GREEN. Microbial Polysaccharides

Transcription

1 GREEN Microbial Polysaccharides CIR EXPERT PANEL MEETING JUNE 11-12, 2012

2 Memorandum To: CIR Expert Panel Members and Liaisons From: Monice M. Fiume MMF Senior Scientific Analyst/Writer Date: June 11, 2012 Subject: Draft Report on the Safety Assessment of Microbial Polysaccharides as Used in Cosmetics Enclosed is the Draft Report on the Safety Assessment of Microbial Polysaccharides as Used in Cosmetics. This is the first time the Panel is seeing this document on these 34 ingredients. The Scientific Literature Review was issued on November 29, Many studies have been conducted with some of the microbial polysaccharides in regard to health claims, immunomodulatory activity, anti-oxidant activity, etc. This safety assessment only includes studies and study-types that relate directly to the safety of the cosmetic use of these ingredients. Quite a bit of published data were available. Additionally, the unpublished data listed below were received and incorporated into the report. These data are included in the data tab of this document: 1. Safety Data Sheet: Xanthan Gum. (Aug 29, 2011) a. Jungbunzlauer Safety data sheet: Xanthan Gum b. Green R Letter from Biopolymer International to the European Commission concerning molecular weight of polymer additives 2. Concentration of Use by FDA Product Category: Microbial Polysaccharides (Oct 26, 2011) 3. Concentration of Us by FDA Product Category: Xanthan Hydroxypropyltrimonium Chloride (Mar 28, 2012) 4. Specifications and Safety data on Sodium Carboxymethyl Beta-Glucan (Aug 22, 2011) a. Mibelle Biochemistry CM-Glucan granulate specifications. b. Klecak J Summary report CM-Glucan (Sodium Carboxymethyl Betaglucan) in skin care products. RCC Project c. Therapy and Performance Research Institute (GTLF) Repeated patch test for skin sensitization and photoallergy of CM-Glucan (700-01) in healthy male and female volunteers. d. Notox BV Evaluation of the mutagenic activity of CM-Glucan in the Salmonella typhimurium reverse mutation assay (with independent repeat). Notox Project e. Life Science Laboratory Primary eye irritation study of CM-Glucan J in rabbits. Test Code No. 99-IXB f. Life Science Laboratory Primary skin irritation study of CM-Glucan J in rabbits. Test Code No. 99-IXA g. Life Science Laboratory Primary skin irritation test for CM-Glucan J in human subjects by closed patch test. Test Code No. 99-XII h. Life Science Laboratory Reverse mutation study of CM-Glucan J in bacteria. Test Code No. 99-VII i. Life Science Laboratory Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method). Test Code No. 99-VIA

3 j. Life Science Laboratory Single dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg). Test Code No. 99-IA k. Eurofins ATS Ocular tolerance test according to HET CAM method: Sodium Carboxymethyl Beta-Glucan. The data profile should be useful in keeping track of the types of data found in the report. If there are no additional data needs, the Panel should be prepared to formulate a tentative conclusion, with the rationale provided for the Discussion, and issue a Tentative Report for public comment. If the data are not sufficient for making a determination of safety, then an Insufficient Data Announcement should be issued, listing the additional data that are needed.

4 Option for Re-review Document for Panel Review **lf Draft Amended Report (DAR) is available, the Panel may choose to review; if not, CIR staff prepares DAR for Panel Review. Expert Panel Decision *The CIR Staff notifies of the public of the decision not to re-open the report and prepares a draft statement for review by the Panel. After Panel review, the statement Is Issued to the Public. SAFETY ASSESSMENT FLOW CHART Distributed for Comment Only -- Do Not Cite or Quote CIR Panel Book Page 1

5 MICROBIAL POLYSACCHARIDES REPORT HISTORY November 29, 2011: Scientific Literature Review The following unpublished data were received and incorporated in the SLR: 1. Safety Data Sheet: Xanthan Gum a. Jungbunzlauer Safety data sheet: Xanthan Gum b. Green R Letter from Biopolymer International to the European Commission concerning molecular weight of polymer additives 2. Concentration of Use by FDA Product Category: Microbial Polysaccharides 3. Specifications and Safety data on Sodium Carboxymethyl Beta-Glucan (Aug 22, 2011) a. Mibelle Biochemistry CM-Glucan granulate specifications. b. Klecak J Summary report CM-Glucan (Sodium Carboxymethyl Betaglucan) in skin care products. RCC Project c. Therapy and Performance Research Institute (GTLF) Repeated patch test for skin sensitization and photoallergy of CM-Glucan (700-01) in healthy male and female volunteers. d. Notox BV Evaluation of the mutagenic activity of CM-Glucan in the Salmonella typhimurium reverse mutation assay (with independent repeat). Notox Project e. Life Science Laboratory Primary eye irritation study of CM-Glucan J in rabbits. Test Code No. 99- IXB f. Life Science Laboratory Primary skin irritation study of CM-Glucan J in rabbits. Test Code No. 99- IXA g. Life Science Laboratory Primary skin irritation test for CM-Glucan J in human subjects by closed patch test. Test Code No. 99-XII h. Life Science Laboratory Reverse mutation study of CM-Glucan J in bacteria. Test Code No. 99-VII i. Life Science Laboratory Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method). Test Code No. 99-VIA j. Life Science Laboratory Single dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg). Test Code No. 99-IA k. Eurofins ATS Ocular tolerance test according to HET CAM method: Sodium Carboxymethyl Beta- Glucan. June 11-12, 2012: Draft Report Additional used data were received from the Council: Concentration of Us by FDA Product Category: Xanthan Hydroxypropyltrimonium Chloride (Mar 28, 2012) CIR Panel Book Page 2

6 Microbial Polysaccharides Data Profile June 2012 Writer, Monice Fiume In Use Constituents/ Impurities Method of Mfg Toxicokinetics Acute Tox Derm Acute Tox Oral Acute Tox Inhalation Acute Tox Parenteral Repeated Dose Dermal Repeated Dose Oral Repeated Dose Inhalation Repeated Dose Parenteral Repro/Dev Tox Genotoxicity Carcinogenicity Dermal Irritation Non Human Dermal Irritation Human Dermal Sens Non Human Dermal Sens Human Photoallergy Human Ocular Irritation Xanthan Gum X X X X X X X X X X X X Hydroxypropyl Xanthan Gum Undecylenoyl Xanthan Gum Dehydroxanthan Gum Xanthan Gum Crosspolymer Xanthan Hydroxypropyltrimonium Chloride X X Gellan Gum X X X X X X X X X X Welan Gum Biosaccharide Gum-1 Biosaccharide Gum-2 Biosaccharide Gum-3 Biosaccharide Gum-4 Biosaccharide Gum-5 Pseudoalteromonas Exopolysaccharides X X X X Dextran X X X X X X Carboxymethyl Dextran Dextran Hydroxypropyltrimonium Chloride X Sodium Carboxymethyl Dextran X X Dextran Sulfate X X X X X Sodium Dextran Sulfate Sclerotium Gum X X Hydrolyzed Sclerotium Gum X X Beta-Glucan X X X X X X X X X X X X X X X X Beta-Glucan Hydroxypropyltrimonium Chloride Beta-Glucan Palmitate Hydrolyzed Beta-Glucan Oxidized Beta-Glucan X Sodium Carboxymethyl Beta-Glucan X X X X X X X X X X X Pullulan X X X X X X X X Myristoyl Pullulan Levan Rhizobian Gum X X Hydrolyzed Rhizobian Gum Alcaligenes Polysaccharides X X X X X indicates that data were available in the category for that ingredient CIR Panel Book Page 3

7 Microbial Polysaccharides Search History Keep Me Posted results received weekly Curdlan Nov 15, 2011 SciFinder 534 hits/ Created Keep Me Posted file Xanthan Hydroxypropyl Trimonium Chloride Nov 15, 2011 SciFinder Search no hits Created Keep Me Posted file Schizophyllan Nov 14, 2011 SciFinder Search searched hits/ 18 papers ordered Created Keep Me Posted file Microbial Polysaccharides Sept 16, 2011 Scifinder Search refined by: Document type 8027 hits These were all then searched with the following qualifiers: adverse effects 1478 hits; 3 of interest reproductive toxicity/teratogenicity 5; 2 of interest carcinogenicity o inhalation dermal oral 71 hits; 10 of interest o other 2117; 8 of interest photosensitization; phototoxicity; dermal sensitization; dermal irritation 62; 7 of interest ocular irritation 15 hits; 6 of interest mutagenicity;genotoxicity 270 hits; 8 of interest toxicity o oral; dermal; inhalation 183 hits; 7 of interest o other 1911 hits; 18 of interest toxicokinetics; pharmacokinetics 3497 hits o dermal penetration; dermal absorption 20 hits; 2 of interest o inhalation 27 hits; 4 of interest 39 papers ordered on Oct 5 24 maybes Created Keep Me Posted file CIR Panel Book Page 4

8 File Name ESIS OECD IPCS ACToR USEPA_SR S CCR ACToR2 IPCS2 USEPA_SR S2 ACToR3 ERMA ACToR4 ACToR5 ACToR6 ESIS2 ACToR7 CCR2 USEPA_SR S3 ACToR8 ACToR9 CCR3 ACToR10 ESIS3 ACToR11 USEPA_SR S4 ACToR12 ACToR13 ACToR14 Web Address F51C 4157 B524 27FC43214F6B rspec=charlie.ccohs.ca%3a9900&querytext2=&search=search&querytext1= arch.do?p_type=srsitn&p_value= D A6E 9CD8134C538E rspec=charlie.ccohs.ca%3a9900&querytext2=&search=search&querytext1= arch.do?p_type=srsitn&p_value= databases/pages/ccid details.aspx?substanceid= B5A3 4CEA A7C D087DCF arch.do?p_type=srsitn&p_value= A7C A206 1AEA08D arch.do?p_type=srsitn&p_value= CIR Panel Book Page 5

9 Report

10 Draft Report On the Safety Assessment of Microbial Polysaccharides as Used in Cosmetics June 11, 2012 The 2012 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel Liebler, 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 CIR Panel Book Page 6

11 TABLE OF CONTENTS Introduction... 1 Chemistry... 1 Definition and Structure... 1 Physical and Chemical Properties... 2 Constituents/Impurities... 2 Method of Manufacture... 2 Use... 3 Cosmetic... 3 Non-Cosmetic... 3 Toxicokinetics... 4 Absorption, Distribution, Metabolism, and Excretion... 4 Dermal... 4 Oral... 4 Parenteral... 6 Toxicological studies... 8 Single Dose (Acute) Toxicity... 8 Inflammatory Response... 8 Cytotoxicity... 8 Repeated Dose Toxicity... 8 Oral Intake by Humans... 8 Industrial Exposure... 9 Reproductive and Developmental Toxicity... 9 Oral... 9 Genotoxicity Carcinogenicity Oral Anti-Tumor Effects Irritation and Sensitization Phototoxicity Adverse Effects Ocular Irritation Summary Discussion Conclusion Tables Table 1. Definition, Function, and Idealized Structure Table 2. Chemical and physical properties Table 3. Constituents/Impurities Table 4. Methods of Manufacture/Microorganisms Used in Production Table 5a. Frequency and concentration of use according to duration and type of exposure Table 5b. Ingredients Not Reported to be Used Table 6. Examples of Non-Cosmetic Uses Table 7. Acute toxicity studies Table 8. Repeated Dose Toxicity Studies Table 9. Genotoxicity studies Table 10. Dermal Irritation and Sensitization Table 11. Ocular Irritation References ii CIR Panel Book Page 7

12 INTRODUCTION This assessment is a review of information relevant to the safety of 34 microbial polysaccharides as used in cosmetic formulations. Reported functions for these ingredients include emulsion stabilizer, film former, binder, viscosity increasing agent, and skin conditioning agent. The Cosmetic Ingredient Review (CIR) Expert Panel has reviewed other non-microbial gums and polysaccharides. In 2012, CIR concluded the Galactomannans, a group of 16 legume polysaccharides, are safe as used in cosmetics. 1 In 2009, the CIR Expert Panel reviewed the safety of Hyaluronic Acid, an amine-derived exopolysaccharide, finding it safe as used. 2 In 1987, the Expert Panel reviewed the safety of Tragacanth Gum (now named Astragalus Gummifer Gum), and concluded that Tragacanth Gum was safe as used; the Panel reaffirmed that conclusion in Other plant-derived gums that the Panel has reviewed include Acacia Catechu Gum, Acacia Farnesiana Gum, Acacia Senegal Gum; in 2005, the Panel concluded that Acacia Catechu Gum is safe as used, but that the data are insufficient to support the safety of Acacia Catechu Gum and Acacia Farnesiana Gum as used in cosmetics. 4 The 34 microbially-produced polysaccharides included in this review are: Xanthan Gum Hydroxypropyl Xanthan Gum Undecylenoyl Xanthan Gum Dehydroxanthan Gum Xanthan Gum Crosspolymer Xanthan Hydroxypropyltrimonium Chloride Gellan Gum Welan Gum Biosaccharide Gum-1 Biosaccharide Gum-2 Biosaccharide Gum-3 Biosaccharide Gum-4 Biosaccharide Gum-5 Pseudoalteromonas Exopolysaccharides Dextran Carboxymethyl Dextran Dextran Hydroxypropyltrimonium Chloride Sodium Carboxymethyl Dextran Dextran Sulfate Sodium Dextran Sulfate Sclerotium Gum Hydrolyzed Sclerotium Gum Beta-Glucan Beta-Glucan Hydroxypropyltrimonium Chloride Beta-Glucan Palmitate Hydrolyzed Beta-Glucan Oxidized Beta-Glucan Sodium Carboxymethyl Beta-Glucan Pullulan Myristoyl Pullulan Levan Rhizobian Gum Hydrolyzed Rhizobian Gum Alcaligenes Polysaccharides Many studies have been conducted with some of the microbial polysaccharides in regard to health claims, immunomodulatory activity, anti-oxidant activity, etc. This safety assessment only includes studies and study-types that relate directly to the safety of the cosmetic use of these ingredients. CHEMISTRY Definition and Structure Microbial polysaccharides are high molecular weight carbohydrate polymers that make up a substantial component of the cellular polymers found in and surrounding most microbial cells. 5 These polysaccharides are produced by a wide variety of microorganisms and are water soluble gums which have novel and unique physical properties. Microbial polysaccharides are generally divided into three groups: exocellular, cell wall, and intercellular. 6 The exocellular polysaccharides are those that constantly diffuse into the cell culture medium and are easily isolated. The cell wall (i.e., structural) and intercellular polysaccharides are integral parts of the cell wall or capsular products, and are more difficult to separate from cell biomass. Microbial polysaccharides may be ionic or non-ionic and are primarily linear molecules to which side chains of varying length and complexity are attached at regular intervals. 5 Most microbial polysaccharides are linear hetero-polysaccharides consisting of three to seven different monosaccharides arranged in groups of 10 or less to form repeating units. The monosaccharides may be pentoses, hexoses, amino sugars, or uronic acids. For example, xanthan gum is a polysaccharide produced by a pure-culture fermentation of a carbohydrate with Xanthomonas campestris, and is composed of glucose, glucuronic acid, 6-acetylmannose, and 4,6-pyruvylated mannose residues, as seen in Figures 1 and 2. 1 CIR Panel Book Page 8

13 Figure 1. Xanthan Gum a polysaccharide composed of glucose, glucuronic acid, 6-acetylmannose, and 4,6-pyruvylated mannose Figure 2. Glucose, glucuronic acid, 6-acetylmannose, and 4,6-pyruvylated mannose, the monosaccharide components of Xanthan Gum. The other ingredients in this report are related by having similar polymeric repeat units. The definitions and polymeric repeat units of the ingredients included in this review are provided in Table 1. Physical and Chemical Properties The available physical and chemical properties are provided in Table 2. The properties of the microbial polysaccharides can vary widely based on, among other parameters, the side groups, the ester substituents, or bacterial strains, but generally speaking, these are very large molecular weight polymers Constituents/Impurities The available constituent and impurity data are provided in Table 3. Method of Manufacture Methods of manufacture for many of the microbial polysaccharides are provided in Table 4. Some of the polysaccharides discussed in this safety assessment can be produced by more than one microorganism. For example, beta-glucans are produced by fungi, yeasts, and grains 11 and levan can be produced by bacteria, yeasts, or fungi CIR Panel Book Page 9

14 USE Cosmetic The microbial polysaccharides named in this report have a variety of reported functions in cosmetics. Reported functions for these ingredients include emulsion stabilizer, film former, binder, viscosity increasing agent, and skin conditioning agent. 13 The Food and Drug Administration (FDA) collects information from manufacturers on the use of individual ingredients in cosmetics as a function of cosmetic product category in its Voluntary Cosmetic Registration Program (VCRP). VCRP data obtained from the FDA in 2011, 14 and data received in response to a survey of the maximum reported use concentration by category conducted by the Personal Care Products Council (Council), 15,16 indicate that 19 of the 34 microbial polysaccharides named in this safety assessment are currently used in cosmetic formulations. Xanthan gum is used in almost every category of cosmetic product, with 2964 reported uses. Biosaccharide gum-1, sclerotium gum, and beta-glucan are reported to be used in 302, 182, and 116 cosmetic formulations, respectively. All other in-use ingredients have less than 60 uses. The ingredient with the highest concentration of use is pullulan; it is used at up to 12% in leave-on formulations and 17% in rinseoff formulations. Both xanthan gum and biosaccharide gum-1 are used at up to 6% in leave-on formulations and xanthan gum crosspolymer and biosaccharide gum-4 are used at 5% in leave-on formulations. All other in-use ingredients are used at concentrations of 3%. In some cases, reports of uses were received in the VCRP, but no concentration of use is available. For example, sodium carboxymethyl dextran is reported to be used in 4 formulations, but no use concentration data were available. In other cases, no reported uses were received in the VCRP, but a use concentration was provided in the industry survey. For example, xanthan gum crosspolymer was not reported in the VCRP to be in use, but the industry survey indicated that it is used in leave-on formulations at up to 5%. It should be presumed that xanthan gum crosspolymer is used. Frequency and concentration of use data are provided in Table 5a. The ingredients not listed in the VCRP or by the Council as being used are listed in Table 5b. Products containing some of the microbial polysaccharides are reported to be used on baby skin, may be applied to the eye area or mucous membranes, or could possible ingested. Some of these ingredients are reported to be used in product types that may possibly be inhaled; for example, dehydroxanthan gum is used in a face and neck spray at 0.2%. In practice, 95% to 99% of the particles released from cosmetic sprays have aerodynamic equivalent diameters in the 10 to 110 µm range. 17,18 Therefore, most particles incidentally inhaled from these sprays are deposited in the nasopharyngeal region and are not respirable. 19,20 Xanthan gum is reported to be used in deodorants at up to 0.6%, and it is not known whether or not these products are sprayed. There is some evidence indicating that deodorant spray products can release substantially larger fractions of particulates having aerodynamic diameters in the range considered to be respirable. 20 However, the information is not sufficient to determine whether significantly greater lung exposures result from the use of deodorant sprays, compared to other cosmetic sprays. All of the microbial polysaccharides named in the report listed in the European Union inventory of cosmetic ingredients. 21 Non-Cosmetic Xanthan gum 22 and gellan gum 23 are approved as direct food additives in gums, chewing gum bases, and related substances. Xanthan gum is also approved as an indirect food additive. 24 Beta-glucan (as curdlan, a specific beta-glucan that is a linear polymer consisting of β-(1 3)-linked glucoside residues) is approved as a direct food additive for multipurpose addition. 25 Dextran is an indirect food additive affirmed as generally recognized as safe (GRAS). 26 The World Health Organization (WHO) concluded that studies on the safety of xanthan gum, 27 gellan gum, 28 and pullulan 29 provided sufficient information to be allocated an acceptable daily intake (ADI) of not specified. Pullulan appears in the Japanese List of Existing Food Additives. 30 Xanthan gum is used as a stabilizer, thickener, and emulsifying agent in water-based pharmaceutical preparations. 31 Dextran, as dextran 70, is an approved active ingredient for over-the-counter (OTC) use as an ophthalmic demulcent at 0.1% when used with another approved polymeric demulcent. 32 Dextran is used as a plasma volume expander (as dextran 70) and as a blood flow adjuvant (as dextran 40). 33 Sodium dextran sulfate is used as a clinical reagent. These uses, and other examples of non-cosmetic, are summarized in Table 6. 3 CIR Panel Book Page 10

