GREEN. Decyl Glucoside and Other Alkyl Glucosides

Transcription

1 GREEN Decyl Glucoside and Other Alkyl Glucosides CIR EXPERT PANEL MEETING JUNE 27-28, 211

2 Administrative

3 Memorandum To: From: CIR Expert Panel Members and Liaisons Monice M. Fiume MMF Senior Scientific Analyst/Writer Date: June 3, 211 Subject: Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics Draft Report Included is the draft report on the Decyl Glucosides and other Alkyl Glucosides as Used in Cosmetics. This is the first time the Panel is seeing this document. The Scientific Literature Review was issued on February 24, 211. Glucoside hydrolases present in human skin may break down these chemicals to their respective starting materials, i.e., glucose and a fatty alcohol. Therefore, summary information from reports on fatty alcohols that have previously been reviewed by the CIR is presented in Table 6. Copies of these reports are available online at The following are included in the data tab: 1. Unpublished information on glucoside ingredients. Memo dated Dec 14, 21. a. Cognis Care Chemicals. 21. Data profile PLANTACARE 81 UP (Caprylyl/Capryl Glucoside); b. Cognis Care Chemicals. 21. Data profile PLANTACARE 818 UP (Coco- Glucoside); c. Cognis Care Chemicals. 21. Data profile PLANTACARE 12 UP (Lauryl Glucoside); d. Cognis Care Chemicals. 21. Data profile PLANTACARE 2 JP (Decyl Glucoside); e. Consumer Product Testing Company Repeat insult patch test of 5% active lauryl glucoside and decyl glucoside; f. Consumer Product Testing Company. 25. Repeat insult patch test of cocoglucoside (1% active). 2. Concentration of use by FDA product category: glucoside ingredients. Memo dated Jan 7, Updated concentration of use by FDA product category: Glucoside ingredients. Memo dated Jan 28, 211.

4 4. Concentration of use by FDA product category: Hexyldecyl d-glucoside and Octadecyl d-glucoside. Memo dated March 15, In Vitro dermal penetration study: Caprylyl/Capryl Glucoside. Memo dated April 8, Comments on the Scientific Literature Review on Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics. Memo dated March 23, FDA raw data. Items 1, 2, and 3 from the above list were included in the SLR. If there are no additional data needs, the Panel should be prepared to formulate a tentative conclusion with rationale and issue a Tentative Report for public comment. If the data are not complete for making a determination of safety, then an Insufficient Data Announcement should be issued listing the additional data that are needed.

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6 History - Decyl Glucosides and Other Alkyl Glucosides Scientific Literature Review: February 24, 211 The following unpublished data were included in the SLR: 1. Unpublished information on glucoside ingredients. Memo dated Dec 14, 21. a. Cognis Care Chemicals. 21. Data profile PLANTACARE 81 UP (Caprylyl/Capryl Glucoside); b. Cognis Care Chemicals. 21. Data profile PLANTACARE 818 UP (Coco-Glucoside); c. Cognis Care Chemicals. 21. Data profile PLANTACARE 12 UP (Lauryl Glucoside); d. Cognis Care Chemicals. 21. Data profile PLANTACARE 2 JP (Decyl Glucoside); e. Consumer Product Testing Company Repeat insult patch test of 5% active lauryl glucoside and decyl glucoside; f. Consumer Product Testing Company. 25. Repeat insult patch test of coco- glucoside (1% active). 2. Concentration of use by FDA product category: glucoside ingredients. Memo dated Jan 7, Updated concentration of use by FDA product category: Glucoside ingredients. Memo dated Jan 28, 211 Draft Report: June 27-28, 211 The following were received after the SLR was announced. The data have been incorporated into the report, and the comments have been addressed. 1. Comments on the Scientific Literature Review on Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics. Memo dated March 23, Concentration of use by FDA product category: Hexyldecyl d-glucoside and Octadecyl d-glucoside. Memo dated March 15, In Vitro dermal penetration study: Caprylyl/Capryl Glucoside. Memo dated April 8, 211 CIR Panel Book Page 2

7 SciFinder Search April 7, 211 History Session began April 7, 211 at 12:35 PM April 7, :36 PM Explore substances by ID: glucoside initiated Explore complete Explore results No answers April 7, :48 PM Explore substances by ID: , , , , , , , , , , , initiated Explore complete Explore results Answer set 2 created with 6 answers from REGISTRY April 7, :57 PM Explore substances by ID: , , , , , , , , , , , , , , , , , , , , , initiated Explore complete Explore results Answer set 3 created with 13 answers from REGISTRY Saved 13 substance answers from Answer set 3 as 'Decyl glucoside 1' Retrieve reference information in 13 substances of Answer set 3 Answer set 4 created with 3,8 answers from CAPLUS 336 answers from MEDLINE Refine Answer set 4 by document type Book, Clinical Trial, Conference, Journal, Letter, Report, Review Answer set 5 created with 1,814 answers from CAPLUS 335 answers from MEDLINE Detailed display from Answer set 5 of Expanding Coverage of the Metabolome for Global Metabolite Profiling Saved 2149 reference answers from Answer set 5 as 'Decyl Gluc 1 Refines by pub type' Refine Answer set 4 by document type Patents only Answer set 6 created with 1,268 answers from CAPLUS April 7, 211 1:3 PM Explore substances by ID: , , , , , C12-18 Alkyl Glucoside, C12-2 Alkyl Glucoside, C12-2 Alkyl Glucoside, C2-22 Alkyl Glucoside, Cetearyl Glucoside, Coco- Glucoside, Octyldodecyl Glycoside initiated Explore complete Explore results Answer set 7 created with 5 answers from REGISTRY Saved 5 substance answers from Answer set 7 as 'decyl gluc 2' CIR Panel Book Page 3

8 Combine the current substance answer set with saved answer sets initiated: decyl gluc 2 OR Decyl glucoside 1 at April 7, 211 1:4 PM Answer set 8 created with 15 answers from REGISTRY Saved 15 substance answers from Answer set 8 as 'Decyl glucoside All' Retrieve reference information in 15 substances of Answer set 8 Answer set 9 created with 3,8 answers from CAPLUS 336 answers from MEDLINE Saved 3416 reference answers from Answer set 9 as 'Decyl Glucoside All' Refine Answer set 9 by document type Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Answer set 1 created with 1,815 answers from CAPLUS 333 answers from MEDLINE Saved 2148 reference answers from Answer set 1 as 'Decyl Glucoside refined by document type' Refine Answer set 1 by research topic sugar surfactant Answer set 11 created with 49 answers from CAPLUS Detailed display from Answer set 11 of Physical properties and inhibitory effects of three-component hybrid liposomes including sugar surfactants Detailed display from Answer set 11 of Alkylpolyglycoside: carbohydrate based surfactant Detailed display from Answer set 11 of Adsorption of n-decyl--d-glucopyranoside and n-decyl--d-maltopyranoside Mixtures at the Liquid-Vapor Interface Explore results Answer set 14 created with 16 answers from CAPLUS 131 answers from MEDLINE Detailed display from Answer set 14 of Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 15 patients Full text accessed for Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 15 patients from Dermatitis : contact, atopic, occupational, drug : official journal of the American Contact Dermatitis Society, North American Contact Dermatitis Group Volume: 21 Issue: 5 Pages: April 7, 211 1:22 PM Explore references by research topic: decyl glucoside initiated, resulting in 2 candidates Explore complete Candidates Selected 413 references were found containing "decyl glucoside" as entered. Automatically removed 6 duplicate MEDLINE answer(s) Explore results Answer set 15 created with 399 answers from CAPLUS 8 answers from MEDLINE April 7, 211 1:23 PM Explore references by research topic: decyl glucoside initiated, resulting in 2 candidates Explore complete Candidates Selected 413 references were found containing "decyl glucoside" as entered. CIR Panel Book Page 4

9 Automatically removed 8 duplicate MEDLINE answer(s) 929 references were found containing the concept "decyl glucoside". Automatically removed 8 duplicate MEDLINE answer(s) Explore results Answer set 18 created with 915 answers from CAPLUS 6 answers from MEDLINE Explore references by research topic: glucoside initiated, resulting in 2 candidates April 7, 211 1:26 PM Explore references by research topic: glucoside initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review, Conference April 7, 211 1:27 PM Explore references by research topic: initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review, Conference Explore complete Candidates Selected 59 references were found containing " " as entered. Automatically removed 3 duplicate MEDLINE answer(s) Explore results Answer set 21 created with 48 answers from CAPLUS 8 answers from MEDLINE April 7, 211 1:29 PM Explore references by research topic: initiated, resulting in candidate April 7, 211 1:31 PM Explore references by research topic: irritancy of glucosides initiated, resulting in 4 candidates Explore complete Candidates Selected 25 references were found where the two concepts "irritancy" and "glucosides" were present anywhere in the reference. Explore results Answer set 22 created with 21 answers from CAPLUS 4 answers from MEDLINE Detailed display from Answer set 22 of Non-irritating cosmetic and pharmaceutical compositions for irritating agents retinoids and - and -hydroxy acids Detailed display from Answer set 22 of Application of surfactants. 51. Alkyl polyglucoside surfactants. 1 Detailed display from Answer set 22 of Lactobionic acid in a natural alkylpolyglucoside-based vehicle: assessing safety and efficacy aspects in comparison to glycolic acid April 7, 211 1:34 PM Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates CIR Panel Book Page 5

10 Explore complete Candidates Selected 145 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 23 duplicate MEDLINE answer(s) Explore results Answer set 23 created with 121 answers from CAPLUS 1 answer from MEDLINE April 7, 211 1:37 PM Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 112 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 22 duplicate MEDLINE answer(s) Explore results Answer set 25 created with 89 answers from CAPLUS 1 answer from MEDLINE Full text accessed for Comparative testing for the identification of skin- of nonionic sugar lipid from Regulatory Toxicology and Pharmacology Volume: 58 Issue: 2 Pages: Full text accessed for Irritant and of eight commonly used in skin cleansers: an evaluation of 15 patients from Dermatitis : contact, atopic, occupational, drug : official journal of the American Contact Dermatitis Society, North American Contact Dermatitis Group Volume: 21 Issue: 5 Pages: Detailed display from Answer set 25 of Zinc and its derivatives: their applications in cosmetic April 7, 211 1:42 PM Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 112 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 22 duplicate MEDLINE answer(s) Explore results Answer set 26 created with 89 answers from CAPLUS 1 answer from MEDLINE Keep Me Posted profile 'sensitization potential of surfactants' created from Answer set 26 Refine Answer set 26 by research topic cosmetics Answer set 27 created with CIR Panel Book Page 6

11 3 answers from CAPLUS 1 answer from MEDLINE April 7, 211 1:44 PM Explore references by research topic: irritation of surfactants initiated, resulting in 5 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 668 references were found containing the two concepts "irritation" and "surfactants" closely associated with one another. Automatically removed 169 duplicate MEDLINE answer(s) Explore results Answer set 28 created with 442 answers from CAPLUS 57 answers from MEDLINE Keep Me Posted profile 'irritation of surfactants' created from Answer set 28 Refine Answer set 28 by research topic cosmetics Answer set 29 created with 152 answers from CAPLUS 7 answers from MEDLINE Detailed display from Answer set 29 of Ocular Irritection: in vitro method for testing ocular irritancy Refine Answer set 29 by research topic glucoside Answer set 3 created with 1 answer from CAPLUS 1 answer from MEDLINE Detailed display from Answer set 3 of Photostability of naturally occurring whitening agents in cosmetic microemulsions Refine Answer set 28 by research topic glucoside Answer set 31 created with 3 answers from CAPLUS 1 answer from MEDLINE Detailed display from Answer set 31 of Evaluation of irritation potential of surfactant mixtures Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_4_7_211_ ris" Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_4_7_211_ ris" Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_4_7_211_ ris" Session began April 7, 211 at 1:5 PM April 7, 211 1:5 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 32 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE CIR Panel Book Page 7

12 April 7, 211 1:54 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 34 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE Answer set 35 created with 4 answers from CAPLUS Detailed display from Answer set 35 of Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants Exported Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants in 'RIS' format as "Cho.ris" Exported Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants in 'RIS' format as "Cho.ris" April 7, 211 1:56 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 36 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE April 7, 211 1:57 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 37 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE April 7, 211 1:57 PM Saved answer set 'Decyl Glucoside All' opened Answer set 38 created with 3,8 reference answers from CAPLUS 336 reference answers from MEDLINE Refine Answer set 38 by document type Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Answer set 39 created with 1,815 answers from CAPLUS 333 answers from MEDLINE April 7, 211 1:58 PM Saved answer set 'Decyl glucoside All' opened Answer set 4 created with 15 substance answers from REGISTRY Retrieve reference information in 15 substances of Answer set 4 Answer set 41 created with 3,8 answers from CAPLUS 336 answers from MEDLINE Refine Answer set 41 by document type Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Answer set 42 created with 1,815 answers from CAPLUS 333 answers from MEDLINE Keep Me Posted profile 'Glucosides Refined by Document Type' created from Answer set 42 April 7, 211 2:1 PM CIR Panel Book Page 8

13 Explore references by research topic: alkyl polyglucosides initiated, resulting in 2 candidates Explore complete Candidates Selected 2649 references were found containing the concept "alkyl polyglucosides". Automatically removed 37 duplicate MEDLINE answer(s) Explore results Answer set 43 created with 2,61 answers from CAPLUS 2 answers from MEDLINE April 7, 211 2:2 PM Explore references by research topic: alkyl polyglucosides initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 359 references were found containing the concept "alkyl polyglucosides". Automatically removed 34 duplicate MEDLINE answer(s) Explore results Answer set 44 created with 323 answers from CAPLUS 2 answers from MEDLINE Keep Me Posted profile 'Alkyl Polygolucosides' created from Answer set 44 Detailed display from Answer set 44 of Profile of irritant patch testing with detergents: sodium lauryl sulfate, sodium laureth sulfate and alkyl polyglucoside Detailed display from Answer set 44 of Toxicology of alkyl polyglycosides Detailed display from Answer set 44 of Alkyl polyglycosides Detailed display from Answer set 44 of Alkyl polyglycosides Full text accessed for from Surfactant Science Series Volume: 98 Issue: Detergency of Specialty Surfactants Pages: Detailed display from Answer set 44 of Nonionic Surfactants: Alkyl Polyglucosides. [In: Surfactant Sci. Ser., 2; 91] Exported 3 reference answers from Answer set 44 in 'RIS' format as "APGs.ris" Detailed display from Answer set 44 of Dermatological properties of alkyl polyglycosides Full text accessed for Dermatological properties of from Alkyl Polyglycosides Pages: Detailed display from Answer set 44 of Alkyl polyglycosides in personal care products Full text accessed for in personal care products from Alkyl Polyglycosides Pages: Detailed display from Answer set 44 of Using alkyl polyglycosides in personal-care products Detailed display from Answer set 44 of Alkylpolyglycosides - a new cosmetic concept for mildness and care Full text accessed for - a new cosmetic concept for mildness and care from Agro-Food-Industry Hi-Tech Volume: 5 Issue: 5 Pages: Detailed display from Answer set 44 of Alkyl polyglycosides: properties and applications Exported 6 reference answers from Answer set 44 in 'RIS' format as "APGs2.ris" April 7, 211 2:18 PM Keep Me Posted searches checked weekly CIR Panel Book Page 9

14 # uses conc data Toxline- Pubmed NLM STN Intl EU FDA ChemPortal Misc NLM NTIS Registry RTECS Merck EU SCCS ECE- TOC SIDS-HPV-IUCL ID IARC NTP EAFUS GRAS OTC NIOSH Misc date searched Oct Decyl Glucoside x 2 x x no no no no no no no no no x Arachidyl Glucoside x x x no no no no no no no no no no Butyl Glucoside no 1 x x no no no no no no no no no x C1-16 Alkyl Glucoside no x x no no no no no no no no no x C12-18 Alkyl Glucoside no x no no no no no no no no no no C12-2 Alkyl Glucoside x x no no no no no no no no no no C2-22 Alkyl Glucoside no no no no no no no no no no no Caprylyl/Capryl Glucoside x 3 x x no no no no no no no no no x Caprylyl Glucoside x x x x no no no no no no no no no x 352 Cetearyl Glucoside x x no no no no no no no no no no Coco-Glucoside x x no no no no no no no no no no Ethyl Glucoside x 2 x x no no no no no no no no no x Isostearyl Glucoside no x x no no no no no no no no no no Lauryl Glucoside x 2 x x no no no no no no no no no x Myristyl Glucoside x 1 x x no no no no no no no no no x Octyldodecyl Glucoside no x no no no no no no no no no no Palm Kernel/Coco Glucoside no x no no no (deleted from report) no Undecyl Glucoside no 1 x x no no no no no no no no no x Tradenames Search 639 In VCRP/but not INCI Hexadecyl D-Glucoside ( ) Octadecyl D-Glucoside ( ) x no no no no no x x no no no no no x References Ordered Whit 25 papers ( ) NTIS 3 documents ( ) CIR Panel Book Page 1

15 Search Terms Distributed for Comment Only -- Do Not Cite or Quote ((DECYL OR ETHYL OR BUTYL OR CAPRYLYL OR UNDECYL OR LAURYL OR MYRISTYL OR ARACHIDYL OR CAPRYL OR ALKYL OR CETEARYL OR COCO OR (PALM AND KERNEL) OR ISOSTEARYL OR ISOOCTADECYL OR OCTYLDODECYL OR OCTYL OR DODECYL OR TETRADECYL OR EICOSYL) AND GLUCOSIDE) OR ((ETHYL OR BUTYL OR OCTYL OR DECYL OR UNDECYL OR DODECYL OR LAURYL OR MYRISTYL OR TETRADECYL OR ARACHIDYL OR EICOSYL OR ISOSTEARYL OR ISOOCTADECYL) AND GLUCOPYRANOSIDE) OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR OR ORAMIX OR PLANTACARE OR PLANTAREN OR ORAMIX OR SUCRAPH OR AVANEL OR ORISTAR OR DESULF OR (TRITON AND SURFACTANT) OR SEPISOFT OR (TEGO AND CARE) SCiFinder Seach Terms C12-18 Alkyl Glucoside C12-2 Alkyl Glucoside C12-2 Alkyl Glucoside C2-22 Alkyl Glucoside Cetearyl Glucoside Coco- Glucoside Octyldodecyl Glycoside Alkyl Glucoside CIR Panel Book Page 11

16 Decyl Glucosides and Other Alkyl Glucosides Data Profile* June 211 Writer, Monice Fiume (updated ) 1 Distributed for Comment Only -- Do Not Cite or Quote Reported Use Method of Manufacture Toxicokinetic Data Animal Tox Acute, Derrmal Animal Tox Acute, Oral Animal Tox, Acute, Inhalation Animal Tox Rptd Dose, Dermal Animal Tox, Rptd Dose, Oral Animal Tox Rptd Dose, Inhalaiton Repro/Dev Tox Genotox Carcinogenicity Dermal Irr/Sens Ocular Irritaton Decyl Glucoside X X X Arachidyl Glucoside X C1-16 Alkyl Glucoside X X C12-18 Alkyl Glucoside C12-2 Alkyl Glucoside X C2-22 Alkyl Glucoside Caprylyl/Capryl Glucoside X X X X X X X X Caprylyl Glucoside X X X Cetearyl Glucoside X Coco-Glucoside X X X Ethyl Glucoside X X X Isostearyl Glucoside Lauryl Glucoside X X X X Myristyl Glucoside X Octyldodecyl Glucoside Undecyl Glucoside Alkyl Polyglucosides (APGs), General Info X X X X Previously Reviewed Fatty Alcohols Yr. Rev. n-butyl Alcohol 28 X X X X X X X X Cetearyl Alcohol 1988 X X Cetyl Alcohol 1988 X X X X X X X X Coconut Alcohol 28 Isostearyl Alcohol 1988 X X X Myristyl Alcohol 1988 X X X X Octyl Dodecancol 1985 X X X X Stearyl Alcohol 1985 X X X X X X X * X indicates that data were available in a category for the ingredient CIR Panel Book Page 12

17 Report

18 Draft Report Decyl Glucoside and Other Alkyl Glucosides as Used in Cosmetics June 27, 211 The 211 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 M. Fiume, Senior Scientific Analyst/Writer, and Bart A. Heldreth, Ph.D., Chemist, CIR. Cosmetic Ingredient Review th Street, NW, Suite 412 " Washington, DC " ph " fax " cirinfo@cir-safety.org

19 TABLE OF CONTENTS Introduction... 1 Chemistry... 1 Definition and Structure... 1 Impurities, Constituents, and Physical and Chemical Properties... 2 Method of Manufacture... 2 Use... 2 Cosmetic... 2 Non-Cosmetic... 3 Toxicokinetics... 3 Absorption, Distribution, Metabolism, and Excretion... 3 Dermal... 3 Oral... 4 Absorption Enhancement... 4 Toxicological studies... 4 Single Dose (Acute) Toxicity... 5 Dermal... 5 Oral... 5 Repeated Dose Toxicity... 5 Dermal... 5 Oral... 7 Reproductive and Developmental Toxicity... 7 Oral... 7 In Vitro Estrogenicity Assays... 8 Genotoxicity... 8 Carcinogenicity... 8 Irritation and Sensitization... 8 Skin Irritation... 9 Non-Human... 9 Human... 9 Sensitization... 9 Non-Human... 9 Human... 9 Mucosal Irritation... 9 Case Studies... 1 Summary... 1 Tables Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment Table 2. Chemical and physical properties Table 3a. Frequency and concentration of use according to duration and type of exposure Table 3b. Ingredients Not Reported to be Used Table 4. Skin irritation and sensitization studies Table 5. Mucosal irritation studies Table 6. Summaries of information on fatty alcohols from previous CIR reports References CIR Panel Book Page 14

20 INTRODUCTION This document is a review of studies relevant to the safety of 17 alkyl glucoside ingredients as used in cosmetic formulations. Most of these ingredients function in cosmetics as surfactants. A few do not function as surfactants, but are used as skin conditioning agents, hair conditioning agents, or emulsion stabilizers. The ingredients included in this review are obtained by the condensation of an alcohol with a cyclic form of glucose (Dglucopyranose). The ingredients included in this group are: Decyl Glucoside Arachidyl Glucoside Butyl Glucoside C1-16 Alkyl Glucoside C12-18 Alkyl Glucoside C12-2 Alkyl Glucoside C2-22 Alkyl Glucoside Caprylyl/Capryl Glucoside Caprylyl Glucoside Cetearyl Glucoside Coco-Glucoside Ethyl Glucoside Isostearyl Glucoside Lauryl Glucoside Myristyl Glucoside Octyldodecyl Glucoside Undecyl Glucoside While the names of these ingredients imply that they are mono-glucosides, these ingredients, however, are not limited to mono-glucosides, but may involve products that are the result of a number of condensed glucose repeat units. Glucoside hydrolases in human skin are likely to break down these chemicals to release their respective fatty alcohols and glucose. Therefore, summary information on the appropriate fatty alcohols that have previously been reviewed by the Cosmetic Ingredient Review (CIR) is presented at the end of this report in the last table (Table 6). CHEMISTRY Definition and Structure This group of ingredients consists of anomerically-alkyl-substituted D-glycopyranosides. Specifically, the alkyl substituents range from 2 to 22 carbons in length, and the D-glycopyranosides consist of glucose-type mono-, di-, tri-, oligo-, or poly-saccharides (e.g., mono = glucose (i.e. D-glucopyranoside) and di = maltose (i.e. D-maltopyranoside)). Regardless of the degree of polymerization (i.e. number of glucose monomers (n); e.g., a degree of polymerization equal to 2 means the diglucose (disaccharide), maltose) these ingredients are simply named glucosides. Although these ingredients are most likely the ß-anomers, the names of these alkyl glucosides are not necessarily specific to either anomer. For example, the general decyl glucoside structure shown in Figure 1 is the ten-carbon, alkyl-chain substituted glycopyranoside, wherein n can be 1 (for a mono-glucoside) or more (for di-, tri-, oligo-, and poly-glucosides): Figure 1. General Decyl Glucoside Structure Therefore, decyl glucoside may be monomeric or polymeric. Poly will be used generically used throughout the rest of this report to refer to di-, tri-, oligo-, poly-glucosides, and mixtures thereof. 1 CIR Panel Book Page 15

21 Decyl glucoside, for example, may be comprised of one or more of the following polyglucosides (in this case maltopyranosides) shown in Figure 2: Distributed for Comment Only -- Do Not Cite or Quote Figure 2. Examples of Decyl Glucoside Forms A decyl glucoside with a degree of polymerization of 1.6, for example, would then be a mixture comprised of decyl glucopyranoside and one of the decyl maltopyranosides (with a slightly higher percentage of the maltose derivative) shown in Figure 2. Because many of these fatty alcohols are supplied from natural feed stocks, the designated length may be the average (e.g., median) length (e.g. decyl glucoside may actually be a mixture of C6, C8, C1, C12, C14, and C16 chain lengths, each anomerically attached to a glucopyranose). 1 The definitions and structures of the ingredients included in this review are provided in Table 1. Impurities, Constituents, and Physical and Chemical Properties These compounds are typically solids, with solubility in both aqueous and organic solutions. The available impurity, constituent, and physical and chemical property information is presented in Table 2. Method of Manufacture The first report of the synthesis of alkyl glucosides, reported by Fischer in 1893, involves reacting glucose with anhydrous ethanol under acidic conditions to produce ethyl glucoside. 2 Though not at all new, alcoholysis of glucose and polysaccharides under acidic conditions is still the method of choice. It is considered to be a green process that can involve the use of natural and renewable sources (e.g., the alcohols can be obtained from coconut oil or palm oil and the glucose or polysaccharide can be obtained from corn, potato, or wheat starch). 3 Of note, the reaction conditions that produce an ether linkage between a fatty alcohol and the anomeric hydroxy group of glucose are known to cause condensation of one molecule of glucose with another molecule of glucose, thereby producing alkyl polyglucosides (APGs) even when an alkyl monoglucosides may be the intended product. USE Cosmetic The alkyl glucosides named in this report function primarily as surfactants. 4 A few do not function as surfactants, and are used as a skin conditioning agents, hair conditioning agents, or emulsion stabilizers. Voluntary Cosmetic Registration Program (VCRP) data obtained in 211 indicate that, of the ingredients reviewed in this safety assessment, decyl glucoside has the highest frequency of uses reported i.e., 492); the majority of these uses i.e., 421), are in rinse-off formulations. 5 Cetearyl glucoside, lauryl glucoside, and coco-glucoside have 477, 399, and 35 2 CIR Panel Book Page 16

22 reported uses, respectively. Cetearyl glucoside is mostly reported to be used in leave-on products. The remaining ingredients that are reported to be used have 75 uses. Based on data from a survey conducted by the Personal Care Products Council (Council), decyl glucoside has the highest leave-on and rinse-off concentrations of use, at 11 and 33%, respectively. 6 Products containing alkyl glucosides are reported to be used on baby skin or applied to the eye area, and mucous membranes may be exposed to these products. Coco-glucoside is reported to be used in a product that could be ingested, and a few of the ingredients are reported to be used in product types that could be inhaled. Since some of the glucosides 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 >8 µm, respectively, and is large compared to respirable particle sizes ( 1 µm). Therefore, because of their size, most aerosol particles are deposited in the nasopharyngeal region and are not respirable. Frequency and concentration of use data are provided in Table 3a. In some cases, reports of uses were received in the VCRP, but no concentration of use is available. For example, decyl glucoside is reported to be used in 25 baby products, but no use concentration was available. In other cases, no reported uses were received in the VCRP, but a use concentration was provided in the industry survey. For example, caprylyl glucoside was not reported to be used in non-coloring hair products, but the industry survey indicated that it was used in such products at 4%. It should be presumed that caprylyl glucoside is used in at least one hair care product. The ingredients not listed in the VCRP or by the Council as in use are listed in Table 3b. All of the glucosides named in the report, with the exception of C2-22 alkyl glucoside, are listed in the European Union inventory of cosmetic ingredients. 11 Non-Cosmetic Caprylyl glucoside and similar alkyl glucosides are effective solubilizers of lipids and proteins below their critical micelle concentrations (CMC), and are used in various biochemical techniques and membrane research. These ingredients also can be used to reconstitute enzymes of other proteins from crude biological preparations. 12 excipient. 13 A C 16/18 -APG named cetearyl glucoside and cetearyl alcohol has been approved by the FDA as a pharmaceutical The use of decyl glucoside as a stabilizer in nanosuspensions for dermal delivery has been investigated; decyl glucoside was effective as a stabilizer with resveratrol 14 and hesperetin ((S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4- methoxy-phenyl)-4h-1-benzopyran-4-one) 15 nanosuspensions. TOXICOKINETICS In an in vitro dermal absorption study using human skin samples, the mean absorbed dose of 1% caprylyl/capryl glucoside was.1%. In an oral study in which female mice were dosed by gavage with a 5% aq. solution of caprylyl [U- 14 C]glucoside, the highest levels of radioactivity at 2 h after dosing were found in the stomach, intestines, liver, and kidneys. The radioactivity in the stomach was primarily unchanged substrate, while only a trace amount found in the liver was unchanged. Labeled glucose was found in all of these organs. In a feeding study in rats in which dietary sucrose was replaced with 1 or 2% ethyl glucoside for 39 days, 6-9% of the ingested ethyl glucoside was recovered in the urine. Absorption, Distribution, Metabolism, and Excretion Dermal Glucoside hydrolases are known to be present in human skin. Therefore, the first step in the metabolism of these ingredients may be breaking down these chemicals to glucose and their respective fatty alcohols. 16 In Vitro Distributed for Comment Only -- Do Not Cite or Quote 3 CIR Panel Book Page 17

