l) + sodium benzoate (100 mg/l) + inoculum, and their respirations were recorded as done for the other flasks. If they were lower than those of the flasks containing: mineral medium + sodium benzoate (100 mg/l) + inoculum, the test chemical can be assumed to be inhibitory to the inoculum used. Test material samples (25 mg, corresponding to 100 mg/l in a 250 ml flask) were weighed in small aluminum boats and added directly to the test flasks of the SAPROMAT, where as reference samples (sodium benzoate) were added as 1.0 ml of a 25 mg/ml solution in mineral medium. All flasks were filled with 250 ml of mineral medium. Samples of test or reference substance or both were added. Then, a volume of suspended sludge corresponding to 7.5 mg dry weight was added. The ph of each flask was measured and adjusted as necessary. About 2 g of soda lime was placed in an attachment of the stopper, then flasks closed and placed in the water bath of the SAPROMAT. After temperature and pressure equilibration, the oxygen meters of the instrument were set to zero (time zero of the experiment). Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring were checked. At the end of the test period (normally 28 days), the ph of each flask was measured again. Nominal concentrations of test substance and reference substance (sodium benzoate) were 100 mg/l. Dry weight of suspended solids was g/l. To obtain a concentration of 30 mg/l (dry weight) in a 250 ml flask, 2.94 ml of sludge was needed (inoculum). Test temperature was 22 C; test duration was 32 days. Study Length: 32 days Regulatory Compliance OECD Guidelines for Testing of Chemicals, Method No. 301F (1992). GLP Yes Summary Under the conditions of this study, test material was readily biodegradable. 100 mg/l readily biodegradable, The test material underwent 90% biodegradation after 32 days (90% after 28 days) in the test conditions. Biodegradation started on day 3 and reached 74% at the end of the 10-day window (days 3-13). Test material should be regarded as readily biodegradable according to the test. (Givaudan,1998) Species/Media : sludge. The inherent biodegradability of the test material was determined in a sealed vessel CO2 production test using an acclimatized inoculum from a modified semicontinuous activated sludge test (SCAS). The SCAS test was used to acclimatize microorganisms to the test substance. A 10 ul aliquot of the test material was added to activated sludge in a SCAS aeration unit and aerated for approximately 23 hours. Next the aeration was stopped, the mixture was settled, and the supernatant liquor was removed. Settled domestic sewage and the test material were added to the settled sludge and the aeration was re-started. The cycle was repeated for 13 weeks during which time conditions favorable for the acclimatization CIR Panel Book Page 300

307 of the microorganisms to the test material were ensured. The test temperature was C. To determine the biodegradability of the test material, the pre-exposed microorganisms were used as inoculum in the sealed vessel test, and the inoculum was filtered through a Whatman filter paper to remove coarse particulate matter. The level of dissolved inorganic carbon (DIC) was reduced by sparging the filtered effluent with nitrogen after prior adjustment of the ph to 6.5. The test was conducted in 160 ml vessels containing 100 ml medium and up to mg/l as organic carbon of test or reference substance (11.7 mg/l as the test material and 8.9 mg/l as carbon). The reference substance was chosen because it had previously been shown to be readily biodegradable in The Sealed Vessel Test. Multiple vessels were prepared and sealed. The headspace in each vessel had a volume of 60 ml and when filled with air, contained approximately 6 times the mass of oxygen required for the complete oxidation of the test material. The sealed vessels were incubated for 4 weeks at C on a rotary shaker, and on day 28, the vessels were removed and the concentration of carbon dioxide in the headspace gas was determined. The seal was broken and the concentration of inorganic carbon in the test medium was also determined. The total inorganic carbon in the vessel was calculated and corrected by subtracting the total inorganic carbon produced in the control vessels, which were identical to the test vessels except for the absence of the test material. As the initial organic carbon concentration added as test material was known, the extent of mineralization could be determined. Regulatory Compliance OECD Guide-line 301 B; EEC Directive 87/548/EEC, Part C-Biodegradation-Modified SCAS Test; OECD Guidelines for Testing of Chemicals, Section 3, Guideline 302A, Inherent Biodegradability, Modified SCAS Test. GLP Yes Summary The average extent of mineralization (after 28 days) in the sealed vessel test using an acclimatized inoculum was 96.4%, which was sufficient to classify the material as inherently ultimately biodegradable mg/l Degradation was seen, The average extent of mineralization (after 28 days) in the sealed vessel test using an acclimatized inoculum was 96.4% (95% C.I ), which was sufficient to classify the material as inherently ultimately biodegradable. (Quest,1997) Species/Media : water. A study was conducted by the United States Geological Survey (USGS) and United States Environmental Protection Agency (USEPA) in 2002 to evaluate the utility of a suite of organic chemical compounds as specific indicators of human fecal contamination. The results were intended to determine which wastewater compounds are commonly found downstream from Waste Water Treatment Plants (WWTPs) and provide insight on their environmental persistence, the initial phase in determining if these compounds are useful chemical indicators of human fecal contamination. The study was designed to explore the correlation between the presence of the chemicals and known human waste sources and not the relation of the chemicals directly to the pathogens that are presumably present within the waste. The study focused on 10 WWTPs across the United States. Site selection was primarily based on the results of previous research activities. Most of the sample sets consisted of one upstream, one effluent and two downstream sampels (DS1 = sites proximal to WWTP discharge and DS2 = sites further downstream from WWTP discharge). The network consisted of 40 sampling sites: 9 upstream samples (one site had no upstream sampling point), 11 WWTP effluent samples (one site had two WWTP discharge points) and 20 downstream samples. The 10 locations represent a variety of climatic conditions, populations served, stream sizes and treatment practices. The distances from the treatment plants to the upstream and downstream locations vary because of sampling accessibiltiy. The discharge from the WWTPs contributed between 10 and 95% of the streamflow at the DS1 site. The samples from the Arizona location are unique in that the stream is composed entirely of wastewater; thus, the "upstream" sample is actually a sample from the CIR Panel Book Page 301