15 TOXICOKINETICS Absorption, Distribution, Metabolism, and Excretion Dermal In Vitro Beta-Glucan A single application of 5 mg/cm 2 of a 0.5% (oat) beta-glucan solution was applied to human abdominal skin. 34 Beta-glucan penetrated the skin into the epidermis and dermis. (No details were provided.) Human Dextran Fluorescent dextrans (FDs) in aq. solution were used to determine the dermal absorption of different molecular weight (MW) dextrans (MW ,000, identified as FD-3 and FD-70, respectively) through skin that has been subjected to mini-erosion (i.e., vacuum suctioning and removal of the roof of the epidermal vesicle). 35 Prior to testing, absorption of FD-20 from a test cell into systemic circulation via a mini-erosion site was verified in two male subjects and one female subject. The largest of the test molecules filtered from the blood into the urine was 20,000 MW, and with increasing erosion diameter, the absorption increasingly occurs via the lymphatic system. Each subject received a single cell containing 0.5 ml solution (5 mmol). After 24 h, a mean of 54.3% of the total FD-20 dose had been absorbed, and 12.6% of the absorbed dose was recovered in the urine. The effect of molecular size on absorption of FDs was then determined. Four cells with 100 μl of FD-3, FD-10, FD- 20, or FD-70 in 0.5 ml isotonic saline were applied for 24 h to 6 mm mini-eroded sites on 4 male and 3 female subjects. A fifth cell, without FD, was applied as a negative control. The FD concentration in each cell was measured using spectrofluorometry at various times. The dextrans were readily absorbed, but absorption decreased with increasing molecular size. The absorption of FD-3 was 37.9%, and for FD-70, it was 20.1%. Further testing using 3 male and 3 female subjects determined that the degree of transdermal absorption was directly related to the area of erosion; 20.5% of FD-3 absorbed through a 3 mm erosion area, while 60.7% of the same molecular size FD absorbed through a 10 mm erosion. It was also determined that skin integrity is a factor; at a control site of intact skin, FD-3 was not absorbed. Oral Non-Human Xanthan Gum Rats were fed a diet containing 2% [ 14 C]xanthan gum that was produced by fermentation of uniformly-labeled glucose with Xanthomonas campestris. 36 No accumulation was found in the tissues. A maximum of 15% of the radioactivity was metabolized to carbon dioxide within 100 h. Fecal analysis indicated that there was no accumulation of the polysaccharide material, except acetate. (Acetate and pyruvate accounted for only 9.8% of the label in the gum used). In the feces, 98% of the radioactivity was attributed to unchanged or slightly modified polysaccharide. In vitro testing indicated that nonenzymatic hydrolysis and fecal microorganisms were responsible for the in vivo breakdown of xanthan gum. (No additional details were provided.) Gellan Gum In animal feeding studies using radiolabeled gellan gum, the majority of the gellan gum that was administered was recovered in fecal matter. 10 This appears to indicate that no endogenous enzymes that are able to break down gellan gum are present in the small intestine. (No additional details were provided.) The absorption, distribution, and excretion of gellan gum was determined in studies using a dually-radiolabeled gum that was prepared in separate fermentations using [ 3 H]glucose and [ 14 C]glucose as carbon sources. 37 (The 3 H product was subjected to multi-stage purifications for a relatively pure [ 3 H]polysaccharide, which was then added to the 14 C fermentation, giving a polysaccharide fraction that was dual-labeled and a non-polysaccharide fraction labeled only with 14 CO 2.) In the first study, one male and one female Sprague-Dawley rat were dosed by gavage with a single dose of 960 mg/kg [ 3 H/ 14 C]gellan gum (4 µci). Expired air was collected for 24 h after dosing, and <0.55% of the dosed radioactivity was detected as 14 C. Four male and three female Sprague-Dawley rats were then given a single dose by gavage of 870 mg/kg [ 3 H/ 14 C]gellan gum ( µci 14 C; µci 3 H). Urine and feces were collected for 7 days. Approximately 86% of the dosed 14 C was excreted in the feces and 2-3% in the urine, and approximately 100% of the dosed 3 H was excreted in the feces and 4% was in the urine. Tissue and carcass 14 C radioactivity was approximately 3-4% of the dose. The 3 H activities in the tissues were below the limits of accurate quantification. 4 CIR Panel Book Page 11

16 In the last study, one male and four female Sprague-Dawley rats were dosed with 1 g/kg [ 3 H/ 14 C]gellan gum by gavage, and blood samples were taken at various intervals over a 7-day period. (It is not stated, but it is assumed that one dose was administered.) The peak level of activity, 0.4%, occurred about 5 h after dosing. Dextran Groups of 5 fasted male Sprague-Dawley rats were given a single dose by gavage of 50 mg/kg fluorescein-labeled dextrans (FD-4, 4400 avg. MW; FD-20, 19,000 avg. MW; or FD-40, 40,500 avg. MW). 38 The dextran solution was prepared as 25 mg/ml in isotonic phosphate buffer. Blood samples were taken at various intervals for up to 4 h after dosing with FD-4, up to 8 h after dosing with FD-20, and for up to 24 h with FD-40. Urine samples were taken at intervals for up to 8 h after dosing with FD-4 and FD-20 and for up to 24 h after dosing with FD-40. None of the dextrans were detected in the serum after oral administration. Small amounts of the dose, ranging from 0.308% with FD-4 to % FD-40, were detected in the urine. The oral bioavailability was 0.398, , and % for FD-4, FD-20, and FD-40, respectively. Dextran Sulfate Two group of 5 male Wistar rats was dosed by gavage with 5 mg/ml of 20 mg/kg dextran sulfate containing 12 μci 3 H-dextran sulfate/kg (8000 avg. MW), and each groups was killed 3 or 24 h after dosing. 39 Most of the radioactivity was detected in the feces; 22.5% of the dose was recovered after 24 h. Metabolites, breakdown products, and 3 H 2 O were not found in the feces, and the researchers stated that it was mostly likely that the dose recovered in the feces was unabsorbed dextran sulfate. Only approximately 10% of the dose was recovered in the urine after 24 h, with 6% recovered after 3 h. Urinary 3 H elution profiles indicated that intermediate molecular weight metabolites or breakdown products were formed and that either intact or partially intact dextran sulfate was absorbed through the epithelium of the GI tract. The 6-24 h samples indicated a marked shift towards smaller molecular weight products. Beta-Glucan Two male Sprague-Dawley rats were dosed orally with 20 mg/kg bw [U-14C]beta-glucan (as curdlan) in water prepared from [U- 14 C]glucose. 40 Most of the radioactivity was recovered in expired CO 2 ; 77% of the dose was recovered in 24 h and 89% in 72 h. A total of 7.7 and 12% of the radioactivity as administered dose was recovered in the feces after 24 and 72 h, respectively, and 2.6 and 3.3% was recovered in the urine after 24 and 72 h, respectively. In another study, three male Wistar rats were dosed orally with 20 mg/kg bw [ 14 C] beta-glucan (as curdlan) in water. 40 Initially (i.e. the first 3 h), the excretion of 14 CO 2 was low, but then increased linearly up to 12 h, plateauing at 39% of the administered radiolabel. A total of 3.4 and 3.8% of the radioactivity as administered dose was recovered in the feces after 24 and 48 h, respectively, and 1.3 and1.4% was recovered in the urine after 24 and 48 h, respectively. Using another group of three male Wistar rats administration of 5 mg/ml tetracycline for 5 days prior to and 2 days following, it was demonstrated that the intestinal microflora are partly responsible for the metabolism of beta-glucan to carbon dioxide. The effect of dose on metabolism was also examined. 40 Three male Wistar rats were given an oral dose of 2.3, 23,or 230 mg/kg bw [ 14 C]beta-glucan (as curdlan) in water. At the two higher doses, excretion of radioactivity as carbon dioxide decreased with increasing dose, while fecal excretion of the radiolabel increased. The researchers stated that this was an indication of limited metabolism at higher doses. Pullulan Five fasted male Wistar rats were dosed by gavage with a 2 ml of a 10% solution of pullulan (49,00 MW) in 0.9% saline. 41,42 The animals were killed 1 h after dosing and the contents of their stomach and small intestines were collected. Approximately 3% of the pullulan had been hydrolyzed; it was not known whether the hydrolysis products were absorbed by the small intestine. Human Dextran Dextran can be depolymerized by α-1-glycosidases (dextranases) that occur in the liver, spleen, kidney, and lower part of the gastrointestinal (GI) tract. 7 Dextran Sulfate Six fasted male subjects were given a single oral dose of 1800 mg dextran sulfate ( MW; 17-20% sulfur) and, after 48 h, a single intravenous (i.v.) dose of 225 mg dextran sulfate in saline infused over 60 min. 43 After oral dosing, no measurable dextran sulfate was found in the plasma using the competitive binding assay, and there was no increase in activated partial thromboplastin time (APTT). Plasma lipolytic activity did not increase the first 3 h after oral dosing; at 3-4 h 5 CIR Panel Book Page 12

17 after oral dosing, it increased by two times the baseline average. Very little dextran sulfate was recovered in the urine after oral dosing. After i.v. dosing, peak plasma concentrations were µg/ml, and the APTT was increased by an average of 6.9 times over the baseline values. The plasma lipolytic activity increased by an avg. of 438 times the baseline value. Dextran sulfate was recovered in the urine after i.v. dosing. Pullulan Pullulan is partially hydrolyzed by salivary and pancreatic amylases of the upper GI tract, and essentially no glucose is released during hydrolysis. 42 Pullulan is largely resistant to digestion in the GI tract because of the occasional presence of 1,3-glycosidic linkages and the high percentage of α-1,6-glycosidic linkages. 29 The degree of digestion appears to be dependent on relative molecular mass. Pullulan is fermented in the colon in vitro and in vivo by intestinal microflora to produce short-chain fatty acids; the degree of fermentation is dependent on the degree of polymerization of pullulan. Six subjects ingested 10 g pullulan (50,000 MW) for 14 days. 42,44 Administered pullulan was fully digested in the intestinal tract and was not detected in the feces. After 14 days, the fecal short chain fatty acid concentration increased from 6 mg/g to 8.8 mg/g. The researchers concluded that pullulan was completely fermented to short-chain fatty acids by intestinal bacteria. Parenteral Non-Human Dextran Rabbits were given a daily i.v. 30 ml dose of a 6% solution of partly hydrolysed bacterial dextran (75,000 avg MW) 6 days/wk for wks. 45 Eleven animals were evaluated. Approximately 25% of the dose was excreted in the urine. The plasma concentration of dextran at study termination (0.50 g/100 ml) did not differ from the value at 2 mos (0.44 g/100 ml), but it was generally greater than the value reported at 24 h after a single 30 ml dose (not given). Moderate dextran storage was observed in the spleen without an increase in the total carbohydrates. However, considerable storage was observed in the liver with a marked increase in carbohydrates. (Additional details and results are provided in the section on Repeated Dose Toxicity. Groups of 5 male Sprague-Dawley rats were given a single i.v. dose of 5 mg/kg FD-4 (4400 avg. MW), FD-20 (19,000 avg. MW), FD-40 (40,500 avg. MW), FD-70 (71,000 avg. MW), or FD-150 (147,800 avg. MW). 38 The dextran solution was prepared as 5 mg/ml in isotonic phosphate buffer. Blood samples were taken at various intervals for up to 4 h after dosing with FD-4, up to 8 h after dosing with FD-20, and for up to 24 h with FD-40, FD-70, and FD-150. Urine samples were taken at intervals for up to 8 h after dosing with FD-4 and FD-20 and for up to 24 h after dosing with FD-40, FD-70, and FD-150. Pharmacokinetic parameters were MW-dependent. Concentrations of the three highest MW dextrans could be detected in serum for up to 12 h after dosing, while FD-20 and FD-4 were not found in the serum after 3 and 1.5 h, respectively. The distribution half-life (t 1/2 α) ranged from to h for FD-4 and FD-150, respectively, and the elimination half-life (t 1/2 β) ranged from h for FD-4 and FD-150, respectively. Male Sprague-Dawley rats were also given a single i.v. dose of 1, 25, or 100 mg/kg FD-4 (avg. MW 4300) and FD- 150 (avg. MW 145,000). 46 The dextran solution was prepared as isotonic phosphate buffer at a concentration that would result in a test volume of 2 ml/kg. Groups of 4 rats each were used for blood, urine, and tissue samples at various intervals for up to 6 h in rats dosed with FD-4 and for up to 96 h in rats dosed with FD-150. Renal excretion was a major excretion pathway for FD-4 but not FD-150. Urinary recovery ranged from 79-82% of the dose with FD-4 and only from % of the dose with FD-150; at each MW, the dose administered did not have a statistically significant effect on the amount excreted. Renal clearance ranged from ml/h/kg with FD-4 and from ml/h/kg for FD-150, and systemic clearance ranged from ml/h/kg for FD-4 and from 8-20 ml/k/kg for FD-150. The highest concentrations of FD-4 were found in the kidneys at 1 min after dosing (9.31%, 10.0%, and 10.4% of the dose with 1, 25, and 100 mg/kg, respectively) was linear with dose. The highest concentrations of FD-150 were found in the liver (68.5% at 5 h, 51.6% at 24 h, and 41.5% of the dose at 24 h with 1, 25, and 100 mg/kg, respectively) and the spleen (11.5% at 12 h, 2.09% at 48 h, and 1.21% of the dose at 96 h with 1, 25, and 100 mg/kg, respectively); recovery of dextran in the liver and spleen was non-linear, with a greater difference seen in the spleen than in the liver. The researchers reported that the MW of recovered FD-4 remained relatively constant, but that of recovered FD-150 changed significantly. The MW of FD-150 recovered in the urine was <40,000, and, in the liver, the avg. MW at 96 h was ~70,000. The estimated MW of the recovered FD-150 in the liver appeared to be dose-dependent with higher doses having a higher avg. MW. The researchers concluded that excretion of lower MW dextrans was independent of dose, while excretion of higher MW dextrans was dose-dependent. 6 CIR Panel Book Page 13

18 Another study also found that MW affected the distribution and excretion of dextran. 47 Female BALB/cCrSlc mice were dosed i.v. with 0.1 ml of 0.1 wt% 125 I-labeled dextran, MW ranging from ,000, in phosphate buffered saline (PBS); dextran was labeled through radioiodination of tyramine residues. Blood samples were taken at various intervals for some groups, and tissues were collected from others. (The number of animals/group was not specified.) High MW dextran remained in the blood longer than lower MW dextran. The t 1/2 β also increased with increasing MW, with a pronounced change occurring around 30,000 MW. After 3 h, 83% of the dose of the lowest MW dextran was excreted, while only 41% of the highest MW dextran was found in excrement. Most of the high MW dextran was found in the liver; after 3 h, 5.2% of the high MW dextran was found in the liver, while only 0.7% of the lowest MW was recovered in that tissue. At 3 h, 3.5 vs. 19% of the dose of the lowest and highest MW dextran, respectively, was recovered in the carcass. Two to 10% of the dose was recovered in the GI tract, but the amount recovered did not appear to be related to MW. Dextran Sulfate A preliminary study was performed in which 2 male Wistar rats were dosed by i.v. injection with 20 mg/kg dextran sulfate 8000 avg. MW) containing 10 μci [ 3 H]dextran sulfate/kg via the penile vein (5 mg/100 μl). 39 The rats were killed 1 or 3 h after dosing. The total 3 H excreted in the urine accounted for approximately 50% of the administered dose. Within 1 h after i.v. administration, rapid excretion of intact dextran sulfate occurred. In the main study, groups of 5 male Wistar rats was dosed by i.v. injection via the penile vein with 5 mg/100 μl of 20 mg/kg dextran sulfate containing 12 μci [ 3 H]dextran sulfate/kg, and the each group was killed 3 or 24 h after dosing. Urine was the major route of excretion following i.v. dosing, with approximately 46% of the dose excreted within 3 h and 51% within 24 h. Approximately 2% of the dose was recovered in the feces after 24 h. Based on the 3 H elution profiles, it was hypothesized that dextran sulfate or its metabolites were incorporated into higher molecular weight compounds, such as glycogen, at 3-6 h and smaller molecular weight products at 6-24 h. In the plasma, the highest concentration of dextran sulfate was found at 3 h; the amount decreased with time. In the tissues, the highest amounts of radioactivity were distributed in the liver, kidney, and spleen. The amount of radioactivity recovered in the tissues following i.v. administration was compared to that found following oral administration. (The oral study was described previously in this safety assessment.) Concentrations of 3 H in all tissues and fluids were statistically significantly higher in the animals dosed by i.v. injection compared to those in animals dosed orally. Beta-Glucan Groups of two male Sprague-Dawley rats were dosed by intraperitoneal (i.p.) injections with 20 mg/kg bw [ 14 C]beta-glucan (as curdlan) in water, and the animals were killed 0.5, 3, 6, or 24 h after dosing. 40 After 24 and 48 h, only 1.8% and 4.1% of the radioactivity was recovered in CO 2, respectively, 0.05 and 0.12% was recovered in the feces, respectively, and 3.5 and 4.1% of the radioactivity as % of dose was recovered in the urine, respectively. Whole-body radioautography showed that the radiolabel was distributed in the intestinal fluids. Pullulan The effect of MW on the distribution and excretion of pullulan was examined. 47 Female BALB/ ccrslc mice were dosed i.v. with 0.1 ml of 0.1 wt% [ 125 I]pullulan, MW ranging from ,000, in PBS; pullulan was labeled through radioiodination of tyramine residues. Blood samples were taken at various intervals for some groups, and tissues were collected from others. (The number of animals/group was not specified.) High MW pullulan remained in the blood longer than lower MW pullulan. The t 1/2 β also increased with increasing MW. After 3 h, 96% of the dose of the lowest MW pullulan was excreted, while only 15% of the highest MW pullulan was found in excrement. At 3h, most of the high MW pullulan, 50% of the dose, was recovered in the liver; only 1% of the dose of the lowest MW pullulan was recovered in the liver after 3 h. The highest percentage of the dose recovered in the GI tract and the carcass was found with mid-level MW pullulan; 5.3% of the dose was recovered in the GI tract with 100,000 MW pullulan and 10.1% of the dose was recovered in the carcass with pullulan 48,000 MW pullulan. Fasted male Wistar rats (number not specified) were injected with a single i.v. dose 2 ml of 0, 6, 12, 18, or 24 mg/kg fluorescein-labeled pullulan (MW 58,200) in saline in the jugular vein. 48 Pullulan was rapidly eliminated from the blood; however, elimination decreased as dose increased. In the liver, hepatic uptake was also dose-dependent; the hepatic uptake clearance of pullulan decreased with increased dose. In the liver, distribution of pullulan in the parenchymal cells was 2.5 times greater than in the nonparenchymal cells. The researchers did state that with a higher MW pullulan, avg. 70,000 MW, uptake was greater in the nonparenchymal cells. 7 CIR Panel Book Page 14

19 Human Dextran Four male and three female subjects received an i.v. injection 100 ml a partially degraded dextran (40,000 avg. MW; 10% w/v in 0.9% saline). 49 Blood samples were taken at various intervals after dosing. There was an initial rapid decrease in the serum in the first hour after dosing. Different fractions of the dextran were eliminated from the plasma at different rates; higher molecular weight fractions remained in the plasma longer. TOXICOLOGICAL STUDIES Single Dose (Acute) Toxicity Acute toxicity studies are summarized in Table 7. The acute toxicity of xanthan gum, gellan gum, beta-glucan, sodium carboxymethyl beta-glucan, and pullulan was assessed orally in mice, rats, and/or dogs, and dextran sulfate and betaglucan were tested by i.p. and i.v. dosing in mice and rats. There was no notable toxicity observed in these studies. In acute inhalation studies the LC 50 of xanthan gum was >21 mg/l in rabbits and of gellan gum was >5.06 mg/l in rats. Inflammatory Response Beta-Glucan and Pullulan The inflammatory response following a single exposure to beta-glucan (as curdlan) was evaluated in guinea pigs. 50 Mostly, no effect or a slight decrease in inflammatory cells in lung lavage was observed. A 4 h inhalation exposure to betaglucan in dust (mass mean aerodynamic diameter [MMAD] 5 µm) by guinea pigs produced a delayed subacute nasal congestion when compared to dust without beta-glucan (MMAD 6.5 µm) and resulted in decreased nasal volume. In humans, inhalation exposure to beta-glucan in dust for 18 h resulted in decreased nasal volume, increased nasal eosinophil cells, and increased lung lymphocytes and macrophages, but did not produce microscopic change in lung tissue. Pullulan The effect of pullulan on inflammation was investigated in ICR mice using the xylene-induced acute inflammatory mouse ear model. 51 Groups of 9 mice were dosed orally with 0, 62.5, 125,or 250 mg/kg pullulan in distilled water 30 min prior to topical application of xylene to one ear. Two h after xylene application, all animals were killed. Compared to xylene-treated controls, ear weights were statistically significantly decreased in a dose-dependent manner. Additionally, histological indicators of inflammation were not observed. Levan An interleukin (IL)-1α release assay was used to determine the anti-inflammation effect of a 5% aq. levan solution on artificial skin. 52 Primary skin irritation was first induced using sodium lauryl sulfate. A dose of 0.01 or 0.05 mg/ml of the solution was applied to the skin. Levan decreased IL-1α release, indicating an anti-inflammatory effect. Cytotoxicity Levan The cytotoxic effect of 5% levan (w/w) was determined using human fibroblasts and keratinocytes. 52 The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability and proliferation after 24 h incubation with levan. Levan, 100 µg/ml, was not cytotoxic to human fibroblasts. Levan had a proliferative effect in keratinocytes; proliferation was >30% at concentrations of >1 mg/ml. Repeated Dose Toxicity Repeated dose toxicity studies are summarized in Table 8. The oral toxicity of xanthan gum was evaluated in rats and dogs, of gellan gum in rats, dogs, and monkeys, of dextran in rats, of beta-glucan in mice, rats, and dogs, and of pullulan in rats. Most of the studies were dietary, and study duration lasted up to 2 yrs. Most observations were related to changes in feed consumption and intestinal effects. Inhalation exposure to 100 µg/ml beta-glucan (as curdlan), 4 h/day, 5 days/wk for 4 wks, did not have an effect on the cells of the lung lavage or cell wall, and there were no microscopic lesions of the lung. With i.p. administration, no toxicity was reported when mice were dose 10 times over 2 wks with 5 mg xanthan gum in 0.5 ml water or mice or guinea pigs were dosed with 250 mg/kg bw beta-glucan for 7 days. Intravenous administration of 40 and 1000 mg/kg bw beta-glucan for 30 days resulted in hepatosplenomegaly in mice. Oral Intake by Humans Xanthan Gum Five male subjects consumed 150 mg/kg bw/day xanthan gum as three measured portions daily for 23 days. 53 Ingestion of xanthan gum had no significant adverse effect on hematology, clinical chemistry, or urinalysis parameters. 8 CIR Panel Book Page 15