23 Caprylyl/Capryl Glucoside The dermal penetration of caprylyl/capryl glucoside, diluted to 1% in Hank s buffered salt solution, ph 6.5, was evaluated in vitro using human skin. 17 Two skin samples from each of three donors were used in the study (n=6). After 24 h, the mean recovery was.52% of caprylyl/capryl glucoside from two tape strips and.3% of caprylyl/capryl glucoside in the further 18 tape strips. The mean absorbed dose of caprylyl/capryl glucoside, as the sum of the amounts found in the viable epidermis, dermis, and receptor medium, was.1%. Oral Non-Human Caprylyl Glucoside Three female NMRI mice were given a single oral dose, by gavage, of 37 MBq/mmol caprylyl [U- 14 C]glucoside in.5 ml of a 5% aq. solution of phophatidylcholine. 18 The animals were killed 2 h after dosing. The highest levels of radioactivity were found in the stomach, intestines, liver, and kidneys, with most of the radioactivity (81-98%) distributed in the aqueous phase. High levels of radioactivity that were not extractable with chloroform were found in the urine, which, according to the researchers, indicated a high rate of degradation to water-soluble metabolites. In the stomach, 75% of the radioactivity was associated with unchanged substrate. In the kidneys and intestines, 5% of the total radioactivity was unchanged substrate, while only a trace amount found in the liver was associated with unchanged substrate. Labeled glucose was detected in all four of these organs. In the stomach, intestines, and kidneys, 13-19% of the radioactivity was contained in the chloroform extract, and most of it was derived from caprylyl [U- 14 C]glucoside. In this extract in the stomach, approximately 2% acylated-labeled substrate was detected. Ethyl Glucoside Groups of 6 male Wistar ST rats were fed, for 39 days, a diet in which sucrose was replaced with 1 or 2% ethyl glucoside, and a control group was fed unaltered (i.e., sucrose-containing) feed. 19 A 24-h urine volume was measured weekly. Approximately 6-9% of the ethyl glucoside ingested by treated animals was recovered in the urine. Absorption Enhancement Caprylyl glucoside has been shown to increase the absorption of poorly absorbed drugs (e.g., insulin), both in vitro across human carcinoma monolayers and in vivo through mucosal membranes. In the in vitro study, the enhancement of the permeability of insulin across T84 and Caco-2 cell monolayers by was caprylyl glucoside concentration-dependent; permeability of insulin was not significantly enhanced at concentrations of.2 and.3%, while it was enhanced with.4 and.5% caprylyl glucoside. 2 In a transmucosal absorption study, the effect of caprylyl glucoside on the nasal, buccal, and rectal absorption of insulin was examined using male Lewis rats. 12 A 5% solution of caprylyl glucoside had an enhancing effect on buccal absorption. The effect of other alkyl glycosides, including decyl and lauryl glucoside, on mucosal penetration was also evaluated. A 5% solution of decyl glucoside also enhanced the buccal absorption of insulin, but 5% lauryl glucoside did not have a significant effect. The researchers stated that there was no consistent relationship between alkyl chain length and penetration enhancement. TOXICOLOGICAL STUDIES In single dose dermal studies with caprylyl/capryl glucoside and C1-16 alkyl glucoside (both 5% a.i., n:1.6) in rabbits, the LD 5 was greater than the 2 mg/kg dose administered. In oral studies with the same test substances, none of the mice dosed with 2 mg/kg caprylyl glucoside and none of the rats dosed with 5 mg/kg C1-16 alkyl glucoside died during the study. In 2-wk repeated dose dermal studies in rabbits with 6% active caprylyl/capryl 4 CIR Panel Book Page 18

24 glucoside, occlusive applications produced testicular effects, while non-occlusive application did not. In the two occlusive studies, one with.9 and 1.8 g a.i./kg and the other with g a.i./kg, an NOEL for testicular effects could not be established. In the non-occlusive study, the NOEL for systemic toxicity was.18 g a.i./kg caprylyl/capryl glucoside. Severe dermal irritation was observed in both occlusive studies, while slight to moderate irritation was reported in the non-occlusive study. In oral repeated dose toxicity studies, moderately-dilated renal tubules were observed in 3 of 6 rats fed 2% ethyl glucoside for 39 days, but in none of the rats fed 1% ethyl glucoside. Kidney weights were statistically significantly increased in the test animals. In rats dosed orally with 25-1 mg/kg C12/16 APG for 13 wks, reversible irritation and ulceration of the stomach mucosa was observed, but there was no systemic toxicity reported for any group. Dermal Caprylyl/Capryl Glucoside Single Dose (Acute) Toxicity Groups of 5 male and 5 female New Zealand White (NZW) rabbits were given a single dermal dose of 2 mg/kg bw caprylyl/capryl glucoside, 5% active ingredient (a.i.) (as C8/C1 APG); the degree of polymerization, n, was to moderate irritant effects, fecal staining, yellowing around the application site, and a few other effects were observed. One animal died of an unrelated infection, and 5 had gross findings at necropsy. C1-16 Alkyl Glucoside Groups of 5 male and 5 female NZW rabbits were given a single dermal dose of 2 mg/kg bw C1-16 alkyl glucoside, 5% a.i. (as C1-16 APG; n:1.6). 21 desquamation were observed. None of the animals died during the study. Oral Caprylyl Glucoside Slight depression, hunched posture, mild to marked erythema, and marked Female NMRI mice were given a single oral dose of 4 mg (2 mg/kg bw) caprylyl glucoside as a suspension in.2 ml of a 5% aq. solution of phosphatidylcholine. 18 No toxic effects were observed during a 2-wk post-dose observation period. Growth and behavior were not affected. Caprylyl/Capryl Glucoside Groups of 5 male and 5 female Sprague-Dawley rats were given a single oral dose of 5 mg/kg bw caprylyl/capryl glucoside (as C8/1 APG; n:1.6, 5% a.i.). 21 C1-16 Alkyl Glucoside None of the animals died during the study. Groups of 5 male and 5 female Sprague-Dawley rats were given a single oral dose of 5 mg/kg bw C1-16 alkyl glucoside (as C1/16 APG; n:1.6, 5% a.i.). 21 groups. One female of the 1.8 g a.i./kg group died after 1 doses, and the death was considered test article-related. Severe 5 Mild None of the animals died during the study. Additionally, no mortality was observed upon dosing of 2 male and 2 female Wistar rats with a single oral dose of 2 mg/kg bw C12/14 APG, n: 1.6 and 6% a.i. Dermal Caprylyl/Capryl Glucoside Repeated Dose Toxicity In a 2-wk study, 1 occluded applications of.15 and.6 g a.i./kg caprylyl/capryl glucoside in distilled water were made to the skin of rabbits. 22 (Additional details were not provided.) Distributed for Comment Only -- Do Not Cite or Quote Slight to moderate irritation was reported. No mortality and no signs of toxicity were observed. Groups of 6 male and 6 female NZW rabbits were dosed with 4 ml/kg of,.9, and 1.8 g a.i./kg (, 22.5, and 45 w/v%, respectively) caprylyl/capryl glucoside (6% active) in distilled water. 23 Ten 6-h occlusive applications were made over a 2-wk period. Treatment-related signs of toxicity, such as ataxia, lethargy, and emaciation, were observed in both test CIR Panel Book Page 19

25 dermal irritation was observed in males and females of both test groups by days 5-6 of the study; slight irritation was observed 1 day after the initial dose. Body weights of treated male and female rabbits were significantly less than those of controls, and mean body weight loss was observed for both groups. Significant changes were observed in some hematology and clinical chemistry values; a dose-response relationship was not observed for most of the hematology changes. Compared to controls, absolute testes weights were significantly lower in treated males of both dose groups. No other compoundrelated changes in organ weights were observed. Microscopic examination of selected male tissues reported very slight to marked testicular degeneration in all rabbits in the.9 g a.i./kg group and slight to marked testicular degeneration in four of the rabbits of the 1.8 g a.i./kg group. 24 Very slight to moderate atrophy of the prostate and accessory sex glands was observed in 3 rabbits of each group. The researchers stated that irritation, inflammation, and stress in these animals were major contributing factors to many, if not all, of the toxicologic effects; however, the researchers also stated that it is possible that caprylyl/capryl glucoside produced some of the effects. 23 A no-observed effect level (NOEL) was not determined. In a similar 2-wk study, but with a non-occlusive application, 2 ml of,.6,.18, or.54 g a.i./kg caprylyl/capryl glucoside (6% active) in distilled water (corresponding to concentrations of, 3, 9, and 27% a.i., respectively) were applied to the intact skin of the backs of 6 male rabbits/group. 25 These doses were selected following a 2-wk pilot study, in which unoccluded exposure to.12,.23, and.45 a.i. g/kg caprylyl/capryl glucoside produced slight to moderate erythema and edema. In the main study, treatment-related signs of toxicity were not observed. Slight dermal irritation was observed in all groups after the initiation of dosing; the irritation became moderate in the high dose group after 3 days of dosing. Body weights of rabbits of the high dose group were slightly, but significantly, decreased compared to controls. Absolute testes weights were slightly, but not significantly, decreased in the high dose group. No treatment-related effects on hematology or clinical chemistry values or organ weights were reported. Microscopically, epithelial hyperplasia, hyperkeratosis, congestion, and eschar formation were observed in the skin of rabbits of the high dose group; these changes were not observed in rabbits of the other test groups. No test article-related microscopic changes were observed in the testes or accessory sex glands at any dose. The NOEL for systemic toxicity was.18 g a.i./kg caprylyl/capryl glucoside. In another 2-wk study, 1 occlusive applications of.14,.41, and 1.25 g a.i./kg caprylyl/capryl glucoside (6% active) in distilled water (, 3.5, 1.4, and 31.1% a.i., respectively) were made to intact skin on the backs of 6 male NZW rabbits/group in order to determine the NOEL for testicular toxicity. 22 Two of the high dose animals died during the study, and the 4 surviving animals had signs of treatment-related toxicity. No treatment-related mortality occurred in the low or mid-dose groups. Dermal irritation, which progressed from slight to severe with time, was observed in all test groups., and slight to moderate irritation was observed in the controls. Changes in some hematology and clinical chemistry values were observed, but were attributed to stress of the occlusive procedure, irritation, and body weight loss. A decrease in the mean absolute testicular weights in animals of the mid- and high dose groups was considered treatment-related. A treatment-related loss in body weight was observed in all test groups, and the mean terminal body weights of rabbits of all test groups were decreased compared to controls. Treatment-related microscopic changes, consisting of an increased incidence and severity of diffuse bilateral testicular atrophy with necrotic spermatocytes, and atrophy of the prostate and vesicular glands, were observed in the testes, epididymides, prostate, and vesicular glands of the mid and high dose group animals. 26 Testicular effects were evident in one rabbit of the low dose group. Changes in the testes and accessory sex glands were attributed to the stresses described above An NOEL was not established CIR Panel Book Page 2

26 Oral Ethyl Glucoside In a study described earlier under Toxicokinetics, in which groups of 6 male Wistar ST rats were fed for 39 days a diet in which sucrose was replaced with 1 or 2% ethyl glucoside, body weight gains, but not final body weights, were statistically significantly decreased in the 2% group when compared to control values. 19 All animals survived until study termination. Total water intake was increased with increased ethyl glucoside consumption. In animals fed ethyl glucoside, kidney weights were statistically significantly increased and epididymal and abdominal fatty pad weights were statistically significantly decreased. The renal tubules of 2 and 4 control rats were not-dilated and slightly dilated, respectively, and the renal tubules of all the rats in 1% group were slightly dilated. In the group fed 2% ethyl glucoside, the renal tubules of 3 rats were slightly dilated,, while the other 3 had moderately-dilated renal tubules. No microscopic damage to renal cells was observed. Alkyl Polyglucosides Groups of 1 male and 1 female Sprague-Dawley rats were dosed orally, by gavage, with, 25, 5 and 1 mg/kg bw C12/16 APG for 13 wks. 21 Distributed for Comment Only -- Do Not Cite or Quote An additional 5 male and 5 female control and high dose rats were used as a recovery group. No treatment-related changes in body weights, organ weights, or biochemistry or hematology parameters were observed. Absolute gonad weights were decreased in all test groups, but the decrease was not considered treatment related by the researchers due to the lack of a dose-response. A dose-dependent, slowly reversible, irritation and ulceration of the forestomach mucosa was observed in animals of the 5 and 1 mg/kg bw groups. Systemic toxicity was not observed in any group. The no-observed adverse effect level (NOAEL) for systemic toxicity was 1 mg/kg bw. The no-observed effect concentration for local compatibility was deduced as 2.5% a.i. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Dermal application of 6% active caprylyl/capryl glucoside, g a.i./kg, under occlusive conditions may affect the testes and accessory sex glands of rabbits; however, it was not clear if the effects were test-article related or due to stress of the occlusive procedure and resulting irritation and weight loss. Lauryl glucoside, 1-1 mg/kg by gavage, did not produce adverse reproductive or developmental effects. Lauryl glucoside,.1-1, nmol, did not have any effects in in vitro estrogenicity assays. Oral Lauryl Glucoside Groups of 24 female Sprague-Dawley CD rats were dosed orally, by gavage, with, 1, 3, or 1 mg/kg bw/day lauryl glucoside (as C1-14 or C1-16, n: 1.4) on days 6-15 of gestation. 27 All animals were killed on day 2 of gestation. No maternal toxicity was observed, and no reproductive or developmental effects were indicated. There were also no differences in external, visceral or skeletal malformations between groups. The NOAELs for maternal toxicity, embryotoxicity/fetotoxicity, and teratogenicity were all 1 mg/kg bw/day. Lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) was given orally, by gavage, to groups of 1 male and 1 female Sprague-Dawley rats at doses of, 1, 3, and 1 mg/kg/day, from 2 wks prior to mating to 4 days after delivery. 27 No signs of general parental toxicity were observed, and relative and absolute weights of the testes, epididymides, and seminal vesicles were similar for treated and control animals. There were no test article-related effects on reproductive parameters, and no effects on neonates. 7 CIR Panel Book Page 21

27 In Vitro Estrogenicity Assays Lauryl Glucoside Lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) was evaluated in the E-Screen assay, in which the induction of cell proliferation in the estrogen-dependent human breast tumor MCF-7 cells is determined, at concentrations of.1-1, nmol/ml β-Estradiol and bisphenol-a were reference substances, and the medium was the negative control. No effects were reported at concentrations up to 1 5 higher than the concurrent controls. The effects of.1-1 nmol/ml lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) were determined in the MCF-7 reporter gene assay, in which the induction of luciferase activity in stable transfected MCF-7 cells is determined. 27 No effects were seen with lauryl glucoside alone, and no anti-estrogenic or other synergistic effects were observed after incubation with.1-1 nmol/ml estradiol:lauryl glucoside (1:1 molar ratio). Alkyl Polyglucosides GENOTOXICITY The mutagenic potential of APGs (chain length not specified) was determined in two Ames tests at concentrations of 8-5 µg/l and 11-9 µg/plate in distilled water, with and without metabolic activation. 21 APGs were not mutagenic. Positive and negative controls gave expected results. The genotoxic potential of C1/16 APG was evaluated in an assay for chromosomal aberrations using Chinese hamster V79 lung fibroblasts, at concentrations of 16 µg/ml with and 16 µg//ml without metabolic activation. 21 C1-16 alkyl glucoside was not clastogenic in this assay. Positive and negative controls gave expected results. Published carcinogenicity studies were not found. Distributed for Comment Only -- Do Not Cite or Quote CARCINOGENICITY IRRITATION AND SENSITIZATION Caprylyl/capryl glucoside, 3% a.i., was slightly irritating to rabbit skin. With APGs of varying chain length (C8/1 to C12/16; 15-7% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from. to 4.6 in rabbits. (A PDII of 2 was considered a positive responder). In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.% a.i.) and soap chamber tests (1.% a.i.), and decyl glucoside was evaluated in an SIOPT (.5% a.i.). At most, these ingredients were slightly irritating. Glucosides with alkyl chain lengths ranging from C8- C1 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of % for intradermal induction, 5-1% for epidermal induction, and 2.5-5% for challenge, and the local lymph node assay (LLNA) at concentrations of %. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitization at concentrations of 8.4%, 5.9%, and.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 2%) and the Magnusson-Kligman protocol (1, 6,and 1% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively). C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. In clinical testing, the sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. aq. coco-glucoside was evaluated in a human repeated insult patch test (HRIPT). These ingredients were not irritating or sensitizing. In alternative system studies for mucosal irritation, decyl, lauryl, C1-16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating. In a HET-CAM study with APG of varying proportions of alkyl chain length, the ocular irritation potential increased with the increased proportion of shorter-chain APGs. In studies using rabbits, neutralized lauryl glucoside produced slight ocular reactions. Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 1% for 3% a.i. caprylyl/capryl glucoside and 5% for 6% a.i. caprylyl/capryl glucoside. Dermal irritation and sensitization studies are summarized in Table 4. 8 CIR Panel Book Page 22

28 Skin Irritation Non-Human Caprylyl/capryl glucoside, 3% a.i., was slightly irritating to rabbit skin With APGs of varying chain length (C8/1 to C12/16; 15-7% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from. to 4.6 in rabbits. 21 (A PDII of 2 was considered a positive responder.) Human In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.% a.i.) and soap chamber tests (1.% a.i.), and decyl glucoside was evaluated in a single-insult occlusive patch test (SIOPT) (.5% a.i.). 32,33 At most, these ingredients were slightly irritating. Sensitization Non-Human Glucosides with alkyl chain lengths ranging from C8-C1 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of % for intradermal induction, 5-1% for epidermal induction, and 2.5-5% for challenge, and the local lymph node assay (LLNA) at concentrations of %. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitizations at concentrations of 8.4%, 5.9%, and.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 2%) and the Magnusson-Kligman protocol (1, 6,and 1% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively). 21 C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. Human The sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. coco-glucoside was evaluated in a human repeat insult patch test (HRIPT). 34,35 These ingredients were not irritating or sensitizing. Mucosal Irritation Mucosal irritation studies, in both alternative and non-human systems, are summarized in Table 5. In the alternative systems studies, decyl, lauryl, C1-16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating. 21,32 In a comparative study assessing the relationship between ocular irritation potential and alkyl chain length in the hen s egg test-chorioallantoic membrane (HET-CAM) assay with four decyl glucosides (with varying proportions of alkyl chain length) and one lauryl glucoside, the ocular irritation potential increased with the increased proportion of shorter-chain APGs (C 6 -C 1 ). 36 The researchers stated that results of a bovine corneal opacity permeability (BCOP) assay suggested that changes in permeability with different alkyl chain lengths could be the main factor for the different eye irritation potentials. In the non-human studies, all of which used rabbits, neutralized lauryl glucoside produced slight reactions. 21 Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 1% for 3% a.i. caprylyl/capryl glucoside and 5% for 6% a.i. caprylyl/capryl glucoside. 24,29-31, CIR Panel Book Page 23

29 Case Studies Decyl Glucoside Case studies with reactions to antiseptic, hair, and sunscreen products that contain decyl glucoside are described in published literature. 1,44-48 Subsequent patch testing with decyl glucoside at.5-1% had positive results in these cases. Patch testing with other glucosides also produced positive results in these patients. SUMMARY The 17 alkyl glucosides reviewed in this report are ingredients that consist of anomerically-alkyl-substituted D- glycopyranosides; alkyl substituents range from 2 to 22 carbons in length and the D-glycopyranosides consist of glucose-type mono-, di-, tri-, oligo-, or poly-saccharides. The alkyl glucosides are synthesized by the alcoholysis of glucose and polysaccharides under acidic conditions. Most of these glucosides function in cosmetics as surfactants; a few function as skin conditioning agents, hair conditioning agents, or emulsion stabilizers. In 211, decyl glucoside was reported to be used in 492 cosmetic formulations, 421 of which are rinse-offs. Decyl glucoside has the highest concentration of use, with 11 and 33% being reported for leaveon and rinse-off formulations. The most frequently use glucoside in leave-on formulations is cetearyl glucoside, with 445 of 477 uses being in leave-on formulations. In an in vitro dermal absorption study using human skin samples, the mean absorbed dose of 1% caprylyl/capryl glucoside was.1%. In an oral study in which female mice were dosed by gavage with a 5% aq. solution of caprylyl [U- 14 C]glucoside, the highest levels of radioactivity at 2 h after dosing were found in the stomach, intestines, liver, and kidneys. The radioactivity in the stomach was primarily unchanged substrate, while only a trace amount found in the liver was unchanged. Labeled glucose was found in all of these organs. In a feeding study in rats in which dietary sucrose was replaced with 1 or 2% ethyl glucoside for 39 days, 6-9% of the ingested ethyl glucoside was recovered in the urine. In single dose dermal studies with caprylyl/capryl glucoside and C1-16 alkyl glucoside (both 5% a.i., n:1.6) in rabbits, the LD 5 was greater than the 2 mg/kg dose administered. In oral studies with the same test substances, none of the mice dosed with 2 mg/kg caprylyl glucoside and none of the rats dosed with 5 mg/kg C1-16 alkyl glucoside died during the study. In 2-wk repeated dose dermal studies in rabbits with 6% active caprylyl/capryl glucoside, occlusive applications produced testicular effects, while non-occlusive application did not. In the two occlusive studies, one with.9and 1.8 g a.i./kg and the other with g a.i./kg, an NOEL for testicular effects could not be established. In the non-occlusive study, the NOEL for systemic toxicity was.18 g a.i./kg caprylyl/capryl glucoside. Severe dermal irritation was observed in both occlusive studies, while slight to moderate irritation was reported in the non-occlusive study. In oral repeated dose toxicity studies, moderately-dilated renal tubules were observed in 3 of 6 rats fed 2% ethyl glucoside for 39 days, but in none of the rats fed 1% ethyl glucoside. Kidney weights were statistically significantly increased in the test animals. In rats dosed orally with 25-1 mg/kg C12/16 APG for 13 wks, reversible irritation and ulceration of the stomach mucosa was observed, but there was no systemic toxicity reported for any group. Dermal application of 6% active caprylyl/capryl glucoside, g a.i./kg, under occlusive conditions may affect the testes and accessory sex glands of rabbits; however, it was not clear if the effects were test-article related or due to stress of the occlusive procedure and resulting irritation and weight loss. Lauryl glucoside, 1-1 mg/kg by gavage, did not produce adverse reproductive or developmental effects. Lauryl glucoside,.1-1, nmol, did not have any effects in in vitro estrogenicity assays. 1 CIR Panel Book Page 24

30 APGs (chain length not specified), tested at 8-5 µg/l and 11-9 µg/plate in distilled water, were not mutagenic in Ames tests with or without metabolic activation. C1-16 APG, tested at concentrations of 16 µg/ml with and 16 µg/ml without metabolic activation, was not clastogenic. Caprylyl/capryl glucoside, 3% a.i., was slightly irritating to rabbit skin. With APGs of varying chain length (C8/1 to C12/16; 15-7% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from. to 4.6 in rabbits. (A PDII of 2 was considered a positive responder). In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.% a.i.) and soap chamber tests (1.% a.i.), and decyl glucoside was evaluated in an SIOPT (.5% a.i.). At most, these ingredients were slightly irritating. Glucosides with alkyl chain lengths ranging from C8-C1 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of % for intradermal induction, 5-1% for epidermal induction, and 2.5-5% for challenge, and the local lymph node assay (LLNA) at concentrations of %. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitization at concentrations of 8.4%, 5.9%, and.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 2%) and the Magnusson-Kligman protocol (1, 6, and 1% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively). C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. In clinical testing, the sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. aq. coco-glucoside was evaluated in a human repeated insult patch test (HRIPT). These ingredients were not irritating or sensitizing. In alternative system studies for mucosal irritation, decyl, lauryl, C1-16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating. In a HET-CAM study with APG of varying proportions of alkyl chain length, the ocular irritation potential increased with the increased proportion of shorter-chain APGs. In studies using rabbits, neutralized lauryl glucoside produced slight ocular reactions. Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 1% for 3% a.i. caprylyl/capryl glucoside and 5% for 6% a.i. caprylyl/capryl glucoside 11 CIR Panel Book Page 25

31 TABLES Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Ethyl Glucoside Ethyl Glucoside is the product obtained from the condensation of ethyl alcohol and glucose. Skin- Conditioning Agents - Humectant Butyl Glucoside Butyl Glucoside is the product obtained by the condensation of butyl alcohol with glucose. Surfactants - Cleansing Agents H H O O O O OH CH 3 Caprylyl Glucoside Caprylyl Glucoside is the product obtained by the condensation of caprylic alcohol with glucose. Surfactants - Cleansing Agents OH n Decyl Glucoside Decyl Glucoside is the product obtained from the condensation of decyl alcohol with glucose. Surfactants - Cleansing Agents Undecyl Glucoside Undecyl Glucoside is the product obtained by the condensation of undecyl alcohol with glucose. Surfactants - Cleansing Agents Lauryl Glucoside Lauryl Glucoside is the product obtained by the condensation of lauryl alcohol with glucose. Surfactants - Cleansing Agents Myristyl Glucoside Myristyl Glucoside is the product obtained by the condensation of myristyl alcohol with glucose. Surfactants - Cleansing Agents 12 CIR Panel Book Page 26

32 Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Arachidyl Glucoside Arachidyl Glucoside is the product obtained by the condensation of Arachidyl Alcohol with glucose. Surfactants - Cleansing Agents Mixtures Caprylyl/ Capryl Glucoside Caprylyl/Capryl Glucoside is the product obtained by the condensation of a mixture of caprylic and decyl alcohols with glucose. Surfactants - Cleansing Agents C1-16 Alkyl Glucoside C1-16 Alkyl Glucoside is the product obtained by the condensation of C1-16 alcohols with glucose. Surfactants - Emulsifying Agents wherein R = an alkyl chain 8 or 1 carbons long C12-18 Alkyl Glucoside C12-18 Alkyl Glucoside is the product obtained by the condensation of C12-18 alcohols with glucose. Emulsion Stabilizers wherein R = an alkyl chain 1 to 16 carbons long C12-2 Alkyl Glucoside C12-2 Alkyl Glucoside is the product obtained by the condensation of C12-2 alcohols with glucose. Surfactants - Emulsifying Agents wherein R = an alkyl chain 12 to 18 carbons long Cetearyl Glucoside Cetearyl Glucoside is the product obtained by the condensation of cetearyl alcohol with glucose. Surfactants - Emulsifying Agents wherein R = an alkyl chain 12 to 2 carbons long C2-22 Alkyl Glucoside C2-22 Alkyl Glucoside is the product obtained by the condensation of C2-22 alcohols with glucose. wherein R = an alkyl chain 16 or 18 carbons long Coco- Glucoside Coco-Glucoside is the product obtained by the condensation of coconut alcohol with glucose. Surfactants - Cleansing Agents wherein R = an alkyl chain 2 to 22 carbons long wherein R = alkyl chain residue of fatty alcohols derived from Coconut Acid 13 CIR Panel Book Page 27

33 Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Branched Isostearyl Glucoside Isostearyl Glucoside is the product obtained by the condensation of isostearyl alcohol with glucose. Distributed for Comment Only -- Do Not Cite or Quote Surfactants - Emulsifying Agents one example of an iso Octyldodecyl Glucoside Octyldodecyl Glucoside is the product obtained by the reaction of Octyldodecanol with glucose. Surfactants - Emulsifying Agents 14 CIR Panel Book Page 28

34 Table 2. Chemical and physical properties Property Description Reference Appearance molecular weight active substance boiling point melting point critical micelle concentration Viscosity Density log P may contain (as Plantacare 2 UP): magnesium oxide free fatty alcohol sulfate ash (as APG 81): free fatty acid ash molecular weight boiling point melting point Density log P Decyl Glucoside cloudy, viscous aq. solution (as Plantacare 2 UP) (of poly) light yellow aq. solution (as APG 81) (of poly) 34.2 (of mono) 39 g/mol (as Plantacare 2) (of poly) 51-55% (as Plantacare 2 UP) (of poly) 5% (as APG 81) (of poly) C (of mono) C (of mono) 2-3 mm (of poly, specifically the maltopyranoside) 1-6 mpas (2 C) aq. solution (as Plantacare 2 UP) (of poly) 5 mpas (2 C) aq. solution (as APG 81, a polyglucoside) (of poly) 1.14 g/cm 3 (at 2 C) (of mono) 2.92 (at 25 C) (of mono) max. 5 ppm (of poly) max. 1.% (of poly) max. 3.% (of poly) 1% (of poly) 2% (of poly) C C molecular weight boiling point melting point Density log P Appearance 1.4 g/cm 3 (at 2 C) (at 25 C) 412. C C molecular weight active substance boiling point melting point critical micelle concentration Density log P may contain (as Plantacare 81 UP): fatty alcohol 1.3 g/cm 3 (at 2 C) (at 25 C) Ethyl Glucoside (mono) Butyl Glucoside (mono) Caprylyl Glucoside white solid yellowish, slightly cloudy and viscous aq. solution (as Plantacare 81 UP) (of poly) 62-65% (as Plantacare 81 UP) (of poly) C (sic) 2-25 mm 1.18 g/cm 3 (at 2 C).887 (at 25 C).7% (of poly) CIR Panel Book Page 29

35 Table 2. Chemical and physical properties Property Description Reference molecular weight boiling point Density log P Appearance C 1.13 g/cm 3 (at 2 C) (at 25 C) Undecyl Glucoside (mono) Lauryl Glucoside viscous pale yellow aq. solution (as APG 1214,) (of poly) molecular weight (of mono) 42 (as Plantacare 12 UP) (of poly) active substance boiling point critical micelle concentration Viscosity Density log P may contain (as Plantacare 129 UP) fatty alcohol ash (as APG 1214): free fatty acid ash 5-53% (as APG 1214 and as Plantacare 12 UP) (of poly) C (of mono).13 mm (of poly, specifically the maltopyranoside) 2 mpas (2 C) aq. solution (as APG 1214) (of poly) 1.12 g/cm 3 (at 2 C) (of mono) (at 25 C) (of mono).8% (of poly) 2%(of poly) 1% (of poly) 2%(of poly) molecular weight boiling point Density log P C molecular weight boiling point Density log P Appearance 1.9 g/cm 3 (at 2 C) (at 25 C) C 1.4 g/cm 3 (at 2 C) 7.46 (at 25 C) Myristyl Glucoside (mono) Arachidyl Glucoside (mono) Coco-Glucoside (poly) cloudy, viscous aq. solution (as Plantacare 818 UP) cloudy, viscous pale yellow aq. solution (as APG 814, a polyglucoside) % active 51-53% (as Plantacare 818 UP) 5% (as APG 814, a polyglucoside) Viscosity may contain (as Plantacare 818 UP) magnesium oxide free fatty alcohol sulfate ash may contain (as APG 814): free fatty acid ash 25-6 mpas (2 C) aq. solution (as Plantacare 818 UP) 2 aq. solution (as APG 814, a polyglucoside) max. 5 ppm magnesium max. 1.% max. 3.% 1% 2% , CIR Panel Book Page 3

36 Table 3a. Frequency and concentration of use according to duration and type of exposure Decyl Glucoside Arachidyl Glucoside C12-2 Alkyl Glucoside # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 Totals* Duration of Use Leave-On Rinse Off Diluted for Use NR NR NR NR Exposure Type Eye Area Possible Ingestion NR NR NR NR NR NR Inhalation NR.5-.6 NR NR NR NR Dermal Contact Deodorant (underarm) NR NR NR NR NR.6 Hair - Non-Coloring NR Hair-Coloring NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR Bath Products NR NR NR NR Baby Products 25 NR NR NR NR NR Caprylyl/Capryl Glucoside Caprylyl Glucoside Cetearyl Glucoside # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 Totals* NR Duration of Use Leave-On NR Rinse Off NR NR Diluted for Use NR NR NR NR 1 NR Exposure Type Eye Area NR NR Possible Ingestion NR NR NR NR NR NR Inhalation NR NR NR NR 3.6 Dermal Contact NR NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR Hair-Coloring NR 3 NR NR NR.2 Nail NR NR NR NR 2 NR Mucous Membrane 6 NR NR NR 5.3 Bath Products NR NR NR NR 1 NR Baby Products NR.6 NR NR 3 NR Coco-Glucoside Ethyl Glucoside Lauryl Glucoside # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 Totals* Duration of Use Leave-On Rinse Off Diluted for Use 14 NR NR NR Exposure Type Eye Area NR 5 Possible Ingestion NR.5 NR NR NR NR Inhalation 1.4 NR NR NR 8 Dermal Contact Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR Hair-Coloring NR NR Nail NR NR NR NR NR NR Mucous Membrane NR Bath Products 14 NR NR NR Baby Products 11 NR NR NR 5 NR 17 CIR Panel Book Page 31