308 channel immediately downsteam from a wastewater treatment plant. For all the data interpretation, the Arizona location was considered to have two WWTP samples and no upstream sample. In addition to the 10 WWTP influenced locations, samples were collected from 2 remote sites in Michigan and Montana in areas having minimal direct impact from human wastewater. These samples were not included when calculating frequencies of detection or any other statistical analysis. All of the samples were collected by USGS personnel. For the stream samples, standard width- and depth-integrating techniques were used to ensure a representative water sample. The effluent sample was collected as a grab sample from the discharge. The collected water samples were placed in baked amber glass bottles and shipped on ice for analysis to the USGS National Water Quality Laboratory in Colorado and the USGS Organic Geochemistry Research Laboratory in Kansas for analysis. Three different analytical methods were used because of the differences in the physiochemical properties of the compounds. The three methods were referred to as the "pharmaceutical method", "wastewater method", and the "antibiotic method". Microbial analysis was also conducted. Samples were collected in presterilized Teflon bottles and shipped overnight on ice for analysis to USEPA, Office of Development's National Exposure Research Laboratory in Ohio for analysis. Three different aliquots (1, 10 and 100 ml) were analyzed in triplicate by two different USEPA methods: the modified E. coli method (modified from method ;mTEC) and modified enterococci method (method 1600;MEI). For quality control, compound concentrations were blank corrected to zero if the concentrations in the environmental samples were less than 10 times that measured in the associated laboratory blanks. In addition, two replicate field blanks were collected and processed after collection of a surface water sample comprised wholly of treated wastewater effluent at a site in Arizona. Field blank samples were also used to blank correct the associated field samples, using the same correction level of 10 times the detected concentration. Results included the median concentration, maximum concentration and frequency of detection (%) for test material investigated in the study. Data was also evaluated by sample site. Summary The frequency of detection of the test material = 72.5%; maximum concentration = 0.52 ug/l; median concentration = ug/l; reporting level = 0.5 ug/l. The frequency of detection of the test material = 72.5%; maximum concentration = 0.52 ug/l; median concentration = ug/l; reporting level = 0.5 ug/l. (Glassmeyer,2005) Species/Media : sludge. The presence of various organic contaminants in final clarifier effluents of wastewater treatment plants was analyzed. Effluent samples were collected from a rural and an urban treatment plant during dry weather, and placed in 10-liter glass bottles containing sodium azide to preserve the samples. The samples were filtered through cellulose acetate membranes, and the filtrates were divided into smaller volumes (0.1 1 liter) and stored at 4oC until analysis by gas chromatography/mass spectroscopy (GC/MS). The concentration of various substances identified in the urban and rural filtrates was reported. The concentration in the urban effluent was 5.6 ug/l, but could not be quantified in the rural effluent. (Gulyas,1997) Species/Media : water. During 2001, 76 water samples were collected upstream and downstream of select towns and cities in Iowa during high-, normal- and low-flow conditions to determine the contribution of urban centers to concentrations of pharmaceuticals and other organic wastewater contaminants (OWCs) in streams under varying flow conditions. Water samples from 23 stream locations situated upstream and downstream of 10 cities in Iowa were sampled three times during high, normal and low streamflow conditions. For some cities, two upstream sites were necessary because of the presence of multiple tributaries. During the low-flow sampling period, four additional urban centers were included. For one of these added urban areas, however, there was CIR Panel Book Page 302

309 no flow at the upstream site at the time of sample collection. The 14 towns and cities ranged in population from approximately 2000 to 200,000. All samples were collected by US Geological Survey personnel using consistent protocols and procedures designed to obtain a represnetative water sample using standard depth and width integrating techniques. At each site, a composite water sample was collected from approximately 4 to 6 verticla profiles through a stream cross section. The composite sample was subsequently split into pre-cleaned, amber, glass bottles and prepared in duplicate. The duplicate samples were used for backup purposes and for laboratory replicates. Those samples requiring filtration were passed through a 0.7 um, baked glass-fiber filter. Upon collection, all samples were immediately chilled and shipped to the laboratory for analysis. To minimize contamination of smaples, use of personal care items (i.e. insect repellents, colognes, perfumes), caffeinated products, pharmaceuticals and tobacco were discouraged during sample collection and processing. A total of 105 OWCs were analyzed in each water sample collected using three analytical methods developed by the US Geological Survey. 25 antibiotic compounds were extracted and analyzed by tandem solid-phase extraction (SPE) and single quadrapole, liquid chromatography/mass spectrometry with electro-spray ionization set in positive mode and selected ion monitoring (SIM). 21 human prescrption and non-prescription drugs and their selected metabolites were extracted by SPE and analyzed by high performance liquid chromatography (HPLC) using a polar reverse-phase octylsilane (C8) HPLC column. 67 other wastewater-releated compound were extracted by capillary-column gas chromatography/mass spectrometry. Six field blanks were collected during the study to determine if field conditions were introducing target analytes to the environmental samples. These blanks were prepared from laboratory grade organic-free water and were subject to the same sample processing, handling and equipment as the stream samples. At least one fortified laboratory spike and one laboratory blank was analyzed with each set of environmental samples All detections in the field blanks were near their respective reporting levels indicating that sample collection procedures did not systematically introduce target compounds into environmental samples. Environmental concentrations within 5 times the values observed in the corresponding field blank were reported as less than the reporting level. Positive effects, test material was detected by continuous liquid-liquid extraction with gas chromatography/mass spectrometry (CLLE) in Low-flow samples (n=30) at maximum concentration of 0.17 ug/l and frequency of 16.7%. Test material was not detected in High-flow samples (n=23) or Normalflow samples (n=23). (Kolpin,2004) Analytical methodology. Can also refer to physical properties such as boiling temperature, flash point, etc. Givaudan,1998a. The partition coefficient of triethyl citrate was determined. Alvarez,2004. the concentration of test material was measured in a stream that receives agricultural, municipal and industrial wastewaters using two sampling techniques. Environmental monitoring. Studies in which the presence of test material is evaluated in samples taken from streams, soils or other environmental samples. Barber,2007. Organic wastewater contaminants detected in the Metropolitan Wastewater Treatment Plant and effluent treated with XAD8 resin. Stackelberg,2007. Compounds detected in source water or solid samples. Environmental studies, tests or effects. Definition: fate and effect studies that include biodegradation, bioaccumulation, biotransformation, bioconcentration, soil adsorption, soil migration, sediment and water studies, etc. CIR Panel Book Page 303