20 Gellan Gum Five male and five female subjects consumed a daily dose of 175 mg/kg gellan gum for 7 days and 200 mg/kg/day for the next 16 days. 53 No adverse dietary or physiological effects and no allergenic effects were reported Dextran and Pullulan No adverse effects were seen in a study in which 10 g pullulan, dextran, and soluble starch were ingested by 8 male volunteers. 40,54 Each ingredient was administered for 14 days, and there was a 14-day wash-out period between treatments. Beta-Glucan Six male subjects ingested milkshakes with or without beta-glucan (as curdlan) for 28 days. 40 The test subjects were given 6 g/day for 5 days, 35 g/day for 2 wks, and 50 g/day from day No evidence of toxicity was observed. Pullulan In a tolerance study, 13 subjects ingested 10 g/day of pullulan (50,000 MW) for 14 days. 42,44 There were no effects on clinical chemistry parameters, and no adverse effects were reported. Industrial Exposure Xanthan Gum The relationship between the handling of xanthan gum powder and adverse symptoms was examined using exposure groups based on average percentage of time spent in a plant that uses a fermentation process to manufacture xanthan gum, not on the basis of expected intensity of exposure to xanthan gum. 55 The exposure of interest was to the employees exposed to milling, blending, and packaging of the product. Analysis of the results found no significant acute or chronic effects in pulmonary function in an of the exposure groups. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Oral Xanthan Gum A three-generation reproductive toxicity study was performed in which albino rats were fed dietary levels of 0, 0.25, and 0.5 g/kg bw/day xanthan gum. 56 Ten males and 20 females were used for the first generation, and 20 males and 20 females were used in the next two generations. Rats were mated to produce two litters per generation, and the successive generations were selected from weanlings of the second litter. Survival and reproductive parameters were similar for treated and control parental rats. Body weights of treated parental rats were slightly decreased compared to controls in each generation. There were no significant differences in developmental parameters between test and control litters, and no malformations were observed in any of the offspring. Gellan Gum Groups of 25 gravid female Sprague-Dawley rats were fed a diet containing 0, 2.5, 3.8, or 5.0% gellan gum (varied degree of acetylation; 58.5% polysaccharide) on days 6-15 of gestation. 37 No fetotoxic or teratogenic effects were reported. (No other details were provided.) Beta-Glucan A developmental study was performed with 0, 5, or 15% beta-glucan (as curdlan) with a control group of 40 male and 80 female CD rats and test groups of 20 male and 40 female rats. 40 The animals, which were mated twice, were fed the test diet throughout the study. Twenty of the treated dams nursed their own litters; the other 20 treated dams switched litters with the control dams so that treated animals would nurse control pups and control animals would nurse test pups. The F 1a offspring were killed prior to the second mating. No changes in mortality, behavior, or appearance were observed. Male parents of the 15% beta-glucan group had reduced growth compared to controls, and males and females of the 15% group had decreased feed consumption. At birth, there were no differences in fertility or lactation among the groups, and no abnormalities were reported. However, survival of the F 1a, but not the F 1b, pups of the 5% group was statistically significantly decreased compared to controls. Weight gain of all F 1a litters of treated dams that nursed their own pups was statistically significantly decreased compared to controls; for the F 1b litters, the difference was statistically significant only in the 15% group. Statistically significant decreases in weight gain were also observed for pups of treated dams that were nursed by control dams, but the effect was reduced. Statistically significant decreased weight gain at some intervals was also observed for control pups nursed by treated dams. A no-observable effect level (NOEL) was not established. 9 CIR Panel Book Page 16

21 The researchers also examined whether there would be a reduced weight gain by the pups if dosing was discontinued during lactation. The protocol was similar to that just described, except that all groups consisted of 20 male and 40 female CD rats, and there was no cross-over at lactation. Weight gain by all pups during lactations was similar, although the researchers did state that the pups could have consumed parental diet from day 10+. The NOEL for parental toxicity and embryotoxicity was 15% beta-glucan. A three-generation reproductive study was performed in which groups of 20 male and 40 female CD rats were fed a diet containing 0, 1, 5, or 15% beta-glucan (as curdlan) for 100 days. 40 The F 0 parents were mated twice, with the number of parents was halved after weaning of the first litter. The F 1 parents were mated three times and the F 2 parents were mated twice. The F 1b and F 2b litters were used to produce the next generation. After the third mating of the F 1 parents, half of the F 1 dams were killed on day 13 of gestation, and the remaining dams were killed on day 20 of gestation. Mean growth and feed consumption were slightly decreased in male parental rats of the F 0 and F 1 generations of the 15% group. No gross or microscopic changes were observed in F 2 parents. No treatment-related effects on reproductive and developmental parameters were observed, but body weights of pups in almost all litters in all generations were statistically significantly decreased during lactation in the 15% group. Biologically-significant differences in body weights were not seen in litters of the other dose groups. No gross or microscopic lesions were observed in the F 3b pups of the 15% group. In the F 1 parents killed after the third mating, no reproductive or developmental effects were observed. Mean fetal weights in all groups were statistically significantly decreased compared to controls; however there was no dose-response. The NOEL for parental animals was 5%, based on decreased growth and increased cecal weights at 15% beta-glucan, and the NOEL for embryotoxicity was also 5%, based on decreased weight gain during lactation in the 15% beta-glucan group. The teratogenic potential of beta-glucan (as curdlan) was determined using groups of gravid Dutch belted rabbits. 40 The rabbits were dosed orally with 0, 1, 2, or 5 g/kg bw/day beta-glucan in a gelatine capsule delivered using a syringe. The 5 g/kg dose was administered as two divided doses, and the controls received two empty capsules. The rabbits were killed on day 28 of gestation. None of the controls died, but one, one, and three dams of the 1, 2, and 5 g/kg bw/day groups, respectively, died during the study. Eleven resorptions were observed in the high dose group, as compared to four in the control group, six in the 1 g/kg group, and five in the 2 g/kg group. The researchers stated, however, that the number of dams with resorptions was similar in all groups, and that no teratogenic effects were observed. The NOEL for both maternal and embryotoxicity was 5 g/kg bw/day. GENOTOXICITY Genotoxicity studies are summarized in Table 9. The in vitro genotoxicity of gellan gum ( 20 mg/ml), sodium dextran sulfate ( 25 mg/plate), beta-glucan ( 5000 µg/plate or /ml), sodium carboxymethyl beta-glucan ( 50,000 µg/plate or /ml) and pullulan ( 12 mg/ml) was evaluated in Ames test, chromosomal aberration assays, and/or DNA repair tests, with and without metabolic activation. The results were negative in these tests. The only non-negative result was a weak positive outcome with 20 mg/plate pullulan in a rec assay using Bacillus subtilis. Negative results were also reported in in vivo mouse micronucleus tests with 5000 mg/kg beta-glucan and 1800 mg/kg pullulan. CARCINOGENICITY Oral Gellan Gum Groups 50 male and 50 female Swiss Crl mice were fed a diet containing 0, 1, 2, or 3% gellan gum (varied degree of acetylation; 58.5% polysaccharide) for 96 wks (males) or 98 wks (females). 37 No treatment-related effects on body weights or feed consumption were observed. No neoplastic or non-neoplastic lesions were reported. In another study, groups of 50 male and 50 female F 1 generation Sprague-Dawley rats were exposed in utero to gellan gum, and were maintained on a diet containing 0, 2.5, 3.8, or 5.0% gellan gum (varied degree of acetylation; 58.5% polysaccharide) for 104 wks. 37 Survival of treated male rats was decreased when compared to controls, but survival of treated female rats was better than the concurrent controls. Male rats of the 3.8 and 5.0% test groups had decreased body weights compared to controls initially and after 76 wks. However, the researchers stated that the growth pattern of these test animals was the same as that of the controls, and the lower body weighs were not indicative of toxicity. There were no neoplastic or non-neoplastic lesions associated with dosing, and gellan gum was not carcinogenic when fed to Sprague-Dawley rats. 10 CIR Panel Book Page 17

22 Dextran A group of 15 male and 15 female ACI rats were fed a diet containing 2.5% dextran (21,500 MW) for 480 days, and the control group of 20 males and 20 females was fed a basal diet. 57 Body weights gains of treated male rats were statistically significantly decreased compared to controls. An increased incidence in tumors was not reported, and no intestinal tumors were found. Sodium Dextran Sulfate A number of studies have demonstrated that oral exposure to sodium dextran sulfate produces colon carcinogenesis in rats; the mechanism is not genotoxic. Oral administration has been shown to induce colonic inflammation, and a 2-day study 58 in which female Fischer 344 rats were given 3 or 6% sodium dextran sulfate in the drinking water indicated that oxidative DNA damage occurred in the colonic mucosa. The molecular weight of sodium dextran sulfate has been found to be a factor in carcinogenic activity. While an extensive number of studies are available in the published literature, just a few are summarized below. An example of an inflammation-related mouse colon carcinogenesis models is described, with an indication of strain-dependency. In one study evaluating the carcinogenic potential of sodium dextran sulfate (54,000 avg. MW; sulfur content 18.9%) in ACI rats, 10 males were fed a diet containing 10% sodium dextran sulfate, 14 males and 12 females were fed 5% in the diet, and a control group of 9 males and 9 females were given a basal diet. 59 All animals were necropsied at natural death or when killed due to moribund condition. All animals in the 10% group died 6-14 days after initiation of dosing, and all had severe acute nephrosis. Two animals of the 5% group died on day 14, but most of the remainder of this group lived for more than 130 days. Blood was observed on the surface of the stools of these animals at 2.5 mos. The weight gain of animals of this group was decreased compared to controls. Of the 23 rats that survived for more than 130 days, 15 rats developed intestinal tumors; tumors included 5 adenomas, 5 adenocarcinomas, and 3 papillomas in the colon and 6 adenomas and 2 adenocarcinomas in the cecum. While most rats had a single tumor upon gross observation, microscopic examination found multicentric foci of atypical hyperplasia of the glandular epithelium. No intestinal tumors were reported in the control group. In a second study testing the same sodium dextran sulfate, 15 male and 15 female ACI rats were fed 1% in the diet for 660 days; the avg. daily intake was 0.15 g/day/animal. 60 A control group of 10 males and 10 females were fed a basal diet. All but 2 treated male rats survived for 350+ days. Body weight gains of the test group were similar to that of the controls. Intestinal tumors were observed in 22 treated rats; 16 papillomas, 4 squamous cell carcinomas, 2 adenomas, and 5 adenocarcinomas were reported in the colon and rectum and 1 adenocarcinoma was found in the cecum. Thirteen tumors were reported at miscellaneous sites. Again, no intestinal tumors were reported in the control group. To examine the effect of molecular weight, three groups of 15 male and female ACI rats were fed for 480 days a diet containing 2.5% of a sodium dextran sulfate, MW 520,000, 54,000,or 9500; each sodium dextran sulfate had a sulfur content of 18-19%. 57 (The 54,000 MW substance was synthesized using the dextran described previously.) A control group of 20 male and 20 female rats was fed a basal diet. There was no significant difference in survival time between any of the groups. Body weights gains of male rats of the 54,000 MW diet were statistically significantly decreased compared to control males; body weight gains in the other two groups were similar to controls. The 54,000 MW sodium dextran sulfate had the strongest carcinogenic activity, with tumors being reported similar to the studies described previously. The other two MW substances did not show significant carcinogenic activity; only 2 colorectal adenocarcinomas in the 520,000 MW group. Colorectal squamous metaplasia was observed in most rats in all test groups. Other miscellaneous tumors were reported, but there was no statistically significant difference between treated and control groups. A colitis-related mouse colon carcinogenesis model was developed. 61 When 8 male Crj:CD-1 mice were given a single i.p. injection of 10 mg/kg azoxymethane (AOM), followed by 7 days of 2% sodium dextran sulfate in the drinking water 1 wk later, there was a 100% incidence of colonic adenocarcinomas and 38% of adenomas at wk 20. No adenocarcinomas were observed in any of the mice dosed with AOM and sodium dextran sulfate simultaneously, 1 wk of sodium dextran sulfate and then AOM, AOM only, or sodium dextran sulfate only, and only 2 adenomas (in 10 mice) were observed when AOM was given during sodium dextran sulfate administration. All of the adenocarcinomas were positive for β-catenin, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (inos) and negative for immunoreactivity of p53. Strain-sensitivity to the above model was then examined. 62 In testing with male Balb/c, C3H/HeN, C57BL/6N, and DBA/2N mice, it was found that Balb/c mice were very sensitive to the model. C57BL/6N mice also developed colonic adenocarcinomas, but to a lesser extent. C3H/HeN and DBA/2N mice developed only a few colonic adenomas. However, 11 CIR Panel Book Page 18

23 the greatest inflammation response was observed in C3H/HeN mice, followed by Balb/c mice. The researchers stated hypothesized that the strain differences in susceptibility of colon carcinogenesis induced by AOM and sodium dextran sulfate might be influenced by the response to nitrosation stress due to inflammation as determined by the genetic background. Anti-Tumor Effects Xanthan Gum Groups of C57BL/6 mice were inoculated subcutaneously (s.c.) with 1 x 10 6 B16K b melanoma cells. 63 A suspension of 10 mg/ml xanthan gum, 100 µl, or PBS was given by gavage once every 5 days, starting 1 day prior to inoculation. Rapid tumor growth occurred in PBS-treated mice, but tumor growth was statistically significantly reduced in xanthan gum-treated mice. Spleen cell composition was analyzed 22 days after inoculation. There was no change in overall cellular composition, but natural killer (NK) cells and tumor-specific cytotoxic T-lymphocyte (CTL) activity were increased by xanthan gum. All PBS-treated mice died by day 46 after inoculation; 40% of the xanthan gum-treated mice were alive at day 100. The researchers demonstrated that the anti-tumor effect of xanthan gum was highly dependent on Toll-like receptor (TLR) 4-mediated signaling. Pullulan Male Balb/c mice were used to determine the anti-tumor and anti-metastatic potential of pullulan (water-soluble low-molecular weight [LMW] β-(1 3)- with 50-80% branched β-(1 6); MW 100,000). 64 Colon-26 cells were implanted into the spleens of mice on day 0, and groups of mice were dosed orally with 25 or 50 mg/kg or i.p. with 5 or 15 mg/kg pullulan for 14 consecutive days, starting 12 h after implantation. Control groups consisted of sham-operated mice (not implanted with colon-26 cells) and mice implanted with the cells but given physiological saline or distilled water instead of pullulan. Splenic tumor weights were reduced with the 50 mg/kg oral and 15 mg/kg i.p. doses of pullulan, but not with the other doses. Liver metastasis was significantly inhibited with 50 (but not 25) mg/kg oral and 5 and 15 mg/kg i.p. pullulan. Levan The anti-tumor activity of levan produced from four different microorganisms, i.e., Gluconoacetobacter xylinus, Rahnella aquatilis, Zymomonas mobilis, and Microbacterium laevaniformans (G-levan, R-levan, Z-levan, and M-levan, respectively) was evaluated using female ICR mice. 65 The molecular weights of these levans were 40,000, 380,000, 570,000, and 710,000, respectively. On day 0, 0.2 ml (equiv. to 3 x 10 6 cells) of sarcoma-180 tumor cells were implanted s.c. into the mice. Groups of mice were then dosed daily on days 1 to 7 with 200 mg/kg of the levans in distilled water, and killed on day 26. The tumor growth inhibition ratio (I.R.) ranged from % for the four levans. M-Levan had the highest I.R. value, but it was not statistically significant. The I.R. for R- and Z-levan were comparable, and the G-levan was significantly less effective. IRRITATION AND SENSITIZATION Non-human and human dermal irritation and sensitization studies are summarized in Table 10. The dermal irritation and sensitization potentials of xanthan gum, beta-glucan, and sodium carboxymethyl beta-glucan were evaluated in animal studies. Xanthan gum, up to 5% aq., and beta-glucan, concentration not specified, were not irritating to rabbit skin. Neither xanthan gum, tested at 0.1%, nor beta-glucan (concentration not specified) were sensitizers in guinea pigs. Sodium carboxymethyl beta-glucan was at most a slight skin irritant in guinea pigs at a concentration of 50% aq.; a 10% aq. solution was not a sensitizer in guinea pigs. In humans, neither beta-glucan nor sodium carboxymethyl beta-glucan were irritants or sensitizers. The test concentration of beta-glucan was not specified. Sodium carboxymethyl beta-glucan was applied neat in the irritation study and as a 2% aq. solution in the sensitization study. Phototoxicity A human photoallergenicity study is also summarized in Table 10. A 2% aq. solution of sodium carboxymethyl beta-glucan was not photosensitizing in clinical studies. Adverse Effects Dextran Over a 5-yr period, 12,646 dextran 70 units were administered to 5745 patients (mean of 2.2 units/patient) undergoing gynecological surgery or a Cesarean section. 66 Fifteen immediate anaphylactoid reactions were reported, with an incidence of one reaction/383 patients treated. Life-threatening reactions occurred in 7 of these patients. 12 CIR Panel Book Page 19