37 Table 3a. Frequency and concentration of use according to duration and type of exposure Myristyl Glucoside Hexadecyl D-Glucoside** Octadecyl D-Glucoside** # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 # of Uses 5 Conc of Use (%) 6 Totals* % 1 NR Duration of Use Leave-On NR Rinse Off 1 NR NR NR NR NR Diluted for Use NR NR NR NR NR NR Exposure Type Eye Area 2.4 NR NR NR NR Possible Ingestion NR NR NR NR NR NR Inhalation NR NR NR NR NR NR Dermal Contact NR Deodorant (underarm) NR NR NR NR NR 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 NR NR NR NR Bath Products NR NR NR NR 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 my not equal the sum of total uses. ** These ingredients are included in the VCRP, but are not listed in the International Cosmetic Ingredient Dictionary and Handbook NR none reported Table 3b. Ingredients Not Reported to be Used Butyl Glucoside C1-16 Alkyl Glucoside C12-18 Alkyl Glucoside C2-22 Alkyl Glucoside Isostearyl Glucoside Octyldodecyl Glucoside Undecyl Glucoside 18 CIR Panel Book Page 32

38 Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference IRRITATION STUDIES NON-HUMAN Caprylyl/Capryl Glucoside 3% a.i. rabbits; no. not specified not provided slightly irritating,; PII C8/1 APG 15% a.i 5 rabbits 4 h application; semi-occlusive patch PDII=. C8/1 APG 35% a.i.; n:1.6 3 rabbits 4 h application; semi-occlusive patch PDII = 1.3; no signs of systemic toxicity C8/1 APG 7% a.i.; n:1.6 3 rabbits 4 h application; semi-occlusive patch PDII =.8; no signs of systemic toxicity C1/16 APG 2% a.i.; n:1.4 4 rabbits 4 h application; occlusive patch PDII=.4; no signs of systemic toxicity C1/16 APG 6% a.i.; n:1.4 4 rabbits 4 h application; occlusive patch PDII=4.6; erythema and edema in all animals; 24/48/72 h mean erythema score- 2.9, mean edema score-2.1 C12/16 APG 5% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.7; no signs of systemic toxicity; erythema and edema in all animals; 24/48/72 h mean erythema score-2.2, mean edema score-1.6 C12/16 APG 5% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.; no signs of systemic toxicity; erythema in all animals and edema in 1 animal; 24/48/72 h mean erythema score- 2.1, mean edema score-.9 C12/16 APG 5% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.; no signs of systemic toxicity; C8/1 + C12/16 APG C8/1, 21% a.i. C12/16, 35% a.i. erythema in all animals and edema in 1 animal; 24/48/72 h mean erythema score- 1.9, mean edema score rabbits 4 h application; semi-occlusive patch PDII=2.7; erythema and edema in all animals; 24/48/72 h mean erythema score- 1.8, mean edema score-.8 HUMAN Decyl Glucoside 2.% a.i., ph subjects epicutaneous patch test; 75 µl, 24 h occlusive application Decyl Glucoside 1.% a.i., ph subjects soap chamber test; 1 µl applied occlusively to the ventral for 24 h on day 1 and 6 h on days 2-5 Decyl Glucoside a.i. not stated; tested at.5% aq. 15 subjects; 14.3% were atopic patients SIOPT; 4 µl was applied for 48 h using Haye s test chambers very slightly irritating slightly irritating AII=.46; non-irritating Lauryl Glucoside 2.% a.i., ph subjects epicutaneous patch test; 75 µl, 24 h occlusive application Lauryl Glucoside 1.% a.i., ph subjects soap chamber test; 1 µl applied occlusively to the ventral for 24 h on day 1 and 6 h on days 2-5 slightly irritating slightly irritating CIR Panel Book Page 33

39 Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference AII=.46; non-irritating 33 Lauryl Glucoside a.i. not stated; tested at.5% aq. 15 subjects; 14.3% were atopic patients SIOPT; 4 µl was applied for 48 h using Haye s test chambers Coco-Glucoside 2.% a.i., ph subjects epicutaneous patch test; 75 µl, 24 h occlusive application Coco-Glucoside 1.% a.i., ph subjects soap chamber test; 1 µl applied occlusively to the ventral for 24 h on day 1 and 6 h on days 2-5 C8-C1 glucoside a.i. not stated; tested at % in acetone/olive oil (4:1) C8-C1 glucoside a.i. not stated; tested at 5% - intraderm induction 5% - epiderm induction 2.5 and 5% - challenge C1-C14 glucoside a.i. not stated; tested at % in DMF C1-C14 glucoside a.i. not stated; tested at C12/16 APG (may be similar to C12-18 alkyl 5% - intraderm induction 5% - epiderm induction 2.5 and 5% - challenge glucoside) C12/16 APG (may be similar to C12-18 alkyl glucoside) C12-C18 glucoside a.i. not stated; tested at 2.5- (granules) 1% in DMF C12-C18 glucoside a.i. not stated; tested at (granules) 1% - intraderm induction 5% - epiderm induction 5 and 1% - challenge C12-C18 glucoside (flakes) a.i. not stated; tested at % in DMF C12-C18 glucoside (flakes) a.i. not stated; tested at SENSITIZATION NON-HUMAN slightly irritating slightly irritating 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 1% was an irritant (based on ear thickness); not a sensitizer guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 not an irritant or sensitizer 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 not an irritant or sensitizer guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 a.i. not provided 2 guinea pigs Buehler method; 2% tested at induction and challenge a.i. not provided 2 guinea pigs Magnusson-Kligman study; 1% used for intracutaneous and 6% for epidermal induction; 1% for epidermal challenge 1% - intraderm induction 5% - epiderm induction 5 and 1% - challenge C14 glucoside a.i. not stated; tested at % in DMF not an irritant or sensitizer not a sensitizer; one very weak reaction during induction and challenge for one guinea pig not a sensitizer; no positive reactions 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 not an irritant or sensitizer guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 not an irritant or sensitizer 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 may cause skin sensitization guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 not an irritant or sensitizer 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 all concentrations were irritants (based on ear thickness); may cause skin sensitization CIR Panel Book Page 34

40 Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference not an irritant or sensitizer 61 C14 glucoside a.i. not stated; tested at 1.25%-intraderm induction 5% - epiderm induction 25 and 5% - challenge C16-C18 glucoside a.i. not stated; tested at 2.5-1% in DMF C16-C18 glucoside a.i. not stated; tested at 1% - intraderm induction 1% - epiderm induction 5 and 1% - challenge >C18 glucoside a.i. not stated; tested at 2.5-1% in DMF >C18 glucoside a.i. not stated; tested at 5% - intraderm induction 1% - epiderm induction 2.5 and 5% - challenge C18 branched glucoside a.i. not stated; tested at 2.5-5% in DMF C18 branched glucoside a.i. not stated; tested at 2.5% - intraderm induction 1% - epiderm induction 6.25 and 12.5% - challenge Decyl Glucoside; tested under 4 tradenames guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm CBA/j mice LLNA; application volume of.25 g/cm 2 not an irritant or sensitizer guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 not an irritant or sensitizer 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 not an irritant or sensitizer guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 not an irritant or sensitizer 4-5 CBA/j mice LLNA; application volume of.25 g/cm 2 all concentrations were irritants (based on ear thickness); may cause skin sensitization guinea pigs; 1 treated, 5 controls GPMT with FCA and SLS; occlusive induction patch application volume of.625 g/cm 2 HUMAN 5% a.i. 49 subjects HRIPT;.2 ml, 24-h semi-occlusive; 3x/wk for 3 wks; 1 applications; challenge performed after 2-wk non-treatment period not an irritant or sensitizer not an irritant or a sensitizer Lauryl Glucoside; tested under 5 tradenames 5% a.i. 49 subjects HRIPT, as above not an irritant or a sensitizer 34 Coco-Glucoside 52% a.i. 2% aq. Tested 213 subjects HRIPT;.2 ml, 24 h occlusive; 3x/wk for 3 wks; 9 applications; challenge performed after 2-wk non-treatment period C8-C1 glucoside a.i. not stated; tested at 5% aq. C1-C14 glucoside a.i. not stated; tested at 5% aq. C12-C18 glucoside a.i. not stated; tested at 5% (granules) aq. C12-C18 glucoside (flakes) a.i. not stated; tested at 1% aq. C14 glucoside a.i. not stated; tested at 5% aq. 5 subjects HRIPT; 2 µl, 9 occlusive applications, not an irritant or a sensitizer irritation index during induction=.4; no application volume of.4 g/cm 2 positive reactions at challenge 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction = ; no application volume of.4 g/cm 2 positive reactions at challenge 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction =.1; no application volume of.4 g/cm 2 positive reactions at challenge 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction =.15; no application volume of.4 g/cm 2 positive reactions at challenge 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction =.62; no application volume of.4 g/cm 2 positive reactions at challenge CIR Panel Book Page 35

41 Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference C16-C18 glucoside a.i. not stated; tested at 5% aq. 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction =.3; no application volume of.4 g/cm 2 positive reactions at challenge >C18 glucoside a.i. not stated; tested at 5% 5 subjects HRIPT; 2 µl, 9 occlusive applications, aq. C18 branched glucoside a.i. not stated; tested at 6% 5 subjects HRIPT; 2 µl, 9 occlusive applications, irritation index during induction =.21; no application volume of.4 g/cm 2 positive reactions at challenge irritation index during induction =.4; no application volume of.4 g/cm 2 positive reactions at challenge Abbreviations: a.i. active ingredient; AII average index of skin irritation; APG alkyl polyglucoside; DMF dimethyl formamide; FCA Freund s complete adjuvant; HRIPT human repeat insult patch test; LLNA local lymph node assay; n degree of polymerization; PDII primary dermal irritation index; SIOPT single insult occlusive patch test; SLS sodium lauryl sulfate positive responder: irritation score = CIR Panel Book Page 36

42 Table 5. Mucosal irritation studies Ingredient/Chain Length % a.i.; ph; conc. tested Animals Method Results Reference ALTERNATIVE STUDIES Decyl Glucoside 1.% a.i. in PBS; ph 7 RBC not irritating Decyl Glucoside 3.% a.i.; ph 6.5, aq. soln HET-CAM assay slightly irritating Decyl Glucoside.6% a.i.; ph 7., aq. soln ocular tissue model not irritating Lauryl Glucoside 1.% a.i. in PBS; ph 7 RBC slightly irritating Lauryl Glucoside 3.% a.i.; ph 6, aq. soln HET-CAM assay slightly irritating Lauryl Glucoside.6% a.i.; ph 7., aq. soln ocular tissue model not irritating Caprylyl/Capryl Glucoside, as C8/C1 APG not specified HET-CAM assay highly irritating 21 C1-16 Alkyl Glucoside, as ph 7 HET-CAM assay produced slight reactions C1/16 APG C1-16 Alkyl Glucoside, as ph 11.5 HET-Cam assay produced slight reactions C1/16 APG Coco-Glucoside 1.% a.i. in PBS; ph 7.4, aq. Soln RBC not irritating Coco-Glucoside 3.% a.i.; ph 6.5, aq. soln HET-CAM assay slightly irritating Coco-Glucoside.6% a.i.; ph 7., aq. soln ocular tissue model not irritating NON-HUMAN STUDIES Lauryl Glucoside, neutralized 12.5% a.i. 6 NZW rabbits Draize test; eyes were not rinsed very slight reactions, in one animal (subsided in 48 h); medium to mild conjunctival irritation was observed in all rabbits; effects were reversible within 7 days in all but one of the rabbits Caprylyl/Capryl Glucoside 3% a.i. 9 rabbits.1 ml instilled into the conjunctival sac; eyes of 3 rabbit rinsed 2-3 sec after dosing Caprylyl/Capryl Glucoside 3% a.i. 9 rabbits.1 ml instilled into the conjunctival sac; eyes of 3 rabbit rinsed 2-3 sec after dosing Caprylyl/Capryl Glucoside 3% a.i. 9 rabbits.1 ml instilled into the conjunctival sac; eyes of 3 rabbit rinsed 2-3 sec after dosing Caprylyl/Capryl Glucoside 3% a.i. 9 rabbits.1 ml instilled into the conjunctival sac; eyes of 3 rabbit rinsed 2-3 sec after dosing Caprylyl/Capryl Glucoside 3% a.i. 9 rabbits.1 ml instilled into the conjunctival sac; eyes of 3 rabbit rinsed 2-3 sec after dosing Caprylyl/Capryl Glucoside 3% a.i.;.1-5% tested 2 rabbits/group; 4 additional rabbits dosed w/5 and 1% severely irritating; rinsing reduced the intensity and duration severely irritating; rinsing reduced the intensity and duration severely irritating severely irritating severely irritating.1 ml instilled into the conjunctival sac.1,.5, 1., 5.%: non or inconsequential irritant 1%: conjunctival irritation in 6/6 at 4 h; subsided in 4/6 by 72 h; moderate irritant 2%: moderate irritant 5% substantial to severe irritant Irritation threshold determined to be 1% (v/v); equivalent to 3% solids CIR Panel Book Page 37

43 Table 5. Mucosal irritation studies Ingredient/Chain Length % a.i.; ph; conc. tested Animals Method Results Reference Caprylyl/Capryl Glucoside 6% a.i. rabbits details not provided severely irritating 42 Caprylyl/Capryl Glucoside 6% a.i.; conc. of.5, 1., 5., and 1.% tested 6% a.i.; conc. of 2 and 1.% tested 6 rabbits/group.1 ml instilled into the conjunctival sac.5 and1.%: no irritation 5%: conjunctival irritation at 4 h, cleared by 72 h 1%: moderate irritation in 6/6, cleared by 72 h 2 rabbits/group.1 ml instilled into the conjunctival sac 2 and 5%: severely irritating Irritation threshold determined to be 5.% Caprylyl/Capryl Glucoside 7% a.i.; 4% solution 6 rabbits/study.1 ml instilled into the conjunctival sac; two studies performed moderately to highly irritating (both studies) Caprylyl/Capryl Glucoside 7% a.i. not provided not provided; two studies performed moderately to highly irritating (both studies) Caprylyl/Capryl Glucoside 7% a.i.; 4% solution 6 rabbits.1 ml instilled into the conjunctival sac; two moderately to highly irritating (both studies) studies performed C12/16 APG (may be similar to C12-18 alkyl glucoside) 5% a.i, aq. Solution 4 albino rabbits OECD Guideline 45 24/48/72 h mean scores for the cornea, conjunctival erythema, and iris:.5/4, 2.8/3, and.25/2, respectively; moderate to strong reactions in the conjunctivae did not completely subside within 21 days in 2 of the animals, persistent corneal effects did not subside in 1 of these rabbits Abbreviations: a.i. active ingredient; HET-CAM hen s egg test-chorioallantoic membrane; PBS phosphate buffered saline; RBS red blood cell test CIR Panel Book Page 38

44 Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference n-butyl Alcohol ADME can be absorbed through the lungs, gastrointestinal tract, the cornea, and the skin; mainly metabolized by alcohol dehydrogenase and eliminated rapidly from the 64 blood; dogs given i.v. n-butyl alcohol eliminated 15% of the does in CO 2(none unchanged) and 2.7% in the urine animal toxicology dermal LD 5 (rabbits), 4.2 g/kg; oral LD 5 (rats), g/kg short-term oral: 6.9% n-butyl alcohol and 25% sucrose given in drinking water for 3 wks produced some changes in hepatic mitochondria inhalation: results in irritation of the mucous membranes, intoxication, restlessness, ataxia, prostration, and narcosis; high concentrations can be fatal dermal irritation/sensitization no data mucosal irritation repro/developmental toxicity Genotoxicity Carcinogenicity clinical assessment of safety important Discussion items Conclusion 15% n-butyl alcohol produced an ocular irritation score of <5/2 and a 4% solution produced a score of >5/2 in rabbit eyes fetotoxicity has been demonstrated at maternally toxic levels (1 mg/kg); no significant behavioral or neurochemical effects were seen in offspring following either maternal or paternal exposure to 3 or 6 ppm negative in an Ames test, did not induce sister chromatid exchange.1 or (15% aq.) or micronuclei formation, and did not impair chromosome distribution in mitosis no data a nail color containing 3% n-butyl alcohol was not a significant irritant or sensitizer in HRIPTs, and this product was not a phototoxin or photoallergen; negative for non-immunological urticaria occupational exposure: n-butyl alcohol (alone or with other solvents) produced complaints of ocular irritation, headache and vertigo, slight irritation of the nose and throat, and dermatitis of the hands and fingers at air concentrations of >5 ppm uses in products other than nail products are at very low concentrations, so there were no toxicity concerns safe as used Cetearyl Alcohol animal toxicology no data 65 dermal irritation/sensitization mucosal irritation repro/developmental toxicity Genotoxicity Carcinogenicity clinical assessment of safety important Discussion items Conclusion Distributed for Comment Only -- Do Not Cite or Quote formulation w/3%, mildly irritating (rabbits) formulation w/3%, not irritating no data no data no data formulation w/3%: not a sensitizer no relevant items identified safe as used Cetyl Alcohol ADME in general, long-chain aliphatic alcohols, such as cetyl alcohol, are oxidized to their corresponding fatty acids in mammalian tissues; in rats administered radioactive cetyl alcohol by either stomach tube or thoracic duct fistulas, most of the radioactivity was found in the thoracic duct lymph, indicating good absorption; some of the cetyl alcohol was eliminated unchanged in waste products, but most of the cetyl alcohol was oxidized to palmitic acid and incorporated into triglycerides and phospholipids animal toxicology oral LD 5(rats): >8.2 g/kg; formulations w/ 4%, no toxic effects; dermal LD 5: >2.6 g/kg; formulation w/5%, 2 g/kg; inhalation: 6-h exposure, 26 ppm (rats, mice, guinea pigs), slight irritation of mucous membranes, but no signs of systemic toxicity or mortality; 6 h exposure, 222 mg/m 3, 1% mortality short-term dermal: 2 day, 11.5%, 5x/day, exfoliative dermatitis, parakeratosis, hyperkeratosis (rabbits); 3 day, 3% in methyl alcohol and propylene glycol, dermal infiltrates of histocytes 3 mos dermal study: formulations w/2%, well-defined erythema, mild edema, no systemic toxicity (rabbits) dermal irritation/sensitization undiluted, minimally to slightly irritating; formulations w/2-4%, no to well-defined erythema and edema mucosal irritation formulations w/ 6.36%, mostly non-irritating mucosal irritation 2%: not irritating to genital mucosa of rabbits repro/developmental toxicity no data Genotoxicity negative, Ames test Carcinogenicity no data clinical assessment of safety 1%: not irritating; formulations w/2-11.5%,:at most, mild irritants formulations w/1-8.4%, not sensitizers 3%: 11.2% of eczema patients (pop. 33) had allergic reactions formulations w/1-4%, not photosensitizers important Discussion items no relevant items identified Conclusion safe as used CIR Panel Book Page 39

45 Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference Coconut Alcohol animal toxicology no data 66 dermal irritation/sensitization mucosal irritation repro/developmental toxicity Genotoxicity Carcinogenicity clinical assessment of safety important Discussion items Conclusion no data no data no data no data no data no data toxicity and use profiles expected to be similar to coconut oil, coconut acid, hydrogenated coconut oil, hydrogenated coconut acid; addressed use in inhalation products; possible issues with botanicals safe as used Isostearyl Alcohol animal toxicology oral LD 5: >2 g/kg (rats); formulations w/25-27%, >15 g/kg 65 dermal irritation/sensitization mucosal irritation repro/developmental toxicity Genotoxicity Carcinogenicity clinical assessment of safety important Discussion items Conclusion Distributed for Comment Only -- Do Not Cite or Quote formulation w/5%: mild irritant (rabbits); formulation w/25-27%: barely perceptible erythema.2-5%: not a sensitizer formulations w/5 and 1%, transient irritation; formulations w/25-27%, minimal to mild irritation no data no data no data 1%: not irritating; formulations w/25-28%, not irritating; deodorant formulation w/ 5%, severe irritation in a 21-day cumulative study 25% in 95% isopropyl alcohol: not a sensitizer; formulations w/5%: sensitization reactions occurred no relevant items identified safe as used Myristyl Alcohol animal toxicology oral LD 5 (rats): >8 g/kg; formulation w/.8%, >5 g/kg; dermal LD 5: formulation w/.8%, >2 g/kg inhalation: 3%, 1 h, ataxia and moderate nasal irritation in all animals 1 min after exposure, no mortality dermal irritation/sensitization formulation w/.8%, non-irritating (rabbits) mucosal irritation formulation w/.8%: not irritating; formulation w/3%: mildly irritating (rinsed eyes), moderately irritating (unrinsed eyes) repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety formulations w/.1-.25%, not irritants; formulations w/.25-.8%, not irritating in a 4-wk clinical study formulations w/.1-.25%, not sensitizers formulation w/.1%, not a photosensitizer important Discussion items no relevant items identified Conclusion safe as used Octyl Dodecanol animal toxicology oral LD 5 (rats): >5 g/kg, undiluted; formulation w/1.2%, >25 g/kg; dermal LD 5: >3 g/kg dermal irritation/sensitization 1%: irritation score of -1.13/4 (rabbits); 3%: irritation score /4 (rabbits); formulations w/4 and 1.2%, mild irritation, at most; technical grade: moderate to severe irritation (rabbits, guinea pigs, rats), no irritation (swine, humans) mucosal irritation 1%: irritation score of 1 or 4/11 (24 h) repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety 1%: mild irritation in 1/4 subjects; undiluted technical grade: no irritation; formulations w/3-1.2%: essentially non-irritating screening patch tests for contact sensitization in large populations: incidence rate of.36% (6/1664) formulation w/1.2%: not phototoxic or photoallergenic important Discussion items Conclusion no Discussion safe as used CIR Panel Book Page 4

46 Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference Stearyl Alcohol ADME found naturally in various mammalian tissues; readily converted to stearic acid, another common constituent of mammalian tissues; results from several studies indicate that stearyl alcohol is poorly absorbed from the GI tract animal toxicology oral LD 5: >8 g/kg; 3 mos dermal study: formulations w/8%,some dermal effects,, no systemic toxicity (rabbits) dermal irritation/sensitization 1%: minimal to mild primary skin irritant (rabbits) mucosal irritation repro/developmental toxicity Genotoxicity Carcinogenicity clinical assessment of safety important Discussion items Conclusion Distributed for Comment Only -- Do Not Cite or Quote formulation w/24%: not a sensitizer 1%: mildly irritating no data negative: Ames test did not promote tumor formation in mice when tested with dimethylbenz[a]anthracene 1%: produced mild irritation in 1/8 subjects; formulations w/14-24% were nonto slightly irritating formulations w/14-2%, not sensitizers screening patch tests for contact sensitization in large population: incidence rate of.51% (19/374) Discussion not included in report safe as used CIR Panel Book Page 41

47 REFERENCES 1. Le Coz, C. J. and Meyer, M. T. Contact allergy to decyl glucoside in antiseptic after body piercing. Contact Dermatitis. 23;48:(5): Fischer E. Uber die Glucoside der Alkohole. Berichte. 1893;26: Barel AO, Paye M, and Maiback HI (eds). Handbook of Cosmetic Science and Technology. 3 ed Gottschalck T.E. and Bailey, J. E. eds. 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. Updated concentration of use by FDA product category: Glucoside ingredients Unpublished data submitted by the Council on Jan, 28, 211. (5 pp) Available from CIR. 7. James AC, Stahlhofen W, Rudolf G,, and et al. Deposition of inhaled particles. Annals of the ICRP. 1994;24:(1-3): Oberdorster G, Oberdorster E, and Oberdorster J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Helath Perspect. 25;113:(7): Bower D. Unpublished information on hair spray particle size provided at the September 9, 1999 CIR Expert Panel meeting Johnson MA. The influence of particle size. Spray Technology and Marketing. 24;November: European Commission.European Commission Health and Consumers Cosmetics - Cosing - Database Accessed Aungst, B. J. Site-dependence and structure-effect relationships for alkylglycosides as transmucosal absorption promoters for insulin. Int.J.Pharm. 1994;15:(May 9): Savic S, Lukic M, Jaksic I, Reichl S, Tamburic S, and Müller-Goymann C. An alkyl polyglucoside-mixed emulsifer as stabilizer of emulsion systems: The influence of colloidal structure on emulsions skin hydration potential. J Colloid Interface Sci. 211;358: Kobierski, S., Ofori-Kwakye, K., ller, R. H., and Keck, C. M. Resveratrol nanosuspensions for dermal application-- production, characterization, and physical stability. Pharmazie. 29;64:(11): Mishra, P. R., Al, Shaal L., ller, R. H., and Keck, C. M. Production and characterization of Hesperetin nanosuspensions for dermal delivery. Int J Pharm. 29;37:(1-2): Glucoside Hydrolase Activity of Normal and Glucosylceramidotic Cultured Human Skin Fibroblasts. J Biological Chem. 1977;252:(3): Across Barriers GmbH. In vitro permeation and penetration of Capryl glucoside (INCI: Caprylyl/Capryl Glucoside) from the Product Plantacare 81 UP in human skin under GLP conditions. GLP Study Report STP 44/. Science Park Saarbruecken Germany, Across Barriers GmbH Weber, N. and Benning, H. Metabolism of orally administered alkyl β-glycosides in the mouse. J Nutr. 1984;114:(2): CIR Panel Book Page 42

48 19. Mishima, T., Katayama, Y., Takagi, Y., Ozeki, K., Hayakawa, T., and Tsuge, H. Ethyl alpha-d-glucoside increases urine volume and causes renal morphologic changes in rats. J Nutr Sci Vitaminol.(Tokyo). 25;51:(1): Tirumalasetty, P. P. and Eley, J. G. Permeability enhancing effects of the alkylglycoside, octylglucoside, on insulin permeation across epithelial membrane in vitro. J Pharm Pharm Sci. 26;9:(1): Ecology and toxicologly of alkyl polyglycosides. 24. Chapter: 18. Handbook of Detergents, Part B. Environmental Impact. New York NY: Marcel Dekker. 22. Rohm and Haas Company. 2-Week repeat dermal toxicity study in rabbits of Triton CG-11 (6%). Protocol No. 85P-144. Report No. 85R NTIS No. OTS Rohm and Haas Company. 2-Week dermal toxicity study in rabbits on Triton CG-11 (6%). Protocol NO. 82P- 25. Report No. 82R NTIS No. OTS Rohm and Haas Company. Histopathology report of a 2-week repeat dermal toxicity study in rabbits of Triton CG- 11 (6%). Protocol No. 82P-25. Report No. 83R NTIS No. OTS Rohm and Haas Company. 2-Week repeat dermal toxicity study in male rabbits of Triton-CG-11 (6%). Protocol No. 83P-533. Report no. 84R NTIS No. OTS Rohm and Haas Company. Histopathology report of a 2-week repeat dermal toxicity study in rabbits of Triton CG- 11 (6%). Protocol No. 85P-144. Histopathology report no. 85R NTIS No. OTS Messinger, H., Aulmann, W., Kleber, M., and Koehl, W. Investigations on the effects of alkyl polyglucosides on development and fertility. Food Chem Toxicol. 27;45:(8): Yang, J. H., Lu, Y., and Zhang, J. H. [Research on spermicidal action of octyl-beta-d-glucopyranoside (OGP) in vitro] (English abstract.). Shengzhi.Yu Biyun.(Reproduction and Contraception) 21.Jun.;21(3): ;(Reproduction and Contraception 21 Jun;21(3):173-5). 29. Rohm and Haas Company. Ocular irritation study on Triton CG-11, 3% a.i.. Report No. 8R NTIS No. OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 3% a.i.. Report No. 8R OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 3% a.i.. Report No. 8R NTIS No. OTS Mehling A, Kleber M, and Hensen H. Comparative studies on the ocular and dermal irritation potential of surfactants. Food Chem Toxicol. 27;45: Corazza M, Lauriola MM, Bianchi A, Zappaterra M, and Virgili A. Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 15 patients. Dermatitis. 21;21:(5): Consumer Product Testing Company. Repeat insult patch test on decyl glucoside (5% active) and lauryl glucoside (5% active) under numerous trade names. Experiment Refn. No. C Unpublished data submitted by the Council on Dec. 14, 21. (34 pp) Available from CIR. 35. Consumer Product Testing Company. Repeated insult patch test of coco-glucoside. Experiment Ref. No. C Unpublished data submitted by the Council on Dec 14, 21 (21 pp) Available from CIR. 36. Cho, Sun A., Han, Ju Hee, An, Susun, Lee, Key Hyun, Park, Jae Hak, Kim, Han Kon, and Lee, Tae Ryong. Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants. Cutaneous and Ocular Toxicology. 21;29:(1): CIR Panel Book Page 43

49 37. Rohm and Haas Company. Ocular irritation study on Triton CG-11, 3% surfactant. Report No. 81R NTIS No. OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 3% surfactant. Report No. 81R NTIS No. OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 7% a.i. Report No NTIS No. OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 7% a.i. Report No NTIS No. OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 7% a.i. Report No OTS Rohm and Haas Company. Ocular irritation study on Triton CG-11, 6% surfactant. Report No. 82R NTIS No. OTS Rohm and Haas Company. Triton CG-11 (6%): Rabbit eye irritation titration studies. Report No. 82P-289. Protocol No. 82R NTIS No. OTS Andersen, K. E. and Goossens, A. Decyl glucoside contact allergy from a sunscreen product. Contact Dermatitis. 26;54:(6): Andrade, P., alo, M., and Figueiredo, A. Allergic contact dermatitis to decyl glucoside in Tinosorb M. Contact Dermatitis. 21;62:(2): Blondeel A. Contact allery to the mild surfactant decylglucoside. Contact Dermatitis. 24;49: Horn, H. M., Murray, C., and Aldridge, R. D. Contact allergy to decyl glucoside. Contact Dermatitis. 25;52:(4): Krehic, M. and Avenel-Audran, M. Allergic contact dermatitis from decyl glucoside in an antiseptic lotion. Contact Dermatitis. 29;61:(6): Cognis. Data profile on Plantacare 2 UP (decyl glucoside). 21. Submitted by the Counicl on Dec 14, 21. (2 pp) Available from CIR. 5. Pharmachem.Technical data sheet on alkyl polyglucoside surfactants Accessed ACD/Labs. Advanced Chemistry Development (ACD/Labs) Software (C). 52. Joshi VY. Indian Journal of Chemistry, Section B: Organic. 53. Cognis.Data sheet for Plantacare 2 UP Accessed Takahashi Y. JP B Pigman W. Journal of the American Chemical Society. 1951;73: STN International. 21. Distributed for Comment Only -- Do Not Cite or Quote 57. Cognis. Data sheet on Plantacare 81 UP (caprylyl/capryl glucoside). 21. Submitted by the Council on Dec 14, 21 (2 pp) Available from CIR. 58. Cognis. Data profile on Plantacare 12 UP (lauryl glucoside). 21. Submitted by the Council on Dec 14, 21. (2 pp) Available from CIR. 3 CIR Panel Book Page 44