310 Yun,1994. References Abraham M.H. and Rafols C. (1995) Factors that influence tadpole narcosis. An LFER analysis. Journal of the Chemical Society - Perkin Transactions, 2(10), Alvarez D.A., Stackelberg P.E., Petty J.D., Huckins J.N., Furlong E.T., Zaugg S.D. and Meyer M.T. (2004) Comparison of a passive sampler to standard water-column sampling techniques for organic contaminants associated with wastewater effluents. SETAC 25th Annual Meeting in North America, November, Portland, Oregon, 296. Baker R.A., Massey ED and Smith G. (2004) An overview of the effects of tobacco ingredients on smoke chemistry and toxicity. Food and Chemical Toxicology, 42S, S53-S83. Baker R.R., Pereira da Silva JR and Smith G. (2004a) The effect of tobacco ingredients on smoke chemistry. Part I: Flavourings and additives. Food and Chemical Toxicology, 42S, S3-S37. Barber L.B., Lee K.E., Swackhamer D.L. and Schoenfuss H.L. (2007) Reproductive responses of male fathead minnows exposed to wastewater treatment plant effluent, effluent treated with XAD8 resin, and an environmentally relevant mixture of alkylphenol compounds. Aquatic Toxicology, 82(1), Blackburn K., Stickney J.A., Carlson-Lynch H.L., McGinnis P.M., Chappell L. and Felter S.P. (2005) Application of the threshold of toxicological concern approach to ingredients in personal and household care products. Regulatory Toxicology and Pharmacology, 43(3), Bouma K. and Schakel D.J. (2002) Migration of phthalates from PVC toys into saliva simulant by dynamic extraction. Food Additives and Contaminants, 19(6), Cosmetic Ingredient Review (1976) Sensitization potential of acetyl triethyl citrate, tributyl acetylcitrate, and triethyl citrate as tested in guinea pigs. Unpublished. August 24 August 24a August 24b August 24d August 24e Cosmetic Ingredient Review (1978) Repeated insult patch test with acetyl triethyl citrate, tributyl acetylcitrate, and triethyl citrate. Unpublished. February 24 February 24a Cronin M.T.D. and Basketter D.A. (1994) Multivariate QSAR analysis of a skin sensitization database. Environmental Research, (SAR QSAR) 2(3), DeGroot A.C. (1994) Patch Testing. Test Concentrations and Vehicles for 3700 Allergens. In: Test Concentrations and Vehicles for 3700 Allergens, Devillers J. (2000) A neural network SAR model for allergic contact dermatitis. Toxicology Methods, 10(3), Ekwall B., Nordensten C. and Albanus L. (1982) Toxicity of 29 plasticizers to HeLa cells in the MIT-24 system. Toxicology, 24, Federation of American Societies for Experimental Biology (1977) Evaluation of the health aspects of citric acid, sodium citrate, potassium citrate, calcium citrates, ammonium citrate, triethyl citrate, isopropyl citrate, and stearyl citrate as food ingredients. FDA ; NTIS PB ; FDA/BF-78/96. Finkelstein M. and Gold H. (1959) Toxicology of the citric acid esters: Tributyl citrate, acetyl CIR Panel Book Page 304