24 Ocular Irritation Ocular irritation studies are summarized in Table 11. Xanthan gum, 1%, and up to 0.8% gellan gum were not ocular irritants in rabbit eyes, and up to 0.5% gellan gum was not irritating to human eyes. The ocular irritation potential of sodium carboxymethyl beta-glucan was described as weakly irritating in a HET-CAM assay, but the ingredient was practically nonirritating in rabbit eyes. SUMMARY Microbial polysaccharides can be produced intercellularly, by the cell wall, or exocellularly. Exocellular polysaccharides constantly diffuse into the cell culture medium and are easily isolated, while cell wall and intercellular polysaccharides are more difficult to separate from cell biomass. Many of the 34 microbial polysaccharides discussed in this safety assessment are produced exocellularly. They are reported to have numerous functions in cosmetics, including emulsion stabilizer, film former, binder, viscosity increasing agent, and skin conditioning agent. The same microbial polysaccharides can often be produced by more than one microorganism. For example, levan can be produced by bacteria, yeasts, or fungi. The properties of the microbial polysaccharides can vary widely based on, among other parameters, the side groups, the ester substituents, or bacterial strains. These polysaccharides are generally very large molecules, and the molecular weight of each ingredient can vary considerably. Xanthan gum is reported to be used in almost every category of cosmetic product, with 2964 reported uses. Biosaccharide gum-1, sclerotium gum, and beta-glucan are reported to be used in 302, 182, and 116 cosmetic formulations, respectively. All other in-use ingredients have less than 60 uses. The ingredient with the highest concentration of use is pullulan; it is used at up to 12% in leave-on formulations and 17% in rinse-off formulations. Both xanthan gum and biosaccharide gum- 1 are used at up to 6% in leave-on formulations and xanthan gum crosspolymer and biosaccharide gum-4 are used at 5% in leave-on formulations. All other in-use ingredients are used at concentrations of 3%. Xanthan gum, gellan gum, and beta-glucan are approves as direct food additives, and xanthan gum and dextran are approved indirect food additives. Xanthan gum and dextran also have pharmaceutical applications. Xanthan gum, orally administered, is slowly broken down in the gut by enzymatic and non-enzymatic mechanisms, and can be absorbed in some form to some extent. The absorbed fraction does not accumulate in the tissues and can be completely metabolized to CO 2. Gellan gum, orally administered, does not breakdown to any substantial extent in the gut, and is only very poorly absorbed. Dextrans, 3,000-20,000 MW, are not absorbed through intact skin in humans. They are absorbed through the dermis (up to 38% for 3000 MW) if the epidermis is removed (e.g., via mini-erosion); absorption in this case is inversely proportional to MW. Orally administered dextrans, 4400 to 40,500 MW, are not absorbed to any appreciable extent in the gut (very low bioavailability). If dextrans were absorbed to a significant extent through the skin, animal studies in which dextans were administered i.v. indicate that their half-lives in blood plasma would be directly related to the MW; they would be excreted in the urine to an extent that is inversely related to the MW; the lower MW dextrans would tend to be readily excreted unchanged in the urine; and the higher MW dextrans would have the potential to accumulate and to be broken down in the liver and other tissues, and would be more likely to be excreted in the urine in a dose-dependent manner than the lower MW dextrans. Dextran sulfate, 7000 to 8000 MW, orally administered, is only very poorly absorbed in the human GI tract. It is absorbed to some extent in the GI tract, most likely as intact or partially intact molecules. After i.v. administration, it is rapidly excreted, mostly intact, in the urine, although at least some of it can accumulate in the tissues (to a substantially greater extent than observed after oral exposure), and some of it appears to be incorporated into glycogen and other substances in the body. Beta-glucan in a topically applied solution can penetrate into the epidermis and dermis. There appears to be no information about its absorption through the skin into the bloodstream. Orally administered, it is readily metabolized to CO 2, at least partially by microflora in the gut. Beta-glucan is not well absorbed or eliminated after i.p. injection. Pullulan, orally administered, is hydrolyzed to some small extent in the gut. It is partially hydrolyzed by amylases in the upper GI tract of human subjects, and is subject to breakdown by intestinal microflora to form short-chain fatty acids; the rate of the latter depends on the degree of polymerization. However, pullulan can be essentially completely broken down to short-chain fatty acids in the human gut. It has not been determined to what extent, if any, the products of hydrolysis can be absorbed. The acute toxicity of xanthan gum, gellan gum, beta-glucan, sodium carboxymethyl beta-glucan, and pullulan was assessed orally in mice, rats, and/or dogs, and dextran sulfate and beta-glucan were tested by i.p. and i.v. dosing in mice and 13 CIR Panel Book Page 20

25 rats. There was no notable toxicity observed in these studies. In acute inhalation studies, the LC 50 of xanthan gum was >21 mg/l in rabbits and of gellan gum was >5.06 mg/l in rats. The inflammatory response following a single exposure to beta-glucan (as curdlan) and to pullulan was also evaluated in guinea pigs. Mostly, no effect or a slight decrease in inflammatory cells in lung lavage was observed. A 4 h inhalation exposure to beta-glucan in dust by guinea pigs produced a delayed subacute nasal congestion when compared to dust without beta-glucan and resulted in decreased nasal volume. In humans, inhalation exposure to beta-glucan in dust for 18 h resulted in decreased nasal volume, increased nasal eosinophil cells, and increased lung lymphocytes and macrophages, but did not produce microscopic change in lung tissue. Industrial exposure to xanthan gum powder did not appear to cause significant acute or chronic pulmonary effects. Using artificial skin, 5% levan had an anti-inflammatory effect in irritated skin. Repeated dose oral toxicity of xanthan gum was evaluated in rats and dogs, of gellan gum in rats, dogs, and monkeys, of dextran in rats, of beta-glucan in mice, rats, and dogs, and of pullulan in rats. Most of the studies were dietary, and study duration lasted up to 2 yrs. Most observations were related to changes in feed consumption and intestinal effects. Inhalation exposure to 100 µg/ml beta-glucan (as curdlan), 4 h/day, 5 days/wk for 4 wks, did not have an effect on the cells of the lung lavage or cell wall, and there were no microscopic lesions of the lung. With i.p. administration, no toxicity was reported when mice were dose 10 times over 2 wks with 5 mg xanthan gum in 0.5 ml water or mice or guinea pigs were dosed with 250 mg/kg bw beta-glucan for 7 days. Intravenous administration of 40 and 1000 mg/kg bw beta-glucan for 30 days resulted in hepatosplenomegaly in mice. No toxic effects were observed in human subjects with oral ingestion of 150 mg/kg/day xanthan gum or mg/kg/day gellan gum for 23 days, 10 g of dextran or pullulan for 14 days, or 6-50 g/day beta-glucan for up to 28 days. No significant or chronic effects in pulmonary function were reported in groups exposed occupationally to xanthan gum. Dietary reproductive and developmental toxicity studies were conducted with xanthan gum, gellan gum, and betaglucan. No reproductive or developmental effects were reported in a three-generation reproductive study in which rats were fed diets containing up to 5 g/kg bw/day xanthan gum. Gellan gum, up to 5%, did not have a fetotoxic or teratogenic effect on rats. Dietary administration of beta-glucan in rats in reproductive and developmental studies did not have any reproductive effects, but there were statistically significant decreases in body weights and body weight gains in offspring and parental animals. In a teratogenicity study in which rabbits were dosed with 5 g/kg bw/day beta-glucan, an increase in resorptions in the 5 g/kg group was considered similar to the other test groups and the controls, and 5 g/kg beta-glucan was not teratogenic in rabbits. The in vitro genotoxicity of gellan gum ( 20 mg/ml), sodium dextran sulfate ( 25 mg/plate), beta-glucan ( 5000 µg/plate or /ml), sodium carboxymethyl beta-glucan ( 50,000 µg/plate or /ml) and pullulan ( 12 mg/ml) was evaluated in Ames test, chromosomal aberration assays, and/or DNA repair tests, with and without metabolic activation. Results were negative in all of these tests. The only non-negative result was a weak positive result with 20 mg/plate pullulan in a rec assay using Bacillus subtilis. Negative results were also reported in in vivo mouse micronucleus tests with 5000 mg/kg betaglucan and 1800 mg/kg pullulan. Dietary studies examining the carcinogenic potential of 5% gellan gum and 2.5% dextran reported that neither of these ingredients caused an increase in tumors. However, a number of studies have demonstrated that oral exposure to sodium dextran sulfate produces colon carcinogenesis in rats, and the mechanism is non-genotoxic. In one study, the molecular weight of sodium dextran sulfate was a factor in carcinogenic activity; a 54,000 MW sodium dextran sulfate produced colorectal tumors, but 9500 and 520,000 MW sodium dextran sulfate did not have significant carcinogenic activity. Oral administration has been shown to induce colonic inflammation, and a 2-day study in which female Fischer 344 rats were given 3 or 6% sodium dextran sulfate in the drinking water indicated that oxidative DNA damage occurred in the colonic mucosa. An inflammation-related mouse colon carcinogenesis model indicated that the development of colonic tumors is strain-dependent, and that Balb/c mice were very sensitive to the model. C57BL/6N mice also developed tumors, but to a lesser extent, while C3H/HeN and DBA/2N mice only developed a few tumors. Oral administration of 10 mg/ml xanthan gum had an anti-tumor effect in mice inoculated with melanoma cells, and 50 mg/kg pullulan, but not 15 mg/kg, significantly inhibited tumor growth in mice following implantation of colon-26 cells. Levan did not significantly inhibit tumors growth following sarcoma-180 tumor cell implantation in mice. The dermal irritation and sensitization potentials of xanthan gum, beta-glucan, and sodium carboxymethyl betaglucan were evaluated in animal studies. Xanthan gum, up to 5% aq., and beta-glucan, concentration not specified, were not 14 CIR Panel Book Page 21

26 irritating to rabbit skin, and neither xanthan gum, tested at 0.1%, nor beta-glucan (concentration not specified) were sensitizers in guinea pigs. Sodium carboxymethyl beta-glucan was at most a slight skin irritant in guinea pigs at a concentration of 50% aq.; a 10% aq. solution was not a sensitizer in guinea pigs. In humans, neither beta-glucan or sodium carboxymethyl beta-glucan were irritants or sensitizers. The test concentration of beta-glucan was not specified. Sodium carboxymethyl beta-glucan was applied neat in the irritation study and as a 2% aq. solution in the sensitization study. A 2% aq. solution of sodium carboxymethyl beta-glucan was not photosensitizing in clinical studies. Xanthan gum, 1%, and gellan gum, up to 0.8%, were not ocular irritants in rabbit eyes, and up to 0.5% gellan gum was not irritating to human eyes. The ocular irritation potential of sodium carboxymethyl beta-glucan was described as weakly irritating in a HET-CAM assay, but the ingredient was practically non-irritating in rabbit eyes. To be developed. To be determined. DISCUSSION CONCLUSION 15 CIR Panel Book Page 22

27 TABLES Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Xanthan Gum a high molecular weight heteropolysaccharide gum produced by a pure-culture fermentation of a carbohydrate with Xanthomonas campestris; 13 composed of glucose, glucuronic acid, 6-acetylmannose, and 4,6- pyruvylated mannose residues 67 binder; emulsion stabilizer; skin conditioning agent-misc.; surfactantemulsifying agent; viscosity increasing agent-aq. (given below definition) Hydroxypropyl Xanthan Gum the hydroxypropyl ether of xanthan gum, 13 wherein ether substitution occurs primarily at 6-position of the backbone sugar residues emulsion stabilizer; film former; viscosity increasing agent-aq. 16 CIR Panel Book Page 23

28 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Undecylenoyl Xanthan Gum the condensation product of undecylenic acid chloride and xanthan gum, 13 wherein esterification occurs primarily at the 6-position of backbone sugar residues emulsion stabilizer; hair conditioning agent OR OR O wherein R = H or CH 3 O O H O O OH HO OH O OH CH 3 C(O)O O O O HO HO O HO O O HO O H 3 C O O Dehydroxanthan Gum [dehydro is co-listed under this CAS No. with Xanthan Gum] HO OH HO the product obtained by the dehydration of xanthan gum 13 OH emulsion stabilizer; film former; hair fixative; suspending agent-nonsurfactant; viscosity increasing agentaq. Xanthan Gum Crosspolymer xanthan gum crosslinked with disodium sebacate 13 skin protectant; skin conditioning agent-misc. Crosslinked with: 17 CIR Panel Book Page 24

29 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Xanthan Hydroxypropyl Trimonium Chloride H 3 C CH 3 HO N CH 3 the quaternary ammonium salt formed by the reaction of Xanthan Gum with 2,3-epoxypropyltrimethylammonium chloride OR OR hair conditioning agent; skin conditioning agent-humectant; viscosity increasing agent-aq wherein R = H or Cl O O H O O OH HO OH O OH CH 3 C(O)O O O O HO HO O HO O O HO O H 3 C O O HO OH HO OH Other Gums Gellan Gum a high molecular weight heteropolysaccharide gum produced by pure-culture fermentation of a carbohydrate with Pseudomonas elodea; 13 consists of the basic backbone 3)-β-D-Glc-(1 4)-β-D-GlcA-(1 4)-β- D-Glc-(1 4)-α-L-Rha-(1 ); 8 composed of glucose (some of which is glyceryl and/or acetyl esterified), glucuronic acid, and rhamnose residues 9 emulsion stabilizer; viscosity increasing agent-aq. CH 3 O O OH OH O H 3 C O O O O HO O O O OH H O O HO OH HO OH HO OH O OH OH 18 CIR Panel Book Page 25

30 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Welan Gum a gum produced by the fermentation of Alcaligenes; 13 consists of the basic backbone 3)-β-D-Glcp-(1 4)- β-d-glcpa-(1 4)-β-D-Glcp-(1 4)-α-L-Rhap- ( α-l-rhap or α-l-manp; composed of glucose, glucuronic acid, rhamnose and mannose residues, with rhamnopyranosyl and/or L-mannopyranosyl side chains 68 binder; emulsion stabilizer; film former; viscosity increasing agentaq. wherein R is CH 3 or CH 2 OH and R is H or C(O)CH 3 Sclerotium Gum the polysaccharide gum produced by the fungus, Sclerotium rolfsii; 13 consists of the basic backbone of β-d-(1 3)-glucopyranosyl units with a β-d-glucopyranosyl unit (1 6) linked to every third unit 69 emulsion stabilizer; skin conditioning agent-misc.; viscosity increasing agent-aq. Hydrolyzed Sclerotium Gum the partial hydrolysate of sclerotium gum derived by acid, enzyme, or other method of hydrolysis 13 film former; skin protectant; skin conditioning agent-humectant 19 CIR Panel Book Page 26

31 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Rhizobian Gum [for Rhizobium Gum] Distributed for Comment Only -- Do Not Cite or Quote the polysaccharide gum produced by the fermentation by Rhizobium bacterium; 13 composed of glucose, glucuronic acid, galactose and pyruvylated galactose residues, with some acylation 70 film former; hair fixative; plasticizer; suspending agent-nonsurfactant; viscosity increasing agent-aq Hydrolyzed Rhizobian Gum Biosaccharide Gum the partial hydrolysate of Rhizobian Gum derived by acid, enzyme, or other method of hydrolysis 13 a fermentation gum derived from sorbitol; described as a polymer of α-l-fucose(1 3)α-D-galactose(1 3)-α- D-galacturonic acid trisaccharides; 13 composed of fucose, galactose, and galacturonic acid residues film former skin conditioning agent-misc. 20 CIR Panel Book Page 27

32 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Biosaccharide Gum a fermentation gum derived from sorbitol; described as a polymer of α-l-rhap(1 3)-β-D-Galp-(1 2)-α-L- Rhap-(1 4)-β-D-GlepA (1 3)-[α-L-Rhap-(1 2)-]- α-d-galp-(1; 13 composed of rhamnose, galactose, and glucuronic acid residues skin conditioning agent-misc. Biosaccharide Gum a fermentation gum derived from sorbitol; described as a polymer of α-l-fucose(1"3)-α-d-galactose(1"3)-α- D-galacturonic acid trisaccharides, and is characterized by a smaller degree of polymerization and molecular weight in comparison to biosaccharide gum skin conditioning agent-misc. 21 CIR Panel Book Page 28

33 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Biosaccharide Gum a fermentation gum derived from sorbitol; it is a deacetylated branched polymer consisting of L-fucose, 2-D-glucose and glucuronic acid repetitive units 13 skin conditioning agent-misc. Biosaccharide Gum-5 a fermentation gum derived from sorbitol; described as a polymer of glucose-β-(1-->2)-rhamnose-α-(1-->4)- glucose-α-(1-->3)-rhamnose-β-(1-->4), with rhamnose α-(1-->3) branching 13 skin conditioning agent-misc. Pseudoalteromonas Exopolysaccharides the exopolysaccharide excreted by the fermentation of Pseudoalteromonas skin conditioning agent-misc. 22 CIR Panel Book Page 29

34 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Dextran a high molecular weight glucose polymer produced by the action of the bacteria, Leuconostoc mesenteroides, on a sucrose substrate 13 ; the degree of branching is dependent on the source of dextrans and can vary from % 71 binder; bulking agent Carboxymethyl Dextran the carboxymethyl derivative of dextran 13 film former; viscosity increasing agent-aq. Dextran Hydroxypropyltrimonium Chloride the quaternary ammonium compound formed by the reaction of dextran with glycidyltrimethylammonium chloride 13,72 hair conditioning agent 23 CIR Panel Book Page 30

35 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Sodium Carboxymethyl Dextran H O the sodium salt of a carboxymethyl dextran 13 O Distributed for Comment Only -- Do Not Cite or Quote binder; emulsion stabilizer; viscosity increasing agent-aq. RO OH RO OR H O O O wherein R = H, Na O, or a branch of RO O RO OR Dextran Sulfate H O RO the sulfuric acid ester of dextran 13 O OH binder; skin conditioning agent-misc. RO OR H O O O wherein R = H, HO S, or a branch of RO O O RO OR Sodium Dextran Sulfate the sodium salt of the sulfuric acid ester of dextran 13 suspending agent-nonsurfactant 24 CIR Panel Book Page 31

36 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Beta-Glucan [CAS No. is specific to (1 3)(1 4)] [CAS No. is specific to (1 3)(1 6)] [sic; this CAS No. is for Glucanase, the enzyme] a polysaccharide consisting of β(1-3), β(1-4), and β(1-6) linked glucose units 13 bulking agent; skin conditioning agent-misc Beta-Glucan Hydroxypropyltrimonium Chloride the quaternary ammonium compound formed by the reaction of beta-glucan with glycidyltrimethylammonium chloride anti-static agent; hair conditioning agent; skin conditioning agent - misc Beta-Glucan Palmitate the product obtained by the condensation of betaglucan with palmitoyl chloride 13 skin conditioning agent - misc Hydrolyzed Beta-Glucan the partial hydrolysate of beta-glucan, derived by acid, enzyme or other method of hydrolysis skin conditioning agent - misc 25 CIR Panel Book Page 32

37 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Oxidized Beta-Glucan the product obtained by the oxidation of beta-glucan, 13 wherein the 6-position is oxidized to the acid 73 anti-acne agent; anti-dandruff agent; anti-fungal agent; antimicrobial agent; deodorant agent; exfoliant; skin conditioning agent - misc Sodium Carboxymethyl Beta-Glucan [CAS No. specific to (1 3) β-d-glucan] the sodium salt of a carboxymethyl ether of betaglucan, wherein the ether substitution occurs primarily at the 6-position 74 ; 3 of 4 glucose-units of the β-1,3-glucopyranose are carboxymethylated 75 binder; viscosity increasing agent - misc Pullulan an extracellular polysaccharide produced from starch by cultivating the yeast, Aureobasidium pullulans; composed of 1 6 linked maltitriose residues (a maltitriose is a block of three 1 4 linked, glucose resides) 76 binder; film former Myristoyl Pullulan the reaction product of myristoyl chloride and pullulan 13 film former; hair fixative; hair-waving/straightening agent 26 CIR Panel Book Page 33

38 Table 1. Definition, Function, and Idealized Structure Ingredient; CAS No. Definition Reported Function(s) 13 Idealized Structure Levan a polyfructose (fructofuranose), with some branching, produced by enzymatic action on sucrose 76 ; the degree of branching varies by organism, but has been reported as high as 20% 77 film former; skin protectant; viscosity increasing agent aq. Alcaligenes Polysaccharides [for the pure disaccharide repeat unit] the polysaccharides produced by a bacterial culture of Alcaligenes latus; composed of mannose and fucose 78 emulsion stabilizer; humectant; skin conditioning agent humectant; viscosity increasing agent-aqueous 27 CIR Panel Book Page 34

39 Table 2. Chemical and physical properties Property Description Xanthan Gum appearance cream-colored, odorless, free-flowing powder 33 white/beige powder with a characteristic odor 79 molecular weight 1,000,000 10,000,00 31 varies within a very wide range 80 solubility dissolves readily in water with stirring to give highly viscous solutions at low concentrations 33 completely soluble in water, forming colloidal solution; insoluble in alcohol 79 readily soluble in hot or cold water to form neutral, viscous, and nonthixotropic solutions that have relatively high viscosity 31 stability resistant to heat degradation 33 ionic nature anionic 6,76 ph (1% solution, 25 C) 79 Gellan Gum appearance off-white powder types native, deacetylated, clarified (i.e., filtered deacetylated gum) 81 molecular weight varies within a very wide range 80 ~500, >70,000, with 95% above 500, solubility soluble in hot or cold deionized water 82 soluble in water; insoluble in ethanol 81 viscosity can exhibit high viscosity in solution 8 high-acyl gellan gum is viscous; deacylated gellan gum (treated with an alkali) has relatively low solution viscosity 10 cold dispersions of native gellan gum provide extremely high viscosities, and the solutions are highly thiotropic; the viscosity decreases with heating as the gum hydrates; hot native gum solutions are more viscous than deacylated gellan gum solutions 10 gelling property forms a weak gel in water in its native state, but forms a rigid gel after treatment with an alkali 9 ionic nature anionic 6,76 hydration native (acylated) gellan gum will swell in deionized water forming a very thick particulate system, and the hydration temperature is reached at ~70 C; the swelling behavior and hydration temperature is altered in the presence of ions 10 deacylated gum will only partially hydrate in cold deionized water, with hydration occurring with a heated deionized water temperature of ~70 C; also hydration is poor in mildly acidic conditions and in the presence of divalent ions 10 Welan Gum molecular weight 865, , viscosity non-gelling polysaccharide forming highly viscous aq. solutions 83 ionic nature anionic 6 Biosaccharide Gum-1 molecular weight (avg) 84 Biosaccharide Gum-4 molecular weight (avg) 84 Dextran appearance Dextran 1: white to off-white powder 33 molecular weight Dextran 1: ~1000 (avg) 85 Dextran 40: ~25,000 (solution has an avg MW of 40,000) 66 Dextran 70: ~40,000 (solution has an avg MW of 70,000) 66 molecular weight distribution is dependent on the source of the dextran 71 solubility Dextran 1: very soluble in water; sparingly soluble in alcohol 33 degree of solubility decreases with an increase in the degree of branching; dextrans with >43% branching are insoluble 71 ionic nature nonionic 6,76 stability stable under mild acidic and basic conditions 71 ph Dextran 1: (15% aq. solution) 85 specific rotation Dextran 1: between +148 and +164 at 20, for an aq. solution 85 Dextran 40: between +195 and Dextran 70: between +195 and Dextran Sulfate molecular weight , CIR Panel Book Page 35