50 59. Cognis.Product data sheet: Plantacare 818 UP Accessed Personal Care Products Council. Concentration of use by FDA product category: hexadecyl d-glucoside and octadecyl d-glucoside Unpublished data submitted by the Council on March 15, 211. (2 pp). 61. Garcia C, Ball N, Cagen S, Carrillo J-C, Certa H, Eigler D, Esch H, Graham C, Haux C, Kreiling R, and Mehling A. Comparative testing for the indentification os skin-sensitizing potentials of nonionic sugar lipid surfactants. Regul Toxicol Pharmacol. 21;58: Ecology and toxicology of alkyl polyglycosides. 24. Chapter: 18. Handbook of Detergents, Part B. Environmental Impact. New York, NY: Marcel Dekker. 63. Rohm and Haas Company. Triton CG-11: Rabbit eye irritation tirtration studies. Report No. 81R-131. Protocol No. 8P NTIS No Andersen, FA (ed). Final report of the addendum to the safety assessment of n-butyl Alcohol as used in cosmetics. Int J Toxicol. 28;27:(Suppl 2): Elder, RL (ed). Final report o the safety assessment of Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl Alcohol. J Am Coll Toxicol. 1988;7:(3): Diamante, CD, Andersen, FA, and Cosmetic Ingredient Review Expert Panel. Amended safety assessment of Cocos Nucifera (Coconut) Oil, Coconut Acid, Hydrogenated Coconut Acid, Hydrogenated Coconut Oil, Ammonium Cocomonoglyceride Sulfate, Butylene Glycol Cocoate, Caprylic/Capric/Coco Glycerides, Cocoglycerides, Coconut Alcohol, Coconut Oil Decyl Esters, Decyl Cocoate, Ethylhexyl Cocoate, Hydrogenated Coco-Glycerides, Isodecyl Cocoate, Lauryl Cocoate, Magnesium Cocoate, Methyl Cocoate, Octyldodecyl Cocoate, Pentaerythrityl Cocoate, Potassium Cocoate, Potassium Hydrogenated Cocoate, Sodium Cocoate, Sodium Cocomonoglyceride Sulfate, Sodium Hydrogenated Cocoate, and Tridecyl Cocoate Elder, RL (ed). Final report on the safety assessment of Stearyl Alcohol, Oleyl Alcohol, and Octyl Dodecanol. J Am Coll Toxicol. 1985;4:(5): CIR Panel Book Page 45

51 Data

52 Please Please Please Please Where What It COSMETIC the Distributed for Comment Only -- Do Not Cite or Quote Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (Cifi) FROM: John Bailey, Ph.DZIt j- Q_-._ 3(23 1 Industry Liaison to the CIR Expert Panel t DATE: March 23, 211 SUBJECT: Comments on the Scientific Literature Review on Decyl Glucoside and Other Alkyl Glucosides as Used in Cosmetics Why does this report have two covers? The first cover indicates that the report was prepared by Bart Heldreth. The second cover indicates that the report was prepared by Monice Fiume and Bart Heidreth. Is there a reason why this report does not include section summaries? p.3 - Please name the ingredient for which no uses were reported to the VCRP, but uses were reported in the Council concentration of use survey. The cosmetic use information is presented in Tables 3a and 3b not 4a and 4b as stated in the text. p.4- - indicate what is meant by and a other toxic effects were observed. p.7 - provide the doses or concentrations tested in the genotoxicity studies of C 1-16 Alkyl Glucoside. p.8 - also note that irritation was dependent on the concentration of the ingredient tested. It would be helpful to note that all the non-human studies were done in rabbits. p.8 - include the concentrations that were tested in the alternative eye irritation methods. It would be helpful to be more specific - non-animal mucosal irritation studies were all rabbit eye irritation studies. p.9 - is the Summary of this report? p.1-12, Table 1 - Please include the reference(s) with this table. p.17, Table 4-As all of the studies summarized in Table 4 were completed with rabbits, rabbits could be stated once in the title of the table rather than 1 times in the third row. What solvent was used to dilute the test substances? p.18, Table 5 - solvent was used to dilute the test substances? As all the non-human studies were done in rabbits, rabbits should be in the heading of the table. In the results for Lauryl Glucoside, neutralized, is something missing as it currently states e reversible within 7 days in all but one of the rabbits? p.19, Table 5 - would be helpful if the % active ingredient column included the concentrations that were actually tested. What solvent was used to dilute the test substances? th Street, N.W, Suite 3OO Washington, D.C (fax) CIR Panel Book Page 46

53 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Personal Care Memorandum Products Council Committed to Safety, ua ty nnovation TO: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) FROM: John Bailey, 12 I c4 1 Industry Liaison to the CIR Expert Panel DATE: December 14, 21 SUBJECT: Unpublished Information on Glucoside Ingredients Cognis, Care Chemicals. 21. Data profile PLANTACARE 81 UP (Caprylyl/Capryl Glucoside). Cognis, Care Chemicals. 21. Data profile PLANTACARE 818 UP (Coco-Glucoside). Cognis, Cre Chemicals. 21. Data profile PLANTACARE 12 UP (Lauryl Glucoside). Cognis, Care Chemicals. 21. Data profile PLANTACARE 2 JP (Decyl Glucoside). Consumer Product Testing Company Repeat insult patch tes. of 5% active Lauryl Gluoside anc Decyl Glucoside. Experiment Reference Number: C (Note: In the past, Plantaren was the trade name for product produce tin the United States. Now, the trade namc: worldwide is Plantacare. The former US product has thc same specifications as pmvided for the Pla tacare products.) Consumei Product Testing Company. 25. Repeated insult patch Iest of Coco-Glucoside (t% act ie). Experiment Reference Number: C th Street, N.W., Suite 3OO Washington, D.C (fax) CIR Panel Book Page 47

54 COGMS NATURAL MDFIED Distributed for Comment Only -- Do Not Cite or Quote l( )O j carechemicals Data Profile PLANTACARE 81 UP Address Producer I Supplier Cognis GmbH Care Chemicals D Düsseldorf Tel.: Fax.: General characterisation Chemical description C8-1 fatty alcohol glucoside Raw material basis vegetable (coconut/palm kernel oil, glucose) Labeling information INCI name(s) Caprylyl/Capryl Glucoside (EU + CTFA) Composition hints for finished product label Capryl Glucoside > 6-7 % Aqua >3-4% Ingredient information Ingredient CASR-No. EINECSIELINCS-No. Caprylyl/Capryl Glucoside no longer polymer Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at higher temperatu-e. Cognis, Care Chemicals PLANTACARE 81 UP CIR Panel 1/3Book Page 48 Rev. 3. ZJs

55 1.5 Distributed for Comment Only -- Do Not Cite or Quote Product properties Appearance PLANTACARE 81 UP is a yellowish, slightly cloudy and viscous liquid. Example of use The tirbidity of the product is attributable to a combination of its magnesium oxide content (max.,1 % magnesium) and the ph value at which it is supplied. This ttibidity has no negative effects on the products properties and disappears if the ph value is adjusted to below 8.5. PLANTACARE 81 UP is a nonionic sixfactant with excellent solubility, stability, sirface and interfacial activities. PLANTACARE 81 UP shows excellent solubilizing properties in highly concentrated sulactant solutions also in the presence of salt and alkalies. Solubility according to Ph. EUR. Ethylether Solubility: practically insoluble Chloroform Solubility: sparingly soluble Petrolether Solubility: practically insoluble Ethyalcohol Solubility: practically insoluble Paraffinoil Solubility: practically insoluble Water Solubility: practically insoluble Characteristic values 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.) Water % DGF H-Ill 3A Active matter % Difference: 1 % - % Water ph value (1% in 15% 11,5-12,5 DGF H-Ill 1 Isopropanol) Additional product descriptive data (Data vvtiich is proven statistically but not determined regularly.) Fatty alcohol <=,7 % Density (4, C) 1,13-1,14 g/cm3 Viscosity (dynamic) (4 C) 5 - mpas Cognis, Care Chemicals PLANTACARE 81 UP CIR Panel 2/3Book Page 49 Rev. 3. ZJs

56 Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present Storage and transportation Shelf life 24 months Storage temperature <+ 4 Store frost-free. Storage conditions In original sealed containers and protected from moistixe. Additional information Upon storage of PLANTACARE 81 UP separation of the product may occt-, iich has no negative effects on the product quality. The product should be stirred until uniform before use. Containers and storage tanks made of V2A steel (material no ) are of only limited use for storing PLANTACARE 81 UP, because corrosion may occir. Storage containers made of V4A (material no ) should therefore be preferred. PLANTACARE 81 UP has a high ph-value and for this reason the product contains no preservatives (suffix UP = unpreserved). 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 pu-poses 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 pu-poses. 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, Care Chemicals 5 PLANTACARE 81 UP CIR Panel 3/3 Book Page 5 Rev. 3. IIIG1:

57 COGNIS NATURAL MODIFIED carechemicals Data Profile PLANTACARE 818 UP Address Producer I Supplier Cognis GmbH Care Chemicals D Düsseldorf Tel.: Fax.: General characterisation Chemical description C8-1 6 fatty alcohol glucoside Mol weight 39 g/mol Raw material basis vegetable (coconut/palm kernel oil, glucose) Labeling information lncl name(s) Coco-Glucoside (EU + CTFA) Composition hints for finished product label Coco-Glucoside > 5-6 % Aqua >4-5% Ingredient information Ingredient CASR-No. EINECS!ELINCS-No. Coco-Glucoside no longer polymer Officially listed in I Quality conforms to JCIC: Alkyl (8-16) Glucoside (Ingredient Code: 5237) Bra Miljöval: approved for use in products eco-labelled Good Environmental Choice (in Swedish: Bra Miljoval) by the Swedish Society for Nature Conservation Cognis, Care Chemicals PLANTACARE 818 UP CIR Panel Book Page 51 1/3 Rev. 12.

58 Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at increased temperature. Product properties Appearance PLANTACARE 818 UP is a cloudy, viscous, aqueous solution of a C8-C16 fatty alcohol glycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 5 ppm magnesium) and the ph value at which it is supplied. This turbidity has no negative effects on the products properties and disappears if the ph value is adjusted to below 7. PLANTACARE 818 UP is a nonionic surfactant with a balanced combination regarding the foam volume and the excellent dermatological properties. Therefore it is suitable for use as a base surfactant or a co-surfactant in cosmetic cleansing preparations. Characteristic values 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 Water % DGF H-Ill 3A Active matter % Difference: 1 %-%water Viscosity (dynamic) (2 C) mpas DIN 5315 ph value (2% in 15% Isopropanol) 11,5-12,5 QP157.1 Additional product descriptive data (Data which is proven statistically but not determined regularly.) Free fatty alcohol <= 1, % Cognis Method Sulphate ash <= 3, % DAB 1 Cognis, Care Chemicals PLANTACARE 818 UP CIR Panel Book Page 52 2/3 Rev. 12.

59 Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present Storage and transportation Shelf life 24 months Storage temperature <+ 4 C Storage conditions In original sealed containers and protected from moisture. Additional information The product should not be stored at temperatures below 15 C. If PLANTACARE 818 UP is stored at temperatures below 15 C crystallization may occur. The product should be heated and stirred until uniform before use. Upon storage also some sedimentation can occur which has no negative effects on the quality. It is recommended to homogenize the product before use by stirring. Containers and storage tanks made of V2A steel (material no ) and of V4A (material no ) are suitable for storing PLANTACARE 818 UP. PLANTACARE 818 UP has a high ph-value and for this reason the product contains no preservatives (suffix UP = unpreserved). Disclaimer All products in the text marked with an S 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, Care Chemicals PLANTACARE 818 UP CIR Panel Book Page 53 3/3 Rev. 12.

60 COGNIS NATURAL MODIFIED carechemicals Data Profile PLANTACARE 12 UP Address Producer I Supplier Cognis GmbH Care Chemicals D Düsseldorf Tel.: Fax.: General characterisation Chemical description C fatty alcohol glucoside Mol weight 42 g/mol Raw material basis Vegetable (coconut / palm kernel oil, glucose) Labeling information INCI name(s) Lauryl Glucoside (EU + CTFA) Composition hints for finished product label Lauryl Glucoside > 5-6 % Aqua >4-5% Ingredient information Ingredient CASR-No. EINECSIELINCSIECIEU NLP-No. Lauryl Glucoside Officially listed in I Quality conforms to Bra Miljöval: approved for use in products eco-labelled Good Environmental Choice (in Swedish: Bra Miljöval) by the Swedish Society for Nature Conservation Cognis, Care Chemicals PLANTACARE 12 UP CIR Panel 1/3Book Page 54 Rev ñis

61 Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at increased temperature. Product properties Appearance PLANTACARE 12 UP is a cloudy, viscous, aqueous solution of a C12-C16 fatty alcohol polyglycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 6 ppm magnesium) and the ph value at which it is supplied. This turbidity has no negative effects on the products properties and disappears if the ph value is adjusted to below 7. PLANTACARE 12 UP is a nonionic surfactant with good dermatological compatibility and viscosity enhancing effects. Therefore it is suitable for use as an additive or a co-surfactant in cosmetic surfactant cleansing preparations. Solubility according to Ph. EUR. Ethylether Solubility: practically insoluble Chloroform Solubility: sparingly soluble Petrolether Solubility: practically insoluble Ethyalcohol Solubility: slightly soluble Paraffinoil Solubility: slightly soluble Water Solubility: sparingly soluble Characteristic values 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 Visual appearance versus standard Water Active matter ph value (2 Weight% in isopropanol/water 15 Weight%) corresponds to the standard corresponds to the standard 47, - 5, % 5, - 53, % 11,5-12,5 Cognis Method Cognis Method Cognis Method Additional product descriptive data (Data which is proven statistically but not determined regularly.) Fatty alcohol Ash Density 4 C Viscosity (dynamic) 4 C <=,8 % <= 2, % 1,7-1,9 g/cm mpas Cognis Method Cognis Method DIN 51757V4 DIN 5315 Cognis, Care Chemicals PLANTACARE 12 UP CIR Panel 2/3Book Page 55 Rev. 12. This

62 Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present Storage and transportation Shelf life 24 months Storage temperature <+ 45 C Storage conditions In original sealed containers and protected from moisture. Additional information If the product is stored at temperatures below 38 C crystallization may occur. Depending on the storage time sedimentation may occur. Therefore, the product should be heated and stirred until uniform before use. Containers and storage tanks made of V2A steel (material no ) and of V4A (material no ) are suitable for storing PLANTACARE 12 UP. PLANTACARE 12 UP has a high ph-value and for this reason the product contains no presevatives (suffix UP = unpreserved). For easier handling the product can be warmed up to max. 5CC for a short duration without influence onto the product specification. 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, Care Chemicals PLANTACARE 12 UP CIR Panel 3/3 Book Page 56 Rev. 12. js

63 4551 Distributed for Comment Only -- Do Not Cite or Quote COGNIS NATURAL MODIFIED carechemicals Data Profile PLANTACARE 2 UP Address Producer! Supplier Cognis GmbH Care Chemicals D - Düsseldorf Tel.: Fax.: General characterisation Chemical description C8-1 6 fatty alcohol glucoside Mol weight 39 g/mol Raw material basis vegetable (coconut oil, palm kernel oil, glucose) Labeling information INCI name(s) Decyl Glucoside (EU + CTFA) Composition hints for finished product label Decyl Glucoside > 5-6 % Aqua >4-5% Cognis, Care Chemicals PLANTACARE 2 UP CIR Panel 1/4 Book Page 57 Rev c gnis

64 Ingredient information Ingredient CASR-No. EINECS/ELINCS-No no longer polymer no longer polymer Officially listed in I Quality conforms to JCIC: Alkyl (8-16) Glucoside (Ingredient Code 5237) Bra Miljäval: approved for use in products eco-labelled Good Environmental Choice (in Swedish: Bra Miljöval) by the Swedish Society for Nature Conservation Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at higher temperature Product properties Appearance PLANTACARE 2 UP is a cloudy, viscous, aqueous solution of a C8-C1 6 fatty alcohol polyglycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 5 ppm magnesium) and the ph value at which it is supplied. This turbidity has no negative effects on the product properties and disappears if the ph value is adjusted to below 7. PLANTACARE 2 UP is a nonionic surfactant with excellent foaming capacity and good dermatological compatibility. Therefore it is suitable for use as a base surfactant or a co-surfactant in cosmetic surfactant cleansing preparations. Solubility according to Ph. EUR. Ethylether Solubility: practically insoluble Chloroform Solubility: sparingly soluble Petrolether Solubility: practically insoluble Ethyalcohol Solubility: practically insoluble Paraffinoil Solubility: practically insoluble Water Solubility: soluble Cognis, Care Chemicals PLANTACARE 2 UP CIR Panel Book Page 58 2/4 Rev Is

65 Characteristic values 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 Water % DGF H-Ill 3A Active matter % Difference: 1% - %water Viscosity (dynamic) (2 C) mpas DIN 5315 Color number < 3 DIN ISO 463 ph value (2% in 15% isopropanol) 11,5-12,5 QP157.1 Additional product descriptive data (Data which is proven statistically but not determined regularly.) Fatty alcohol < 1, % Sulphate ash < 3, % Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present Cognis, Care Chemicals PLANTACARE 2 UP CIR Panel Book Page 59 3/4 Rev *qnis

66 Storage and transportation Shelf life 24 months Storage temperature Temperatures between + 1 C and + 4 C Storage conditions In original sealed containers and protected from moisture. Additional information Upon storage of PLANTACARE 2 UP separation of the product may occur, which has no negative effects on the product quality. The product should be stirred until uniform before use. Containers and storage tanks made of V2A steel (material no ) and of V4A (material no ) are suitable for storing PLANTACARE 2 UP. PLANTACARE 2 UP has a high ph-value and for this reason the product contains no preservatives (suffix UP = unpreserved). 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, Care Chemicals PLANTACARE 2 UP CIR Panel 4/4Book Page 6 Rev. 12.2

67 - - Distributed for Comment Only -- Do Not Cite or Quote /A Consumer Product Testing Company lncorported 12 Spielman Road Fairfield, New Jersey 74 (21) FAX: (21) FINAL REPORT CLIENT: Henkel Corporation 131 Jefferson Street Hoboken, New Jersey 73 ATI ENTION: Barry A. Salka Technical Manager COSPHA Division TEST: Repeat Insult Patch Test 1 1l (I TEST MATERIALS: 1. VIII 48A 8. VIII 48H EXPERIMENT REFERENCE NUMBER: VII I-48A-P1 antaren 48B Pi dli Lctreii 48C-P1 antaren 48D-APG E-APG-2 UP 48F-APG-2 icuu br 12 48G-P1antaren 2 UP c:dr48h-p1afltarefl 2 48J-APG-2 UP-BF 48K-APG-2 BF G jc t48l-p1antarefl 148M-P1antaren 48N Plantaren 48- P1 antaren C 5 93 act.) -!_. i-ijcy/ &/5 II 1: UP-BF 2. VIII 48E 9. VIII 48J 3. VIII 48C 1. VIII 48K 4. VIII 48D 11. VIfl 48L 5. VIII 48E 12. VIII 48M 6. VIII 48F 13. VIII 48N 7. VIII 48G 14. VIII 48 1c/(- -j (ijce,d exl..t Geral N Wachs, M D,F A C P Medical Director 3FinkR.N. xecutive Vice President BF i Clinical Evaluations UP-BF /_ci-. l Glcc,cq BF / j,4._&_t s+cire f)1-ec ic frjc>tf Date )4jrr1 JF/cr Oliver Shapiro, Director Quality Assurance and Regulatory Compliance This report is submitted for the exclusive use of the person. parmershlp. or corporation to whom It is addressed, arid neither the report nor the named these Laboratories nor of any member of It s staff. may be used k connection with the advertising or sale of any product or process CIR Panel Book Page 61

68 Consumer Product Testing Co. OUALITY ASSURANCE UNIT STATEMENT Study No.: C-5-93 The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical laboratory studies. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study on the date(s) listed below. Studies lasting six months or more are inspected at time intervals to assure the integrity of the study. The findings of these inspcctions have been reported to management and the Clinical Director. All materials and data pertinent to this study will be stored in the Archives Facility unless otherwise requested by the sponsor. Date of InspectIon: March 1, 1993 Professional personnel Involved: Joy Frank; R.N. - Executive Vice President Clinical Evaluations Beverly Pinsky - Clinical Laboratory Supervisor Caryl K. Wood - Director of Photobiology Susan O Connor, RN. - Clinical Research Associate, Group Leader Sandra J. Keeler, B.S. - Clinical Research Associate, Group Leader Michele Kouzis, M.T. - Photobiology Study Coordinator Charlotte Mosher - Clinical Research Associate Marie Hoick - Clinical Research Associate Beatrice Karolis Clinical Research siöeiàte Diane T. Margo - Clinical Research Associate Anita Przyborowski - Clinical Research Associate Diana Martin - Clinical Research Associate Johanna Erdmann - Clinical Research Associate Dawn M. Berkenbush, B.S. - Quality Assurance Associate 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 study protocol as well as government regulations regarding such procedures and protocols as outlined in the Federal Register (Vol. 46, No. 17 of Tuesday, January 27, 1981). 1 2 Spiclrnan Road Fairfield. New Jersey ) 8M-7 I I I Fax: (2() 1) CIR Panel Book Page 62

69 (-5-93 Page 3 oorao Objective: To determine by epidermal contact the primary or cumulative irritation and/or sensitization potential of test materials. Participants: A panel comprised of fifty-six (56) subjects, ranging in age from 22 to 69 years, who qualified were selected for this study. Forty-nine (49) subjects completed this evaluation. The remaining subjects discontinued their participation for various reasons, none of which were related to the use of the test materials. It should be noted that Subject #26 exhibited extensive diffuse reactions to the test articles. Upon further investigation this subject revealed that she had a history of numerous allergies which she had not mentioned when filling out her medical history. It is the laboratory s opinion that she is a reactive subject and will not be used in future studies. Her data is not included in the results but is shown for completeness. The criteria for selection was: 1. Willingness to cooperate, 2. Absence of any visible skin disease which might be confused with skin reactions from the test materials, 3. Avoidance of use of topical or systemic steroids and/or antihistamines for several days prior to study initiation, 4. Dependability and intelligence in following directions, and 5. Reading, understanding and signing an informed consent contracl Test Materials: 1. VUI-48A 8. Vffl-48H 2. VuI-48B 9. VllI-48J 3. Vffl-48C 1. Vffl-48K 4. VIfl-48D 11. VUI-48L 5. VIII-48E 12. Vffl-48M 6. VUI-48F 13. Vfll-48N 7. VflI-48G 14. VUT-48 Study Schedule: Initiation Date Completion Date January 11, 1993 February 19, 1993 CIR Panel Book Page 63

70 CrA C:-5-93 Page 4 Methodology: The upper back between the scapulae served as the treatment site. Approximately.2 ml of each test material was applied to the 1 x 1 gauze portion of a webril adhesive dressing.* This was then applied to the appropriate treatment site to form semi-occluded patches. This procedure was followed three times per week: Monday, Wednesday, and Friday for a total of ten applications. 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. The sites were marked to ensure the continuity of patch application. The participants were instructed to remove these patches after twenty-four hours. The evaluation of each site was made just prior to re-application. Rest periods consisted of twenty-four hours following the Tuesday and Thursday removal, and forty-eight hours following the Saturday removal. At the conclusion of a rest period of approximately fourteen days following the tenth application, a challenge patch was applied to the original site and to a virgin site. Each site was evaluated at twentyfour and forty-eight hours after application. The volar forearm served as the virgin test site. Evaluation Key: - No visible reaction 1+ - Mild erythema 2+ - Well-defined erythema, possible presence of barely perceptible edema 3+ - Erythema and edema 4+ - Erythema and edema with vesiculation and ulceration Results: The results of each participant are appended. Observations of all treated areas remained negative throughout the test interval. Summary: Under the conditions of this study, the following test materials do not indicate a potential for dermal irritation or sensitization: 1. VII1-48A 8. VIfl-48H 2. VUI-48B 9. VUI Vffl-48C 1. VIll-48K 4. Vffl-48D 11. Vffl-48L 5. VllI-48E 12. VIlI-48M 6. VIII-48F 13. Vlll-48N 7. VIII-48G 14. VIfl-48 *Manufactured by Professional Medical Products, Inc., Greenwood, SC CIR Panel Book Page 64

71 crporaor. C-5-93 Page 5 Table I Individual Results Vffl-48A Original Virgin Subject Induction Exposures Site Site Number omo. DID NOT COMPLETE STUDY omo -DiD NOT COMPLETE STUDY om -DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY PATCHING DISCONT1NUED* m = Makeup day * =seetext,p.3 CIR Panel Book Page 65

72 , Original Distributed for Comment Only -- Do Not Cite or Quote C-OO593 Page 6 p Table 1 (continued) Individual Results Vffl-48A Virgin Subject Induction Exposures Site Site Number m DID NOT COMPLETE STUDY omo o o omo omo A U A V 1. J I V U DID NOT COMPLETE STUDY om A U oo U omo A V Oom DID NOT COMPLETE STUDY 56 m = Makeup day n V U U V o A CIR Panel Book Page 66

73 :-ee: Corporation C-5-93?age 7 Table 2 Individual Results IllI-48B. Original Virgin Subject Induction Exposures Site Site Number DID NOT COMPLETE STUDY orno DID NOT COMPLETE STUDY oooooooooom DID DID NOT COMPLETE STUDY NOT COMPLETE STUDY PATCHING DISCONTINUED* m=makeup day * =seetext,p.3 CIR Panel Book Page 67

74 . Distributed for Comment Only -- Do Not Cite or Quote *ene.. Corporaon (-5-93 Page 8 Table 2 (continued) Individual Results VIll-48B Original Virgin Subject Induction Exposures Site Site Number An I U o omo m omo omo m Q111Q a a a. a n a a U U -U U V U U U omo DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY a 11 om --DID NOT COMPLETE STUDY- o m = Makeup day CIR Panel Book Page 68

75 ,, C-5-93 Page 9 Table 3 Individual Resulis VllI-48C Original Virgin Subject Induction Exposures Site Site Number DID NOT COMPLETE STUDY m mo 17 ooo DID NOT COMPLETE STUDY 2 ooooooooo 21 DiD NOT COMPLETE STUDY DID NOT COMPLETE STUDY PATCHING DISCONTINUED* m = Makeup day * =seetext,p.3 CIR Panel Book Page 69

76 C-5-93 Page 1 Table 3 (continued) Individual Results V1I1-48C. Original Virgin Subject - -- Induction Exposures-- Site Site Number om DID NOT COMPLETE STUDY omo omo AG flft. Gmo (1 fl I Vt Sr Vt Vt omo omo - ( Vt Il Vt -DID NOT COMPLETE STUDY omo Vt omo 1 Vt DID NOT COMPLETE STUDY Vt SI VP m = Makeup day CIR Panel Book Page 7

77 Henkel Corporation C-5-93 Page 11 Table 4 Individual Results VIfl-48D Original Virgin Subject Induction Exposures Site Site Number o o DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY O - -DID NOT COMPLETE STUDY- DID NOT COMPLETE SThDY PATCHING DISCONTINTJED* m =Makeup day * =seetext,p.3 CIR Panel Book Page 71

78 Henkel Corporation C-5-93 Page 12 Table 4 (continued) Individual Results VIll-48D. Original Virgin Subject Induction Exposures Site Site Number m DID NOT COMPLETE STUDY m 33 DID NOT COMPLETE STUDY 34 m 36 m 37 m 38 OOOOOO OOm ooom 49 OGG m 5 oooo 51 m DID NOT COMPLETE STUDY 56 m = Makeup day CIR Panel Book Page 72

79 :-en.e: orporaon C:-5-93 Page 13 Table 5 Individual Results VIII-48E Original Virgin Subject - ---Induction Exposures Site She Number I LL omo m DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY- -DID NOT COMPLETE STUDY- DID NOT COMPLETE STUDY PATCHING DISCONTINUED * m = Makeup day * =seetext,p.3 CIR Panel Book Page 73

80 :n. oporan C -O593 Page 14 Table 5 (continued) Individual Results VIU-48E Original Virgin Subject Induction Exposures Site Site Number m DID NOT COMPLETE STUDY 3 31 o 32 m 33 DID NOT COMPLETE STUDY 34 m 35 o 36 oom 37 m moo oomo 47 m 48 4.n &..n.n_n fi flfl fla fl A A A 5 m 51 m DID NOT COMPLETE STUDY 56 m = Makeup day CIR Panel Book Page 74

81 :enke.. (..orpora;on C-OO593 Page 15 Table 6 Individual Results VIII-48F Original Virgin Subject Induction Exposures Site Site Number LL DID NOT COMPLETE STUDY 111 omo DID NOT COMPLETE STUDY-- DID NOT COMPLETE STUDY oom -ijijjinji LLt1.iLJL)X PATCHING DISCONTINUED* m = Makeup day * =seetext,p3 CIR Panel Book Page 75

82 ;cne.. or2ora.ofl c Page 16 Table 6 (continued) Individual Results VI1I-48F. Original Virgin Subject -- -Induction Exposures Site Site Number A Urn DID NOT COMPLETE STUDY rn omo rn A A A DID NOT COMPLETE STUDY rn m omo omo orno m -DID NOT COMPLETE STUDY- 9 m = Makeup day CIR Panel Book Page 76

83 Hene1 Corporation C-5-93 Page 17 Table 7 Individual ResulLs Vffl-48G Original Virgin Subject Induction Exposures Site Site Number DID NOT COMPLETE STUDY OO OOOOOOOO DID NOT COMPLETE STUDY m 2 DID NOT COMPLETE STUDY 22 DID NOT COMPLEFb SIUDY PATCHING DISCONTIN1JED* rn =Makeup clay * =seetext,p.3 CIR Panel Book Page 77

84 :enke.. orporaon (-5.93 Page 18 Table 7 (continued) Individual Results Vffl-48G. Original Virgin Subject Induction Exposures - Site Site Number AO omo m omo A A A,,,,J m omo A I., S ---DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY A A A A A A fl A t I I V omo. G DiD NOT COMPLETE STUDY- m = Makeup day CIR Panel Book Page 78

85 C-5-93 Page 19 Table 8 Individual Results VIII-48H Original Virgin Subject Induction Exposures Site Site Number LL omo omo DID NOT COMPLETE STUDY m DID NOT COMPLETE STUDY flv S,fl.i, flt%s S1%T V% St --i)ll) 1VILi I Ui-I I DID NOT COMPLETE STUDY PATCHING DISCONTINUED* m = Makeup day * =seetext,p.3 CIR Panel Book Page 79

86 C-OO593 Pace 2 Table 8 (continued) Individual Results VllI-48H Original Virgin Subject Induction Exposures Site Site Number AG m = Makeup day m omo m omo omo omo DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY On o o o o OfllO o o o o ( r A A o omo om o o DID NOT COMPLETE STUDY CIR Panel Book Page 8