311 tributyl citrate, triethyl citrate and acetyl triethyl citrate. Toxicology and Applied Pharmacology, 1(3), Gaworski C.L., Dozier M.M., Heck J.D., Gerhart J.M., Rajendran N., David R.M., Brennecke L.H. and Morrissey R. (1998) Toxicologic evaluation of flavor ingredients added to cigarette tobacco: 13-week inhalation exposures in rats. Inhalation Toxicology, 10(4), Givaudan (1998) Ready biodegradability of triethyl citrate. Unpublished. October 15 Givaudan (1998a) Partition coefficient n-octanol/water of triethyl citrate. Unpublished. Glassmeyer S.T., Furlong E.T., Kolpin D.W., Cahill J.D., Zaugg S.D., Werner S.L., Meyer M.T. and Kryak D.D. (2005) Transport of chemical and microbial compounds from known wastewater discharges: Potential for use as indicators of human fecal contamination. Environmental Science & Technology, 59(14), Golaz M., Guess W.L. and Autian J. (1967) Mechanistic toxicology of triethyl citrate in mouse fibroblast cells by liquid scintillation techniques. Journal of Pharmaceutical Sciences, 56(10), Groten J.P., Butler W., Feron V.J., Kozianowski G., Renwick A.G. and Walker R. (2000) An analysis of the possibility for health implications of joint actions and interactions between food additives. Regulatory Toxicology and Pharmacology, 31(1), Guess W.L. and Haberman S. (1968) Toxicity profiles of vinyl and polyolefinic plastics and their additives. Journal of Biomedical Materials Research, 2, Guiot P., Ryan M.A. and Hull M.H. (1992) Citrates and their applications. Inf. Chim., 343, Gulyas H. (1997) Discharge of Organic Contaminants to Rivers with Treated Municipal Wastewaters. In: Water Pollution IV: Modelling, Measuring and Prediction, Int. Conf. on Water Pollution, 4th, Haggard B.E., Galloway J.M., Green W.R. and Meyer M.T. (2006) Pharmaceuticals and other organic chemicals in selected north-central and northwestern Arkansas streams. J. Environ. Qual., 35(4), Hull E.H. and Mathur K.K. (1984) Citric acid esters as plasticizers for medical-grade PVC. Modern Plastics, 61(5), 66-68, 70. Hyakutake M., Nakajima H., Machida Y., Fujiyama Y. and Mitsui T. (1985) Correlation between permeability of stratum corneum and primary skin irritancy. Journal of the Society of Cosmetic Chemists Japan Japan, 19(1), Interagency Coordinating Committee on the Validation of Alternative Methods (1999) Corrositex: An in vitro test method for assessing dermal corrosivity potential of chemicals. NIH Publication No International Flavors and Fragrances (1977) Rabbit skin irritation test with triethyl citrate. Unpublished. International Flavors and Fragrances (1977a) Rabbit skin irritation test with triethyl citrate. Unpublished. May 03 International Flavors and Fragrances (1977b) Rabbit skin irritation test with triethyl citrate. Unpublished. May 06 International Flavors and Fragrances (1977c) Rabbit eye irritation test with triethyl citrate. CIR Panel Book Page 305

312 Unpublished. May 16 International Flavors and Fragrances (1977d) Rabbit eye irritation test with triethyl citrate. Unpublished. May 11 International Flavors and Fragrances (1977e) Rabbit eye irritation test with triethyl citrate Unpublished. May 03 IFF Incorporation (1977f) Sensitization study with triethyl citrate (citroflex 2) in human subjects. Unpublished. IFF Incorporated (1977g) Sensitization study with triethyl citrate (citroflex 2) in human subjects. Unpublished. IFF Incorporated (1977h) Repeated insult patch test with triethyl citrate (citroflex 2). Unpublished. IFF Incorporated (1977i) Repeated insult patch test with triethyl citrate (citroflex 2). Unpublished. IFF Incorporated (1977j) Repeated insult patch test with triethyl citrate (citroflex 2). Unpublished. Jones A.B., Guess W.L. and Autian J. (1968) Mechanistic toxicology of triethyl citrate in cultured mammalian cells by cinematography. Journal of Pharmaceutical Sciences, 57(2), Kannikoski A., Mannermaa S. and Marvola M. (1984) The effects of some tablet film coatings on the adherence of drug products to the isolated porcine oesophagus. Acta Pharmaceutica Fennica, 93, Kolpin D.W., Skopec M., Meyer M.T., Furlong E.T. and Zaugg S.D. (2004) Urban contribution of pharmaceuticals and other organic wastewater contaminants to streams during differing flow conditions. Science of the Total Environment, 328(1-3), Krebs K., de Jesus V., Mason M. and Liu X. (2003) Source characterization of air fresheners. Presented at the Indoor Air Quality Conference August 21-23, Research Triangle Park, NC. Lanzet M. (1986) Comedogenic effects of cosmetic raw materials. Cosmetics and Toiletries, 101, Lipnick R.L. (1988) A quantitative structure-activity relationship study of Overton's data on the narcosis and toxicity of organic compounds to the tadpole, Rana temporaria. ASTM Spec. Technical Publication, 1007(11th Vol.), Likacs A., Korting H.C., Braun-Falco O. and Stanzl K. (1991) Efficacy of a deodorant and its components: Triethylcitrate and perfume. Journal of the Society of Cosmetic Chemists Japan, 42(3), Maibach H.I., Akerson J.M., Marzulli F.N., Wenninger J., Greif M., Hjorth N., Andersen K.E. and Wilkinson D.S. (1980) Test concentrations and vehicles for dermatological testing of cosmetic ingredients. Contact Dermatitis, 6, Meyers D.B., Autian J. and Guess W.L. (1964) Toxicity of plastics used in medical practice. II. Toxicity of citric acid esters used as plasticizers. Journal of Pharmaceutical Sciences, 53(7), Munro I.C. and Kennepohl E. (2001) Comparison of estimated daily capita intakes of flavouring substances with no-observed-effect levels from animal studies. Food and Chemical Toxicology, 39(4), CIR Panel Book Page 306