40 Table 2. Chemical and physical properties Property Description Sodium Dextran Sulfate appearance white powder 33 molecular weight , solubility freely soluble in water 33 Sclerotium Gum solubility disperses easily in water at room temperature; refined grades dissolve readily in hot and cold water 5 ionic nature nonionic 6,76 Beta-Glucan appearance white to pale yellow powder with a slight odor 86 white to nearly white powder (as curdlan) 82 molecular weight ~500,000 (native state) 87 solubility as curdlan: insoluble in water, alcohol, and most organic solvents; soluble in alkaline solutions 5 ionic nature nonionic 6 Oxidized Beta-Glucan molecular weight continuum of ~30,000 to >70, Sodium Carboxymethyl Betaglucan appearance white/brown solid 88 amber, white granulate with a characteristic isopropyl alcoholic odor 75 molecular weight ~100, x 10 5 (avg) 74 degree of substitution 0.75 ±1 75,88 (as a 2% aq. solution) solubility solubility up to 98% 74 ph (2% aq. solution) ~7 88 Pullulan appearance white to slightly yellowish powder; tasteless; odorless (food grade) 89 tasteless, odorless fine white powder 90 molecular weight can vary considerably; a commercially available product has a molecular mass of 200,000 Da >2,000,000; approx. 200,000 (mean) 89 solubility highly soluble in cold or hot water; not soluble in organic solvents, except dimethylformamide or dimethyl sulfoxide (DMSO) 30,89 stability stable in aq. solution over a wide ph range; decomposes upon dry heating, carbonizing at C 29 viscosity dissolves in water producing a stable viscous solution; does not gel; viscosity is proportional to molecular weight 89 solutions are of relatively low viscosity; viscosity is stable to heating, changes in ph, and most metal ions 91 ionic nature nonionic 6,76 ph (food grade) 89 refractive index significant positive linear correlation of concentration and refractive index at 20 and 45 C 89 Levan molecular weight up to several million Daltons; typically 2x10 6 to usually >0.5 million, and can be as high as 40,000, particle size partially forms nanoparticles in water; nm in water and nm in ethanol 52 solubility highly soluble in water at room temperature 12 water soluble; does not swell in water 77 viscosity exceptionally low intrinsic viscosity 77 ionic nature nonionic 76 Rhizobian Gum molecular weight 1,500,000 (native molecule, in the fermentation broth) 70 melting point ~60 C; gets lower after sterilization 92 viscosity with a 10 g/l solution, the viscosity decreases as the ph increases 70 Alcaligenes Polysaccharides molecular weight 64, CIR Panel Book Page 36

41 Table 3. Constituents/Impurities Ingredient Xanthan Gum Constituents/Impurities nitrogenous constituents equal to approx.. 1% nitrogen by wt; approx.. half of the nitrogenous matter is proteinaceous and contains amino acid residues in the same proportions as other food-grade gums; the remainder is present as amino sugars, nucleic acids, and nucleotides 27 food-grade: contains D-glucose and D-mannose as the dominant hexose units, and D-glucuronic acid and pyruvic acid; NMT 2 mg/kg lead; NMT 0.075% ethanol and isopropyl alcohol, singly or combined % pyruvic acid, present as side chains 31 Gellan Gum usually contains a small amount of nitrogen-containing compounds as a result of the fermentation procedure 81 < 2 mg/kg lead; <3% nitrogen;,750 mg/kg isopropyl alcohol 81 native: can contain 10% protein and 7% ash; deacetylated: can contain 17% protein and 8% ash 81 gellan gum is characterized by the polysaccharide content, percent of o-acetylated substitution, and protein content (including nucleic acid residues and other organic nitrogen sources) 37 Dextran Sulfate can contain polymers of various molecular weights and degrees of sulfation 39 Beta-Glucan 85% β-1,3-1,6-glucan, sucrose, yeast extract, minerals, Auerobasidium pullulans 86 Sodium Carboxymethyl Betaglucan Distributed for Comment Only -- Do Not Cite or Quote as curdlan: 90% carbohydrate; 10% water 5 as food grade curdlan NMT 0.5 mg/kg lead; microbial limits, aerobic plate count NMT 1000 CFU/g; Escherichia coli, negative in 1 g 94 ~90% sodium carboxymethyl betaglucan; <0.5% nitrogen (protein); <1.0% glycolic acid; <0.05% chloroacetic acid; ~10% volatile matter 75 Pullulan 90% glucan on a dried basis; main impurities are mono-, di-, and oligosaccharides from the starting material 29 >90% pullulan; <10% mono-, di-,and oligosaccharides; <0.1 ppm lead; <2 ppm arsenic; <5 ppm heavy metals (food grade) 89 Table 4. Methods of Manufacture/Microorganisms Used in Production Ingredient Method Xanthan Gum pure-culture fermentation of glucose or corn syrup from the bacterium Xanthomonas campestris; the polysaccharide is recovered by precipitation and purification with isopropyl alcohol, followed by drying and milling 31,55 Gellan Gum aerobic submerged fermentation using the bacterium Pseudomonas elodea; small seed fermentation is followed by pasteurization; the gum is recovered by precipitation with isopropyl alcohol, followed by drying and milling 95 pure culture fermentation of carbohydrates by Pseudomonas elodea, purified by recovery with isopropyl alcohol, dried, and milled 81 gellan gum can be recovered using alcohol precipitation (high acyl gum) or with alkali (deacylated gum) 10 produced by an organism that appears to belong to the Auromonas (ATCC 31461) genus; glycerate substitution predominates over acetate 8 Sphingomonas paucimobilis produce gellan gum 81 production is affected by media components, carbon source, nitrogen source, precursors, agitation rate, ph, temperature, and oxygen 81 Welan Gum fermentative production by Alcaligenes CGMCC produced by an Alcaligenes species (ATCC 31555) 8,83 Dextran Dextran 40; Dextran 70: obtained by the controlled hydrolysis and fractionation of polysaccharides elaborated by the fermentative action of certain appropriate strains of Leuconostoc mesenteroides on a sucrose substrate 85 the fermentation process for reaching high MW dextran takes place at 25 C; at lower temperatures, the amount of low MW dextran increases; at >25 C, higher branching occurs 7 different strains of the same bacterium produce dextrans with differing branched structures 66 the sucrose concentration also affects branching; increased sucrose content the degree of branching and the yield of high MW dextran decreases 7 dextran can be synthesized by dextrinase of different Gluconobacter species 7 dextran can be produced enzymatically using cell-free culture supernatants that contain dextransucranase 7 dextran can be synthesized chemically via a cationic ring-opening polymerization of levoglucosan 7 Carboxymethyl Dextran carboxymethylation of dextran in water/organic solvent mixtures using monochloroacetic acid under strong alkaline conditions 7 Sclerotium Gum produced by Sclerotium glucanicum 76 or Sclerotium rolfsii 5 Beta-Glucan extraction of extracellular β-glucan produced by the black yeast Aureobasidium pullulans 86 produced by fungi, yeasts, and grains (such as oat and barley); beta-glucans present in cereal bran are commonly produced as agricultural by-products 11 as curdlan: pure-culture fermentation of a carbohydrate by a nonpathogenic and nontoxigenic strain of Agrobacterium biobar 1 (formerly Alcaligenes faecalis var. myxogenes) or Agrobacterium radiobacter CIR Panel Book Page 37

42 Table 4. Methods of Manufacture/Microorganisms Used in Production Ingredient Oxidized Beta-Glucan Method oxidation of beta-glucan is performed using phosphoric acid and sodium nitrite, with the actual oxidant being NO 2 gas; extent of oxidation was typically 10-20% 87 Sodium Carboxymethyl Betaglucan derived from the inner cell walls for baker s yeast (Saccharomyces cerevisiae); 3 of 4 glucose units of the β 1,3- glucopyranose are carboxymethylated 75,88 particulate glucan and sodium hydroxide are mixed, the sodium salt of monochloroacetic acid in 95% ethanol is added and stirred, excess sodium hydroxide is neutralized, the product is washed with 80% ethanol and dried 74 Pullulan fermentation of liquefied corn starch by Aureobasidium pullulans; the fungal biomass is removed by microfiltration, the filtrate is heat-sterilized, and the pigments and other impurities are removed by adsorption and ion-exchange chromatography 29 one company reports the following raw materials: ammonium sulfate; beer yeast extract; calcium hydroxide; caustic soda; corn syrup; diatomaceous earth; diammonium phosphate; dipotassium phosphate; hydrochloric acid; ion exchange resin; magnesium sulfate; salts; silicone oil; sodium glutamate; zinc carbon chloride 89 produced by Pullularia pullulans IFO 6353, Dermatium pullulans IFO 4464, etc in a culture medium containing a carbon source (such as glucose, fructose, etc) under anaerobic conditions 96 Levan produced extracellularly from sucrose- and raffinose-based substrates by levansucrase from a wide range of taxa, such as bacteria, yeasts, and fungi, but mainly be bacteria 12 fermentation of Zymomonas mobilis in a medium that contains 10% sucrose and 1% yeast extract, centrifugation via ultrafiltration, precipitation by the addition of ethanol, resuspension with distilled water, and drying 52 sources include Erwinia herbicola, Aerobacter lavanicum, Streptococcus salivarius, Pseudomonas prunicola, Arthrobacter acetigenum, Bacillus polymxa, Bacilus subtilis, Actinomyces sp. 76 Rhizobian Gum produced by fermentation of Rhizobium sp. strain 70 bacterial strain YAS 34 is isolated from the rhizosphere of the sunflower plant; selection of the isolates is carried out on high carbon:nitrogen ratio liquid media; the culture broth was inoculated and fermented; the exopolysaccharide is recovered with a multi-step downstream processing that includes heating and centrifugation; diafiltration is used to eliminate fermentation residue, followed by further purification by alcoholic precipitation 97 Alcaligenes Polysaccharides Distributed for Comment Only -- Do Not Cite or Quote neutral polysaccharide: culture broth was precipitated with ethanol and redissolved in hot water, filtration was used to remove the water-insoluble cells and acidic polymers, and the polysaccharide was recovered by ethanol precipitation and further purified CIR Panel Book Page 38

43 Table 5a. Frequency and concentration of use according to duration and type of exposure # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 Xanthan Gum Dehydroxanthan Gum Xanthan Gum Crosspolymer Totals* NR Duration of Use Leave-On NR Rinse-Off NR NR Diluted for (Bath) Use NR NR NR NR Exposure Type Eye Area NR NR NR Incidental Ingestion NR NR NR NR Incidental Inhalation-Spray 124 a a ; 0.05 b NR 0.1 a -0.2 NR NR Incidental Inhalation-Powder NR NR NR NR Dermal Contact a NR Deodorant (underarm) 1 c c ; NR NR NR NR Hair - Non-Coloring NR NR Hair-Coloring NR NR NR NR Nail NR NR NR NR Mucous Membrane NR NR Baby Products NR NR NR NR Gellan Gum Biosaccharide Gum-1 Biosaccharide Gum-2 Totals* Duration of Use Leave-On Rinse Off 1 NR NR Diluted for (Bath) Use NR NR 2 NR NR NR Exposure Type Eye Area NR Incidental Ingestion NR 0.08 NR NR Incidental Inhalation-Spray NR NR 3 a a NR NR Incidental Inhalation-Powder NR NR NR NR Dermal Contact Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR Hair-Coloring NR NR 1 NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR Baby Products NR NR NR NR NR NR Biosaccharide Gum-4 Dextran Sodium Carboxymethyl Dextran Totals* NR Duration of Use Leave-On NR Rinse-Off NR NR NR Diluted for (Bath) Use NR NR NR 0.2 NR NR Exposure Type Eye Area NR NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR 1 a 1 a 0.01 a NR NR Incidental Inhalation-Powder 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 0.2 NR NR Baby Products NR NR NR NR NR NR 32 CIR Panel Book Page 39

44 Table 5a. Frequency and concentration of use according to duration and type of exposure # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 Dextran Sulfate Sodium Dextran Sulfate Sclerotium Gum Totals Duration of Use Leave-On Rinse Off NR NR NR NR Diluted for (Bath) Use NR NR NR NR Exposure Type Eye Area 10 NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR NR NR NR 13 a Incidental Inhalation-Powder NR NR NR NR 4 NR Dermal Contact Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR Hair-Coloring NR NR NR NR NR 0.8 Nail NR NR NR NR Mucous Membrane NR NR NR NR Baby Products NR NR NR NR 4 NR Hydrolyzed Sclerotium Gum Beta-Glucan Sodium Carboxymethyl Beta-Glucan Totals* NR Duration of Use Leave-On NR Rinse-Off NR NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR 8 NR Incidental Ingestion NR NR 2 NR NR NR Incidental Inhalation-Spray NR NR NR NR NR NR Incidental Inhalation-Powder NR NR 6 NR NR 0.02 Dermal Contact NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR 0.04 Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR 0.1 Baby Products NR NR 8 NR NR 0.02 Pullulan Rhizobian Gum Hydrolyzed Rhizobian Gum Totals* NR Duration of Use Leave-On NR NR Rinse-Off NR NR 1 NR Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area NR NR 3 Incidental Ingestion 5 17 NR NR NR NR Incidental Inhalation-Spray NR NR NR NR NR NR Incidental Inhalation-Powder NR NR NR NR NR NR Dermal Contact NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR 12 NR NR NR NR Hair-Coloring NR 0.03 NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane 5 17 NR NR NR NR Baby Products NR NR NR NR NR NR 33 CIR Panel Book Page 40

45 Table 5a. Frequency and concentration of use according to duration and type of exposure # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 # of Uses 14 Max. Concs. of Use (%) 15 Alcaligenes Polysaccharides Totals* Duration of Use Leave-On Rinse-Off Diluted for (Bath) Use NR NR Exposure Type Eye Area 2 NR Incidental Ingestion NR NR Incidental Inhalation-Spray NR NR Incidental Inhalation-Powder NR NR Dermal Contact Deodorant (underarm) NR NR Hair - Non-Coloring NR NR Hair-Coloring NR NR Nail NR NR Mucous Membrane NR NR Baby Products 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. NR no reported uses a includes suntan products, in that it is not known whether or not the reported product is a spray b this product is a pump spray c it is not known whether or not the product is a spray Table 5b. Ingredients Not Reported to be Used Hydroxypropyl Xanthan Gum Undecylenoyl Xanthan Gum Xanthan Hydroxypropyltrimonium Chloride Welan Gum Biosaccharide Gum-3 Biosaccharide Gum-5 Pseudoalteromonas Exopolysaccharides Carboxymethyl Dextran Dextran Hydroxypropyltrimonium Chloride Beta-Glucan Hydroxypropyltrimonium Chloride Beta-Glucan Palmitate Hydrolyzed Beta-Glucan Oxidized Beta-Glucan Myristoyl Pullulan Levan Table 6. Examples of Non-Cosmetic Uses Ingredient Non-Cosmetic Use Xanthan Gum direct food additive 22 ; indirect food additive 24 ; stabilizer, thickener, and emulsifying agent in water-based pharmaceutical preparations 31 ; used in oil and gas drilling and completion fluids 33 ; stabilizer in the agrochemical industry and in water based paints and water-based printing inks, and other industrial uses. 98 Gellan Gum direct food additive 23 ; thickener and gelling agent in the food industry 99 ; novel drug-delivery vehicle, film formation for transdermal drug delivery, component in controlled-release systems 10 ; alternative to agar for microbiological media 8,99,100 and plant tissue culture 99 Welan Gum thermostable thickener for industrial and oilfield application 8 ; suspending, stabilizing, emulsifying, and thickening agent for food, coating materials, medicine, concrete additives, and enhanced oil recovery 68 Dextran GRAS indirect food additive 26 ; approved active ingredient for OTC use (as dextran 70) as an ophthalmic demulcent at 0.1% when used with another approved polymeric demulcent 32 ; a plasma volume expander (as dextran 70) and as a blood flow adjuvant (as dextran 40) 33 ; 99m Tc-labeled dextran is used as a tracer in lymphoscintigraphy 47 Sodium Dextran Sulfate clinical reagent 33 Sclerotium Gum pharmaceutical applications include use in table coatings, ophthalmic solutions, and injectable antibiotic solutions; thickening in the oil industry; drilling s mud and enhanced oil recovery; preparation of adhesives, water colors, printing inks; preparation of liquid animal feed 5 Beta-Glucan direct food additive (as curdlan) 25 Pullulan Levan agricultural applications 12 glazing agent, as a film-forming agent, as a thickener, and as a carrier in the production of capsules for dietary supplements, coatings for coated tablets, and edible flavored films 29 ; can be used in wound-healing compositions; denture adhesive; photographic, lithographic, and electronic applications CIR Panel Book Page 41

46 Table 7. Acute toxicity studies Ingredient Animals No./Group Vehicle Concentration/Dose/Protocol LD 50 /Results Reference ORAL Xanthan Gum mice not specified water not specified >1 g/kg 101 Xanthan Gum rats not specified not specified 5 g/kg, max >5 g/kg 102 Xanthan Gum dogs not specified not specified 20 g/kg, max >20 g/kg 102 Gellan Gum rats, M/F not specified not specified administered in diet and by gavage; >5 g/kg 37 non-acetylated; >95% polysaccharide Beta-Glucan (as mice, M/F not specified water tested as a 10% suspension >10 g/kg 40 curdlan) Beta-Glucan (as rats, M not specified water tested as a 10% suspension >10 g/kg 40 curdlan) Beta-Glucan (highly pure extract of S. cerevisiae) rats 5M/5F water 100 mg/ml suspension administered at 2 g/kg bw (20 ml/kg bw) by gavage >2 g/kg 103 Sodium Carboxymethyl Beta-Glucan (>90% pure) ddy mice 6M/6F purified water 2 g/kg of a 20 aq. solution >2 g/kg; no signs of toxicity Pullulan mice not specified olive oil not specified >14 g/kg INHALATION Xanthan Gum albino rabbits 5 none calculated chamber conc of 21 mg/l; 1 h exposure Gellan Gum rats, M/F not specified not specified non-acetylated; >95% polysaccharide; duration of exposure not stated Beta-Glucan (as curdlan) Distributed for Comment Only -- Do Not Cite or Quote guinea pigs 5 not specified 100 µg/ml curdlan for 4 h; continuous flow aerosol >21 mg/l; no toxicity; no gross lesions at necropsy >5.09 mg/l no inflammatory cell response Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) guinea pigs 3 NaOH 1 µg/ml; continuous flow for 4 h decrease in inflammatory cells with curdlan alone, increase in cells, especially neutrophils with NaOH guinea pigs 5 not specified 100 µg/ml; continuous flow for 4 h slight decrease in macrophages; (almost significant) decrease in lymphocytes guinea pigs not specified dust ffoice dust spiked with 1% beta-glucan (as curdlan; 280 g office dust was spiked with 2.8 g curdlan) for 4 h in a whole-body exposure chamber; necropsied 5 or 18 h after exposure humans 36 dust addition of 10 mg beta-glucan (as curdlan) to office dust (10 mg curdlan/g dust); subjects were exposed to the dust in a climate chamber for 180 min Pullulan guinea pigs 5 not specified 100 µg/ml curdlan for 4 h; continuous flow aerosol; PARENTERAL Dextran Sulfate, avg MW of 7500, 47,000, and 458,000 Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (soluble; extract of S. cerevisiae) Beta-Glucan (soluble; extract of S. cerevisiae) glucan-spiked dust produced a delayed subacute nasal congestion in guinea pigs compared to dust without beta-glucan.; at 18 h after exposure, there was a significant decrease in in nasal volume compared to clean dust, nasal volume decreased, swelling in the nasal turbinate increased, and nasal eosinophil cell concentration increased no inflammatory cell response mice and rats not specified not specified g/kg, i.v. and i.p. some animals died immediately; the largest MW dextran was more toxic mice. M/F not specified saline 5% suspension, i.p. males: 2.75 g/kg females: 2.5 g/kg rats, M not specified saline 5% suspension, i.p g/kg; adhesions of beta-glucan to liver and spleen ICR/HSD mice Sprague- Dawley rats not specified not specified 1 g/kg, i.v. >1 g/kg not specified not specified 0.5 g/kg, i.v. >0.5 g/kg CIR Panel Book Page 42