87 C-5-93 Page 21 :a;.: Table 9 Individual Results VIJI-483 Original Virgin Subject Induction Exposures Site Site Number m m DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY 21 DID NOT COMPLETE STUDY m TT% )TI1 1 I.. AThT TT V C l l T17 4. LJ1LJ 4.J i JIYU A A.,.) I JL) PATCHING DISCONTINUED* m = Makeup day * =seetext,p.3 CIR Panel Book Page 81

88 zerle.. c.otpora:.o. C Page 22 Table 9 (continued) IndivduaI Results VIII-48J Original Virgin Subject -----Induction Exposures Site Site Number A om DID NOT COMPLETE STUDY m DID NOT COMPLETE STUDY O omo omo omo A (i A A A A A A A A n A,.,., t, I. S ¼? omo m m DID NOT COMPLETE STUDY in = Makeup day CIR Panel Book Page 82

89 :-ene;, Corpora.on C-oO5S3 Page 23 Table 1 Individual Results VllI-48K Original Virgin Subject -Induction Exposures Site Site Number hh omo DID NOT COMPLETE STUDY omo DID NOT COMPLETE STUDY m PATCHING DISCONTINUED*.. --DID NOT COMPLETE STUDY --Liii) f1ul LUM1 LLIt i 1)1.)! m = Makeup day * =seetext,p.3 CIR Panel Book Page 83

90 :-en;e,, (-5-93 Page 24 c.oporaon Table 1 (continued) Individual Results VIU-48K. Original Virgin Subject Induction Exposures ---- Site Site Number Afl A I o m = Makeup day o omo omo O O O A A A V V Id om A J A - om m A A A A., U I omo om DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY A V U V A CIR Panel Book Page 84

91 C Page 25 cpo:a.o. Table 11 Individual Results Vffl-48L Original Virgin Subject Induction Exposures Site Site Number JO omo DID NOT COMPLETE STUDY omo o o o o o o D1D NOT COMPLETE STUDY. -DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY PATCHING DISCONTINUED* - m m =Makeup day * =seetext,p.3 CIR Panel Book Page 85

92 (-5-93 Page 26 Table 11 (continued) Individual Results VIU-48L. Original Virgin Subject --Induction Exposures Site Site Number q C) m DID NOT COMPLETE STUDY omo omo m omo o G DID NOT COMPLETE STUDY m m.o C) OmO om - DID NOT COMPLETE STUDY- A m = Makeup day CIR Panel Book Page 86

93 C-5-93 Page 27 Table 12 Individual Results VllI-48M Original Virgin Subject Induction Exposures Site Site Number hh o omo omo DID NOT COMPLETE STUDY m -DID NOT COMPLETE STUDY --LJUJPiUI LUMkJ11ULJX DID NOT COMPLETE STUDY o PATCHING D1SCONTINUED* m =Makeup day * =seetext,p.3 CIR Panel Book Page 87

94 C-5-93 Page 28 Table 12 (continued) Individual Results VUI-48M Virgin Subject ----Induction Exposures Site Site. Original Number m DID NOT COMPLETE STUDY 3 31 m oooo DID NOT COMPLETE STUDY 34 m OOOOOOO 37 m 38 moo oom m m OOO DID NOT COMPLETE STUDY 56 m = Makeup day CIR Panel Book Page 88

95 C-5-93 Page 29 Table 13 Individual Results VllI-48N Original Virgin Subject Induction Exposures Site Site Number omo mb DID NOT COMPLETE STUDY. m DID NOT COMPLETE STUDY rtr Mrvr ((AT 9t L DTTTh J 1.dALf a%. A 4.flAVAA S14_. I_PS %_PS S. DID NOT COMPLETE STUDY PATCHING DISCONTINUED* m = Makeup day * =seetextp.3 CIR Panel Book Page 89

96 C-5-93 Page 3 Table 13 (continued) Individual Results VIll-48N. Originai Virgin Subject Induction Exposures -- Site Site Number _._49 (I m = Makeup day omo amo DID NOT COMPLETE STUDY -DID NOT COMPLETE STUDY m O O omo omo omo fl C) A C). omo m ooo DID NOT COMPLETE STUDY CIR Panel Book Page 9

97 C-5-93 Page 31 Table 14 1ndivdual Results VIll-48. Original Virgin Subject Induction Exposures Site Site Number DID NOT COMPLETE STUDY omo omo DID NOT COMPLETE STUDY. m DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY PATCHING DISCONTINUED* in = Makeup day * =seetext,p.3 CIR Panel Book Page 91

98 C-5-93 Page 32 Table 14 (continued) Individual Results VIll-48 Original Virgin Subject Induction Exposures- Site Site Number (I o omo omo O omo omo a omo ff DII) NOT COMPLETE STUDY -DID NOT COMPLETE STUDY O om A om DID NOT COMPLETE STUDY---. o o o o o o o o o o o o o o o o m = Makeup day CIR Panel Book Page 92

99 opoaon C-5-93 Page 33 Table 15 Subject Data Subject Number Initials Age Sex 1 WC 41 M 2 A.H 61 F 3 HB 47 F 4 IH 61 F 5 DD 63 F 6 IM 41 F 7 SF 65 F 8 LR 59 F 9 JC 64 F 1 PL 63 F 11 NV 32 F 12 PA 65 M 13 MA 64 F 14 AM 4 F 15 PS 46 F 16 LH 56 F 17 LG 3 F 18 DR 26 F 19 LO 24 M 2 CA 5 F 21 CP 22 F 22.BW 33 M 23 in 37 F TY JI 25 WH 62 M 26 DO 29 F 27 MP 47 F 28 LG 4 F CIR Panel Book Page 93

100 :-ene: Corporaton C-5-93 Page 34 Table 15 (continued) Subject Data Subject Number Initials Age Sex 29 TM 28 F 3 GB 53 F 31 MP 45 F 32 LH 31 F 33 MM 41 F 34 BH 53 M 35 CE 33 F 36 JR 58 F 37 RR 61 M 38 LI 31 F 39 GB 44 M 4 MP 41 F 41 RC 46 F 42 SC 54 M 43 LD 25 F 44 BR 36 M 45 TK 48 F 46 RD 32 F 47 IC 57 F 48 LH 48 F 49 MW 38 F 5 CP 24 F 51 KI 31 M 52 SV 3 F 53 MD 69 F 54 MS 53 F 55 DM 46 F 56 RM 43 F CIR Panel Book Page 94

101 I. 75 Consumer Product Testing Co. FINAL REPORT Cognis Deutsehiand GmbH & Co. KG. Henkelstr. D-4551 Duesseldorf, Germany ATTENTION: Mr. Peter Wierich Repeated Insult Patch Test Protocol No.: TEST MATERIAL: C-SAT 515 Coco- 1Qcos;c e 8/ EXPERIMENT I Vf REFERENCE NUMBER: C I IC) Richard R. Eisenberg, M i). Board Certified Dermatologist Michael Traudt, PlLDc Senior Director, Clinical Evaluations / Jank, RN. fe6tive Vice President, Clinical Evaluations This report is submitted for the exclusive use of the persor:, partnership, or corporation to whom it is addressed. ard neither tie report nor the name of these Laboratories nor any member of its staff may be used in connection with the advertising or sate of any product or process without written authorization. 7 New l.)utch LaIR- Rtirflelci. New Jersey (973) ax (973) CIR Panel Book Page 95

102 IST Ic75 Consumer Product Testing Co. QUALITY ASSURANCE UNIT STATEMENT Study No.: C monitor the conduct and reporting of clinical laboratory studies. These studies have been performed with adherence to the applicable ICH Guideline The objective of the Quality Assurance Unit (QAU) is E6 for Good Clinical Practice and requirements provided for in to 21 CFR parts 5 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: I: < 1 duality Assurance J3ate 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 government regulations regarding such procedures and protocols. as 7 Nc\ L)ith Ljnc (l1nical Fairfield, Nu\V FOXi(Ol( Jersey gy 4 (973) 8S-7 71> ic>1 ( hcmni5trv \I1iI\ CIR Panel Book Page \ikrobiolog\ Fax (973)

103 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 and/or allergic contact sensitization. Participants: Two hundred twenty-seven (227) qualified subjects, male and female, ranging in age from 16 to 79 years, were selected for this evaluation. Two hundred thirteen (213) 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 16a 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 515 Study Schedule: Panel# Initiation Date Completion Date March 23, 25 March 28, 25 March 28, 25 March 3, 25 May 5, 25 May 5, 25 May 6, 25 May 5, 25 awith parental or guardian consent CIR Panel Book Page 97

104 Cognis Deutschland GmbH & Co. KG. C5-247.O1 Page 4 Methodology: Prior to the initiation of this study, the test material was prepared as follows: The ph was adjusted to 6.5 (±.5) with HCL. prepared. (i!)e.-.l vic lp) A 2% dilution was then The upper back between the scapulae served as the treatment area. Approximately.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 occlusive patch. The test material was refrigerated, when not in use. 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. ChaI1erne 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 98

105 Cognis Deutschland GmbH & Co. KG. C Page 5 Evaluation Key: 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 erytherna, possible edema 4 = Severe erythema, possible edema, vesiculation, bullae andlor ulceration Results: The results of each participant are appended (Table 1). Observations remained within normal limits throughout the test interval. Summary: Under the conditions of this study, test material, C-SAT 515, did not indicate a potential for dermal irritation or allergic contact sensitization. CIR Panel Book Page 99

106 Distributed for Comment Only -- Do Not Cite or Quote Cognis Deutschland GmbH & C Page 6 Co. KG. Table Panel #25143 Individual Results C-SAT Subject Number 24*hr Virgin Challenge Induction PhaseSite *hr 72 hr DID NOT COMPLETE STUDY DII) NOT COMPLETE STUDY 24* = Supervised removal of 1 Induction and Challenge Patch CIR Panel Book Page 1

107 Cognis Deutsehiand GmbH & Co. KG. C Page 7 Table 1 (continued) Panel # Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72hr 29 3 DID NOT COMPLETE STUDY om DNC * = Supervised removal of 1st Induction and Challenge Patch m = Additional makeup day granted at the discretion of the clinic supervisor DNC = Did not complete study CIR Panel Book Page 11

108 Cognis Deutschland GmbH & Co. KG. C Page 8 Table 1 (continued) Panel #25147 Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72br DID NOT COMPLETE STUDY * = Supervised removal of 1c Induction and Challenge Patch DNC = Did not complete study CIR Panel Book Page 12

109 Cois Deutschland GmbH & Co. KG. C Page 9 Table 1 (continued) Panel # Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72hr * = Supervised removal of Induction and Challenge Patch CIR Panel Book Page 13

110 Cognis Deutschland GmbH & Co. KG. C Page 1 Table 1 (continued) Panel #25155 Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*1w *hr 72hr DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY * = Supervised removal of 1st Induction and Challenge Patch - = Subject not present for supervised removal CIR Panel Book Page 14

111 Cois Deutschland GmbH & Co. KG. C I Page 11 Table I (continued) Panel #25155 Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72 hr DID NOT COMPLETE STUDY 57 24* = Supervised removal of 1 Induction and Challenge Patch - = Subject not present for supervised removal CIR Panel Book Page 15

112 Cognis Deutschland GmbH & Co. KG. C5-247.i Page 12 Table 1 (continued) Panel #2516 Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*hr *hr 72 hr DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY * = Supervised removal of l Induction and Challenge Patch CIR Panel Book Page 16

113 Cois Deutschland GrnbH & Co. KG. C Page 13 Table 1 (continued) Panel #2516 Individual Results C-SAT 515 Virgin Challenge Subject Induction Phase Site Number 24*1w _8 9 24*1w 721w DID NOT COMPLETE STUDY DID NOT COMPLETE STUDY 57 24* = Supervised removal of 1 Induction and Challenge Patch CIR Panel Book Page 17

114 Cognis Deutschland GmbH & Co. KG. C Page 14 Table 2 Panel # Subject Data Subject Number Initials Age Sex 1 GV 32 F 2 SH 46 F 3 JR 42 F 4 KS 61 F 5 PS 61 M 6 VR 33 F 7 MK 66 F 8 NE 57 M 9 JS 36 F 1 WE 73 M 11 JC 55 F 12 DZ 41 F 13 MT 21 F 14 CS 43 F 15 SB 5 F 16 MP 17 F 17 CW 74 F 18 SN 43 M 19 IP 38 F 2 ES 58 F 21 CR 46 F 22 FR 55 M 23 DG 3 F 24 JH 5 M 25 NH 5 F 26 RG 25 M 27 MC 49 F 28 DQ 52 M CIR Panel Book Page 18

115 Cognis Deutschland GmbH & Co. KG. C Page 15 Table 2 (continued) Panel # Subject Data Subject Number Initials Age Sex 29 KI 44 M 3 JT 24 F 31 KS 55 F 32 PM 43 F 33 RH 49 M 34 PW 38 F 35 AW 62 F 36 CH 65 F 37 ES 49 F 38 BF 23 F 39 MH 66 F 4 DJ 74 M 41 MS 6 F 42 CV 49 F 43 EC 45 M 44 RD 48 F 45 KP 46 F 46 RC 3 F 47 DC 49 F 48 EH 58 F 49 FS 63 M 5 NR 46 F 51 AC 5 F 52 MA 27 F 53 EM 69 F 54 BR 73 F 55 HT 72 M 56 KL 45 F CIR Panel Book Page 19

116 Cognis Deutschland GmbH & Co. KG. C Page 16 Table 2 (continued) Panel #25147 Subject Data Subject Number Initials Age Sex 1 NM 42 M 2 BM 4 F 3 LR 71 F 4 PD 65 F 5 PL 75 F 6 JF 7 F 7 HF 74 M 8 HP 75 F 9 LS 49 M 1 LS 48 F 11 LL 76 F 12 AL 78 M 13 BS 49 F 14 JO 64 M 15 DS 74 M 16 PR 28 F 17 JD 58 F 18 GM 55 M 19 SK 18 M 2 JS 67 F 21 EC 42 F 22 AS 79 M 23 ES 76 F 24 EN 47 F 25 DW 51 F 26 GP 71 F 27 RN 56 F 28 SH 43 F 29 RM 61 F CIR Panel Book Page 11

117 Cognis Deutschland GmbH & Co KG. C Page 17 Table 2 (continued) Panel #25147 Subject Data Subject Number Initials Age Sex 3 KP 45 F 31 MG 58 M 32 HF 42 M 33 KC 35 F 34 RS 65 F 35 MR 44 F 36 OB 56 F 37 KU 23 F 38 MC 4 F 39 GM 42 F 4 DR 38 F 41 AD 7 F 42 AD 75 M 43 MA 57 F 44 CF 4 F 45 KF 21 M 46 LL 45 F 47 EM 63 F 48 MV 19 F 49 WL 66 F 5 RF 68 F 51 JO 69 M 52 MP 59 F 53 VL 46 F 54 RG 32 F 55 MM 42 F 56 SC 41 F 57 RL 57 F CIR Panel Book Page 111

118 Cognis Deutschland GmbH & Co. KG. C5.247.l Page 18 Table 2 (continued) Panel #25155 Subject Data Subject Number Initials Age Sex 1 LN 35 M 2 YG 29 F 3 BL 35 F 4 RG 54 M 5 RR 62 F 6 LA 51 F 7 PA 53 M 8 DM 39 F 9 CC 68 F 1 PR 47 M 11 AV 58 F 12 JA 54 F 13 PS 46 F 14 DH 64 M 15 EB 6 F 16 NT 43 F 17 KR 19 F 18 JT 42 F 19 CO 61 F 2 MG 37 F 21 TR 17 F 22 DC 47 F 23 CS 23 F 24 PC 63 F 25 EL 66 F 26 FS 16 M 27 AJ 31 F 28 RC 29 F 29 RV 21 M CIR Panel Book Page 112

119 Cognis Deutschland GmbH & Co. KG. C Page 19 Table 2 (continued) Panel # bject Data Subject Number Initials Age Sex 3 SG 7 M 31 PV 6 M 32 AA 47 M 33 AA 53 F 34 MM 71 F 35 SH 38 F 36 DB 56 F 37 EB 58 M 38 JP 62 M 39 CL 46 F 4 JA 42 M 41 GC 4 F 42 AC 39 M 43 HH 64 F 44 RL 47 M 45 LS 45 F 46 DR 59 F 47 ES 53 F 48 NG 5 F 49 EB 45 F 5 ZP 41 F 51 CP 47 F 52 DP 46 M 53 CB 26 F 54 RW 38 F 55 MV 47 F 56 AV 47 M 57 JG 31 M CIR Panel Book Page 113

120 Cois Deutschland GmbH & Co. KG. C Page 2 Table 2 (continued) Panel #2516 Subject Data Subject Number Initials Age Sex 1 MK 41 M 2 LO 61 F 3 SV 55 F 4 TB 55 M 5 MS 56 F 6 MO 21 F 7 MF 56 M 8 JB 55 F 9 SS 4 F 1 KL 41 F 11 CO 39 F 12 RM 66 M 13 MP 59 F 14 HF 23 F 15 JA 45 F 16 LC 52 F 17 LA 5 M 18 LC 59 F 19 CM 58 F 2 VS 31 F 21 JS 68 M 22 DB 38 F 23 MS 21 F 24 MC 69 F 25 ER 41 F 26 DR 38 F 27 AD 4 M 28 DM 62 F 29 LH 71 F CIR Panel Book Page 114

121 Cois Deutsehiand GmbH & Co. KG. C Page 21 Table 2 (continued) Panel #2516 Subject Data Subject Number Initials Age Sex 3 IM 65 F 31 FR 66 M 32 MD 47 F 33 DW 65 F 34 RW 68 M 35 AS 72 F 36 JS 72 M 37 ES 41 M 38 AB 48 F 39 IA 55 M 4 CD 73 F 41 MD 76 M 42 DP 33 F 43 NM 6 F 44 LC 38 F 45 ES 71 F 46 FN 71 F 47 RM 7 F 48 YH 26 F 49 SS 4 F 5 MK 51 F 51 FR 76 F 52 JM 66 F 53 NM 57 F 54 CS 59 F 55 TD 35 F 56 CB 23 F 57 SB 5 M CIR Panel Book Page 115

122 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CLR Expert Panel DATE: January 7, 211 SUBJECT: Concentration of Use by FDA Product Category: Glucoside Ingredients th Street, N.W., Suite 3O Washington, D.C (fax) CIR Panel Book Page 116

123 Concentration of Use by FDA Product Category Decyl Glucoside, Arachidyl Glucoside, Butyl Glucoside, C1O-16 Alkyl Glucoside, C12-18 Alkyl Glucoside, C12-2 Alkyl Glucoside, C2-22 Alkyl Glucoside, CaprylyL/Capryl Glucoside, Caprylyl Glucoside, Cetearyl Glucoside, Coco-Glucoside, Ethyl Glucoside, Isostearyl Glucoside, Lauryl Glucoside, Lauroyl Ethyl Glucoside, Myristoyl Ethyl Glucoside, Myristyl Glucoside, Octyldodecyl Glucoside, Oleoyl Ethyl Glucoside, Palm Kernel/Coco Glucoside, Phytosteryl Glucoside, Rapeseed Glucoside, Safflower Glucoside, Tallowoyl Ethyl Glucoside, Tocopheryl Glucoside and Undecyl Glucoside* Ingredient Product Category Concentration of Use Decyl Glucoside Bubble baths.5-.8% Decyl Glucoside Other bath preparations 1% Decyl Glucoside Eyeliner.2% Decyl Glucoside Eye lotion.2-1% Decyl Glucoside Eye makeup remover.3-6% Decyl Glucoside Mascara.5% Decyl Glucoside Hair conditioners.4-6% Decyl Glucoside Hair sprays (aerosol fixatives).6% Decyl Glucoside Shampoos (noncoloring) 1-7% Decyl Glucoside Tonics, dressings and other hair grooming aids.2-2% Decyl Glucoside Other hair preparations (noncoloring) 1% Decyl Glucoside Hair dyes and colors (all types requiring caution 2-8% statement and patch tests) Decyl Glucoside Foundations 2% Decyl Glucoside Other makeup preparations.2% Decyl Glucoside Bath soaps and detergents.3 7% Decyl Glucoside Other personal cleanliness products 2-3% Decyl Glucoside Shaving cream (aerosol, brushless and lather) 2% Decyl Glucoside Skin cleansing (cold creams, cleansing lotions,.8-33% liquids and pads) Decyl Glucoside Face and neck creams, lotions and powders.2-.5% Decyl Glucoside Body and hand creams, lotions and powders.5-11% Decyl Glucoside Body and hand sprays.5% Decyl Glucoside Moisturizing creams, lotions and powders.7% Decyl Glucoside Night creams, lotions and powders.9% Page 1 of 5 CIR Panel Book Page 117

124 Decyl Glucoside Paste masks (mud packs) 2% Decyl Glucoside Skin fresheners.2% Decyl Glucoside Other skin care preparations.6-2% Decyl Glucoside Suntan gels, creams and liquds.2% Decyl Glucoside Indoor tanning preparations.8% Arachidyl Glucoside Eye shadow.8% Arachidyl Glucoside Tonics, dressings and other hair grooming aids.5% Arachidyl Glucoside Skin cleansing (cold creams, cleansing lotions,.5% liquids and pads) Arachidyl Glucoside Face and neck creams, lotions and powders.2-.6% Arachidyl Glucoside Body and hand creams, lotions and powders.2-.5% Arachidyl Glucoside Moisturizing creams, lotions and powders.5% Arachidyl Glucoside Night creams, lotions and powders.5% Arachidyl Glucoside Indoor tanning preparations.5% C 12-2 Alkyl Glucoside Eye shadow.8% C 12-2 Alkyl Glucoside Other hair preparations (non-coloring) 1% C 12-2 Alkyl Glucoside Deodorants (underarm).6% C12-2 Alkyl Glucoside Skin cleansing (cold creams, cleansing lotions,.1% liquids and pads) C 12-2 Alkyl Glucoside Face and neck creams, lotions and powders.6% Cl 2-2 Alkyl Glucoside Other skin care preparations 1% CaprylyllCapryl Glucoside Other baby products.6% CaprylyllCapryl Glucoside Eye lotion.2% Caprylyl/Capryl Glucoside Eye makeup remover.3% Caprylyl/Capryl Glucoside Hair conditioners.3% Caprylyl/Capryl Glucoside Shampoos (noncoloring) 3% Caprylyl/Capryl Glucoside Tonics, dressings and other hair grooming aids.5% Caprylyl/Capryl Glucoside Hair dyes and colors (all types requiring caution 3% statement and patch test) Caprylyl/Capryl Glucoside Skin cleansing (cold creams, cleansing lotions,.9% liquids and pads) Page 2 of 5 CIR Panel Book Page 118

125 Caprylyl/Capryl Glucoside Face and neck creams, lotions and powders.8% Caprylyl/Capryl Glucoside Body and hand creams, lotions and powders.3% Caprylyl/Capryl Glucoside Night creams, lotions and powders.8% CaprylyllCapryl Glucoside Suntan gels, creams and liquids.3% Caprylyl Glucoside Other hair preparations (noncoloring) 4% Cetearyl Glucoside Eye shadow 1-2% Cetearyl Glucoside Eye lotion.6-2% Cetearyl Glucoside Other fragrance preparations.6% Cetearyl Glucoside Hair conditioners.3-.6% Cetearyl Glucoside Hair dyes and colors (all types requiring caution.2% statement and patch testing) Cetearyl Glucoside Bath soaps and detergents.3% Cetearyl Glucoside Aftershave lotions.2-.6% Cetearyl Glucoside Skin cleansing (cold creams, cleansing lotions,.3-.9% liquids and pads) Cetearyl Glucoside Depilatories 3% Cetearyl Glucoside Face and neck creams, lotions and powders.6-2% Cetearyl Glucoside Body and hand creams, lotions and powders.2-7% Cetearyl Glucoside Moisturizing creams, lotions and powders.6% Cetearyl Glucoside Night creams, lotions and powders.2-.8% Cetearyl Glucoside Paste masks (mud packs).6-.8% Cetearyl Glucoside Other skin care preparations.3-.7% Cetearyl Glucoside Suntan gels, creams and liquids.2% Cetearyl Glucoside Indoor tanning preparations.2-.6% Cetearyl Glucoside Other suntan preparations.3% Coco-Glucoside Eyeliner 2% Coco-Glucoside Eye makeup remover 3% Coco-Glucoside Hair conditioners.2% Coco-Glucoside Shampoos (noncoloring).4-8% Coco-Glucoside Hair dyes and colors (all types requiring caution 5% statement and patch testing) Page 3 of 5 CIR Panel Book Page 119

126 Coco-Glucoside Other hair coloring preparations.3% Coco-Glucoside Dentifrices (aerosol, liquid, pastes and powders).5% Coco-Glucoside Bath soaps and detergents.4-3% Coco-Glucoside Other personal cleanliness products.7-8% Coco-Glucoside Shaving cream (aerosol, brushless and lather).6% Coco-Glucoside Skin cleansing (cold creams, cleansing lotions,.4-8% liquids and pads) Coco-Glucoside Face and neck creams, lotions and powders.2-2% Coco-Glucoside Body and hand creams, lotions and powders.6-2% Coco-Glucoside Other skin care preparations 2% Ethyl Glucoside Eye makeup remover.2% Ethyl Glucoside Rouges.2% Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions,.5% liquids and pads) Ethyl Glucoside Body and hand creams, lotions and powders.4% Ethyl Glucoside Moisturizing creams, lotions and powders.3% Ethyl Glucoside Paste masks (mud packs).5% Lauryl Glucoside Bath oils, tablets and salts.5% Lauryl Glucoside Bubble baths.3-4% Lauryl Glucoside Eye shadow 5% Lauryl Glucoside Hair conditioners.4% Lauryl Glucoside Shampoos (noncoloring) 1-5% Lauryl Glucoside Hair dyes and colors (all types requiring caution.3-2% statement and patch test) Lauryl Glucoside Hair color sprays (aerosol) 8% Lauryl Glucoside Hair shampoos (coloring) 2% Lauryl Glucoside Other makeup preparations.3% Lauryl Glucoside Bath soaps and detergents.3-8% Lauryl Glucoside Other personal cleanliness products.6-2% Lauryl Glucoside Skin cleansing (cold creams, cleansing lotions,.6-1% liquids and pads) Page 4 of 5 CIR Panel Book Page 12

127 Lauryl Glucoside Face and neck creams, lotions and powders 2% Lauryl Glucoside Body and hand creams, lotions and powders.3% Lauryl Glucoside Moisturizing creams, lotions and powders 2% Lauryl Glucoside Other skin care preparations.2-2% Lauroyl Ethyl Glucoside Bath soaps and detergents 1% Lauroyl Ethyl Glucoside Other personal cleanliness products 2% Lauroyl Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions 2% liquids and pads) Myristyl Glucoside Eye lotion.4% Myristyl Glucoside Face and neck creams, lotions and powders.5-.6% Myristyl Glucoside Night creams, lotions and powders.6% Safflower Glucoside Hair conditioners.1% Safflower Glucoside Hair sprays (aerosol fixatives).5% Safflower Glucoside Other hair preparations (noncoloring).1% *Ingredients included in the title of the table but not found in the body of the table were incluoed in the concentration of use survey, but no uses were reported..6% in a leave-on baby product Information collected in 21 Table prepared January 6, 211 Page 5 of 5 CIR Panel Book Page 121

128 Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel DATE: January 28, 211 SUBJECT: Updated Concentration of Use by FDA Product Category: Glucoside Ingredients th Street, N.W., Suite 3 Washington, D.C (fax) CIR Panel Book Page 122

129 Concentration of Use by FDA Product Category Decyl Glucoside, Arachidyl Glucoside, Butyl Glucoside, C1O-16 Alkyl Glucoside, C12-18 Alkyl Glucoside, C12-2 Alkyl Glucoside, C2-22 Alkyl Glucoside, CaprylyllCapryl Glucoside, Caprylyl Glucoside, Cetearyl Glucoside, Coco-Glucoside, Ethyl Glucoside, Isostearyl Glucoside, Lauryl Glucoside, Lauroyl Ethyl Glucoside, Myristoyl Ethyl Glucosidc, Myristyl Glucoside, Octyldodecyl Glucoside, Oleoyl Ethyl Glucoside, Palm Kernel/Coco Glucoside, Phytosteryl Glucoside, Rapeseed Glucoside, Safflower Glucoside, Tallowoyl Ethyl Glucoside, Tocopheryl Glucoside and Undecyl Glucoside Ingredient Product Category Concentration of Use Decyl Glucoside Bubble baths.5-.8% Decyl Glucoside Other bath preparations 1% Decyl Glucoside Eyeliner.2% Decyl Glucoside Eye lotion.2-1% Decyl Glucoside Eye makeup remover.3-6% Decyl Glucoside Mascara.5% Decyl Glucoside Hair conditioners.4-6% Decyl Glucoside Hair sprays (aerosol fixatives).6% Decyl Glucoside Shampoos (noncoloring) 1-7% Decyl Glucoside Tonics, dressings and other hair grooming aids.2-2% Decyl Glucoside Other hair preparations (noncoloring) 1% Decyl Glucoside Hair dyes and colors (all types requiring caution 2-8% statement and patch tests) Decyl Glucoside Foundations 2% Decyl Glucoside Other makeup preparations.2% Decyl Glucoside Bath soaps and detergents.3 7% Decyl Glucoside Other personal cleanliness products 2-3% Decyl Glucoside Shaving cream (aerosol, brushless and lather) 2% Decyl Glucoside Skin cleansing (cold creams, cleansing lotions,.8-33% liquids and pads) Decyl Glucoside Face and neck creams, lotions and powders.2-.5% Decyl Glucoside Body and hand creams, lotions and powders.5-11% Decyl Glucoside Body and hand sprays.5% Decyl Glucoside Moisturizing creams, lotions and powders.7% Decyl Glucoside Night creams, lotions and powders.9% Page 1 of 5 CIR Panel Book Page 123

130 Decyl Glucoside Paste masks (mud packs) 2% Decyl Glucoside Skin fresheners.2% Decyl Glucoside Other skin care preparations.6-2% Decyl Glucoside Suntan gels, creams and liquds.2% Decyl Glucoside Indoor tanning preparations.8-1% Arachidyl Glucoside Eye shadow.8% Arachidyl Glucoside Tonics, dressings and other hair grooming aids.5% Arachidyl Glucoside Skin cleansing (cold creams, cleansing lotions,.5% liquids and pads) Arachidyl Glucoside Face and neck creams, lotions and powders.2-.6% Arachidyl Glucoside Body and hand creams, lotions and powders.2-.5% Arachidyl Glucoside Moisturizing creams, lotions and powders.5% Arachidyl Glucoside Night creams, lotions and powders.5% Arachidyl Glucoside Indoor tanning preparations.2-.5% C 12-2 Alkyl Glucoside Eye shadow.8% C 12-2 Alkyl Glucoside Other hair preparations (non-coloring) 1% C 12-2 Alkyl Glucoside Deodorants (underarm).6% C 12-2 Alkyl Glucoside Skin cleansing (cold creams, cleansing lotions,.1% liquids and pads) C 12-2 Alkyl Glucoside Face and neck creams, lotions and powders.6% C 12-2 Alkyl Glucoside Other skin care preparations 1% C 12-2 Alkyl Glucoside Suntan gels, creams and liquids.2% C12-2 Alkyl Glucoside Indoor tanning preparations.3% C 12-2 Alkyl Glucoside Other suntan preparations.5% Caprylyl/Capryl Glucoside Other baby products.6% Caprylyl/Capryl Glucoside Eye lotion.2% Caprylyl/Capryl Glucoside Eye makeup remover.3% Caprylyl/Capryl Glucoside Hair conditioners.3% Caprylyl/Capryl Glucoside Shampoos (noncoloring) 3% Caprylyl/Capryl Glucoside Tonics, dressings and other hair grooming aids.5% CaprylyllCapryl Glucoside Hair dyes and colors (all types requiring caution 3% Page 2 of 5 CIR Panel Book Page 124