313 Nicolau G., Dahlin D.C., Kohlbrenner M., Chan P.S., Ronsberg M.A., Saunders T.K., Yacobi A. and Cervoni P. (1989) Skin metabolism and transdermal absorptin of viprostol, a synthetic PGE2 analog, in the rat: Effect of vehicle. Skin Pharmacology, 2(1), Phillips P.J., Stinson B., Zaugg, S. D., Furlong E. T., Kolpin D.W., Esposito K.M., Bodniewicz B., Pape R. and Anderson J. (2005) A multi-disciplinary approach to the removal of emerging contaminants in municipal wastewater treatment plants in New York State, WEFTEC, Quest International (1995) The biodegradability of perfume ingredients in the sealed vessel test. Unpublished. July 20 Quest International (1997) Assessment of the inherent biodegradability of triethyl citrate in a sealed vessel CO2 production test using acclimatised effluent from a modified semicontinuous activated sludge test. Unpublished. January 30 Ramsey J.D., Lee T.D., Osselton M.D. and Moffat A.C. (1980) Gas-liquid chromatographic retention indices of 296 non-drug substances on SE-30 or OV-1 likely to be encountered in toxicological analyses. Journal of Chromatography A, 184, Rastogi S.C., Lepoittevin J.P., Johansen J.D., Frosch P.J., Menne T., Bruze M., Dreier B., Andersen K.E. and White I.R. (1998) Fragrances and other materials in deodorants: Search for potentially sensitizing molecules using combined GC-MS and structure activity relationship (SAR) analysis. Contact Dermatitis, 39(6), Research Institute for Fragrance Materials, Inc, (1975). Report on human maximization studies. Report to RIFM. Unpublished report 1798 from Epstein W.L. August 15B Research Institute for Fragrance Materials, Inc, (1975). Acute toxicity studies in rats, mice, and rabbits. Report to RIFM. Unpublished report 2024 from Levenstein I. May 30 Stackelberg P.E., Gibs J., Furlong E.T., Meyer M.T., Zaugg S.D. and Lippincott R.L. (2007) Efficiency of conventional drinking-water-treatment processes in removal of pharmaceuticals and other organic compounds. Science of the Total Environment, 377(2-3), Stull D.R. (1947) Vapor pressure of pure substances organic compounds. Industrial and Engineering Chemistry, 39(4), Takenaka T., Hasegawa E., Takenaka U., Saito F. and Odaka T. (1986) Fundamental studies of safe compound perfumes for cosmetics. Part 1. The primary irritation of compound materials to the skin. Unknown Source, pp Verrett M.J., Scott W.F., Reynaldo E.F., Alterman E.K. and Thomas C.A. (1980) Toxicity and teratogenicity of food additive chemicals in the developing chicken embryo. Toxicology and Applied Pharmacology, 56(2), Yoshida M., Ikumo H. and Suzuki O. (1971) Evaluation of available energy of aliphatic chemicals by rats. An application of bioassay of energy to mono-gastric animal. Agricultural and Biological Chemistry, 35(8), Yun S., Teraguchi T., Zhu X. and Iwashima K. (1994) Identification and analysis of dissolved organic compounds in Tamagawa River. Kankyo Kagaku, 4(2), No data added since 13-Dec-10 CIR Panel Book Page 307