47 Table 8. Repeated Dose Toxicity Studies Ingredient Animals/Group Study Duration Vehicle Dose/Concentration Results Reference Oral Xanthan Gum rats (#/group not stated) 18 days in diet 7.5% paired feeding study wt gains were similar to controls Xanthan Gum 25 rats 30 days in diet 1.5% avg. wt of test animals was greater than controls; no gross lesions Xanthan Gum albino rats, 5M 91 days in diet 3, 6, or 15% reduced weight gain in the 15% group; no effect on organ wts; no microscopic lesions at necropsy in the 15% group Xanthan Gum rats, 5M/5F 110 days in diet 0, 2.5, 5.0, or 10% (the gum product consisted of drum-dried whole fermentation medium (beer)); no significant toxic effects Xanthan Gum CD rats, 30M/30F 2 yr in diet 0, 0.25, 0.50, or 1.0 g/kg/day no significant differences in growth rate, survival, hematology and clinical chemistry parameters, or organ weight were observed between treated and control animals; while not statistically significant (stat. sig.), there was an increase in uterine polyps in the high dose groups compared to controls, 5 in the high dose animals vs. 2 in controls Xanthan Gum beagle dogs, 3M/3F 12 wks in diet 0, 0.25,or 0.5 g/kg bw/day growth of high-dose males was slightly less than controls; the noobservable adverse effect level (NOAEL) was 0.25 g/kg bw/day Xanthan Gum beagle dogs, 2M/2F 12 wks in diet 0, 1,or 2 g/kg bw/day; positive controls were given 20 g/kg bw/day powdered cellulose immediate and persistent diarrhea in the 2 g/kg group; body wt loss was observed in treated and control animals, with the wt loss greatest in the 2 g/kg group; red blood cell counts, hemoglobin, and serum cholesterol levels were decreased in high dose animals; adrenal glands were slightly enlarged in the 2 g/kg group; no treatment-related microscopic lesion were observed at necropsy Xanthan Gum beagle dogs, 4M/4F 107 wks in diet 0, 0.25, 0.37, and 1.0 g/kg/day no significant differences in survival, body weight gain, feed consumption, organ weights, hematology parameters, or gross or microscopic lesions were observed between treated and control animals; a doserelated increased in urinary specific gravity was observed; ophthalmic findings were not considered treatment-related Gellan Gum 20 rats 28 days in diet 5% no changes in urinalysis values; no gross lesions at necropsy Gellan Gum; nonacetylated; >95% polysaccharide Sprague-Dawley rats, 20M/20F 13 wks in diet 0-6% no mortality; no signs of toxicity Gellan Gum; varied degree of acetylation; 58.5% polysac. Gellan Gum; varied rhesus monkey, 2M/2F 28 days not provided 0, 1, 2,or 3 g/kg, by gavage no signs of toxicity degree of acetylation; 58.5% polysac. Dextran albino rats, 6M 62 days in diet 15% wt gain was normal Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) beagle dogs, 5M/5F 52 wks in diet 0, 3, 4.5, or 6% no mortality; feed consumption was greater in treated animals compared to controls; no adverse effects were observed CD-1 mice, 10M/10F 8 wk in diet 0, 1, 5, 10, 20, or 30%; equiv to 0, 1.4, 7.1, 14, 29, and 43 g/kg bw, respectively CD-1 mice, 100M/100F wks (until survival was 20%) one female of the 30% group died; body wt gains of male mice of the 30% group were decreased compared to controls; no gross abnormalities; differences in stools and cecal wts were reported; the NOEL was 5% based on an increase in full cecal wts and large stools at higher doses in diet 0, 1, 5, or 15% no treatment-related differences in survival or body wts; feed consumption was decreased by ~13% in 15% females; NOEL of 5% due to decreased feed consumption CIR Panel Book Page 43

48 Table 8. Repeated Dose Toxicity Studies Ingredient Animals/Group Study Duration Vehicle Dose/Concentration Results Reference Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (as mushroom beta-glucan from Ganoderma lucidum) Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) Sprague-Dawley Ta rats; 5M 20 Sprague-Dawley rats, males Sprague-Dawley rats, 10M/10F CD (SD) IGS rats, 12M,12F 4 wks in diet 0, 3, 10, or 30%; equiv to 0, 2.5, 8.5,or 30 g/kg bw 30% powder agar group no difference in body wts; dose-related increase in neutrophils, with a stat. sig. increase in the 10% group; occult blood was present in the urine 4 10% rats, 1 30% rat, and 1 fed agar; relative (rel.) kidney wts were statistically significantly decreased in all groups; rel. liver weights were statistically significantly decreased and pituitary wts were stat. sig. increased in the 30% group 8 wks in diet 0, 1, 5, or 15% no mortality or signs of toxicity; body wt of animals of the 15% dose group were stat. sig. decreased; feed consumption in this group was slightly decreased 3 mos in diet 0, 5, 10, or 20%; equiv to 0, 4.4, 9, and 19 g/kg bw (males) and 0, 5.5, 12, and 24 g/kg bw (females) 3 mos sterile water; by gavage 0, 500, 1000, or 2000 mg/kg bw (10 ml/kg dosing volume) body wt gains decreased with increasing dose, being stat. sig. in the 20% group; a stat. sig. decrease in platelet count in males and in total protein and globulin concs. in males and females of the 10 and 20% groups; stat. sig. increase in body wt of males of the 10 and 20% group; stat. sig. decreases in absolute liver wts in 20% males, absolute (abs.) kidney wts of 10 and 20% males, abs. and rel. ovary wts in 20% females, abs. and rel. pituitary wts of in all females of all doses; and rel. adrenal wts in 10 and 20% males; differences in stools and cecal wts were reported; the NOEL was 5% based on fecal changes, diarrhea, and cecal wts and large stools at higher doses no test article-related adverse effects on mortality, toxicity; opthalmoscopy, body wts or body wt gains, clinical chemistry, hematology, or urinalysis; statistically significant decreases in testes wt in males of the low dose group and heart wts in females of the high dose group were not considered test-article related; there were no treatment related gross or microscopic lesions, the NOAEL was 2000 mg/kg bw CD rats; 60M/60F 2 yrs in diet 0, 1, 5, or 15% no changes in mortality, behavior, appearance, or ophthalmic parameters; wt gain was decreased by ~10%, which was not stat. sig., in the 15% group; feed consumption was also reduced; no microscopic lesions were found the NOEL was 5% based on decreased feed consumption, body wt gain and increased cecal wts CD rats; 60M/60F, of the F1a generation of a reproductive tox. study wks (20% survival) in diet 0, 1, 5, or 15% no changes in mortality, behavior, appearance, or ophthalmic parameters; body wts of the 15% group were stat. sig. decreased, and body wts were stat. sig. decreased in 5% males until wk 65; feed consumption was decreased in the 15% group; no changes in hematology or urinary parameters, but some stat. sig. changes were reported in blood chemistry; stat. sig increase in gross and microscopic incidences of uterine polyps in the 15% groups the incidences were 0/450, 3/50, 4/51, and 7/50 for the 0, 1, 5, and 15% groups, indicating that the polyps were possible treatment-related; the NOEL was 1% based on increased cecal wts, decreased body wts and feed consumption, and the incidence of polyps beagle dogs, 4M/4F 52 wks in diet 0, 1, 5, or 15% one 15% male died at 37 wks (not dose related); no stat. sig. treatmentrelated changes, except for fecal changes and cecal wts; NOEL was 5%based on fecal changes and cecal wts CIR Panel Book Page 44

49 Table 8. Repeated Dose Toxicity Studies Ingredient Animals/Group Study Duration Vehicle Dose/Concentration Results Reference Beta-Glucan (highly pure extract of S. cerevisiae) Beta-Glucan (highpurity extract barley beta-glucan) Beta-Glucan (extracted from Candida albicans ) SPF Fischer rats, 10M/10F 91 days water 0, 2, 33.3, or 100 mg/kg bw/day, volume 0.5 ml/100 g bw, by gavage SPF Wistar rats, 5M/5F 28 days in diet 0, 1, 5, or 10%, supplemented with 10% potato starch Sprague-Dawley rats, 20M/20F Pullulan 8 Wistar rats, males (test and cellulose control groups) Pullulan (200,000 MW) SPF Wistar rats, 10M/10F Pullulan Sprague-Dawley rats, 15M/15F Beta-Glucan (as curdlan) Beta-Glucan (as curdlan) 52 wks sterile saline (using a rubber catheter) 4 or 9 wks in diet 0, 5, 10, 20, or 40%; equiv. to 0, 2500, 5000, 10,000, or 20,000 mg/kg, respectively cellulose controls: 20 or 40% cellulose 13 wks in diet 0, 2.5, 5, or 10%,; equiv to 0, 1960, 4100, or 7900 mg/kg bw/day diet of the groups fed 0, 2.5, or 5% pullulan was supplemented with 10, 7.5, or 5% potato starch, respectively 62 wks( was to be 24 mos; study was terminated because of poor survival due to pneumonia) no mortality; no stat. sig. differences in wt gains, feed consumption, or gross or microscopic lesions; a dose-dependent and stat. sig. increase in clotting time in males, isolated stat. sig. changes in some clinical chemistry parameters, and slight but stat. sig. incr. in absolute liver, kidney, heart, spleen, adrenal, and testicle wts in males and absolute kidney and thymus wts in females were not considered toxicologically significant; NOAEL was 100 mg/kg bw/day no mortality; no sig. effects on body wts, feed consumption, or functional observational battery results; no treatment-related change in hematology or urinalysis values; empty caecum wt was increased 0, 50, 100, or 200 mg/kg/day no sig. effects on mortality, body wts, feed consumption, or hematology, clinical chemistry, or urinalysis parameters; with the exception of cecal enlargement with variable hyperplasia, not gross or microscopic lesions were noted; the NOEL was 100 mg/kg/day body wt gains were decreased by day 10 in rats of the 20 and 40% groups when compared to untreated controls; wt gains of animals of the 5 and 10% pullulan group were lower than untreated controls after 7 wks, but this difference was not stat. sig.; similar decreases were observed in the animals fed cellulose; diarrhea was observed with 40% pullulan; rel. wts of the stomach, small intestine, and large intestine were increased in treated animals no mortality; no dose-related clinical sign; stat. sig. reduced motor activity in females of the 5 and 10% groups was observed and appeared treatment related, but as a physiological phenomenon rather than a toxic effect; no difference in hematology parameters between treated and control groups; differences that were observed in clinical chem. parameters were not biologically significant or dose-related; dosedependent, stat. sig. increases in abs. and rel. empty cecal wts were observed in males of the 5 and males and females of the 10% group; no microscopic changes were observed in diet 0, 1, 5, and 10% no treatment-related effects on body wts, feed consumption or organ wts (except for cecal wts); changes in hematology or clinical chemistry parameters and microscopic lesions that were observed were not considered treatment-related; the NOAEL was 10% in the diet (equiv. to 4450 mg/kg bw/day) INHALATION guinea pigs, 16F 5 wks distilled water 100 µg/ml; 4 h/day, 5 days/wk no significant change in lung lavage cells, but there was a decrease in the number of lymphocytes in the cell wall; slight but not statistically significant increase in macrophages and eosinophils in the lung wall cells; no lesions observed at microscopic examination of the lungs guinea pigs, 6 5 wks saline 100 µg/ml; 4 h/day, 5 days/wk; continuous flow exposure with a dose of 8 pg; animals were examined 24 h after last dose PARENTERAL Xanthan Gum mice 2 wks water 5 mg in 0.5 ml water; 10 i.p. injections over 2 wks the only effect on lung lavage cells was a slight decrease in neutrophils no toxicity; there was no xanthan gum in the abdominal cavity at necropsy , CIR Panel Book Page 45

50 Table 8. Repeated Dose Toxicity Studies Ingredient Animals/Group Study Duration Vehicle Dose/Concentration Results Reference Dextran, partly hydrolysed, bacterial, 75,000 avg MW Beta-Glucan (soluble; extract of S. cerevisiae) Beta-Glucan (soluble; extract of S. cerevisiae) Beta-Glucan (soluble; extract of S. cerevisiae) Beta-Glucan (soluble; extract of S. cerevisiae) wks physiological saline 30 ml of 6% solution, i.v. 4 animals died and one was killed in moribund condtion; wt gain was similar to that of controls; increases in absolute heart and adrenal wts were probably statistically significant; statistically significant increase in liver and spleen wts; no microscopic lesions mice 7 days not specified 250 mg/kg bw, i.p. no effect on weight gain guinea pigs 7 days not specified 250 mg/kg bw, i.p. sig. 10% decrease in weight gain ICH/HSD mice, M 30 days not specified 1000 mg/kg bw, i.v. sig. toxicity leading to hepatosplenomegaly in the 40 and 1000 mg/kg bw groups ICH/HSD mice, M 60 days not specified 1000 mg/kg bw, i.v. dose-dependent increase in hepatosplenomegaly; was stat. sig. at 1000 mg/kg Table 9. Genotoxicity studies Ingredient Concentration Vehicle Procedure Test System Results Reference IN VITRO Gellan Gum (non-acetylated; >95% polysacc.) Gellan Gum (non-acetylated; >95% polysacc.) Gellan Gum (non-acetylated; >95% polysacc.) Sodium Dextran Sulfate (54,000 MW) Sodium Dextran Sulfate (54,000 MW) Sodium Dextran Sulfate (54,000 MW) 10, 30, 100, 300, and 1000 µg/plate not provided Ames test, with and without metabolic activation Salmonella typhimurium TA98, TA100, TA1537, TA1538, TA1535 negative 3, 5, 10, and 20 mg/ml not provided DNA repair test (details not provided) rat hepatocytes negative 3, 5, 10, and 20 mg/ml not provided V79/HGRPT (details not provided) Chinese hamster lung fibroblasts negative 1.0, 7.5, 25 mg/plate not provided Ames test; appropriate positive controls were used S. typhimurium TA100, TA98 negative 0, 10, 100 µg distilled water hepatocyte primary culture (HPC)/DNA repair test; appropriate positive controls were used 0, 10, 100 µg distilled water intestinal mucosal cell (IMC)/DNA repair test; appropriate positive controls were used rat hepatocytes negative intestinal mucosal cells from rat ileum or colon and rectum Beta-Glucan (as curdlan) µg/plate sterile water Ames test, with and without metabolic activation S. typhimurium TA98, TA100, TA1537, TA1538, TA1535 Beta-Glucan (as mushroom beta-glucan from Ganoderma lucidum) μg/plate not provided Ames test, with and without metabolic activation S. typhimurium TA98, TA100, TA102, TA1535, TA1537 Beta-Glucan (as curdlan) µg/ml tissue culture medium Beta-Glucan (as mushroom beta-glucan from Ganoderma lucidum) chromosomal aberration assay, with and without metabolic activation μg/ml culture medium chromosomal aberration assay, with and without metabolic activation negative negative Chinese hamster ovary (CHO) cells negative CHO cells negative CIR Panel Book Page 46

51 Table 9. Genotoxicity studies Ingredient Concentration Vehicle Procedure Test System Results Reference Beta-Glucan (as curdlan) µg/ml tissue culture tk locus test, with and without metabolic activation mouse lymphoma L518Y cells negative medium Sodium Carboxymethyl 0, µg/plate Milli-Q water Ames test, with and without metabolic activation, S. typhimurium TA98, TA100, TA1537, TA1535 negative Beta-Glucan appropriate positive controls were used; Sodium Carboxymethyl Beta-Glucan Sodium Carboxymethyl Beta-Glucan Sodium Carboxymethyl Beta-Glucan Carboxymethyl-Glucan 12.5, 25, 50, 100, and 200 µg/ml 0, µg/plate Milli-Q water Ames test, with and without metabolic activation; appropriate positive controls were used 0, ,000 µg/ml purified water Ames test, with and without metabolic activation; appropriate positive controls were used; S. typhimurium TA98, TA100, TA1537, TA1535 negative S. typhimurium TA98, TA100, TA1537, TA1535; Escherichia coli WP2 uvra 0, ,000 µg/ml purified water Ames test, with and without metabolic activation; S. typhimurium TA98, TA100, TA1537, TA1535; negative appropriate positive controls were used Escherichia coli WP2 uvra electrophoresis test in single-cell gel (comet assay) CHO-k1 cells negative Pullulan 10,000 µg/plate not provided Ames test, with and without metabolic activation; appropriate positive controls were used S. typhimurium TA98, TA100, TA1537, TA1535; Escherichia coli WP2 uvra Pullulan 12 mg/ml not provided chromosomal aberration assay Chinese hamster lung fibroblasts negative Pullulan 20 mg/plate distilled water rec assay; with and without metabolic activation Bacillus subtilis weak positive IN VIVO Beta-Glucan (as curdlan) 0, 500, 1000, or 2000 water micronucleus test; dosed by gavage at 24 h intervals male and female CD-1 mice negative mg/kg sterile water micronucleus test; dosed by gavage CD-1 mice, 5/sex/group negative Beta-Glucan (as mushroom beta-glucan from Ganoderma lucidum) 0, 1250, 2500, and 5000 mg/kg bw Pullulan 1800 mg/kg bw. not provided micronucleus test; one i.p. injection ddy mice negative Pullulan 4x1000 mg/kg bw not provided micronucleus test; four i.p. injections in 24 h ddy mice negative negative negative Table 10. Dermal Irritation and Sensitization Ingredient Animals/Subjects/Gp Dose/Conc/Vehicle Procedure Results Reference IRRITATION - NON-HUMAN Xanthan Gum rats 1%; vehicle not provided solution was applied daily for 15 days (details not provided) not an irritant 36 Xanthan Gum 6 rabbits 0.5 ml of a 1% solution primary cutaneous irritation test; occlusive patches on intact and non-irritant; primary irritation index (PII) abraded skin Xanthan Gum 6 rabbits 0.5 ml of a 1% solution primary cutaneous irritation test; occlusive patches on intact and non-irritant; PII abraded skin Xanthan Gum 3 rabbits 2 ml/animal of a 1% solution 6-wk cumulative cutaneous irritation test, 5 applications/wk very well tolerated; mean max. irritation index Xanthan Gum 3 rabbits 1% 6-wk cumulative cutaneous irritation test, 5 applications/wk very well tolerated; MMII Xanthan Gum rabbits 5% aq. daily applications to shaved skin (details not provided) localized irritation with bleeding and cracking 101 Beta-Glucan rabbits not provided primary skin irritation test ( details not provided) not an irritant 86 Beta-Glucan rabbits not provided repeated skin irritancy test (details not provided) not an irritant 86 Sodium Carboxymethyl Beta-Glucan (>90% pure) Japanese white rabbits, 6M 0.5 g moistened with 0.2 ml distilled water occlusive patches were applied to intact and abraded shaved skin for 24 h non- to mildly irritating; primary irritation index (PII) of 0.33/8 at test site and 0.29/8 for adhesive control; no irritation was reported for intact skin CIR Panel Book Page 47