131 statement and patch test) Caprylyl/Capryl Glucoside Skin cleansing (cold creams, cleansing lotions,.9% liquids and pads) Caprylyl/Capryl Glucoside Face and neck creams, lotions and powders.8% CaprylyllCapryl Glucoside Body and hand creams, lotions and powders.3% Caprylyl/Capryl Glucoside Night creams, lotions and powders.8% CaprylyllCapryl Glucoside Suntan gels, creams and liquids.3% Caprylyl Glucoside Other hair preparations (noncoloring) 4% Cetearyl Glucoside Eye shadow 1-2% Cetearyl Glucoside Eye lotion.6-2% Cetearyl Glucoside Other fragrance preparations.6% Cetearyl Glucoside Hair conditioners.3-.6% Cetearyl Glucoside Hair dyes and colors (all types requiring caution.2% statement and patch testing) - Cetearyl Glucoside Bath soaps and detergents.3% Cetearyl Glucoside Aftershave lotions.2-.6% Cetearyl Glucoside Skin cleansing (cold creams, cleansing lotions,.3-.9% liquids and pads) Cetearyl Glucoside Depilatories 3% Cetearyl Glucoside Face and neck creams, lotions and powders.6-2% Cetearyl Glucoside Body and hand creams, lotions and powders.2-7% Cetearyl Glucoside Moisturizing creams, lotions and powders.6% Cetearyl Glucoside Night creams, lotions and powders.2-.8% Cetearyl Glucoside Paste masks (mud packs).6-.8% Cetearyl Glucoside Other skin care preparations.3-.7% Cetearyl Glucoside Suntan gels, creams and liquids.2% Cetearyl Glucoside Indoor tanning preparations.2-.6% Cetearyl Glucoside Other suntan preparations.3% Coco-Glucoside Eyeliner 2% Coco-Glucoside Eye makeup remover 3% Coco-Glucoside Hair conditioners.2% Page 3 of 5 CIR Panel Book Page 125

132 Coco-Glucoside Shampoos (noncoloring).4-8% Coco-Glucoside Hair dyes and colors (all types requiring caution 5% statement and patch testing) Coco-Glucoside Other hair coloring preparations.3% Coco-Glucoside Dentifrices (aerosol, liquid, pastes and powders).5% Coco-Glucoside Bath soaps and detergents.4-3% Coco-Glucoside Other personal cleanliness products.7-8% Coco-Glucoside Shaving cream (aerosol, brushless and lather).6% Coco-Glucoside Skin cleansing (cold creams, cleansing lotions,.4-8% liquids and pads) Coco-Glucoside Face and neck creams, lotions and powders.2-2% Coco-Glucoside Body and hand creams, lotions and powders.6-2% Coco-Glucoside Body and hand sprays.4% Coco-Glucoside Other skin care preparations 2% Coco-Glucoside Suntan, gels, creams and liquids.7% Coco-Glucoside Indoor tanning preparations.4% Coco-Glucoside Other suntan preparations 1% Ethyl Glucoside Eye makeup remover.2% Ethyl Glucoside Rouges.2% Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions,.5% liquids and pads) Ethyl Glucoside Body and hand creams, lotions and powders.4% Ethyl Glucoside Moisturizing creams, lotions and powders.3% Ethyl Glucoside Paste masks (mud packs).5% Lauryl Glucoside Bath oils, tablets and salts.5% Lauryl Glucoside Bubble baths.3-4% Lauryl Glucoside Eye shadow 5% Lauryl Glucoside Hair conditioners.4% Lauryl Glucoside Shampoos (noncoloring) 1-5% Lauryl Glucoside Hair dyes and colors (all types requiring caution.3-2% statement and patch test) Page 4 of 5 CIR Panel Book Page 126

133 Lauryl Glucoside Hair color sprays (aerosol) 8% Lauryl Glucoside Hair shampoos (coloring) 2% Lauryl Glucoside Other makeup preparations.3% Lauryl Glucoside Bath soaps and detergents.3-8% Lauryl Glucoside Other personal cleanliness products.6-2% Lauryl Glucoside Skin cleansing (cold creams, cleansing lotions,.6-1% liquids and pads) Lauryl Glucoside Face and neck creams, lotions and powders 2% Lauryl Glucoside Body and hand creams, lotions and powders.3% Lauryl Glucoside Moisturizing creams, lotions and powders 2% Lauryl Glucoside Other skin care preparations.2-2% Lauroyl Ethyl Glucoside Bath soaps and detergents 1% Lauroyl Ethyl Glucoside Other personal cleanliness products 2% Lauroyl Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions 2% liquids and pads) Myristyl Glucoside Eye lotion.4% Myristyl Glucoside Face and neck creams, lotions and powders.5-.6% Myristyl Glucoside Night creams, lotions and powders.6% Safflower Glucoside Hair conditioners.1% Safflower Glucoside Hair sprays (aerosol fixatives).5% Safflower Glucoside Other hair preparations (noncoloring).1% *Ingredients included in the title of the table but not found in the body of the table were incluued in the concentration of use survey, but no uses were reported. 1.6% in a leave-on baby product Information collected in 21 Table prepared January 6, 211 Table updated January 28, 211 (Decyl Glucoside: added high concentration to Indoor tanning preparations; Arachidyl Glucoside: added low concentration to indoor tanning preparations; C 12-2 Alkyl Glucoside: added 3 tanning product categories; Coco-Glucoside: added Body and hand sprays and 3 tanning product categories) Page 5 of 5 CIR Panel Book Page 127

134 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Persona Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: DATE: SUBJECT: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CW) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel March 15, 211 Concentration of Use by FDA Product Category: Hexyldexyl d-glucoside and Octadecyl d-glucoside th Street, N.W., Suite 3OO Washington, D.C (fax) CIR Panel Book Page 128

135 Concentration of Use by FDA Product Category Hexadecyl d-glucoside and Octadecyl dg1ucoside* Ingredient Product Category Concentration of Use Hexadecyl d-glucoside Moisturizing creams, lotions and powders I 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. Information collected in 211 Table prepared March 14, 211 CIR Panel Book Page 129

136 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Personal Care Memorandum Products Council Committed to Safety, Quality & Innovation TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel DATE: April 8, 211 SUBJECT: In Vitro Dermal Penetration Study: Caprylyl/Capryl Glucoside Across Barriers GmbH. 29. In vitro permeation and penetration of Capryl glucoside (INCI: Caprylyl/Capryl Glucoside) from the Product Plantacare 81 UP in human skin under GLP conditions. GLP Study Report STP 44/. Report number: C th Street, N.W., Suite 3 Washington, D.C (fax) CIR Panel Book Page 13

137 In vitro permeation and penetration of Capryl glucoside from c yiii/l... 1/ the Product Plantacare 81 UP in human skin under GLP conditions GLP Study Report STP 441 (/icscie) Sponsor: Study Monitor: Test Facility: Cognis GmbH Henkelstral3e 4589 Düsseldorf GERMANY Annette Mehling Phone: ± Fax: ± annette.mehling@cognis.com Across Barriers GmbH Science Park Saarbruecken GERMANY Report number: c-i Study Director: Responsible Scientist: Dr. Monika Kaca Phone: ±49681/ Fax: ±49681/ m.kaca@acrossbarriers.de Dr. Iris Zengerly Author: Dr. Iris Zengerly Date of report: CIR Panel Book Page 131

138 )RIGINAL Authentication The undersigned study director herewith confirms that the present study was performed in ac cordance to GLP as defined in the German Chemikaliengesetz and the OECD Principles of Good Laboratory Practice with the methods described. The final report includes the correct and complete results of the study. Tabular timeline of study report STP 44/ Draft final report: Final report: Saarbruecken, Q Oj Y( Saarbruecken, OOsaarbruecken, (jg I ( Dr. Eleónore Haitner-Ukomadu Tawuik Jalal Dr. Monika Kaca Mnaging Director Head of Quality Assurance Study Director Further staff involved in the study: Function: Scientist Name: Nazende Gunday Scientist Scientist Laboratory assistant Laboratory assistant Laboratory assistant Alesandro Marangon Nicole Kiet3ling Natalie Lenz Phillip Koch Jessica Gimmler 9O429FinalreportClO335244llO7STPO44.doc CIR Panel Book Page 132 GLP Study SW 44-

139 Across Barriers GmbH Seite 2 von 71 Intelligent Drug Profiling Contents 1. Summary Objectives Summarized results HPLCmethod In vitro permeation study In vitro penetration study Conclusion Materials and methods Materials Equipment and software Buffers and media Methods HPLC method development and validation Sample preparation for HPLC method Liquid Scintillation counting Preparation of test solution 1% CG (application solution) Determination of CG content in the test solution In vitro experiments Permeation studies with the test product Penetration experiments with the test product Establishment of the extraction method MEA: Qualification of skin membranes Calculations Results HPLCmethod System suitability test Selectivity Linearity in KRB-/- ph Accuracy and Precision Stability of the test product in donor and acceptor media Determination of CG content in the test solution Permeation study and penetration studies Establishment of extraction method for penetration study Results from permeation and penetration studies Quality control of the utilized skin samples (MEA) Discussion HPLC method development and validation Determination of CG content in the test solution Establishment of the extraction method for penetration study Permeation study Penetration study Quality control of the utilized skin (MEA) References Related guidelines 59 N:\5..*abor\5_]_Auftraege\5.J1.i _Iaufende_Auftraege\c_1 335_ \Berichte\neuere_VersionenBericht\lrZe_9429_Beric ht_c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 133

140 Across Barriers GmbH Seite 3 von 71 Intelligent Drug Profiling 7. Data storage and archiving 6 8. QAU Statement Appendix HPLCmethod Establishment of the extraction method Establishment of the extraction method with MeOH!water (5:5, v/v %) Establishment of the extraction method with water Franz cell volumes and areas Thickness of the human skins Quality control of the human skin (MEA) Transport of Caffeine (1%) through skin Transport studies with the test formulation (permeation results) Transport of CG from test solution through 3 different skin donors into the receptor medium 71 N :\5_Labor\5_1 _Auftraege \5_1 _1 _Iaufende_Auftraege\C_1 335_244_1 17 \Berichte\neuereversionen_Bericht\lrZe_9429Beric ht_c1 335_244_1 1 7.doc GLP Study STP 44 CIR Panel Book Page 134

141 Across Barriers GmbH Seite 4 von 71 Intelligent Drug Profiling Abbreviations A ACB-ID ACC TC BMI c D CG DS E eq. FZ HBSS HBSS- HPLC ID KLP KRB KRB-/- KS LSC MEA mean MeOH MW n OECD Papp PDA Q rpm RT area of exposed skin Across Barriers identification number accuracy Test compound Body mass index concentration dermis Capryl glucoside deeper skin Epiderms equation Franz diffusion cell Hank s buffered salt solution HBSS without glucose high pressure liquid chromatography inner diameter Calibration sample Krebs Ringer bicarbonate buffer KRB without glucose and HEPES Cryo-cuts Liquid scintillation counting initial mass in donor compartment Multiple endpoint analysis arithmetic mean methanol molecular weight number of repeats within an experiment Organization for Economic Co-operation and Development apparent permeability coefficient Photo diode array transported mass rotations per minute room temperature N:\5_La bor5_1 _Auftraege s_i_i _Iaufende_Auftraege\c_1 335_244_i 17 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beilc ht_c_i 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 135

142 Across Barriers GmbH Seite 5 von 71 Intelligent Drug Profiling RSD SC SCCP SD SOP SST STB T t TC TS UV V VA /xqi/xt relative standard deviation Stratum corneum Scientific Committee on Cosmetic Products standard deviation Standard operating procedure System suitability stability temperature time Tenside concentration Tape strip Ultra violet absorption donor volume acceptor volume permeability rate (transported mass of the substrate against the time) N :\5_Labor\5_1 _Auftraege\51 _1 _Iau[ende_Auftraege\c_1 335_244_1 ] 7 \Berichte\neuere_Versionen_Bericht\frZe_9429Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 136

143 Across Barriers GmbH Seite 6 von 71 Intelligent Drug Profiling 1. Summary 1.1. Objectives The aim of the present study was to investigate the in vitro permeation and penetration of capryl glucoside (GC) from the product Plantacare 81 UP at human skin of 3 different donors in vitro according to SCCP requirements and to OECD Guideline 428. The product provided by the cus tomer was diluted with HBSS- buffer to the concentration of 1 % CG and the ph was adjusted to 6.5. This formulation was used as the test solution in all experiments. The first step of the study was to establish an analytical method for quantification of GC. The described analytical method was developed to quantify the compound in the used biological acceptor and extraction media. The method was validated under GLP conditions for this purpose. At the same time the stability of CG from the test solution was tested over 48 hours at 32 ± 2 C, 25 ± 5 C and 4 ± 2 C in biological acceptor medium (KRB buffer without HEPES and glucose) and in the donor medium (HBSS without glucose) used for the permeation study. Fur thermore the stability of the test compound was also investigated in the extraction medium em ployed in the penetration studies (MeOH/water, 5:5 v/v, %). At the beginning of the study, the CG content in the test solution without skin contact in tripli cate and after skin contact (one-fold) was determined. The permeability of CG from the test solution on human skin was investigated on fresh human skin from 3 donors in two fold (n=2) for each donor (totalizing n=6) conformable the SOP M 5 at Across Barriers. The skin specimen was internally encoded according to the corresponding SOP A 5 at Across Barriers. Dermatomized (to approximately 5 pm) skin was used. The skin thickness was measured immediately before performing the studies. 8 samples were taken from the acceptor medium during a period of 24 hours to obtain information about permeability of CG. At the end of the permeation experiment the remaining CG content in the test solution was determined. For that goal the test formulation left on the skin surface was collected with cotton swabs and transferred to the falcon tube with the extraction medium, this is the so-called wash procedure. After removing residual formulation, the concentration of CG in the skin, in the Stratum corneum and deeper skin layers, was quantified. The upper corneous layer of the skin was stripped off and the residual skin was cryo-sectioned. After the in vitro study a mass recovery was carried out to determine the mass balance and local distribution of CG in the different skin compartments. For that goal a quotient of total mass of CG at the end of the study on the skin surface in test solution, in Stratum corneum, Epidermis/Dermis and acceptor compartment versus the applied amount of CG in the formulation at the start of the study was calculated. The permeability of N :\5Labor\5_1 _Auftraege\5_1 htc_1 335_ doc 1 _Iau[ende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\IrZe_9429Beric CIR Panel Book Page 137 GLP Study STP 44-

144 Across Barriers GmbH Seite 7 von 71 Intelligent Drug Profiling Caffeine (MEA: multiple endpoint analysis) was carried out at a concentration of 1 mgl 1 in Krebs-Ringer-buffer (KRB) at ph 7.4 (n=2 for 3 skin donors) for the study C-i Since the same skin donors were employed in the present study, the permeability of caffeine was not performed. Caffeine is a recommended marker molecule of the OECD Guideline for the quality control of human skin. The results of the Caffeine permeation were compared with the permeability coefficients of previous studies on historical human skin membranes of different origin at Across Barriers. N:\5_Labor\5_1 _Auftraege\5_1 _1 _Iaufende_Auftraege\C_1 O335244_1 17 \Berichte\neuere..YersionenBericht\IrZe_9429_Beric ht_c_1 335_244_1 17.doc GL.P Study STP 44 CIR Panel Book Page 138

145 Across Barriers GmbH Seite 8 von 71 Intelligent Drug Profiling 1.2. Summarized results HPLC method The results of the analytical method development and validation are summarized in the following three tables. Tab. 1: Analytical parameters. System Column Mobile Phase Flow Mode Temperature Injection volume MS detection Agilent 11 [C MSD Waters Atlantis C18 3 pm 2.1 x 5 mm (Length. ID); ACB ID: S387 A: Water ±.1% formic acid B: Methanol ±.1% formic acid.4 mlmin 1 Gradient 3 C (Column), 3 C (Autosampler) 2 pl ESI positive SIR m/z Tab. 2: Gradient profile for CG analytical method. Time [mini A [%] B [%] Flow ImL min] N;\5_Labor\5.i_AuItraege\5_1.1 _Iaufende_Auftraege\C_1 335_244_1 1 O7\Berichte\neuere_Versionen_Berich\IrZe_O9O429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 139

146 b pg Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 9 von 71 Intelligent Drug Profiling Tab. 3: Summarized results of the HPLC method validation. Parameter Detail limits Result Compliance System suitability test 6 injections of standard solution RSD = 1 % 2.18 % yes Visual comparison of No interferences No interferences chromatograms of the test with eluent mixture, with eluent mix- Selectivity compound in eluent mix- extraction medium ture, extraction yes ture, extraction medium and acceptor me- medium and ac and acceptor medium dium ceptor medium 2 Order regression model y + m x + o x2, weighting: 1/x 2 mm. 5 calibration levels = yes - Theoretical val ues:.986- Linearity in KRB* buffer ph 7.4 Linear range ml 1 yes measured values: pg ml Deviation conc. Meas- LLOQ:± 2% ured/ theor. conc.: Others: ± 15% LLOQ: 7.7 % Others: until 14.55% 3 concentration levels Recovery Recovery: Accuracy in accep- CON1-CON3 1 ± 2 CON1: % tor medium KRB* n=3 (LLOQ) yes ph7.4 CON2:88.95% deviation mean value/true 1 ± 15 % value (others) CON3: % yes Precision in acceptor medium KRB* 3 concentration levels RSD: RSD (n=3) CON1-CON3 CON1:.58% ± 15% (others) n==3 CON2: 2.12 % ± 2 % (LLOQ) RSD% CON3:12.86% yes Measured value: S/N: = 5:1.88 pg ml 1 concentration near LLOQ (CON3) accuracy: S/N: = 5:1 Limit of 1 ±2% I,..,3 accuracy: quantification precision: 86.59% S/N RSD=2% precision: accuracy/precision RSD = 8.58 % yes * without HEPES and glucose N :\5_Labor\5_1 _Auftraege\5_1 _1 jaufende..auftraege\c_1 335_244_] 17 \Berichte\neuere_Versionen_Bericht\frle_O9O429Beric ht_c_ _1 1 7.doc GLP Study STP 44- CIR Panel Book Page 14

147 .. Recovery.. 4 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 1 von 71 Tab. 3: Summarized results of the HPLC method validation (continuation), Parameter Detail Limits Result Compliance Recovery Recovery Acceptor medium > 85 4 C124h: 95.5 % in acceptor medium: RT, 4 C and 32 C over 24h Precision < 15% RT/24h: 96.5 % yes n=3 32 C/24h: 91.5 % Robustness (sta bility of the test compound in KRB* 7.4) Recovery Recovery in acceptor medium: Recovery > 85 % Acceptor medium RT, 4 C and 32 C over Precision < 15 % C/48h: 89.3 % yes 48h RTI48h: 89.2% n=3 32 C/48h: 91.8 % Robustness (St a bility of the test compound in HBSS** 6.5) Recovery Recovery Recovery > 85 % Donor medium in donor medium: RT 4 C124h: 89.3 i es 32CC and 4 C over 24h Precision < 15 /o y RTI24h: 95. % n=3 32C/24h: 91. % Recovery Recovery Donor medium Recovery > 85 % C/48h 87.8 % in donor medium: RT 32CC and 4 C over 48h Precision < 15 RT/48h: 86.8 % yes n=3 32 C/48h: 91.8 % * without HEPES and glucose ** without glucose N :\5_Labor\5_1 _Auftraege\5_1 _1 _Iaulende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\IrZe_9429Beric htc_ doc GLP Study STP 44- CIR Panel Book Page 141

148 Across Barriers GmbH Intelligent Drug Profiling Seite 11 von In vitro permeation study Tab. 4: Summarized results from the permeation study Theoretical content CG transport into the Skin Test product Test substance of CG in the donor acceptor after 24 h number 1mg] [%] Test solution 1% CG CG 3.9.* CG 3.9.* CG 3.9,* * Values below analytical LLOQ (.88 iig ml 1). N:\5_Labor\5_1 _Auftraege\5..j _1 Jaufende_Auftraege\C_1 335_244_1 1 7\Berichte\neuereVersionen_Bericht\lrZe_9429_Beric htc_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 142

149 Across Barriers GmbH Intelligent Drug Profiling Seite 12 von In vitro penetration study The results of the in vitro transport studies are summarized in Tab. 5 and Tab. 6. Tab. 5: Mean amount [pg cm- 2] of CG in the samples from the three in vitro experiments. Cumulative amount [pg. cm2j of CG in the samples Sample Skin Skin Skin All skins used Mean SD Mean SD Mean SD Mean SD dose ap plied receptor.*..*..*..*. skin wash Tape strips Tape strips * cryocuts.* * Values below the LLOQ oi the analytical method. Tab. 6: Mean recovery rate and dose absorbed [%] of CG in the samples from the three in vitro experi ments. Recovery and dose absorbed [%1 of CG in the samples Sample Skin Skin Skin All skins used Mean SD Mean SD Mean SD Mean SD receptor,*..*..*..*.* skinsurface Tape strips Tape strips cryocuts.*..4.4.*..1.3 Dose absorbed.*.*.4.4.*.*.1.3 Total recovery * Values below the LLOQ of the analytical method. N:\5_Lbor\5_1 _Auftrnege\5_1 _1 _Iaufende_Auftraege\C_1 335_ O7\Berichte\neuereVersionen_Bericht\IrZe_O9O429Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 143

150 Across Barriers GmbH Seite 1 3 von 71 Intelligent Drug Profiling 1.3. Conclusion The HPLC method was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRB buffer without HEPES and glucose). Furthermore, the test compound has demonstrated good stability over the period of 48 hours at RT and 4 C in the extraction medium employed for the penetration experiments (MeO H/water (5:5, v/v %)) and good stability in the acceptor (KRB-/-) and donor media (HBSS-) at 32 C, 4 C and RT over the period of 48 hours, which covers the experiment and measurements duration. The content of CG in the test solution without skin contact was determined in triplicate and the mean value was ± 1.56 %. The test solution was also applied to skin and the recovery reached 9.1 %. This complies to the acceptance criteria of 1 ± 1 %. The results from the extraction method has shown that water/meoh (5:5 v/v, %) was most suitable medium for performing the penetration experiments presenting mean recovery values which has ranged between % and %, which complies to the acceptance limit of 1±2%. The permeated amounts of CG through three different human skins (no , and ) have presented values in the receptor below the LLOQ of the vali dated analytical method. This low absorption was confirmed through the mass recovery calcula tion, where % (mean value for 6 Franz cells) of surfactant were determined in the test solution which remained at the skin surface after 24 hours. The mass recovery calculations present amounts and percentages of compound which perme ated, penetrated and remained in the donor compartment. The mean amount of CG removed from the skin surface (skin wash) ranged from % to % of the dose applied. The mean recovery (mean value for 6 Franz cells) in the two first tape strips was.52 % during all performed experiments. In the further 18 tape strips a mean recovery of.3 % was docu mented. The mean absorbed dose of CG, sum of the amounts found in the viable epidermis, dermis and receptor medium, were considered as.1 %. The mean Papp values measured for Caffeine for the human skins no , and are in good agreement with app values determined for a variety of skin specimens from different donors under comparable conditions. The result are reported in detail in the report C-i N:\5_Labor5_1 _Auftraege\51 _1 _Iaufende_Auftraege\C_1 335_ \Berichte \neuere_versionen_bericht\irze_942 9_Beric ht_c_1335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 144

151 2 2 2 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 14 von Materials and methods 2.1. Materials Tab. 7: Skin sample parameters used for the CG in vitro studies and quality control experiments. Species Gender Age Region BMI Storage Skin number Human Female 48 Abdomen Human Female 43 Abdomen Human Female 3 Abdomen C C C Human Female 38 Abdomen C The skin samples were excised during surgical operations. The skin was not removed to provide samples for these in vitro investigations. The hospital had the prior consent of the patients that the tissue could be used for scientific research. Tab. 8: Test product. Name Compound ACB-ID Supplier Batch Plantacare 81 OUP Capryl glucoside K8828 Cognis GmbH CE73514 Tenside content 62.8 % Tab. 9: Name Test compound from test product. Capryl glucoside Molecular weight g!mol Molecular structure n = 1,6; R C8-1 N:\5_Labor\5_1 _Aultraege\5_1 _1 _Iaufende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_versionenBericht\IrZe_94 29_Benc ta_c_i 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 145

152 . stripping - - Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 1 5 von 71 Intelligent Drug Profiling Tab. 1: Further chemicals and reagents used. Compound I reagent Batch ACB-ID Ethanol K L9781 KRB ph 74* - Methanol 4487 L9454 Formic acid K L8986 HBSS ph 55** - Caffeine 66K85 [87 [3H] Caffeine 819 L8689 *without HEPES and gi ucose **without glucose Tab. 1 1: Other consumables. Article Application Specifications glass diffusion chambers for the acceptor volume: approx. 2 ml Franz cells.. transport 2 assays diffusion area: approx. 3 cm donor volume: variable Franz cells diffusion chambers for the transport assays (MEA) glass acceptor volume: approx. 12 ml diffusion area: approx cm2 donor volume: variable Syringes sampling single use fine dosage Needles sampling disposable Plates Scintillation counting 4 x 6 wells Disposable scalpels cutting of skin Bayha sterile surgical blade of stratum cor-. Tape film 19 mm wide, Beiersdorf, product number 5733 neum HPLC vials sampling - N:\5_Laboi5_1 _Auttraege\5_1 ht_c_1 335_244_1 17.doc 1 _Iaufende_AuItraege\C_1 335_ O7\Berichte\neuereVersionen_Bericht\lrZe_O9O429_Beric CIR Panel Book Page 146 GLP Study STP 44-

153 Across Barriers GmbH Seite 16 von 71 Intelligent Drug Profiling 2.2. Equipment and software lab. 12: HPLC equipment and software. Device I accessories Specification Supplier HPLC Agilent 1 1 [C Agilent Detectors MSD Agilent Software HP G271 [C/MS ChemStation Software Agilent lab, 13: Utilized equipment. Device Type I Specification Manufacturer. SBC22/BCBC1 Analytical Balance d*=,1/.1 mg) Scaltec Centrifuge 4K1 5C Sigma Cryo microtome MEV SLEE Dermatom GA 63 Aesculap Fridge-freezer Santo (4 C/-2 C) AEG Heating cabinet Equ.Nr Heraeus Magnetic stirrer Vario-Mag Mono grau H±P [abortechnik Magnetic stirrer Stirring Drive HP 15 H P [abortechnik Mini Shaker MS2 IKA [abortechnik ph-meter 4-7 model 33 Orion 1-1 p[ Piston-stroke pipettes 5-2 p[ p[ p[ Eppendorf Saarbruecken model developed by Prof. H. [oth - Shaker KS-i 6 Edmund BUhIer Shaker KS-i 5 Edmund Buhler Skin thickness gage ND 221 B Heidenhain Steaming cabinet 2174 / 32 C Heraeus SG Wasseraufbereitung + Ultra-pure water system Ultra Clear. Regenerierstation Ultrasonic bath Sonorex Super RK1O6 Bandelin N:\5_Labor\5_1 _Auftrege\5_1 _1 _Iaufende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\IrZe_ Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 147

154 Across Barriers GmbH Seite 1 7 von 71 Intelhgent Drug Profiling 2.3. Buffers and media Krebs Ringer buffer without HEPES and glucose, ph 7.4 The Krebs Ringer buffer without HEPES and glucose (KRB-!-) was prepared referring to SOP M 3 and used as modified form for acceptor medium in permeation experiments. Tab. 14: Preparation of the Krebs Ringer buffer ph 7.4 without HEPES and glucose (KRB-/-) Substance Weighted sample [g] per 1 1 of water MgCI 26H2.224 CaCI 2 6H2.274 NaCI KCI.375 Na 2HPO 42H2.294 NaH 2PO42H2.47 NaHCO The ph value was adjusted with formic acid to 7.4. HBSS buffer without glucose, ph 6.5 The content of the HBSS without glucose (HBSS-) buffer is summarized below. Tab. 15: HBSS- bufferatph 6.5 Substance Weighed sample [g] per 1 1 of water MgSO 4,98 CaCI 2 6 H2.29 NaCI KCI.4 Na 2HPO 42 H2.69 K2HPO 42H2.11 MES The ph-value was adjusted with formic acid to the value of 6.5. N:\5_Labor\5i_Au[trege\5_1 _1 _Iaufende_Auftraege\C.1 335_244_1 1 O7\Beriche\neuere_Versionen_Bericht\IrZe_O9O429_Beric ht_c_ l 335_244_1 1 O7.doc GLP Study STP 44. CIR Panel Book Page 148

155 Across Barriers GmbH Seite 18 von 71 Intelligent Drug Profiling Mobile phase A Water ±.1% formic acid Mobile phase B Methanol ±.1% formic acid 2.4. Methods HPLC method development and validation The first step of the study was to establish an analytical HPLC method for quantification of CG in the used acceptor media (KRB-!- buffer). The HPLC method development was not carried out under GLP conditions. The validation of the method was investigated in accordance with the FDA Guideline,,Bioanalytical Method Validation (21) and it was performed under GLP conditions. System suitability test A system suitability test is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system. This test was performed for one reference substance CG in six fold determination. Relative standard deviation below 1% was accepted. Selectivity The specificity of the method was investigated by means of eluent mixture, acceptor medium and extraction media. The relevant chromatograms with and without CG were compared and scanned for possible interferences with extraneous components by visual inspection. Linearity The linearity was checked for KRB-!- buffer with 8 concentration levels of the test compound. The absorption values of the standard solutions were plotted against their corresponding theo retical concentrations. The purity of the analytical standard was considered for the calculation of its concentration. The data were subjected to regression analysis to provide information on the slope, the intercept, the determination coefficient (r 2), and the back-calculated calibration stan dard concentrations. N :\5Labor 51 _Auftraege\5_1 _1 _Iaufende_Aultraege\c_1 335_244_1 17 \Berichte\neuere_versionen_Bericht\lrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 149