314 CIR Panel Book Page 308

315 CIR Panel Book Page 309

316 CIR Panel Book Page 310

317 CIR Panel Book Page 311

318 CIR Panel Book Page 312

319 CIR Panel Book Page 313

320 CIR Panel Book Page 314

321 CIR Panel Book Page 315

322 CIR Panel Book Page 316

323 CIR Panel Book Page 317

324 CIR Panel Book Page 318

325 CIR Panel Book Page 319

326 CIR Panel Book Page 320

327 CIR Panel Book Page 321

328 CIR Panel Book Page 322

329 CIR Panel Book Page 323

330 CIR Panel Book Page 324

331 CIR Panel Book Page 325

332 CIR Panel Book Page 326

333 CIR Panel Book Page 327

334 CIR Panel Book Page 328

335 CIR Panel Book Page 329

336 CIR Panel Book Page 330

337 CIR Panel Book Page 331

338 CIR Panel Book Page 332

339 CIR Panel Book Page 333

340 CIR Panel Book Page 334

341 CIR Panel Book Page 335

342 CIR Panel Book Page 336

343 CIR Panel Book Page 337

344 CIR Panel Book Page 338

345 CIR Panel Book Page 339

346 CIR Panel Book Page 340

347 CIR Panel Book Page 341

348 CIR Panel Book Page 342

349 CIR Panel Book Page 343

350 CIR Panel Book Page 344

351 CIR Panel Book Page 345

352 CIR Panel Book Page 346

353 CIR Panel Book Page 347

354 CIR Panel Book Page 348

355 CIR Panel Book Page 349

356 CIR Panel Book Page 350

357 CIR Panel Book Page 351

358 CIR Panel Book Page 352

359 CIR Panel Book Page 353

360 CIR Panel Book Page 354

361 CIR Panel Book Page 355

362 CIR Panel Book Page 356

363 CIR Panel Book Page 357

364 CIR Panel Book Page 358

365 CIR Panel Book Page 359

366 CIR Panel Book Page 360

367 CIR Panel Book Page 361

368 CIR Panel Book Page 362

369 CIR Panel Book Page 363

370 CIR Panel Book Page 364

371 CIR Panel Book Page 365

372 CIR Panel Book Page 366

373 CIR Panel Book Page 367

374 CIR Panel Book Page 368

375 CIR Panel Book Page 369

376 CIR Panel Book Page 370

377 CIR Panel Book Page 371

378 CIR Panel Book Page 372

379 CIR Panel Book Page 373

380 CIR Panel Book Page 374

381 CIR Panel Book Page 375

382 CIR Panel Book Page 376

383 CIR Panel Book Page 377

384 CIR Panel Book Page 378

385 CIR Panel Book Page 379

386 CIR Panel Book Page 380

387 CIR Panel Book Page 381

388 CIR Panel Book Page 382

389 CIR Panel Book Page 383

390 CIR Panel Book Page 384

391 CIR Panel Book Page 385

392 CIR Panel Book Page 386

393 CIR Panel Book Page 387

394 CIR Panel Book Page 388

395 CIR Panel Book Page 389

396 CIR Panel Book Page 390

397 CIR Panel Book Page 391

398 CIR Panel Book Page 392

399 CIR Panel Book Page 393

400 CIR Panel Book Page 394

401 CIR Panel Book Page 395

402 CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID 33 01A - Baby Shampoos 10 01B - Baby Lotions, Oils, Powders, and Creams 69 01C - Other Baby Products 35 02A - Bath Oils, Tablets, and Salts 92 02B - Bubble Baths 2 02C - Bath Capsules 53 02D - Other Bath Preparations 65 03A - Eyebrow Pencil B - Eyeliner 56 03C - Eye Shadow 40 03D - Eye Lotion 14 03E - Eye Makeup Remover 10 03F - Mascara 54 03G - Other Eye Makeup Preparations 24 04A - Cologne and Toilet waters 22 04B - Perfumes 1 04C - Powders (dusting and talcum, excluding aftershave talc) 17 04E - Other Fragrance Preparation A - Hair Conditioner 6 05B - Hair Spray (aerosol fixatives) 10 05C - Hair Straighteners 5 05D - Permanent Waves 11 05E - Rinses (non-coloring) 1,006 05F - Shampoos (non-coloring) G - Tonics, Dressings, and Other Hair Grooming Aids 1 05H - Wave Sets I - Other Hair Preparations A - Hair Dyes and Colors (all types requiring caution statements and patch tests) 1 06B - Hair Tints 10 06C - Hair Rinses (coloring) 20 06D - Hair Shampoos (coloring) 3 06G - Hair Bleaches 5 06H - Other Hair Coloring Preparation 6 07A - Blushers (all types) 24 07B - Face Powders 33 07C - Foundations 3 07D - Leg and Body Paints E - Lipstick 5 07F - Makeup Bases 2 07H - Makeup Fixatives 57 07I - Other Makeup Preparations 10 08A - Basecoats and Undercoats 6 08B - Cuticle Softeners 4 08C - Nail Creams and Lotions E - Nail Polish and Enamel 3 08F - Nail Polish and Enamel Removers 17 08G - Other Manicuring Preparations 2 09A - Dentifrices 9 09B - Mouthwashes and Breath Fresheners 6 09C - Other Oral Hygiene Products A - Bath Soaps and Detergents 22 10B - Deodorants (underarm) 7 10C - Douches E - Other Personal Cleanliness Products 31 11A - Aftershave Lotion 5 11E - Shaving Cream 3 11G - Other Shaving Preparation Products A - Cleansing 12 12B - Depilatories C - Face and Neck (exc shave) D - Body and Hand (exc shave) 1 12E - Foot Powders and Sprays F - Moisturizing 56 12G - Night 42 12H - Paste Masks (mud packs) CIR Panel Book Page 396

403 CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE CITRIC ACID MONOHYDRATE 53 12I - Skin Fresheners J - Other Skin Care Preps 3 13A - Suntan Gels, Creams, and Liquids 71 13B - Indoor Tanning Preparations 4 13C - Other Suntan Preparations 2 02A - Bath Oils, Tablets, and Salts 5 02B - Bubble Baths 2 02D - Other Bath Preparations 3 05A - Hair Conditioner 8 05F - Shampoos (non-coloring) 1 05G - Tonics, Dressings, and Other Hair Grooming Aids 2 05I - Other Hair Preparations 1 07I - Other Makeup Preparations 1 08E - Nail Polish and Enamel 7 10A - Bath Soaps and Detergents 8 10E - Other Personal Cleanliness Products 6 12A - Cleansing 9 12C - Face and Neck (exc shave) 1 12D - Body and Hand (exc shave) 14 12F - Moisturizing 2 12G - Night 1 12H - Paste Masks (mud packs) 1 12I - Skin Fresheners 4 12J - Other Skin Care Preps 1 13B - Indoor Tanning Preparations ALUMINUM CITRATE ALUMINUM CITRATE ALUMINUM CITRATE 1 10A - Bath Soaps and Detergents 1 12D - Body and Hand (exc shave) 2 12I - Skin Fresheners Calcium Citrate 0 Copper Citrate 0 Disodium Cupric Citrate 0 FERRIC CITRATE FERRIC CITRATE FERRIC CITRATE FERRIC CITRATE FERRIC CITRATE FERRIC CITRATE 1 05A - Hair Conditioner 1 05F - Shampoos (non-coloring) 2 05G - Tonics, Dressings, and Other Hair Grooming Aids 1 10A - Bath Soaps and Detergents 1 12F - Moisturizing 1 12J - Other Skin Care Preps MAGNESIUM CITRATE MAGNESIUM CITRATE 6 05A - Hair Conditioner 3 05F - Shampoos (non-coloring) Manganese Citrate 0 MONOSODIUM CITRATE MONOSODIUM CITRATE MONOSODIUM CITRATE 13 02A - Bath Oils, Tablets, and Salts 1 02C - Bath Capsules 2 12A - Cleansing POTASSIUM CITRATE POTASSIUM CITRATE POTASSIUM CITRATE POTASSIUM CITRATE POTASSIUM CITRATE POTASSIUM CITRATE 1 03G - Other Eye Makeup Preparations 1 05A - Hair Conditioner 2 05C - Hair Straighteners 1 07E - Lipstick 1 10A - Bath Soaps and Detergents 2 10E - Other Personal Cleanliness Products CIR Panel Book Page 397