52 Table 10. Dermal Irritation and Sensitization Ingredient Animals/Subjects/Gp Dose/Conc/Vehicle Procedure Results Reference Sodium Carboxymethyl Beta-Glucan (>90% pure) 3 guinea pigs 2, 10, or 50% (w/v) in distilled water 24-h occlusive patch test; this test was used to determine test concentration for the maximization study described below IRRITATION - HUMAN Beta-Glucan not provided not provided occlusive patch test no an irritant Sodium Carboxymethyl Beta-Glucan (>90% pure) 40 subjects; 27M/13F applied neat; small amount of vaseline was used for adhesion slight skin irritation was observed at a concentration of 50% 24-h occlusive patch test; 0.1 g of test material was applied not a primary skin irritant; no irritation was observed SENSITIZATION NON-HUMAN Xanthan Gum guinea pigs, 18M 0.1%; vehicle not provided intradermal challenge test; test solution was injected intracutaneously not a sensitizer 3x/wk for 10 injections; the challenge was performed after a 10-day non-treatment period Beta-Glucan guinea pigs not provided skin sensitization test (details not provided) not a sensitizer Sodium Carboxymethyl Beta-Glucan (>90% pure) Beta-Glucan (as Curdlan) Sodium Carboxymethyl Beta-Glucan Sodium Carboxymethyl Beta-Glucan Hartley guinea pigs, 6F 10% in distilled water maximization study using Freund s complete adjuvant and sodium lauryl sulfate; 0.1% aq. 2,4-dinitro-1-chlorobenzenze was used as the positive control dose volumes were 0.1 ml for intradermal induction, 0.2 ml for dermal induction, and 0.1 ml for challenge SENSITIZATION - HUMAN 213 subjects; M dose not provided; was an aq. paste 32 subjects; 8M/24F induction and challenge: 2% in distilled water 8 male/24 female subjects induction and challenge: 2% in distilled water modified Draize multiple insult patch test; occlusive patches were applied every other day for 10 applications; a 48-h challenge was performed after a day non-treatment period induction: 9 24-h occlusive patches were applied challenge: occlusive patch applied after a day non-treatment period to a previously unpatched site reactions were graded on a scale of 0-4 PHOTOALLERGY- HUMAN induction: 6 24-h patches were applied to non-tanned skin; the test site was irradiated with 2x MED UVB within 10 min after patch removal challenge: applied after a day non-treatment period, a 24-h patch was applied to a previously unpatched site; the site was irradiated with 18 J/cm 2 UVA within 10 min after patch removal MULTITESTER light source was used; 0-4 scale used for scoring not a sensitizer trace, insignificant, irritation observed during induction; not a sensitizer not a sensitizer during induction, 2 subjects had doubtful reactions and one had a grade 1 reaction not photoallergenic 31 subjects had grade 1 skin reactions, and on had a grade 2 reaction, to UVB exposure during induction 1 subject had a doubtful reaction after challenge CIR Panel Book Page 48

53 Table 11. Ocular Irritation Ingredient Animals/Subjects/Group Concentration/Vehicle Procedure Results Reference Sodium Carboxymethyl Beta-Glucan 5% stock solution containing 2% Sodium Carboxymethyl Beta-Glucan ALTERNATIVE STUDIES Ocular tolerance test using HET-CAM method weakly irritant NON-HUMAN Xanthan Gum rabbits 1% ocular irritation test non-irritating; acute ocular irritation index (AOII) /110 Xanthan Gum rabbits 1% ocular irritation test non-irritating; AOII 5.83/110 Xanthan Gum rabbits 1% instilled in the conjunctival sac of rabbit eyes for 5 days (details were not provided) not irritating not irritating Gellan Gum New Zealand White rabbits, 3M 0.2 or 0.3% Draize study; 50 μl of the test solution was instilled into the conjunctival sac of the eye three times a day for 10 days Gellan Gum albino rabbits 0.8% details not provided not irritating Sodium Carboxymethyl Beta-Glucan (>90% pure) Japanese white rabbits, 3M undiluted 0.1 mg were instilled into the conjunctival sac of one eye, and the contralateral eye served as an untreated control; the eyes of one group were rinsed 1 min after instillation HUMAN Gellan Gum 3 subjects %; vehicle not specified 25 µl of a gel formulation was instilled in the conjunctival sac of the eye, remained in contact with the eye for 9-52 min practically non-irritating; slight redness of the conjunctiva in both washed and unwashed eyes at 1 h, considered due to the powder not irritating CIR Panel Book Page 49

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55 19. Rothe H, Fautz R, Gerber E, Neumann L, Rettinger K, Schuh W, and Gronewold C. Special aspects of cosmetic spray safety evaluations: Principles on inhalation risk assessment. Toxicol Lett. 2011;205:(2): Bremmer HJ, Prud'homme de Lodder LCH, and Engelen JGM. Cosmetics Fact Sheet: To assess the risks for the consumer; Updated version for ConsExpo Report No. RIVM /2006. pp European Commission. Health and Consumers Cosmetic Cosing Database Date Accessed Food and Drug Administration (FDA). 21CFR arch= Date Accessed Food and Drug Administration (FDA). 21CFR arch= Date Accessed Food and Drug Administration (FDA). 21CFR arch= Date Accessed Food and Drug Administration (FDA). 21CFR Date Accessed Food and Drug Administration (FDA). 21CFR earch= Joint FAO/WHO Expert Committee on Food Additives (JEFCA). Evaluation of certain food additives and contaminants. Thirtieth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series World Health Organization (WHO): Geneva Date Accessed Joint FAO/WHO Expert Committee on Food Additives (JEFCA). Evaluation of Certain Food Additives and Contaminants. WHO Technical Report Series 806. Thirty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization: Geneva Date Accessed Joint FAO/WHO Expert Committee on Food Additives (JEFCA). Evaluation of certain food additives. WHO Technical Report Series 934. Sixty-fifth report of the joint FAO/WHO Committee on food additives. World Health Organization (WHO): Geneva Date Accessed Chaen, H. Pullulan. Food Stab., Thickeners Gelling Agents CAPLUS AN 2010:484781(Conference; General Review). 31. Leung AY and Foster S. Encyclopedia of Common Natural Ingredients Used in Foods, Drugs, and Cosmetics. 2nd ed. New York: John Wiley & Sons, Inc, Food and Drug Administration (FDA). 21CFR rch= Date Accessed Merck & Co., Inc. The Merck Index. px?formgroupid=200000&appname=themerckindex&allowfullsearch=true&keeprecordcounts ynchronized=false&searchcriteriaid=5&searchcriteriavalue= &currentindex= Date Accessed CIR Panel Book Page 51

56 34. Pillai R, Redmond M, and Röding J. Anit-wrinkle therapy: Significant new findings inthe non-invasive cosmetic treatment of skin wrinkles with beta-glucan. J Cosmet Sci. 2005;56:(May/Jume): Svedman P, Lundin S, Hoeglund P, HammarlundC, Malmros C, and Pantzar N. Passive drug diffusion via standardized skin mini-erosion; methodological aspects and clinical findings with new device. Pharmaceutical Research. 1996;13:(9): Inchem. Xanthan Gum Date Accessed Lin FSD. Gellan Gum - First Draft Date Accessed Mehvar R and Shepard TL. Molecular-weight-dependent pharmacokinetics of fluorescein-labeled dextrans in rats. Journal of Pharmaceutical Sciences. 1992;81:(9): Foster BC, Gallicano KD, Whitehouse LW, McGilveray IJ, and Khan SR. Dextran sulfate disposition in the rat. Biopharmaceutics & Drug Disposition. 1990;11:(7): Joint FAO/WHO Expert Committee on Food Additives (JEFCA). Safety Evaluation of Certain Food Additives and Contaminants. WHO Food Additive Series 44. Curdlan Date Accessed Oku T, Yamada K, and Hosoya N. Effect of pullulan and cellulose on the gastrointestinal tract of rats. Eiyo to Shokuryo (Nutr.Diets). 1982;32: Dixon B, Abbott PJ, Verger P, and DiNovi M. Pullulan - First draft Date Accessed Lorentsen KJ, Hendrix CW, Collins JM, Kornhauser DM., Petty BG, Klecker RW., Flexner C, Eckel RH, and Lietman PS. Dextran sulfate is poorly absorbed after oral administration. Annals of Internal Medicine. 1989;111:(7): Yoneyama M, Okada K, Maddai T, Aga H, Sakai S, and Ichikawa T. Effects of pullulan intake in humans. Starch Chemistry (Denpun Kagaku). 1990;37:(3): Friberg U, Graf W, and Åberg B. Effects of prolonged dextran administration to rabbits. Acta Pharmacol Toxicol. 1953;9: Mehvar R, Robinson MA, and Reynolds JM. Dose dependency of the kinetics of dextrans in rats: Effects of molecular weight. Journal of Pharmaceutical Sciences. 1995;84:(7): Yamaoka T, Tabata Y, and Ikada Y. Body distribution profile of polysaccharides after intravenous administration. Drug Delivery. 1993;1:(1): Kaneo Y, Tanaka T, Nakano T, and Yamaguchi Y. Evidence for receptor-mediated hepatic uptake of pullulan in rats. J Control Release. 2001;70:(3): Arturson E. and Wallenius G. The intravascular persistence of dextran of different molecular sizes in normal humans. Scandinavian Journal of Clinical and Laboratory Investigation. 1964;16:(1): Straszek SP, Adamcakova-Dodd A, Metwali N, Pedersen OF, Sigsgaard T, and Thorne PS. Acute Effect of Glucan- Spiked Office Dust on Nasal and Pulmonary Inflammation in Guinea Pigs. Journal of Toxicology and Environmental Health, Part A. 2007;70:(22): Kim H, Cho H, Moon S, and et al. Effects of -glucan from Aureobasidium pullulans on acute inflammation in mice. Arch Pharm Res. 2007;30:(3): CIR Panel Book Page 52

57 52. Kim KH, Chung CB, and et al. Cosmeceutical properties of levan produced by Zymomonas mobilis. J Cosmet Sci. 2005;56:(-): Eastwood MA, Brydon WG, and Anderson DMW. The dietary effects of xanthan gum in man. Food Additives & Contaminants. 1987;4:(1): Mitsuhashi M, Yonetama M, and Sakai S. Growth promoting agent for bacteria containing pullulan with or without dextran (EP B1):Secondary reference in INCHEM (2005). 55. Sargent EV, Adolph J, Clemmons MK, Kirk GD, Pena BM, and Fedoruk MJ. Evaluation of flu-like symptoms in workers handling xanthan gum powder. Journal of occupational medicine.: official publication of the Industrial Medical Association. 1990;32:(7): Woodard G, Woodard MW, McNeely WH, Kovacs P, and Cronin MTI. Xanthan gum. Safety evaluation by twoyear feeding studies in rats and dogs and a three-generation reproduction study in rats. Toxicology and Applied Pharmacology. 1973;24:(1): Hirono I, Kuhara K, Yamaji T, Hosaka S, and Golberg L. Carcinogenicity of dextran sulfate sodium in relation to its molecular weight. Cancer Letters (Shannon, Ireland). 1983;18:(1): Tardieu D., Jaeg JP, Cadet J, Embvani E, Corpet DE, and Petit C. Dextran sulfate enhances the level of an oxidative DNA damage biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in rat colonic mucosa. Cancer Letters (Shannon, Ireland). 1998;134:(1): Hirono I, Kuhara K, Hosaka S, Tomizawa S, and Golberg L. Induction of intestinal tumors in rats by dextran sulfate sodium. JNCI, Journal of the National Cancer Institute. 1981;66:(3): Hirono I, Kuhara K, Yamaji T, Hosaka S, and Golberg L. Induction of colorectal squamous cell carcinomas in rats by dextran sulfate sodium. Carcinogenesis (London). 1982;3:(3): Tanaka T, Kohno H, Suzuki R, Yamada Y, Sugie S, and Mori H. A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. Cancer Science. 2003;94:(11): Suzuki R, Kohno H, Sugie S, Nakagama H, and Tanaka T. Strain differences in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon carcinogenesis in mice. Carcinogenesis. 2006;27:(1): Takeuchi A, Kamiryou Y, Yamada H, Eto M, Shibata K, Haruna K, Naito S, and Yoshikai Y. Oral administration of xanthan gum enhances antitumor activity through Toll-like receptor 4. International Immunopharmacology. 2009;9:(13-14): Kimura Y, Sumiyoshi M, Suzuki T, and Sakanaka M. Antitumor and antimetastatic activity of a novel water-soluble low molecular weight -1, 3-D-glucan (branch -1,6) isolated from Aureobasidium pullulans 1A1 strain black yeast. Anticancer Research. 2006;26:(-): Yoo SH, Yoon EJ, Cha J, and Lee HG. Antitumor activity of levan polysaccharides from selected microorganisms. International Journal of Biological Macromolecules. 2004;34: Paull, J. D. Dextrans. Developments in biological standardization. 1987;67: Ott CM and Day DF. Modification of Natural Gums pp EBSCO Publishing. 68. Li H, Xu H, and et al. Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428. Bioprocess Biosyst Eng. 2011;34:(1): Manjanna KM, Kumar TMP, and Shivakumar B. Natural polysaccharide hydrogels as novel excipients for modified drug delivery systems: A review. International Journal of ChemTech Research. 2010;2:(1): CIR Panel Book Page 53

58 70. Bresin A, Sanhajl G, and Reynaud R. Rhizobium gum: A novel cosmetic ingredient from soil to the skin. Cosmetics & Toiletries Magazine :(4): pp Mehvar R. Dextrans for targeted and sustained delivery of therapeutic and imaging agents. Journal of Controlled Release. 2000;69:(1): Miyake M and Kakizawa Y. Morphological study of cationic polymer-anionic surfactant complex precipitated in solution during the dilution process. J Cosmet Sci. 2010;61:(July/August): Park SY, Bae IY, Lee S, and Lee HG. Physicochemical and Hypocholesterolemic Characterization of Oxidized Oat -Glucan. Journal of Agricultural and Food Chemistry. 2009;57:(2): Ding XL and Wang M. Development of a water-soluble -carboxymethyl- -(1,3)-glucan derived from Saccharomyces cerevisiae pp Mibelle Biochemistry CM-Glucan granulate specifications. Unpublished data submitted by Personal Care Products Council. 76. Kaplan DL, Wiley BJ, Mayer JM, and et al. Biosynthetic Polysaccharides. Chapter: 8. Shalaby SW. 1994: Combie J. Properties of levan and potential medical uses. Chapter: 13. In: Polysaccharides for Drug Delivery and Pharmaceutical Applications. 2006: Nohata Y, Azuma J, and Kurane R. Structural studies of a neutral polysaccharide produced by Alcaligenes latus. Carbohydrate Research. 1996;293:(2): Jungbunzlauer Safety Data Sheet: Xanthan Gum. Unpublished data submitted by Personal Care Products Council. 80. Green R Letter from Biopolymer International to the European Commission concerning molecular weight of polymer additives. Unpublished data submitted by Personal Care Products Council. 81. Bajaj IB., Survase SA, Saudagar PS., and Singhal RS. Gellan gum: Fermentative production, downstream processing and applications. Food Technology and Biotechnology. 2007;45:(4): Food Chemicals Codex. Rockville, MD: The United States Pharmacopeial Convention, Chandrasekaran R, Radha A, and Lee EJ. Structural roles of calcium ions and side chains in welan: an X-ray study. Carbohydrate Research. 1994;252:(93): Solabia. Active Ingredients Catalog [pamphlet] The United States Pharmacopeia, 32nd Revision/The National Formulary, 27th edition. Rockville, MD: The United States Pharmacopeial Convention, ADEKA Corporation. Technical Information. Fermented Beta-Glucan (LQ/Powder) [pamphlet] Cross GG, Jennings HJ, Whitfield DM, and et al. Immunostimulant oxidized -glucan conjugates. International Immunopharmacology. 2001;1:(-): Klecak J Summary report CM-Glucan (Sodium Carboxymethyl Betaglucan) in skin care products. RCC Project Unpublished data submitted by Personal Care Products Council. 89. Hayashibara International Inc. Pullulan GRAS Notification Date Accessed Nakamura S. Pullulan. Journal of Synthetic Organic Chemistry, Japan. 1984;43:(6): CIR Panel Book Page 54

59 91. Leathers TD. Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol. 2003;62:(5-6): Bozou JC, Gautry L, and Pianelli G. A new biopolymer for refreshment. SOFW Journal. 2004;130:(3): Food Chemicals Codex. 8 ed. Rockville, MD: United States Pharmacopeia (USP), Food Chemicals Codex. 8 ed. Rockville, MD: United States Pharmacopeia (USP), Biopolymer International. Xanthan gum - Manufacturing Process Date Accessed Hijiya H and et al. Shaped bodies of pullulan esters and their use ,151:(3,871,892):Okayama, Japan: 97. Crompin JM, Garnier T, Payot T, and DeBaynast R. A new polysaccharide derived from plant rhizosphere: production, purification and physico-chemical properties. Engineering and Manufacturing for Biotechnology. 2002;4:(8): Biopolymer International. Xanthan gum. Product description Date Accessed Bajaj IB., Survase SA, Saudagar PS., and Singhal RS. Gellan gum: Fermentative production, downstream processing and applications. Food Technology and Biotechnology. 2007;45:(4): Biopolymer International. Gellan Gum - Product description Date Accessed Booth AN, Hendrickson AP, and DeEds F. Physiologic effects of three microbial polysaccharides on rats. Toxicology and Applied Pharmacology. 1963;5: McNeely WH and Kovacs P. The physiological effects of alginates and xanthan gum. ACS Symp Ser. 1975;15: Babícek K, Cechova I, Simon RR, Harwood M, and Cox DJ. Toxicological assessment of a particulate yeast (1,3/1,6)- -D-glucan in rats. Food and Chemical Toxicology. 2007;45:(9): Life Science Laboratory Single oral dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg). Test Code No. 99-IA Unpublished data submitted by Personal Care Products Council Juntendo University, Department of Public Hygiene, School of Medicine Report on acute toxicity test on pullulan with mice. Unpublished data reported in Hayashibara International Inc Fogelmark B, Sjostrand M, Williams D, and Rylander R. Inhalation toxicity of (1 3)- D-glucan: recent advances. Mediators of Inflammation. 1997;6:(4): Fogelmark B, Goto H, Yuasa K, Marchat B, and Rylander R. Acute pulmonary toxicity of inhaled -1 3-glucan and endotoxin. Agents and Actions. 1992;35:(1-2): Bonlokke JH, Stridh G, Sigsgaard T, Kjaergaard SK., Loefstedt H, Andersson K, Bonefeld-Jorgensen EC, Jayatissa MN, Bodin L, Juto JE, and Molhave L. Upper-airway inflammation in relation to dust spiked with aldehydes or glucan. Scandinavian Journal of Work, Environment & Health. 2006;32:(5): Walton KW. Investigation of the toxicity of a series of dextran sulphates of varying molecular weight. Brit J Pharmacol. 1954;9: Williams DL, Sherwood ER, Browder IW, McNamee RB, Jones EL, and DiLuzio NR. Pre-clinical safety evaluation of soluble glucan. International Journal of Immunopharmacology. 1988;10:(4): CIR Panel Book Page 55

60 111. Robbins DJ, Moulton JE, and Booth AN. Subacute toxicity study of a microbial polysaccharide fed to dogs. Food and Cosmetics Toxicology. 1964;2:(5): Anderson DMW, Brydon WG, and Eastwood, M. A. The dietary effects of gellan gum in humans. Food Additives & Contaminants. 1988;5:(3): Jonker, D., Hasselwander, O., Tervilae-Wilo, A., and Tenning, P. P. 28-Day oral toxicity study in rats with high purity barley beta-glucan (Glucagel). Food and Chemical Toxicology. 2010;48:(1): Feletti F, De, Bernardi di Valserra M, Contos S, Mattaboni, P, and Germogli R. Chronic toxicity study on a new glucan extracted from Candida albicans in rats. Arzneimittel-Forschung. 1992;42:(11): Kimoto T, Shibuya T, and Shiobara S. Safety studies of a novel starch, pullulan: chronic toxicity in rats and bacterial mutagenicity. Food and Chemical Toxicology. 1997;35:(3/4): Fogelmark B, Sjoestrand M, and Rylander R. Pulmonary inflammation induced by repeated inhalations of (1 3)- D-glucan and endotoxin. International Journal of Experimental Pathology. 1994;75:(2): Rylander R and Holt PG. (1 -Glucan and endotoxin modulate immune response to inhaled allergen. Mediators of Inflammation. 1998;7: Mori H, Ohbayashi F, Hirono I, Shimada T, and Williams GM. Absence of genotoxicity of the carcinogenic sulfated polysaccharides carrageenan and dextran sulfate in mammalian DNA repair and bacterial mutagenicity assays. Nutrition and Cancer. 1984;6:(2): Chen SN, Nan FH, Chen S, Wu JF, Lu CL, and Soni MG. Safety assessment of mushroom -glucan: Subchronic toxicity in rodents and mutagenicity studies. Food and Chemical Toxicology. 2011;49: NOTOX BV Evaluation of the mutagenic activity of CM-Glucan in the Salmonella lyphimurium reverse mutation assay (with independent repeat). Notox Project Unpublished data submitted by Personal Care Products Council Life Science Laboratory Reverse mutation study of CM-Glucan J in bacterial. Test Code No. 99-VII Unpublished data submitted by Personal Care Products Council Magnani M, Calliari CM., de Macedo, Mori MP., de Syllos Colus I, and Castro-Gomez RJH. Optimized methodology for extraction of (1 3)(1 6)- -D-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative. Carbohydrate Polymers. 2009;78:(4): Ishidate M, Sofuni T, and Kishi M. Results of mutagenicity tests of fodd additives (6). Tokishidoroji Foramu (Toxiccoogy Forum). 1985;8: Kuroda K, Yoo YS, and Ishibashi T. Rec-assay on natural food additives. Seikatsu Eisei. 1989;33: Ishidate M, Takizawa Y, Sakabe Y, Ishizaki M Watanabe S, Date M, and Takemoto K. Mutagenicity tests of food additives (9). Tokishidoroji Foramu (Toxiccoogy Forum). 1988;11: Guillot JP, Giauffret JY, Martini MC, Gonnet JF, and Soule G. Safety evaluation of gums and thickeners used in cosmetic formulations. International Journal of Cosmetic Science. 1982;4:(2): Life Science Laboratory Primary skin irritation study of CM-Glucan J in rabbits. Test Code No. 99-JXA Unpublished data submitted by Personal Care Products Council Life Science Laboratory Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method). Test Code No. 99-VIA Unpublished data submitted by Personal Care Products Council. 49 CIR Panel Book Page 56