156 Across Barriers GmbH Intelligent Drug Profiling Seite 19 von 71 Regression model: y=b+nl.x+ox 2,weighting: 1/x 2 Eq. 1 m: Slope first order : Slope second order b: Intercept y: Absorption values I Areas x: Concentration levels / Amount jiglml Accuracy and precision In order to assess the accuracy of the method the mean value of 3 replicate samples was de termined for 3 different concentration levels. The difference between the determined mean value and the true (theoretical) value was calculated and expressed as percent of the true value: %Acc=1%+ CONtheory 1% Eq. 2 % Acc CONtheory CON, accuracy as percent value theoretical concentration value (true value) single determined concentration value of a particular concentration level The precision corresponds with the standard deviation of the 3 replicates of each concentration level. The following concentration levels were prepared: CON 1: high concentration: 7 %-9 % of the highest calibration standard CON2: mean concentration: 3-5-fold value of the LLOQ CON 3: low concentration: close to LLOQ The demanded values at the LLOQ are = 2 % for the precision (% RSD) and 1 ± 2 % for the accuracy. Regarding concentration values above the LLOQ values of 15 % are demanded for the precision, whereas the accuracy has to be within a range of 1 ± 1 5 %. N:\5_Labor t5_1 _Auftraege\5_1 _1 _Iaulende_Aultraege\C_1 335_244_1 17 \Berichte\neuere_VersionenBericflt\IrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 15

157 Across Barriers GmbH Seite 2 von 71 Intelligent Drug Profiling Quantification limit For the determination of the quantification limit 3 samples in the range of the LLOQ were ana lyzed in series. Regarding a signal noise ratio (SIN) 5 : 1, precision values (% RSD) = 2 % and accuracy values of 1 ± 2 % were accepted. A SIN ratio of 3 : 1 is required for the de tection limit. Stability in donor medium (HBSS without glucose) Aliquots of the test compound were dissolved in the donor medium used (HBSS-) stored for 48 h at 4 C ± 2 C, 25 C ± 5 C and 32 C ± 2 C. Subsequently they were analyzed by HPLC. The stability was determined as the recovery difference between three replicates treated sam ples and three untreated samples. Stability in the permeation matrix (KRB without Glucose and HEPES) As biological acceptor medium KRB-I- buffer at a ph of 7.4 was used. For the stability determi nation aliquots of the test compound were dissolved in this matrix, stored for 48 h at 4 C ± 2 C, 25 C ± 5 C and 32 C ± 2 C. Subsequently they were analyzed by HPLC. The stability was determined as the recovery difference between three replicate treated samples and three untreated samples. N:\5Labor\5_1 _Au[traege\5_1 ht_c_1 335_244_1 1 7.doc 1 jaufende_auftraege\c_] 335_244j 1 O7\Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 151 GLP Study STP 44-

158 CON3) Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 21 von 71 Intelligent Drug Profiling HPLC validation plan Tab. 16: Summarized target parameters for HPLC analysis of in vitro sample. Parameter Description Limits SST 6 injections of standard solution RSD = 1 % Comparison of chromatograms. no interferences with eluents and Selectivity Test compound in matrix vs. matrix, extraction medium, slight interference eluents and extraction medium with the acceptor medium Linearity At least 5 concentration levels (external standards) 25t order regression model 1/x 2 deviation conc. measured / theor. Conc.: LLOQ: ± 2 % others: ± 15 % r =.99 Accuracy 3 concentration levels deviation: (CON1 - n=3 LLOQ: 1 ± 2 % deviation mean value! true value others: 1 ± 15 % RSD (n=3): Precision 3 concentration levels (CON1 CON3) n=3, RSD LLOQ: ± 2 % others: ± 15% Concentration near LLOQ S/N: = 5:1 Limit of quantification (CON3) n=3 accuracy: 1 ± 2% accuracy/precision precision: ± 2 % Test conditions: 4 C, 25 C and Stability of the test Recovery > % C for 48 h compound Precision < 15 % N :\5Labor5_1.Auftraege \5_1 j _lautende_auftraege\c_1 335_244_1 17 \Berichte \neuere.versionen_bericht\lrze_94 29Beric ht_c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 152

159 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 22 von Sample preparation for HPLC method All samples for the validation of the HPLC method were diluted to acceptable concentrations (within the calibration range). Sample preparation for the system suitability test The test compound was firstly dissolved in KRB-!- buffer ph 7.4 (stock solutions) and subse quently diluted to appropriate yield concentrations. The purity of the compound was considered in the calculations. Tab. 17: Preparation of the stock solutions for the reference samples. Sample I Purity Weighted Final volume Compound c ACB-ID I Solvent name I I [%i I sample [mgi I I Emil [ig.ml SST1 CG K KRB-/- I buffer I 1i Tab. 18: Preparation of the reference samples. Samples to assess the linearity Tab. 19: Preparation of the stock solutions for the linearity assessment. The standard solution from the test compound was prepared in the range of.986 pgml 1. The solution preparation is presented in the table Tab. 2. N:\5_Labor\5_1_Auftraege\.5_1 _1 jaulende_auftraege\c_1 335_244_1 1 7\Berichte\neuereVersionen$ericht\lrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 153

160 Across Barriers GmbH Seite 23 von 71 Intelligent Drug Profiling Tab. 2: Preparation of the analytical solutions used to assess the linearity Sample name Stock solution Volume SI - Final volume c Diluted with 1 (SL) [ml] [mil [pg-ml I KLP2 1A 2.5 KRB-/- ph KLP3 1A 2 KRB-/- ph KLP4 1A 1.5 KRB-/- ph KLP5 1A 1.5 KRB-/- ph KLP6 2A 2 KRB-/- ph KLP7 2A 2 KRB-/- ph KLP8 7A 3 KRB-/- ph KLP9 7A 1 KRB-/- ph KLP1O 7A 2.5 KRB-/- ph KLP11 7A 1 KRB-/- ph KLP12 8A 1 KRB-/- ph KLP13 8A 1 KRB-/- ph KLP14 8A 1 KRB-/- ph KLP1S 8A 1 KRB-/- ph * Only the analytical solutions KLP6, KLP7, KLP8, KLP9, KLP1 1, KLP1 2, KLP1 4, and KLP1 5 were em ployed for linearity determination. Samples to assess accuracy and precision in acceptor medium For the determination of accuracy and precision in the acceptor medium (KRB-/-) 3 sarrples were prepared and diluted for 3 different concentrations. The preparation of the stock solutions follow the table below: Tab. 21: Summary of samples preparation to assess accuracy in acceptor medium, Stock Compound* Weighted Final volume c ACB-ID Solvent solution sample 1mg] [ml] [ig-ml 1] KRB-/- buffer CONC1 CG K , ph 7.4 KRB-/- buffer CONC2 CG K ph 7.4 KRB-I- buffer CONC3 CG K ph 7.4 N:\5_Labor\5_1 _Auftraege \5_1 _1 _Iau1endejuftraege\C_1 335_244i 17 \Berichte\neuere...Versionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 154

161 Across Barriers GmbH Intelligent Drug Profiling Seite 24 von 71 Each stock solution was diluted to yield 3 different concentrations according to presented on the table below. Tab. 22: Summary of standard solutions prepared from stock solutions for assessment of accuracy and precision. Sample name C Ipg.ml ACC CON ACC CON ACC CON Sample for assess stability in KRB buffer (without glucose and buffer (without glucose) ph 6.5 HEPES) ph 7.4 and HBSS The samples for assessment of stability were prepared and stored at the temperature of 4 C, RT and 32 C during 48 hours. Tab. 23: Preparation of the stock solutions for the reference samples. Sample Compound* Weighted ACB-lD Final volume c Solvent name sample [mgi [mu IpgmL 1l Methanol/wate KRB STB 1 CG K (5:5) HBSS STB 1 CG K [ HBSS** **Without glucose Tab. 24: Preparation of the reference samples. Sample name Stock solution Volume SI Final vol- c Diluted with (SI) [ml] ume [ml] [pg.ml 1] KRB* buffer KRBSTB2 KRBSTB ph 7.4 KRB* buffer KRB STB 3 KRB STB ,3641 ph 7.4 HBSSSTB2 HBSSSTB1 1 HBSS** HBSS STB 3 HBSS STB 2 1 HBSS** HBSS S184 HBSSSTB3 1 HBSS** Without HEPES and glucose. *Without glucose N :\5_Labor\51 _Auftraege\5_1 ht_c_-1 335_244_1 17.doc 1 _Iaufende_Auftraege\c_1 335_ \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 155 GLP Study SIP 44-

162 2 2 2 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 25 von 71 Intelligent Drug Profiling Liquid Scintillation counting Caffeine was quantified by liquid scintillation counting. The samples (3 were transferred into 24 well plates and mixed with 5 liquid scintillation cocktail. After an equilibration pe riod of at least 1 -hour radiation was measured for 2 minutes per well in a scintillation counter. The recorded counts in a blank sample were subtracted from the value of each test sample. Tab. 25: Scintillation counting equipment. Device I accessories Specification Supplier Liquid scintillation counter Microbeta WallacTM Perkin Elmer Scintillation Cocktail Optiphase TM Supermix Perkin Elmer Preparation of test solution 1 % CG (test solution) The test product provided by the customer was diluted with HBSS buffer (without glucose) to concentration of 1 % CG and ph value was adjusted to 65. The HBSS- buffer was employed instead the KRB-/- buffer because HBSS- buffer shows better buffer capacity at the ph 6.5. The term test solution used in this report applies to the 1% CG solution in HBSS (without glu cose) Determination of CG content in the test solution At the beginning of the study the drug content in the test solution was determined by HPLC in triplicate with an appropriate extraction method. The mean recovery of 1 ± 1% was ac cepted. For the determination of CG content, the following solutions were prepared: Compound I Weighted Final Sample name Stock solu- ACB-ID sample I Solvent volume tion (SI) Volume SL Emil IpgmL j SOL1 CG K mg HBSS- ph SOL2 SOL1 - SOL3 SOL2 - SOL4 SOL3 - ml MeOH/water (5:5) ml MeOH/water (5:5) ml MeOH/water (5:5) The SL1 was diluted until to obtain a theoretical concentration of 1.1 ig/ml. This last solu tion was employed for the AS content determination (For sample name see table Tab. 26 CG1, CG 2 and CG 3). Additionally 3 jl of the application solution (1 % CG) were applied to human dermatomized skin (sample named as CG 4), that was clamped into a Franz cell. The tenside was immediately N:\5Labor\5.1 _Auftraege\5_1 _1 _Iaufende_Au[trege\c_1 335_244_1 17 \Berichte\neuere_Versionen_Bercht\lrZe_9429_Beric ht_c_1 335_244_l 1 7.doc GLP Study STP 44- CIR Panel Book Page 156

163 - penetration, - permeation, - resorption, Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 26 von 71 Intelligent Drug Profiling removed with cotton balls and washed twice with 1 ml of water/methanol (5:5, v/v %). The cotton balls and the wash solution were pooled in a 5 ml volumetric flask and completed with water/methanol (5:5, v/v %). 85 pl of this solution were diluted with the extraction medium to 5 ml. The table below summarizes the respective amount in mg and the theoretical surfac tant content. Tab. 26: tion. Summary of corresponding weight of samples and theoretical tenside content in the test solu Test formula- Compound Skin contact Sample name Amount weighed* [mg] Theoretical tenside concentration. ml1 Test solution 1%CG CG CG no CG CG yes CG *Corresponds to 3 p1 of the test solution CG4 ano 2m1 of the test solution CG In vitro experiments As an introduction a definition of the dermal absorption according to SCCP guideline and its adaption and more detailed use to/in the present report is presented. The percutaneous/dermal absorption process is a global term, which describes the passage of compounds across the skin. This process can be divided into three steps: which is the entry of a substance into a particular layer or structure such as the entrance of a compound into the stratum corneum (in the present report entrance of a test compound into the stratum corneum, entrance of a compound into the viable epidermis and into the dermis); which is the penetration through one layer into another, which is both functionally and structurally different from the first layer (in the present report penetration of the test com pound through skin layers into the receptor medium); which is the uptake of a substance into the vascular system (lymph and/or blood vessel), which acts as the central compartment (not investigated in the present study). Introduction anatomy of human skin Human skin comprises the epidermis, the dermis and the underlying subcutaneous tissue, with sebaceous and sweat glands running throughout. The outermost epidermal layer, i.e. the Stra N :\5_Labor\5_1 _Auftraege\5_1 _1 _Iaufende_Au[traege\c_1 335_244_1 17 \Berichte\neuere_versionen_Bericht\IrZe_ Beric ht_c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 157

164 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 27 von 71 turn corneum (SC) or corneous layer is the main barrier to skin permeation for dermally applied drug formulations. The structure of the Stratum corneum itself can be explained in terms of the so-called brick and mortar model in which corneous keratinocytes (corneocytes) represent the bricks while the intercellular lipids and water-retaining natural moisturizing factors act as the mortar. L OOpm r Fig. 1: Cross-section through human skin (stratum corneum and dermis) The main gender-specific difference in abdominal skin is the higher number of hair follicles in male samples. Because it is important not to cut the hair follicles when preparing the subcutis, female hairless abdominal skin is preferable for in vitro studies. Skin membranes During the study human skin biopsies of 3 donors were used. Preparation of the skin Transport and preparation of full-thickness skin The skin was made available in accordance with the relevant statutory and contractual provi sions. The excised skin from the surgery was cooled to 4 C and its surface was dried before being transported. Care was taken to ensure that the subcutaneous fatty tissue does not get into contact with the surface of the skin. Once in the laboratory, the skin was separated from the subcutaneous fatty layer and then issued by Across Barriers with an encrypted identification number. Encryption was performed in accordance with the Across Barriers SOP A 5. The encrypted identification numbers are always used to refer to the skin specimens in laboratory records and N :\5_Labor 5_1 _Auftraege\5_1 _1 _laufende_auftraege\c1 335_244_1 17 \Berichte\neuere_Versionen_BerichtUrZe_94 29_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 158

165 Across Barriers GmbH Seite 28 von 71 Intelligent Drug Profiling reports. Across Barriers assured to the hospital that information on the patients and the surgery can only be accessed via a tile held by Across Barriers. The suitability of the skin biopsies was assessed according to the following criteria: Inclusion criteria: No pathological findings Hospital must have patient s consent that tissue may be used for scientific purposes Exclusion criteria: Skin damage, strongly marked scarring or stretch marks Working under controlled storage conditions (4 C), the subcutaneous fatty layer was removed within two hours after reception of the abdominal skin. Once separated from the subcutaneous fatty tissue, the residual full-thickness skin was stored at -2 C. According to the OECD Guide line, storage of skin at -2 C for a period of up to six months does not alter its permeability. The skin was used immediately after thawing. Repeated freeze-thaw cycles were avoided. Dermatomization of the skin The human skin was cut into 4 cm broad stripes. Subsequently skin sections with a thickness of approximately 5 pm were prepared from the full-thickness skin samples using an Aesculap GA 63 dermatome. The skin surface with intact Stratum corneum is directed towards the dernm tome. The use of the dermatome is described in SOP G 4. Prior to use, the thickness of the dermatomized skin was checked with a Heidenhain thickness gage (SOP G 26) at five different places of each skin sample. The measured thickness was not exactly the 5 pm scheduled in the study plan, as it strongly depends on the skin s elasticity and cannot be estimated prior to dermatomization. The slightly varying thickness, however, has no influence on the permeability of the marker compound Caf feine [Bock et al Because the stratum corneum represents the principle absorption bar rier a significant alteration of the test compounds transport kinetics by remaining deeper skin layers is not expectable Permeation studies with the test product The cylindrical glass Franz cell is a diffusion chamber comprising an upper and a lower part be tween which the skin specimen will be clamped. The two halves of the cell are held together by means of a ball-and-socket clamp. The lower (receptor) chamber has a volume of approximately N :\5_Labor\5_1 _Auftraege\5_1 ht_c_1 335_244_1 17.doc 1 _Iaulende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_versionen_Bericht\IrZe_942 9_Beric CIR Panel Book Page 159 GLP Study STP 44-

166 Across Barriers GmbH intelligent Drug Profiling Seite 29 von 71 2 ml, while the volume of the upper (donor) chamber is variable. The skin specimens was punched out (area of approximately 4 cm2) immediately prior to insertion in the Franz cells. Fig. 2: Franz cell viewed from above (left) and from the side (right). The skin is always inserted with the dermal layer oriented downwards so that the skin s horny layer is uppermost. Franz diffusion cells Franz diffusion cells with a diffusion area of approximately 3 cm2 and an acceptor volume of approximately 2 ml were used. The stirring speed and the temperature was set at 4 rotation per minute and 32 C ± 2 C, respectively. At each sampling point the stirring speed and tem perature were documented. For more details regarding the used Franz cell diffusion area and volume see appendix topic 9.3. Dose 3 jil of the test solution 1 % CG were applied at the diffusion skin area and homogenously spread over the skin surface. In order to avoid any changes in the formulation, Franz diffusion cells were kept covered with Parafllm over the period of 24 hours. The attribution of the particu lar test formulations and the applied amount to the different Franz cells is depicted in Tab. 27. N:\5_Labor\5_1 _Aultraege\5_1 _1 _Iaufende_Aultraege\C_ _1 1 7 \Berichte\neuere_ver onen_bericht\irze_9429_beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 16

167 Across Barriers GmbH Intelligent Drug Profiling Seite 3 von 71 Tab. 27: Assignment of test formulation, applied amounts and skin thickness to the Franz cells. Formulation Thickness of skin Applied vol Theoretical Donor Skin numume per GC content Mean FZ mass* diffusion ber RSD [%] [mg] in doarea [mg. cm- 1pm] nor [p11cm 2] 2] , Test solution , %CG * Value in mg which corresponds to 3 of test solution 1 % CG Acceptor medium As acceptor medium for the studies with the test preparation, KRB buffer at ph 7.4 without glu cose and HEPES was used. The content of this medium is presented in the Tab. 14. Sampling times The permeation study was performed over a period of 24 hours, after.5, 2, 4, 6, 2, 22, 23 and 24 hours. The samples with a volume of 2 pl were taken from the acceptor compart ments and analyzed for the drug content by HPLC. The taken amount was replaced with a fresh medium preheated to the temperature of 32 C. In total 8 samples were analysed for each time point (3 donors, n=2). N:\5_Labor\5_1 _Auftraege\5_1 _] jaufende_au[traege\c_1 335_244_1 1 7\Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44 CIR Panel Book Page 161

168 Across Barriers GmbH Seite 31 von 71 Intelligent Drug Profiling Tab. 28: Sampling times for the in vitro study. Sample ID Run Time [h] Penetration experiments with the test product After removing the formulation from the skin surface, the skin was partitioned into horizontal segments to determine the distribution profile of CG in the skin Establishment of the extraction method Prior to the penetration studies an appropriate extraction method was established under non GLP conditions. In order to detect a possible adsorption of the test compound to the Tape film or to the skin in the extraction matrix, test solution samples were incubated with Tape strips, Tape strips ± Stratum corneum and deeper skin layers, respectively. For penetration experiments a scotch tape purchased from Beiersdorf (Germany, 1 9 mm wide, product number 5733) was used. The skin No was used for the establishment of the extraction method. For this purpose a skin sample was stripped by means of the Saarbruecken model (Fig. 3). Mo reover, Tape strips without skin contact and cryosections of deeper skin layers (epidermis and dermis) were prepared. Two different concentrations of CG (about 3.6 pglml and.9 ig/ml) were added to the samples, which subsequently were agitated for 1 hour with extraction matrix. Two solutions were tested as extraction media: water and Methanol/water 5:5 (v/v, %). Finally the recovery of the test compound was determined by comparing the extracted and the initially applied amounts. An appropriate extraction medium for the scheduled in vitro penetration stud ies was defined in this experiment. Penetration studies The residual formulation on the skin surface (skin wash) was removed and the concentration of the test substance was quantified. Two segmenting techniques, Tape stripping and Cryosection ing, were applied to separate different skin layers parallel to the upper surface of the sample and N:\5_Laboi5_1_Au1traege\.5.1 htc_ ] 1 7.doc 1 _Iaufende_AuItraege\C_1 335_244_1 1 O7\Berichte\neuere_Versionen_Berich\IrZe_O9O429_Beric CIR Panel Book Page 162 GLP Study STP 44-

169 - All Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 32 von 71 Intelligent Drug Profiling thus provide information on the extent to which the test compound has penetrated into the dif ferent skin areas. Fig. 3: Saarbruecken model (SB) used for penetration studies. left: stripping of the Stratum corneum of the skin right: cryo sectioning of deeper skin layer After removing the residual formulation, the skin biopsies were transferred into a stripping appa ratus. Additionally to the SOP M 1 9 the skin sample may be placed onto the Styrofoam for the stripping procedure. The upper corneous layers (Stratum corneum) of the skin were stripped off using Tape film. The stripped skin area was cm2 ± 5 %. In total 2 tape strips were per formed per each skin biopsy (see Tab. 29). The first two strips are always considered separtately due to potential contaminations by residual drug on the surface of the skin. The samples for 2 tape-strips and 1 8 tape-strips were extracted in 3 ml and 5 ml of the extraction medium, re spectively and quantified by HPLC. Methanol/water (5:5, v/v %) was used as an extraction medium. After stripping, the skin biopsies were punched out and frozen at 8 C. The residual skin was cryo-sectioned to determine the amount of test compound, which had penetrated into the deeper skin layers. The skin biopsy was cut into surface parallel sections with a thickness of 25 pm. The surface area of a single cut was approximately cm2. All cuts were collected into one beaker. All samples were extracted in 3 ml of the extraction medium and quantified by HPLC. Methanol/water (5:5 v/v, %) was used as extraction medium. Tab. 29: Samples from the penetration study. Number of tape strips Sample no. Tesa Strip no. Cryosections (total of 2) Rest - cryocuts N:\5_Labor\51 Au[traege\5_1 _1 _!aufende_auftraege\c_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\lrZe_94 29_Beric ht_c1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 163

170 Across Barriers GmbH Intelligent Drug Profiling Seite 33 von 71 Fig. 4 and Fig. 5 give a schematic view of the stripping procedure and cryo-sectioning, respec tively. sample vials I skin 5 * 4 2 stripping apparatus stopwatch Fig. 4: Schematic view of tape stripping (thesis Heike Wagner). Fig. 5: Schematic view of cryo-sectioning (thesis Heike Wagner) MEA: Qualification of skin membranes The skin donors employed in this experiment was the same employed for the last study C , where the quality control for the skin samples were carried out. For this rea son it was not necessary to perform again the skin quality control experiments. The apparent permeability coefficients determined in the present study were compared to the Caffeine Papp measured using dermatomized skins from different donors. The Franz cells em- N :\5_Labor\51 _Auftraege\5_1 _1 _Iaulende_Auftraege\c_ _1 17 \Berichte\neuere_Versionen_Bericht\lrZe_94 29_Beric ht_c_1o335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 164

171 Across Barriers GmbH Seite 34 von 71 Intelligent Drug Profiling ployed for MEA were different in diffusion area and volume from the Franz cells from the per meations studies. These information are detailed in the report C-i Calculations In the calculations the individual values of Franz cells used in the experiments were considered. All calculations were performed using the program EXCEL 2, SP-2 from Microsoft Corpora tion, USA. The calculations were performed using the full number of digits handled by the EXCEL 2 pro gram. The results were rounded only for printing and for the presentation in the report. Apparent permeability coefficient (Papp) The apparent permeability coefficient (Papp) was calculated from the linear part of the slope of the cumulative transport of the substance. The lag phase was estimated out of the transport curves. The Papp was calculated according to Eq. 3. AQIz\t A m V P = VD [cms ]Eq.3 i At m A permeability rate (steady state transport rate) obtained from the profile of the trans ported amount of substrate versus time [s]. Calculated by the linear regression of time and concentration area of the exposed skin [cm 2] initial mass of test compound in the donor compartment [pg] or [dpm] donor volume [cm3] Mass recovery Results of experiments with test item are presented as a mass balance including the amount and percentage of the active compound permeated, penetrated and remaining in the donor compartment after the experiments. Based on the results of the three compartments a mass balance was determined according to Eq. 4 and 5. f 7RC = m4 + rnosc +lfl + lflpq + 1PDS {j.ig] Eq. 4 m m mass of test compound in the three compartments after the experiment [pg] mean value of the amount of test compound permeated through the tissue into the acceptor medium [pg] N :\5_Laboi5_1 _Auftraege\5..1 htcl 335_244_1 17.doc 1 _Iaulende_Auftraege\C1 335j \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 165 GLP Study STP 44-

172 Across Barriers GmbH Seite 35 von 71 Intelligent Drug Profiling m5 mean value of the amount of remaining test compound on the skin in the test formula tion after the expermient [pg] mosc mean value of the amount of test compound content in two first tape strips [jig] mpsc mean value of the amount of test compound penetrated into the Stratum corneum (1 8 tape strips) [jig] m5 mean value of the amount of test compound penetrated into the deeper skin layers [jig] RC=--1OO [%] 111 D RC total recovery of the active compound after the experiment [%] m m mass of test compound in the three compartments after the experiment [jig] initial mass of test compound in the donor compartment [jig] Eq.5 The overall mass recovery in the range of 1 ± 15 % should be reached according to SCCP. Dose absorbed Dose absorbed was calculated according to the Eq. 6. DA A PDS [%] Eq. 6 DA dose absorbed of test compound after the experiment [%] r recovery of test compound permeated through the tissue into the acceptor medium in relation to the applied formulation amount [%] r recovery of test compound penetrated into the deeper skin layers (cryosections) in rela tion to the applied formulation amount [%] N :\5_Labor\5_1 _Au1traege\51 _1 _Iautende_Auftraege\C_1 335_24ki 17 \Berichte\neuere_VersionenBericht\IrZe_9O4?9_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 166

173 Across Barriers GmbH Seite 36 von 71 Intelligent Drug Profiling 3. Results 3.1. HPLC method System suitability test Tab. 3 summarizes the results of the system suitability test. The test was performed by 6 injec tions from the same vial containing standard solution of CG in KRB-/- buffer. Tab. 3: Summarized results of system suitability test (SST). Sample Name lnj. No. Compound Retention time Area SST pg/ml SST 2 1 CG pg/ml SST 3 2 CG pg/ml SST 4 3 CG pg/ml SST 5 4 CG pg/ml SST 6 5 CG 5, pg/ml 6 CG Mean SD RSD% N:\5_Labor\5_1_Aultraege\.5_1 1 _Iaufende.Auftraege\C_1 335_244_1 1 O7\Berichte\neuere_Versonen_Bercht\IrZeO9O429_Berc ht_c_ doc GLP Study STP 44- CIR Panel Book Page 167

174 Across Barriers GmbH Intelligent Drug Profiling Seite 37 von 71 p I S Fig. 6: First SST chromatogram of CO Jg/mL standard solution in matrix. I 4 Fig. 7: Second SST chromatogram of CG pg/ml standard solution in matrix. N:\5_Labor\5_1 _Auftraege\5_1 _1 Jaufende_Aultraege\C_ _1 17 \Berichte\neuere_Versionen_Bericht\lrZe_O9429Beric ht_c cioc GLP Study STP 44- CIR Panel Book Page 168

175 - i-.-. Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 38 von Selectivity Figures compare the MS-chromatograms of the test compound in acceptor medium (KRB without Glucose and HEPES), in MeOH/Water (5:5 v/v, %) in the donor medium and the blank chromatograms (eluents). With exception of the acceptor medium (slight interferences) there were no interferences with the compound. I --- Fig. 7: Chromatogram of GC standard solution in KRB buffer (without Glucose and HEPES). The injec tion volume amounted to 2 jl. 4eooo t4qo- 4 Fig. 8: Blank-chromatogram of KRB 7.4 without Glucose and HEPES. N :\5_Labor\5_1 Auftraege\5_1.1 _IaulendeAuftraege\c_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric ht 1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 169

176 Across Barriers GmbH Intelligent Drug Profiling Seite 39 von 71 ll : I, IL1 L I 1V V Fig. 9: Blank-chromatogram of methanol / water 5:5. ix I =_ 4 ::: Fig. 1: Blank-chromatogram of eluent B (Methanol +.1% formic acid). N:\5Labor\5_1 Au[traege \51 _1 _Iaufende_Aultraege\C_1 335_244_1 17 \Berchte\neuere_Versionen_Bericht\lrZeO9O429_Beric ht_c_1 335_244_1 1 7.cloc GLP Study STP 44- CIR Panel Book Page 17

177 Across Barriers GmbH Seite 4 von 71 Intelligent Drug Profiling I\ I L\f \A\i Fig. 11: Blank-chromatogram of eluent A (Water dest. ±.1% formic acid). N:\5_Labor\5_1 _Aurtraege\5_1 1 _Iaulende_Auftraege\C_1 O335_244_ 17 \Berichte \neuere_versionen_bericht\irze_9429_beric htc_ doc GLP Study STP 44- CIR Panel Book Page 171

178 Across Barriers GmbH Seite 41 von 71 Intelligent Drug Profiling Linearity in KRB-I- ph 7.4 The linearity data for the established HPLC method in KRB-/- ph 7.4 are depicted in the follow ing table and figure. Tab. 31: Statistics of the calibration curve for CG KRB-/- buffer ph 7.4. Sample name Response accord- Theoretical c ing to internal c found Deviation* [pg.ml standard [ig.ml 1] [Area] 1 1%] KLP KLP KLP KLP , KLP KLP KLP KLP Slope second order Slope first order m Intercept b 1.234e6 Correlation coefficient r Determination coefficient r Weighting 1 1x2 * calculated by means of linear 2 regression. N:\5_Labor\5_1 _Auftraege\5...1 ht_c_9 335_ doc ] _Iaufende_Auftraege\C_1 O335_244_ ] 7\Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 172 GLP Study SW 44-

179 1 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 42 von 71 Sn,! r.nw1d 2-3 io y.ijii,h 4 I Fig. 1: Calibration curve CG in KRB-/- ph 7.4. The calibration curve is quadratic in the range pg ml 1. The deviation of the concentration found from the nominal concentration is within the limit (2 % for LLOQ and 1 5 % for other concentrations) Accuracy and Precision The results from determination of accuracy and precision of CG in KRB-/- buffer are presented in the tables below: Tab. 32: Accuracy and precision of the analytical method: CG in KRB without HEPES and glucose ph 7.4. Concentration level CON1 (high) CON2 (mean) CON3 (low) ni IpgmL n2 [pg.ml 1] n3 [pg.ml 1] ,1.71 Mean IpgmLl SD [pq.ml 1] RSD [%J Theoretical concentration [ig.ml 1] Accuracy [%] N :\5Labor\5_1 Auftraege\5_ l ht_c_1 335_244_1 1 7.doc 1 _Iaufende_Au[traege\C_1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 173 GLP Study STP 44-

180 Across Barriers GmbH Seite 43 von 71 Intelligent Drug Profiling The values for precision and accuracy have complied the limits established in the FDA Guideline (RSD ± 15%forCONl -CN3andaccuracyloo ± 15%). Quantification limit The limit of quantification for CG with a signal to noise ratio (SIN) = 5:1 amounts to.88 pg. ml 1 in KRB ph 7.4 without HEPES and glucose for the validated HPLC method. The accuracy at the LLOQ concentration complies the limit of 1 ± 2 % (86.59 %) and precision RSD ± 2% (8.58 %) Stability of the test product in donor and acceptor media The stability of CG was tested over a period of 48 hours and measured at 3 time points (after, 24 and 48 hours) as presented in the tables below: Tab. 33: Stability of CG in KRB buffer ( pg ml 1) without glucose and HEPES at hour. Medium KRB-/- buffer Storage time Lhl Storage temperature 4 C RT 32 C ni IpgmL n2 IpgmL n3 g.ml- 1 2, Mean [pgml 1J SD [pgml 1J RSD I% Theoretical c [igml 1J Recovery (related to theoretical c)_[/o] N :\5_Labor51 _Auftraege\5_1 ht_c1 335_244j 1 7.doc l _Iaufende_Auftraege\c_1 335_244_1 1 7 \Berichte\neuere_VersionenBericht\IrZeO9429_Beric CIR Panel Book Page 174 GLP Study STP 44-

181 Across Barriers GmbH Seite 44 von 71 Intelligent Drug Profiling Tab, 34: Stability of CC in KRB buffer without HEPES and glucose over 24 hours. Medium KRB-/ Storage time [h] 24 Storage temperature 4 C RI 32 C ni [pg ml l J n2 [ig.ml 1J n3[pg.ml Mean [pg.ml 1] SD [pgml 1J RSDI% Initial c IpgmL 1 Recovery (related to initial c) L% Tab. 35: Stability of CC in KRB buffer without HEPES and glucose over 48 hours. Medium KRB-/ Storage time [hj 48 Storage temperature 4 C RI 32 C 1] ni [ig.ml n2 [pg ml 1 1J n3 [pg.ml Mean [pgml 1J SD [pg.ml 1] , , ,5 RSD[%] Initial c IpgmL 1J Recovery (related to initial c) I% N:\5_Labor5_1 _Aultraege\5_1 _1 _Iaufende_Auftraege\C..i j 17 \Berichte\neuere_Versionen_Bericht\IrZe9429_Beric ht_c_ _il 7.doc GLP Study STP 44- CIR Panel Book Page 175

182 Across Barriers GmbH Seite 45 von 71 Intelligent Drug Profiling Tab, 36: Stability of CG in HBSS buffer without glucose at hour. Medium HBSS- buffer Storage time [hi Storage temperature 4 C RT 32 C 1i ni [ig.ml 1J n2 [pg.ml 1] n3 [pg.ml Mean [ig.ml 1i SD [pgml 1i RSD [%] ] Theoretical c [pgml Recovery (related to theoretical c) [%] Tab. 37: Stability of CO in HBSS- buffer without glucose over 24 hours. Medium HBSS- buffer Storage time [hi 24 Storage temperature 4 C RT 32 C ni [ljg.ml 1J 1 n2 IigmL 1] n3 [pg.ml , Mean IpgmL i SD [pg.ml 1i RSD 1%i Initial c IpgmL 1i Recovery (related to initial c) [%j.. N:\5_Labor\5_1_Aultraege\5_1.J1 _Iaulende_Auttraege\C_1 O335_ O7\Berichte\neuere_Versionen_Bercht\IrZe_O9D429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 176

183 Across Barriers GmbH Seite 46 von 71 Intelligent Drug Profiling Tab. 38: Stability of CG in HBSS buffer without glucose over 48 hours. Medium HBSS Storage time IhI 48 Storage temperature 4 C RT 32 C 1J ni [pgml n2 [pgml 1j 1] n3 [pg ml. 1 Mean [pg.ml SD [pg ml 1l ,26 RSDI% Initial c IpgmL 1I Recovery (related to initial c) [%J The results demonstrate, that the test compound was stable over a period of 48 h at 32 C, room temperature and at 4 C in all tested media. N:\5_Labor\5_1 _Aultraege\.5,,1 ht_c_1 335_244_1 17.doc 1 _Iaufende_Auftraege\C_1 335_244j 17 \Berichte\neuereVersionen_Bericht\IrZe_9429_Benc CIR Panel Book Page 177 GLP Study STP 44.