404 SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT SODIUM CITRATE DIHYDRAT 2 01A - Baby Shampoos 6 01B - Baby Lotions, Oils, Powders, and Creams 1 01C - Other Baby Products 3 02A - Bath Oils, Tablets, and Salts 4 02B - Bubble Baths 3 03B - Eyeliner 23 03D - Eye Lotion 5 03E - Eye Makeup Remover 2 03F - Mascara 14 03G - Other Eye Makeup Preparations 7 04E - Other Fragrance Preparation 14 05A - Hair Conditioner 1 05C - Hair Straighteners 4 05D - Permanent Waves 1 05E - Rinses (non-coloring) F - Shampoos (non-coloring) 7 05G - Tonics, Dressings, and Other Hair Grooming Aids 16 05I - Other Hair Preparations 5 06C - Hair Rinses (coloring) 35 07C - Foundations 1 07F - Makeup Bases 3 07H - Makeup Fixatives 3 07I - Other Makeup Preparations 2 09A - Dentifrices 2 09B - Mouthwashes and Breath Fresheners 3 09C - Other Oral Hygiene Products 61 10A - Bath Soaps and Detergents 1 10B - Deodorants (underarm) 4 10C - Douches 11 10E - Other Personal Cleanliness Products 9 11A - Aftershave Lotion 5 11G - Other Shaving Preparation Products 90 12A - Cleansing C - Face and Neck (exc shave) 25 12D - Body and Hand (exc shave) 1 12E - Foot Powders and Sprays F - Moisturizing 24 12G - Night 10 12H - Paste Masks (mud packs) 29 12I - Skin Fresheners 57 12J - Other Skin Care Preps 4 13A - Suntan Gels, Creams, and Liquids 17 13B - Indoor Tanning Preparations 1 13C - Other Suntan Preparations 1 07E - Lipstick 1 07I - Other Makeup Preparations 1 08E - Nail Polish and Enamel 1 08G - Other Manicuring Preparations 4 10A - Bath Soaps and Detergents 1 10E - Other Personal Cleanliness Products 3 12C - Face and Neck (exc shave) 3 12F - Moisturizing 1 12I - Skin Fresheners 2 12J - Other Skin Care Preps ZINC CITRATE ZINC CITRATE ZINC CITRATE 1 07I - Other Makeup Preparations 4 09A - Dentifrices 4 10B - Deodorants (underarm) DIAMMONIUM CITRATE DIAMMONIUM CITRATE 1 03F - Mascara 3 05F - Shampoos (non-coloring) CIR Panel Book Page 398

405 DIAMMONIUM CITRATE DIAMMONIUM CITRATE 1 10A - Bath Soaps and Detergents 1 12J - Other Skin Care Preps STEARYL CITRATE STEARYL CITRATE STEARYL CITRATE STEARYL CITRATE STEARYL CITRATE 1 05A - Hair Conditioner 2 05F - Shampoos (non-coloring) 7 10A - Bath Soaps and Detergents 12 10E - Other Personal Cleanliness Products 1 12C - Face and Neck (exc shave) Isopropyl Citrate 0 ISODECYL CITRATE ISODECYL CITRATE 2 12C - Face and Neck (exc shave) 2 12D - Body and Hand (exc shave) DILAURYL CITRATE 1 12A - Cleansing Distearyl Citrate 0 TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE TRIETHYL CITRATE 1 03C - Eye Shadow 1 03G - Other Eye Makeup Preparations 4 04A - Cologne and Toilet waters 11 04B - Perfumes 10 04E - Other Fragrance Preparation 61 05B - Hair Spray (aerosol fixatives) 1 05F - Shampoos (non-coloring) 11 05G - Tonics, Dressings, and Other Hair Grooming Aids 19 05H - Wave Sets 14 05I - Other Hair Preparations 3 07A - Blushers (all types) 1 07B - Face Powders 6 07C - Foundations 11 07E - Lipstick 2 07I - Other Makeup Preparations 2 10A - Bath Soaps and Detergents 48 10B - Deodorants (underarm) 4 10D - Feminine Deodorants 1 10E - Other Personal Cleanliness Products 6 12A - Cleansing 5 12C - Face and Neck (exc shave) 5 12D - Body and Hand (exc shave) 4 12F - Moisturizing 1 12G - Night 3 12I - Skin Fresheners 3 12J - Other Skin Care Preps 6 13B - Indoor Tanning Preparations TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE 1 01B - Baby Lotions, Oils, Powders, and Creams 5 02A - Bath Oils, Tablets, and Salts 12 02B - Bubble Baths 12 02D - Other Bath Preparations 4 04A - Cologne and Toilet waters 2 04E - Other Fragrance Preparation 1 05A - Hair Conditioner 4 05F - Shampoos (non-coloring) 7 05G - Tonics, Dressings, and Other Hair Grooming Aids 2 05I - Other Hair Preparations 55 06A - Hair Dyes and Colors (all types requiring caution statements and patch tests) 2 08E - Nail Polish and Enamel CIR Panel Book Page 399

406 TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE TRIBUTYL CITRATE 17 10A - Bath Soaps and Detergents E - Other Personal Cleanliness Products 3 12A - Cleansing 1 12C - Face and Neck (exc shave) 5 12D - Body and Hand (exc shave) 7 12F - Moisturizing 4 12J - Other Skin Care Preps TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE TRICAPRYLYL CITRATE 1 03G - Other Eye Makeup Preparations 3 05A - Hair Conditioner 1 07A - Blushers (all types) 1 07B - Face Powders 1 07C - Foundations 3 07E - Lipstick 2 07I - Other Makeup Preparations 2 12D - Body and Hand (exc shave) 2 12F - Moisturizing 2 12G - Night 1 12J - Other Skin Care Preps Trilauryl Citrate 0 TRI-C12-13 ALKYL CITRATE 1 03B - Eyeliner TRI-C14-15 ALKYL CITRATE TRI-C14-15 ALKYL CITRATE TRI-C14-15 ALKYL CITRATE TRI-C14-15 ALKYL CITRATE TRI-C14-15 ALKYL CITRATE 1 03C - Eye Shadow 1 12C - Face and Neck (exc shave) 4 12F - Moisturizing 2 13A - Suntan Gels, Creams, and Liquids 11 13B - Indoor Tanning Preparations Tristearyl Citrate 0 Triisopropyl Citrate 0 Triethylhexyl Citrate (listed as Trioctyl Citrate) 1 12C - Face and Neck (exc shave) Trihexyldecyl Citrate 0 TRIISOCETYL CITRATE TRIISOCETYL CITRATE TRIISOCETYL CITRATE TRIISOCETYL CITRATE TRIISOCETYL CITRATE TRIISOCETYL CITRATE 8 07A - Blushers (all types) 11 07B - Face Powders 2 07C - Foundations 7 07E - Lipstick 3 07I - Other Makeup Preparations 2 12C - Face and Neck (exc shave) TRIISOSTEARYL CITRATE TRIISOSTEARYL CITRATE TRIISOSTEARYL CITRATE TRIISOSTEARYL CITRATE TRIISOSTEARYL CITRATE 1 03D - Eye Lotion 3 05A - Hair Conditioner 39 07E - Lipstick 3 07I - Other Makeup Preparations 1 12C - Face and Neck (exc shave) TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE 1 03B - Eyeliner 6 03C - Eye Shadow 1 03G - Other Eye Makeup Preparations 1 07A - Blushers (all types) 2 07B - Face Powders CIR Panel Book Page 400

407 TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE TRIOCTYLDODECYL CITRATE 1 07D - Leg and Body Paints 37 07E - Lipstick 1 07F - Makeup Bases 1 07G - Rouges 3 07I - Other Makeup Preparations 1 12E - Foot Powders and Sprays 1 12G - Night Trioleyl Citrate 0 Ethyl Citrates 0 Propylene Glycol Citrate 0 Laureth-6 Citrate 0 LAURETH-7 CITRATE 1 05F - Shampoos (non-coloring) Disodium Laureth-7 Citrate 0 Dilaureth-7 Citrate 0 Sodium Dilaureth-7 Citrate 0 PEG-5 Tricapryl Citrate 0 PEG-5 Trilauryl Citrate 0 Trilaureth-9 Citrate 0 PEG-5 Trimyristyl Citrate 0 PEG-5 Tristearyl Citrate TRIPROPYLENE GLYCOL CIT 1 12D - Body and Hand (exc shave) CIR Panel Book Page 401

408 Rather In In Even USP do The Distributed for Comment Only -- Do Not Quote or Cite Persona Care Products Counci Committed to Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CW Expert Panel DATE: June 20, 2011 SUBJECT: Comments on the Scientific Literature Review on Citric Acid and Its Inorganic Salts and Alkyl and Glycol Esters as Used in Cosmetics Although the disclaimer on the Cifi website ( Scientific literature reviews are distributed for comment only -- not cite ) is helpful, the disclaimer should be on every page of draft reports, including SLRs. p.3 - than presented as text, it would be helpful if the method of manufacture information presented on p.3 was included in a table. p.3, 4 - and Food Chemical Codex specifications should be cited directly to USP (in John Krowka s office) and Food Chemicals Codex (in Carol Eisenmann s office), rather than a company s specification sheet. p.4 - In the use section, it would be helpful to state the highest levels of Citric Acid used in a rinse-off compared to a leave-on product. p.5 - In the discussion of the European regulations, it would be helpful to indicate that water-soluble zinc compounds are listed in Annex ifi and limited to 1% zinc. p.8 - the summary of the Reproductive and Developmental Toxicity section, it would be helpful to include the highest doses that were not associated with reproductive or developmental toxicity. p.8-9, Genotoxicity - section summary, and the text in the section should not be exactly the same. There should be more information in the text of the section. If all the information is presented in a table, then perhaps all the section needs is a section summary and a table. p.9 - Please revise the following sentence. The rational for testing is that aluminum is aluminum is listed by the EPA as a drinking water contaminant with a high health research priority. p.9 - the summary of the Irritation and Sensitization section, please provide some indication of the concentrations used in the sensitization studies. In addition to the information in the table, it would also be helpful to provide some indication of concentrations tested in the section. p.10 - It is not clear what is meant by irritation scores of 39.4, 37.1, and Please provide some context to these values, e.g., what are the minimum and maximum scores? p.12 - if dermal penetration studies show that Citric Acid and citrate compounds readily penetrate the skin, it is not clear why dermal reproductive and carcinogenicity data may be needed. As stated earlier in the report, many of these ingredients have been shown to be safe for ingestion th Street, N.W., Suite 3OO Washington, D.C (fax) CIR Panel Book Page 402

409 Please In Please Distributed for Comment Only -- Do Not Quote or Cite and citrate is already found in the blood at a concentration of 25 mg/i. Exposure through cosmetic use is unlikely to significantly increase body burden. If the CIR Expert Panel decides reproductive and carcinogenicity data are needed, studies concerning these endpoints by all routes of exposure should be included in the report. p , Table 1 - include the reference(s) with this table. p.32, Table 7 - provide additional details for the host-mediated assays, such as the strains of bacteria used and the routes of exposure. Please provide the route of exposure used for the dominant lethal assay. p.33, Table 8 -What happened to the 40% and 70% groups in reference 88? p.35, Table 8 - reference 12, was 15% Triethyl Citrate used for both induction and challenge exposures? 2 CIR Panel Book Page 403