184 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 47 von 71 Intelligent Drug Profiling 3.2. Determination of CG content in the test solution The results of the determination of the CG content in the test solution are summarized in the following table. Tab. 39: Summary of CG content in the test solution, amount of 2 p1 test solution 1 % CG (n=3), Sample Weighted Theoretical Theoretical Measured Recovery [%J amount 1mg] amount lmgiml] amount after amount [ig!ml] dilution [pg/mi] n= n= n= , Mean - SD The mean recovery was within the limits of 1 ± 1 %. Therefore the extraction procedure of CG from the test solution (1% CG) was successfully established. Tab. 4: OlD content in the test solution after skin contact, amount of 3 l test solution 1 % CG Sample Weighted amount Theoretical amount Measured amount Recovery 1%] [mgi Ipglmi] EigImi1 n= The recovery of CG after application of 3 pl test solution 1 % CG was lower than in samples without any skin contact, but also within the limit of 1 ± 1 % Permeation study and penetration studies Samples from in vitro experiments with the test item In the Tab. 41 a short explanation of the samples further discussed in the report is depicted. N :\5_Laboi5_1 _Au[traege\5..i ht.c_1 335_244_1 1 7.doc 1 _laulende_auftraege\c_1 335_244_1 17 \Berichte\neuere_Versionen..Bericht\IrZe_9429.Beric CIR Panel Book Page 178 GLP Study STP 44-

185 . test 2: Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Intelligent Drug Profiling Seite 48 von 71 Tab. 41: Samples from the in vitro experiments with the test item. Sample name dose applied receptor skin wash Description amount of the test compound applied at the skin surface cumulative test compound content in the receptor medium after the permeation experiment compound content remaining on the skin surface at the end of the permea tion experiment (formulation) 2 Tape strips test compound content in the two first tape strips (Stratum corneum) 1 8 Tape strips* test compound content in the 18 further tape strips (Stratum corneum) Cryocuts* test compound content in the deeper skin (viable epidermis ± dermis) dose absorbed sum of the test compound content in the receptor medium and in the deeper skin total recovery mass recovery of the test compound content * Tape strip no. 3 epidermis, parts of dermis). the first third of stratum corneum; Cryocuts: residual skin (rest stratum corneum, Establishment of extraction method for penetration study To develop the extraction method for the in vitro penetration experiments, two different extra c tion media were tested: water and MeOH!water (5:5 vlv, %). The experiments were performed under non-glp conditions. The table below presents an explanation from the denomination of the samples investigated. Tab. 42: Denomination of samples employed in the extraction method experiments. Sample name Description 2 2 tape-strips without skin tape-strips without skin 2/SC 2 tape-strips with stratum corneum 1 8/SC 18 tape-strips with skin stratum corneum Rest deeper skin (deeper epidermis ± dermis) Recovery of CG with water as extraction medium The Fig. 11 presents the recovery values of CG at two different concentrations: 3.62.tg/mL and.89 ig/ml after the simulated extraction procedure with water as extraction medium. N :\5_Labor\51 _Auftraege\5_1 ht_c_1 335_244_1 7.doc 1 _Iaufende_Auftraege\c1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 179 GLP Study STP 44-

186 Across Barriers GmbH Intelligent Drug Profiling Seite 49 von l3,622 [pg/ml] D.897 [pg/ml] /Sc 18/Sc Rest Number of strips I skin fraction Fig. 11: Recovery of CG after the simulated extraction procedure with water as extraction medium. The values shown are the arithmetic mean values ± SD (n=2). With water as extraction medium the mean recovery values of CG have shown a variation from % to % at the higher concentration (3.62 ig!ml) whereas the samples at lower concentration (.89.tglmL) the mean recovery valued have varied from %to %. The lowest recovery values were found in the samples with 1 8 tape-strips without skin either at higher or lower concentration. Recovery of CG with MeOHlwater (5:5 v/v. %) as extraction medium The figure below presents the recovery values of CG at concentration.928 ig/ml and ig/ml after the simulated extraction procedure with MeOH/water (5:5 v/v, %). N :\5_Labor\5_1 _Aultraege \5_1 _1 Jaufende_Aultraege\C_ _1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric ht_c_ doc GLP Study STP 44- CIR Panel Book Page 18

187 Across Barriers GmbH Intelligent Drug Profiling Seite 5 von I.. C) [pg/ml] D.928 [pg/ml] /sc 18/Se Rest Number of strips I skin fraction Fig. 12: Recovery of CG after the simulated extraction procedure with MeOH/water (5:5 v/v, %) as extraction medium. The values shown are the arithmetic mean values ± SD (n=2). With MeG H/water (5:5 vlv, %) as extraction medium the mean recovery values of CG have shown a variation from % to 93.7 % for the samples higher concentrated (3.643 ig!ml), whereas the samples at.928 tg!ml have demonstrated recovery mean values from 94.4 % to %. These results have complied to the specified in the SCCP Guide line (1 ±2 %), therefore MeOH/water (5:5 v/v %) was the selected extraction medium for performing the penetration experiments Results from permeation and penetration studies Cumulative transport of CG from test product through skin no , , The CG amounts in the receptor over an incubation period of 24 hours were below of the LLOQ (.88 tg/ml) of the analytical method for the 3 skin donors (The area of he peaks found were similar to the negative control (pure acceptor medium)). This low absorption is confirmed by the recovery values found for the test solution, which remained on the skin surface (skin wash sam N:\5_Labor\5_1 _Aultraege\5_1.1 jaufende_au[traege\c_ \Berichte\neuere_Versionen_Bericht\lrZe_9429_Beric ht_c1 335_ doc GLP Study STP 44- CIR Panel Book Page 181

188 Across Barriers GmbH Intelligent Drug Profiling Seite 51 von 71 pies), which was % (mean of all 6 Franz-cells). This demonstrates that the CG has mostly remained on the skin surface for all the samples. The results of the penetration study of CG from the test solution 1 % CG through skin no are presented in the lab. 43. Tab. 43: Amount [pg cm2] of CG in the samples from the in vitro experiment using skin no Cumulative amount [pg cm2] of CG in the samples Sample FZ12 FZ5 Mean SD Diffusion area [cm2] dose applied receptor..*.*. skin wash Tape strips Tape strips cryocuts.*,*.*. * Values under the LLOQ of the analytical method. The mass recovery of the test compound in the analysed samples is shown in Tab. 44. Tab. 44: Recovery and dose absorbed [%] of CG in the samples from the in vitro experiment using skin no , Recovery and dose absorbed 1%] of CG in the samples from the in vitro experiment using skin no Sample FZ12 FZ5 Mean SD receptor.*.*.* skin wash Tape strips Tape strips cryocuts.*.*,*. Dose absorbed.*.*.*.* Total recovery * Values under the LLOQ of the analytical method The mean total mass recovery of CG after the in vitro experiment using skin no sum up to %. After the 24-hours incubation no quantifiable amount of CG has been found in the receptor medium and cryocuts. The results of the penetration study of CG from the test solution 1 % CG through skin no are presented in the Tab. 45. N:\5_Labor\5_1 AuFtraege\5_1 ht_c_1 335_244_1 17.doc 1 _Iaufende_Auftraege\C_1 335_244_1 1 7 \Berichte\neuere_versionen_Bericht\lrZe_9429_Beric CIR Panel Book Page 182 GLP Study STP 44-

189 Across Barriers GmbH Intelligent Drug Profiling Seite 52 von 71 Tab. 45: Amount [pg cm2] 98, of CG in the samples from the in vitro experiment using skin no Cumulative amount Ipg cm2] of CG in the samples Sample FZ 129 FZ 133 Mean SD Diffusion area [cm 2] dose applied , receptor.* Q,QQ*.*. skin wash Tape strips Tape strips ,19 cryocuts * Values below the LLOQ of the analytical method. The mass recovery of the test compound in the analyzed samples is shown in the table below. Tab. 46: Recovery and dose absorbed [%] of CG in the samples from the in vitro experiment using skin no , Recovery and dose absorbed [%1 of CG in the samples from the in vitro experiment using skin no Sample FZ 129 FZ 133 Mean SD receptor.*.*.*. skin surface , Tape strips Tape strips cryocuts Dose absorbed Total recovery * Values below the LLOQ of the analytical method. The mean mass recovery of CG after the in vitro experiment using skin no sum up to %. After the 24-hours incubation.4 % of CG have been absorbed. The results from the in vitro penetration experiment across skin are depicted in the table below. N:\5.*abor\5_1 _Au[traege\5_ l _1 _Iaulende_Auftraege\C_1 335_244_1 17 \Beiichte\neuereVersionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 183

190 Across Barriers GmbH Intelligent Drug Profiling Seite 53 von 71 Tab. 47: Amount [pg cm2] 98. of CG in the samples from the in vitro experiment using skin no Cumulative amount [pg. cm21 of CG in the samples Sample FZ 125 FZ 14 Mean SD Diffusion area [cm2] Dose applied , receptor.*,*.*. skin wash , lapestrips Tape strips , cryocuts.*.*,*. * Values below the LLOQ of the analytical method. The mass recovery of the test compound in the analyzed samples is shown in table below. Tab. 48: Recovery and dose absorbed [%] of CG in the samples from the in vitro experiment using skin no Recovery and dose absorbed 1%] of CG in the samples from the in vitro experiment using skin no Sample FZ 125 FZ 14 Mean SD receptor,*,*.*. skin surface Tape strips Tape strips cryocuts.*.*.*. Dose absorbed.*.*.*. Total recovery * Values below the LLOQ of the analytical method. The mean mass recovery of CG after the in vitro experiment using skin no sum up to %. After the 24-hours incubation no quantifiable amount of CG has been found in the receptor medium and in the deeper skin layers. N:\5_Labor5_1 _Auftraege\5_1...1 _Iaufende_Au[traege\C_1 335_244_1 17 \Berichte\neuere_versionen_Bericht\IrZe_9429_Beric ht_c_ _1 17.doc GLP Study STP 44- CIR Panel Book Page 184

191 Across Barriers GmbH Intelligent Drug Profiling Seite 54 von Quality control of the utilized skin samples (MEA) The mean apparent permeability coefficients (Papp) measured for the Caffeine reference marker was for skin no in mean 2.39 ± in mean 5.67 ± cms 1 1 Q.8 cm s 1 (n=2), for skin no (n=2) and for skin no in mean 6.38 ±.53 1 cms (n=2). The Papp was calculated under steady state conditions from.5 to 24 hours. The results of the Caffeine transport studies through all three dermatomized human skins are shown figure below. 1, 9. 8, 7, - E b a. 6, - 5, - 4, - 3, 2, 1, O,i - Skin Skin Skin Skin number Fig. 13: Mean apparent permeability coefficients for the transport of Caffeine through human skin no , and The values shown are the arithmetic mean values ± SD (n =2). In an earlier study, the permeability of Caffeine from a 1 % aqueous solution across fullthickness skin, dermatomized skin, heat-separated epidermis and isolated SC had been studied at Across Barriers to establish quality assurance benchmarks for skin integrity [Bock et al., 22]. Subsequent quality control studies have been performed on a range of human skin sam N:\5_Labor\5_1 _Aultraege \5_] _1 _Iau[ende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_VersionenBericht\lrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 185

192 Across Barriers GmbH Intelligent Drug Profiling Seite 55 von 71 pies. The table below provides an overview of Caffeine permeabilities through dermatomized skin specimens measured at Across Barriers and includes the study. values determined in the present Tab. 49: Comparison of apparent permeability coefficients for Caffeine through different dermatomized skin specimens with intact SC. Skin no , and were used in the present study. Skin number Mean Papplcms (n=3) RSD 1%] 1] E E E E E E-D ,82E ,23E E E OOE E8* EO8* 3 *n EO8* 8 The mean Papp values measured for Caffeine in the present study are in good agreement with values determined for a variety of skin specimens from different donors under comparable conditions. This demonstrates the integrity of the skin biopsies used and their suitability for use in transport studies with the test formulation. N:\5.Labor\5_1 Au[traege\5_1 ht_c_1 335_244_1 1 7.doc 9 _Iautende_Au[traege\C _244_1 17 \Berichte\neuere_versionen_Berich\lrZe_9429_Beric CIR Panel Book Page 186 GLP Study STP 44-

193 Across Barriers GmbH Seite 56 von 71 Intelligent Drug Profiling 4. Discussion 4.1. HPLC method development and validation The HPLC method was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRB-/- buffer). There were minor but acceptable interferences with the acceptor medium (KRB-!-). In addition, CG has presented good stability in the acceptor and donor media at 3 different temperatures (4 C, RT and 32 C) over the period of 48 hours Determination of CG content in the test solution The content of CG was determined in triplicate in the test solution without skin contact employed in the experiments. The mean concentration value was.97 ig/ml which corresponds to the recovery mean value of ± The content of the test solution after the skin contact was.91 jiglml, corresponding to 9.1 %. This complies with the acceptance value 1 ± 1% Establishment of the extraction method for penetration study Two extraction media were investigated for the penetration studies: MeOH/water (5:5 v/v, %) and water. With water as extraction medium the mean recovery values of CG has shown a varia tion from % to % (considering all the samples) whereas with MeOH/water (5:5 v/v, %) the mean recovery values of CG have ranged from % to % (considering all the samples). The results demonstrated that MeOH/water (5:5 v/v, %) was the most suitable extraction medium for the penetration experiments, since the recovery values have complied to the limit of 1 ± 2 % specified in the SCCP Guideline Permeation study The results from the in vitro permeation of CG from the test product through skin no , and have demonstrated that the test compound permea tion after 24 hours was below the LLOQ of the analytical method (.88 j.iglml) (The area of he peaks found were similar to the negative control (pure acceptor medium)). Another fact which support this low permeation are the recovery values found for the test product which remained on the skin surface. These values have varied in the 6 Franz-cells from 19.2 % to %, which demonstrated that the CG has mostly remained on the skin surface for all the samples. N :\5_Labor5_1 _Auftraege\5_1 htc_1 335_244_1 17.doc 1 _Iaufende_Aultraege\C_1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 187 GLP Study STP 44-

194 Across Barriers GmbH Intelligent Drug Profiling Seite 57 von Penetration study The mean amounts of CG found in the samples from the three in vitro transport experiments are summarized in the table below. Tab. 5: Mean amount [pg cm- 2] of CG in the samples from the three in vitro experiments. Cumulative amount [pg cm2] of CG in the samples Sample Skin Skin Skin All skins used Mean SD Mean SD Mean SD Mean SD dose ap plied , receptor.*..*..*..*. skin wash , Tape strips Tape strips cryocuts.*.* *.* * Values below the LLOQ of the analytical method. Tab. 51: Mean recovery rate and dose absorbed [%] of CG in the samples from the three in vitro experi ments. Recovery and dose absorbed [%] of CG in the samples Sample Skin Skin Skin All skins used Mean SD Mean SD Mean SD Mean SD receptor.*..*. Q QQ*..*. skin surface Tape strips Tape strips cryocuts.*..4.4,*..1.3 Dose absorbed.*..4.4.*..1.3 Total recovery * Values below the LLOQ of the analytical method. The mean amount of CG removed from the skin surface (skin wash) ranged from % to % of the dose applied in the mean values of the 3 skin donors. This demonstrates that the CG has mostly remained on the skin surface. The amounts in the receptor could not be N :\5_Labor\5_1 _Auftraege \5.1 _1 _Iaulende_Aultraege\C_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 1].doc GLP Study STP 44- CIR Panel Book Page 188

195 Across Barriers GmbH Seite 58 von 71 Intelligent Drug Profiling quantified for the skin donors and since it was below the analytical LLOQ. The recovery value of the dose absorbed for the skin was accounted to.4 %. Together with skin no (comparable P values) skin sample no also exhibited the highest permeation rates for caffeine. The mean recovery in the two first tape strips was.52 % during all performed experiments. In the further 1 8 tape strips a mean recovery of.3 % was documented. The recovery values for the cryocuts have accounted.4 % only for the skin For the other skin donors CG was not detectable in the samples with cryocuts (below the analytical LLOQ). The mean absorbed dose of CG, sum of the amounts found in the viable epidermis, dermis and receptor medium was.] %, which has accounted only the dose absorbed in the skin The calculated total recovery rate of CG for the three different skin donors used was %. The mean recovery values have varied from % until %, which does not comply to the aspired acceptance criteria of 1 ± 1 5 %. The reason for this could be an interference of skin components that liberated during the permeation experiment and disturb the measure ment or the adding up of the deviations of the single analytical validation steps and of the in vitro experiment itself Quality control of the utilized skin (MEA) The transport rates of caffeine demonstrate that the utilized human skins no , and represent intact tight barrier properties, which are congruent to data obtained on other human skin biopsies under comparable study design. N :\5_Labor\5_1 _Auftraege\5_1 ht_c_1 335_244_1 17.doc 1 jau1endejuftraege\c_1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric CIR Panel Book Page 189 GL.P Study STP 44-CO

196 Across Barriers GmbH Seite 59 von 71 Intelligent Drug Profiling 5. References Bock, U., Schmitz, S. and Haltner, E.: In vitro systems to characterize dermal permeation and penetration, in: The essential Stratum corneum edited by Ronald Marks, Jean-Luc [eve que and Rainer Voegeli Martin Dunitz 22, page Kuhn, A. and Neubert, R.: Characterization of mixtures of alkyl polyglycosides (Plantacare) by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrome try, in: Pharmaceutical research, Vol. 21, No. 12, 24, page Wagner, Heike: Charakterisierung des Arzneistofftransportes in Humanhaut unter in-vitro und in vivo Bedingungen unter Berucksichtigung des Einflusses zweier in-vitro Testsysteme, Uni versität des Saarlandes Related guidelines Organization for Economic Co-operation and Development (OECD), OECD Guideline for the test ing of chemicals 428 (24): Skin Absorption: in vitro Method. Organization for Economic Co-operation and Development (OECD), Guidance Document for the conduct of skin absorption studies No. 28, 24. European Commission, The SCCPs (Scientific Committee on Cosmetic Products) Notes of Guid ance for the testing of cosmetic ingredients and their safety evaluation. Adopted at 1th plenary meeting of 1 9 December 26. European Commission, The Scientific Committee on Cosmetic Products (SCCP), Opinion on Ba sis Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients- up dated on March 26. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM): Guidance for Industry: Bioanalytical Method Validation (May 21) N:\5_Labor\5_LAuftraege\5_1 _1 _Iau[ende_Auftraege\c_1 335_244_1 1 O7\Berichte\neuere_versionenBericht\IrZe_O9O429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 19

197 Across Barriers GmbH Seite 6 von 71 Intelligent Drug Profiling Data storage and archiving All documents and raw data will be archived in the project file C-i in print ver sion and in the GLP archive of Across Barriers for 1 5 years. Archived documents at Across Barriers: German study plan and English Translation (md. Amendments) Relevant correspondence between sponsor and Across Barriers Cover sheets of the records of all inspections performed by the QA during the study Exact copy of relevant lab journal pages Method parameters for HPLC Raw data Data calculation and evaluation GLP study report The test product will be archived until expiration date at Across Barriers. N:\5Labor 5_1 _Auftraege\5_1 j _Iaufende_Aultraege\c_1 335_244_1 1 7 \Berichte\neuere.Versionen_Bericht\IrZe_9429..Beric ht_c_1 335_244.i 17.doc GLP Study STP 44- CIR Panel Book Page 191

198 Distributed for Comment Only -- Do Not Cite or Quote Across Barriers GmbH Seite 61 von 71 Intelligent Drug Profiling 7. Time Line Tab. 52: Time line of the study. Date planned Topic Responsibility HPLC method transfer (non GLP) ACB Establisment of the extraction.28 method (non GLP) ACB Validation of analytical method ACB Assay, drug content in test solution ACB In vitro experiments: permeability, CG content in the formulation, CO content in Stra- ACB turn corneurn, CO content in the deeper skin Multiple endpoint analysis (MEA) (see study C ) ACB Studyaudit ACB Draft Report ACB Final Report ACB I Sponsor N:\5_Labor\5_1_Auftraege\5_1 _1 jaufende_aultraege\c_1 335_244_1 1 7\Berichte\neuere_Versionen_Bericht\IrZe_9429Beric htc_1 335_ doc GLP Study STP 44- CIR Panel Book Page 192

199 Across Barriers GmbH Seite 62 von 71 Intelligent Drug Profiling 8. QAU Statement Study Number: STP 44/ Project Numer: C Study Title: In vitro permeation and penetration of Capryl glucoside from product Plantacare 81 UP at human skin under GLP conditions c) The conduct of this study has been inspected and this report has been audited by the Quality Assurance Unit. The dates of inspection are given below. Date of Study Plan Inspection Date of Report to Management Date of Study Inspection Date of Report to Management Date of Final Report Inspection Date of Report to Management The results reported C) flect the raw data. in this study have been checked on the basis of our current SOPs and re Quality Assurance Unit Date TawfikJalal Head of Quality Assurance and Organization 11 n n - J? C) 9O429_FinaIreport_cjo335_24411O7STP_O44.doc CIR Panel Book Page 193 GLP Study STP 44-

200 Across Barriers GmbH Intelligent Drug Profiling Seite 63 von Appendix 9.1. HPLC method Fig. 14: Chromatogram of CG standard solution in KRB buffer (without glucose and HEPES). The injection volume amounted to 2 pl. Fig. 15: Chomatogram of the acceptor matrix (KRB-/-) during permeation study. N :\5_Labor5i Auftraege\5_1 ht_c_1335_244_1 1].doc 1 _Iaulende_Aultraege\C_1 335_244_1 17 \Berichte\neuere_VersionenBericht\IrZe_O9429_Beric CIR Panel Book Page 194 GLP Study STP 44-

201 Across Barriers GmbH Intelligent Drug Profiling Seite 64 von 71 - Fig. 16: Representative permeation chromatogram of the acceptor sample, transport of CG after.5 hours (skin no ). V I ) z Fig. 17: Representative chromatogram of CG sample (skin wash) after the penetration study. N :\5_Labor\51 _Auftraege\5_1 ht_c_1 335_244_1 17.doc 1 _Iaulende_Auftraege\C.1 335_244_1 1 7 \Berichte\neuere_Versionen_Bencht\IrZe_94 29Beric CIR Panel Book Page 195 GLP Study STP 44-

202 Across Barriers GmbH Seite 65 von 71 Intelligent Drug Profiling j j -_ 4 Fig. 18: Representative chromatogram of CG sample extracted with methanol / water (5:5) from the Stratum Corneum 2 TS/ penetration study. F ft S Fig. 19: Representative chromatogram of CG sample extracted with methanol / water (5:5) from the Stratum Corneum 1 8 TS/ penetration study. N:\5_Labor\5_1 _Auftraege\5_1.1 _Iaufende_Auftraege\C_1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe Beric ht.c_1o doc GLP Study STP 44- CIR Panel Book Page 196

203 Across Barriers GmbH Seite 66 von 71 Intelligent Drug Profiling \\\j _11-7 Fig. 2: Representative chromatogram of CG sample extracted with methanol / water (5:5) from the deeper skin layers (cryo-cuts/ penetration study). N :\5_Labor\5_1 Auftraege\5 1 _Iaulende_Auftraege\tLl O \Beriche\neuere_Versionen_Bericht\IrZe_O9O429_Beric ht_cj 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 197

204 Across Barriers GmbH Seite 67 von 71 Intelligent Drug Profiling 9.2. Establishment of the extraction method The following table shows which samples were pooled and extracted together for the establish ment of the extraction medium. Sample schedule Sample name p_ Description Transport solution (Reference solution) 2 2 Tape strips without Stratum corneum Tape strips without Stratum corneum 2ISC 18!SC Rest 2 Tape strips with Stratum corneum 18 Tape strips with Stratum corneum Deeper Skin (Cryo-cuts) Establishment of the extraction method with MeOHlwater (5:5, vlv %) Recovery of CO (3.643 pqlml) with MeOHlwater (5:5, viv %) n=1 n=2 MW(n=2) Sample ro 1 SD L%1 pglml % pglml % 1 i , C ISC Rest , Recovery of CG (.928.igImL) with MeOHIwater (5:5, vlv %) n=1 n=2 MW(n=2) Sample SD 1%] igiml % pglml [ J !SC 1, !SC , Rest N:\5_Labor\5.i Auftraege\5_] _1 _IaufendeAuftraege\C_1 335_244_1 1 7\Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 198

205 Across Barriers GmbH Seite 68 von 71 Intelligent Drug Profiling Establishment of the extraction method with water Recovery of CG (3.622 pglml) with water n=1 n=2 MW(n=2) Sample SD [%] pglml % pglml , ISC C Rest Recovery of CG (.897 pglml) with water n=1 n=2 MW(n=2) Sample pglml % pg!ml SD [% ISC C Rest N:\5_Laboi\5_1 _Aultraege\.5_1 _1 _Iaufende_Auftraege\C_1 335_244_1 17 \Berichte\neuere_Versionen_Bericht\1r1e9429_Beric ht_c_1335_ doc GLP Study STP 44- CIR Panel Book Page 199

206 Across Barriers GmbH Intelligent Drug Profiling Seite 69 von Franz cell volumes and areas Franz cell no. Acceptor volume [ml] Exposed area [cm 2] Thickness of the human skins FZ Measured thickness [pm] of skin no Mean SD RSD [%] FZ Measured thickness [pm] of skin no Mean SD RSD [%J , FZ Measured thickness [pm] of skin no Mean SD RSD [%] N :\5_La bor5_1 _Auftraege\5.1 _1 _Iaufende_Auftraege\C_1 335_244_1 1 7 \Berichte\neuere_Versionen_Bericht\IrZe_9429_Beric htc_1 335_244_1 1 7.doc GLP Study STP 44- CIR Panel Book Page 2

207 Across Barriers GmbH Seite 7 von 71 Intelligent Drug Profiling 9.4. Quality control of the human skin (MEA) Transport of Caffeine (1%) through skin Time [hi Concentration Ipg ml 1l of Caffeine in the acceptor during transport through 3 skin donors All skins Mean SD Mean SD Mean SD Mean SD.5, , Cumulative transport [pg.cm 2] of Caffeine through through 3 skin donors Time [hi All skins Mean SD Mean SD Mean SD Mean SD E- 8.58E- 5.67E- 1.9E- 6.38E- 5.3E- 4.81E- 4.99E- Papp [1 cms N :\5Labor\5_1..Au1traege \5...1 _1 _iaufende_auftraege\c_1 335_244_1 17 \Berichte\neuere,,Versionen_Bericht\IrZe_9429_Beric ht_c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 21

208 Across Barriers GmbH Seite 71 von 71 Intelligent Drug Profiling 9.5. Transport studies with the test formulation (permeation results) Transport of CG from test solution through 3 different skin donors into the re ceptor medium Concentration [jig.ml ] of CG after transport from the test solution through 3 skin donors Time [hi FZ 5 FZ 12 FZ 129 FZ 133 FZ 125 FZ 14 5 * * * * * * 2 QQ* * * * * O 4 QQ* * QQ* * * * 6 * * * * * * 21 * * * QQ* * * 22 * * * * * * 23 * * * O * * 24..*.*.*.*,*.* * Values below LLOQ N:\5_Labor5_ LAuftraege\.5..1 _1 _Iaulende_Auftraege\C_1 335_244_1 1 7\Berichte\neuere_Versionen_Berichi\lrZe_9429_Beric ht.c_1 335_244_1 17.doc GLP Study STP 44- CIR Panel Book Page 22