61 129. Life Science Laboratory Primary skin irritation test for CM-Glucan J in human subjects by closed patch test. Test Code No. 99-Xll Unpublished data submitted by Personal Care Products Council Therapy and Performance Research Institute (GTLF) Repeated patch test for skin sensitization and photoallergy of CM-Glucan (700-01) in healthy male and female volunteers. Unpublished data submitted by Personal Care Products Council Eurofins ATS Ocular tolerance test according to NET CAM method: Sodium Carboxymethyl Beta-Glucan. Unpublished data submitted by Personal Care Products Council Liu Y, Liu J, Zhang X, Zhang R, Huang Y, and Wu C. In situ gelling Gelrite/alginate formulations as vehicles for ophthalmic drug delivery. AAPS PharmSciTech. 2010;11:(2): Ramaiah S, Kumar TMP, and Ravi V. Studies on biopolymers for ophthalmic drug delivery. Journal of Macromolecular Science, Part A: Pure and Applied Chemistry. 2007;44:(2): Life Science Laboratory Primary eye irritation study of CM-Glucan J in rabbits. Test Code No.99-LXB Unpublished data submitted by Personal Care Products Council. 50 CIR Panel Book Page 57

62 Data

63 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 - 11GRED1ENT REVIEW (CIR) Halyna Breslawec, Ph.D. Industry Liaison to the CW Expert Panel DATE: August 29, 2011 SUBJECT: Safety Data Sheet: Xanthan Gum Molecular Weight Letter Jungbunzlauer Safety data sheet: Xanthan Gum. Green R Letter from Biopolymer International to the European Commission concerning molecular weight of polymer additives th Street, N.W., Suite 3O0 Washington, D.C (fax) CIR Panel Book Page 58

64 5:00 Distributed for Comment Only -- Do Not Cite or Quote Jungbunzlauer Product name SAFETY DATA SHEET Xanthan Gum 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION COMMERCIAL PRODUCT NAME Xanthan gum Product codes include all beginning with F for Food grade T for Technical grade except TGD, TGE, TGRD and TGXB COMPANY/SUPPLIER Jungbunzlauer Inc. 7 Wells Avenue Newton Centre, MA Emergency Phone ; 8:30 - M-F Eastern Time 24 Hour Emergency Phone Number CHEMTREC PRODUCT USE Multipurpose thickener and viscosifier, emulsion stabilizer. 2. COMPOSITION/INFORMATION ON INGREDIENTS Chemical name of the substance Polysaccharide (C35 H49 029)n Chemical Name Xanthan Gum EC-No CAS-No Hazardous impurities None 3. HAZARDS IDENTIFICATION Most important Hazard 4. FIRST AID MEASURES General advice Inhalation Skin contact Eye contact consult a specialist. Ingestion Protection of first-aiders Spilled material, when wet, forms very slippery mass. Health injuries are not known or expected under normal use. Ecological injuries are not known or expected under normal use. No hazards which require special first aid measures. If you feel unwell, seek medical advice. Move to fresh air. Consult a physician if necessary. Rinse with water. If skin irritation persists, call a physician. Flush eyes with water as a precaution. If eye irritation persists, Drink water as a precaution. Consult a physician if necessary No special precautions required. DATE DE REVISION: /4- US2O-51 CIR Panel Book Page 59

65 iungbunzlauer Product name SAFETY DATA SHEET Xanthan Gum 5. FIRE FIGHTING MEASURES FLASH POINT no data available FLAMMABLE LIMITS no data available Autoignition temperature >20000 Special characteristics Power forms slippery mass when wet. Suitable extinguishing media water, water spray, dry powder, foam, carbon dioxide (CO2) Extinguishing media which must not be used for safety reasons None Hazardous decomposition products carbon oxides Special protective equipment Use personal protective equipment including self-contained breathing apparatus when fighting fire in enclosed area. Specific methods Standard procedure for chemical fires. 6. ACCIDENTAL RELEASE MEASURES Personal precautions Environmental precautions Methods for cleaning up 7. HANDLING AND STORAGE HANDLING Technical measures/precautions Safe handling advice STORAGE Technical measures/storage conditions Incompatible products Packaging material Use personal protective equipment. Avoid dust formation. Dispose according to federal, state and local authorities. Sweep up and shovel. After cleaning, flush away traces with copious amounts of water. No special technical protective measures required. Immediately sweep up spills. If possible avoid wetting spilled material. If spilled material must be washed down, then copious amounts of water will be necessary. Avoid walking on wet spilled material which will be very slippery. Keep tightly closed in a dry and cool place. No special restrictions on storage with other products. Store in original container. 8. EXPOSURE CONTROLS I PERSONAL PROTECTION EXPOSURE CONTROLS Engineering measures Ensure adequate ventilation, especially in confined areas.. Exposure limit(s) None established for this ingredient, use OSHA PEL, ACGIH TLV for Nuisance dusts of 5 mg/ m3. PERSONAL PROTECTION EQUIPMENT Respiratory protection NIOSH approved dust respirator Hand protection None Eye Protection Safety glasses Skin and body protection Lightweight protective clothing DATE DE REVISION: /4- US2O-51 CIR Panel Book Page 60

66 completely Distributed for Comment Only -- Do Not Cite or Quote iungbunzlauer Product name SAFETY DATA SHEET Xanthan Gum Hygiene measures Handle in accordance with good industrial hygiene and safety practice 9. PHYSICAL AND CHEMICAL PROPERTIES Form powder Color white/beige Odor Characteristic ph,1%solution,25c Vapor pressure not applicable Vapor density not applicable Boiling point not applicable Evaporation rate not applicable Coefficient of water/oil distribution not applicable Melting pointlrange not applicable Decomposition temperature no data available Flash point No data available Autoignition temperature > 200CC Explosive properties No data available Bulk density kg/rn all except ED grades With approx. 400 kg/ m3 Water solubility, 25CC forms colloidal solution - soluble. Solubility in other solvents Alcohol, 25*C Insoluble STABILITY AND REACTIVITY Stability Conditions to avoid Materials to avoid Hazardous polymerization Stable at normal conditions Keep containers dry and tightly closed to avoid moisture absorption and contamination. No special restrictions on storage with other products. Reactivity with strong oxidizers. No decomposition if stored normally. Thermal decomposition can lead to release of irritating gases and vapours. 11. TOXICOLOGICAL INFORMATION Acute toxicity LD5O/p.o./rat = mg/kg (1) Eye irritation None (2) Skin sensitization None (3) Chronic toxicity, oral, rat None < 1000 mg/kg/24h (4) Human experience Health injuries are not known or expected under normal use. References: (1) Jackson, N. N., Woodard, M. W. & Woodard, 0., report from Woodard Research Corporation, Herdon, VA, USA (2) Hendrickson. A.P. & Booth, AN. report from of the Western Regional Research laboratory, USDA, Albany, CA, USA (3) Durloo, R.S. & Johnston, C.P. (1973), report from Woodard research corporation, Herdon, VA, USA (4) Woodard, Get. al. (1973), Toxicol. Appi. Pharmacol., 24, DATE DE REVISION: /4- US2O-51 CIR Panel Book Page 61

67 iungbunzlauer Product name SAFETY DATA SHEET Xanthan Gum 12. ECOLOGICAL INFORMATION Mobility Completely soluble Biochemical oxygen demand within 5 days (BOD5) = 200 mg 02 /g (DIN EN 29408) (1) Readily biodegradable 93 % after 14 days (DIN EN 29888) (1) Bioaccumulation No data available Ecotoxicity LC5O/96h/rainbow trout = 420 mg/i (2) References: (1) Jungbunzlauer, internal report Dec. 99 (2) Sprague, J.B. and Logan, W. J. (1979) Environ. Pollut. 19(4), DISPOSAL CONSIDERATIONS Waste from residues/unused products Any disposal practice must be in compliance with local, state and federal laws and regulations (contact local or state environmental agency for specific rules). 14. TRANSPORT INFORMATION Not classified as dangerous in the meaning of transport regulations REGULATORY INFORMATION FDA approved as a food additive. EU Food Additive E41 5 TSCA Inventory List listed CERCLA (Comprehensive Response Compensation, and Liability Act): Not hazardous SARA Title Ill (Superfund Amendments and Reauthorization Bill): Not Considered Hazardous Foreign Inventory Status Canadian DSL (Domestic Substance List) 16. OTHER INFORMATION HMIS* Rating Health =0, Fire = 0, Reactivity =0 *Hazardous Material Information System of the National Paint and Coating Association The information provided in this Safety Data Sheet is correct to the best of our knowledge, information and belief at the date of its publication. The information given is designed only as a guidance for safe handling, use, processing, storage, transportation, disposal and release and is not to be considered a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other materials or in any process, unless specified in the text. DATE DE REVISION: /4- US2O-51 CIR Panel Book Page 62

68 BIOPOLYMER INTERNATIONAL ASSOCIATION MONDIALE DES PRODUCTEURS DE BIOPOLYMERES ALIMENTAIRES WORLD ASSOCIATION OF FOOD GRADE BIOPOLYMER PRODUCERS (By ) To: Mr Dimitrios Chrysafidis European Commission Directorate General for Health and Consumers 200, Rue de la Loi B-1049 Bruxelles Dear Mr Chrysafidis, Brussels, 14 April 2010 Subject: Biopolymer International response to the European Commission s molecular weight ofpolymer additives request regarding Biopolymer International is an association of world-wide manufacturers of xanthan gum and gellan gum, and it is a member of ELC, the Federation of European Specialty Food Ingredient Industries. Following to your request of 10 March 2010 sent to ELC regarding the molecular weight in specifications of a number of polymer additives, we would like to contribute to this discussion with some comments, in particular as concerns xanthan gum and gellan gum. Specifically the Commission is requesting that for certain food polymer additives a reference is needed on the model of the molecular weight, and where it is difficult or not possible to measure directly the molecular weight, to characterize the polymer by a viscosity measurement, and that in all cases a clear connection between viscosity data and weight average molecular weight is needed. Thus the purpose of such a request does not seem to be based on any food safety concern, but rather on the need for information to better characterize and identify these polymer additives. We believe that the molecular weight is not a very good parameter for characterizing the polymer additives since 1) the molecular weight often would be within a very wide range not being very descriptive (e.g. the current molecular weight range of guar gum from 50,000-8,000,000), and 2) the molecular weight determination (whatever method) is very difficult to determine independently of the various techniques (HPLC and light scattering etc.). Academic projects on molecular weight on such type of polymers were, to the best of our knowledge, far away from establishing a laboratory routine analysis. Moreover the viscosity of a polymer like xanthan gum or gellan gum is influenced by various parameters (see appendix). Indeed there is no clear correlation between the relative viscosity and molecular weight. There is a link between intrinsic viscosity and molecular weight, which is why the intrinsic viscosity is often used to determine the molecular weight. However, this link or relationship is case specific and depends on the shape on the polymer additive in solution and this shape is defined by the source and processing of the polymer as well as the media (application) it is hydrated in. This again underlines the difficulties and emphasizes the low relevance of determining the molecular weight (whatever method) as an absolute measure. It is not possible to propose a universal correlation between viscosity and molecular weight in the specifications of xanthan gum or gellan gum or any of the other polymer additives. If this were possible it would be easy to determine the molecular weight which is not the case as already mentioned. Also, we do not see the need for correlating the viscosity to the molecular weight since it is rather the 1 CIR Panel Book Page 63

69 BIOPOLYMER INTERNATIONAL ASSOCIATION MONDIALE DES PRODUCTEURS DE BIOPOLYMERES ALIMENTAIRES WORLD ASSOCIATION OF FOOD GRADE BIOPOLYMER PRODUCERS viscosity than the molecular weight that is interesting for characterizing a polymer additive and that has a practical importance in connection to the technological function of the polymer. Viscosity is certainly a better parameter to characterize polymer additives compared to the molecular weight, not at least considering the technological function of the polymer additives. Thus Biopolymer International concludes that: It is very uncertain that a universal technique to measure the molecular weight of these polymers can be devised, if it has to remain practically applicable at the same time. A clear correlation between molecular weight and viscosity cannot be established. Relative viscosity is a better parameter than molecular weight to characterize polymer additives. Please see appendix for further technical details. Should you need further clarification, please do not hesitate to contact us. Yours faithfully, Richard Green President Biopolymer International (Cc: Maryse Hervé / ELC) 2 CIR Panel Book Page 64

70 Technical Distributed for Comment Only -- Do Not Cite or Quote BIOPOLYMER INTERNATIONAL ASSOCIATION MONDIALE DES PRODUCTEURS DE BIOPOLYMERES ALIMENTAIRES WORLD ASSOCIATION OF FOOD GRADE BIOPOLYMER PRODUCERS Appendix details on molecular weight of a polymer Numerous techniques exist, which give the molecular mass of a polymer. 1 2 The number average molecular weight (Ma) It can be determined using membrane osmometry, cryoscopy, ebulliometry and vapour pressure osmometry. Membrane osmometry, which is considered the most useful technique, can in practice only. be used when the molecular weight is below 500,000 g mol 1 The weight average molecular weight (Mw) It is measured using light scattering. The molecular weight can be extracted from e.g. a Zimm-plot. This technique requires sophisticated equipment and highly trained staff, and is not practical for routine measurements. The viscosity average molecular weight (My) It is based on viscometry. Viscometry is not an absolute technique and requires calibration for each polymer system. Measured viscosities can be expressed as relative viscosity, specific viscosity, reduced viscosity, inherent viscosity and intrinsic viscosity ([ii]). According to the Kuhn-Mark-Houwink-Sakurada equation, the intrinsic viscosity ([i]) is proportional to the viscosity average molecular weight (Mg). However, conversion requires the establishment for two empirical constants, which differ for each polymer, solvent and temperature. The z-average molecular weight (Mt) It is determined from the sedimentation equilibrium under ultracentrifugation. Correlation between viscosity data and molecular weight (Mw) There exists no clear correlation between viscosity data (in any form) and the weight average molecular weight (Mw). Furthermore intrinsic and extrinsic factors complicate the exact determination of the molecular weights: Intrinsic factors All polysaccharides are a polydisperse mixture of molecular weights (due to natural variation andjor processing). Many polysaccharides have an extremely broad range of molecular weights. Presence of charged groups within the molecule (most food polysaccharides are polyelectrolytes). Presence of alternating or block sequences (true for almost all polysaccharides). Chain branching. Solubility (not all food hydrocolloids, are water soluble or some, e.g. xanthan gum (E415), form very easily intermolecular aggregates). Extrinsic factors The hydration, association and/or radius of gyration of polysaccharides depends on: ph; Temperature; Ion strength of solvent; Presence of specific ions, e.g. calcium or potassium. Consequently, these factors must be carefully defined for each hydrocolloid and a specific buffer must be suggested. Moreover a filtration of particulates and/or an ion-exchange exchange will be necessary for many hydrocolloids. References [1] Arndt, K.-F., Muller, G. Polymer Charakterisierung, München: Hanser, [2] Cui, S. W. Food Carbohydrates, Boca Raton: CRC Press, [3] Harding, S. E.; Vârum, K. M.; Stokke, B.T.; Smidod, 0. Molecular weight determination of polysaccharides. Advances in Carbohydrate Analysis, 1, CIR Panel Book Page 65

71 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Personal Care iproducts Council Committed to Safety, Qua ity & Innovation Memorandum TO: FROM: DATE: SUBJECT: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CW) Halyna Breslawec, Ph.D. Industry Liaison to the CIR Expert Panel October 26, 2011 Concentration of Use by FDA Product Category: Microbial Polysaccharides th Street, N.W., Suite 300 Washington, D.C (fax) CIR Panel Book Page 66

72 Concentration of Use by FDA Product Category* Xanthan Gum Dextran Sulfate Dehydroxanthan Gum Sodium Dextran Sulfate Hydroxypropyl Xanthan Gum Scierotium Gum Undecylenoyl Xanthan Gum Hydrolyzed Scierotium Gum Xanthan Gum Crosspolymer Beta-Glucan Gellan Gum Beta-Glucan Hydroxypropyltrimonium Welan Gum Chloride Biosaccharide Gum-i Beta-Glucan Palmitate Biosaccharide Gum-2 Hydrolyzed Beta-Glucan Biosaccharide Gum-3 Oxidized Beta-Glucan Biosaccharide Gum-4 Sodium Carboxymethyl Beta-Glucan Biosaccharide Gum-5 Pullulan Pseudoalteromonas Exopolysaccharides Myristoyl Pullulan Dextran Levan Carboxymethyl Dextran Rhizobian Gum Dextran Hydroxypropyltrimonium Hydrolyzed Rhizobian Gum Chloride Alcaligenes Polysaccharides Sodium Carboxymethyl Dextran Ingredient Product Category Maximum Concentrations of Use Xanthan Gum Baby shampoo % Xanthan Gum Baby lotions, oils, powders and creams % Xanthan Gum Other baby products 0.2% Xanthan Gum Bubble baths 0.5% Xanthan Gum Other bath preparations 1-3% Xanthan Gum Eye liner % Xanthan Gum Eye shadow 0.1-2% Xanthan Gum Eye lotion % Xanthan Gum Eye makeup remover % Xanthan Gum Mascara % Xanthan Gum Other eye makeup preparations % Xanthan Gum Colognes and toilet waters 0.5% Page 1 of 9 CIR Panel Book Page 67

73 Xanthan Gum Powders (dusting and talcum) 6% Xanthan Gum Other fragrance preparations % Xanthan Gum Hair conditioners % Xanthan Gum Hair straighteners % Xanthan Gum Rinses (noncoloring) 0.01% Xanthan Gum Shampoos (noncoloring) % Xanthan Gum Tonics, dressings and other hair grooming % aids Xanthan Gum Other hair preparations (noncoloring) 0.4% Xanthan Gum Hair dyes and colors (all types requiring % caution statement and patch test) Xanthan Gum Hair tints 0.2% Xanthan Gum Hair rinses (coloring) 3% Xanthan Gum Hair lighteners with color 4% Xanthan Gum Hair bleaches 0.5-6% Xanthan Gum Other hair coloring preparations 0.2% Xanthan Gum Blushers (all types) % Xanthan Gum Face powders 0.2% Xanthan Gum Foundations % Xanthan Gum Lipstick 0.5-2% Xanthan Gum Makeup bases % Xanthan Gum Rouges 0.05% Xanthan Gum Makeup fixatives 0.3% Xanthan Gum Other makeup preparations % Xanthan Gum Cuticle softeners 0.3-3% Xanthan Gum Nail creams and lotions 0.4% Xanthan Gum Nail polish and enamel 1% Page 2 of 9 CIR Panel Book Page 68

74 Xanthan Gum Other manicuring preparations 0.2% Xanthan Gum Dentifrices (aerosol, liquid, pastes and 0.4-1% powders) Xanthan Gum Mouthwashes and breath fresheners % (liquids and sprays) Xanthan Gum Other oral hygiene products % Xanthan Gum Bath soaps and detergents 0.2-2% Xanthan Gum Deodorants (underarm) % Not a spray product 0.4-1% Xanthan Gum Other personal cleanliness products 0.5-4% Xanthan Gum Aftershave lotions % Xanthan Gum Preshave lotions (all types) 0.1% Xanthan Gum Shaving cream (aerosol, brushless and % lather) Xanthan Gum Shaving soap (cakes, sticks, etc.) 0.003% Xanthan Gum Other shaving preparations 0.5% Xanthan Gum Skin cleansing (cold creams, cleansing % lotions, liquids and pads) Xanthan Gum Depilatories 0.2% Xanthan Gum Face and neck creams, lotions and 0.2-2% powders Xanthan Gum Body and hand creams, lotions and 0.1-2% powders Pump spray 0.05% Xanthan Gum Foot powders and sprays 0.4% Xanthan Gum Moisturizing creams, lotions and powders % Xanthan Gum Night creams, lotions and powders % Xanthan Gum Paste masks and mud packs % Xanthan Gum Skin fresheners % Xanthan Gum Other skin care preparations 0.3-3% Page 3 of 9 CIR Panel Book Page 69

75 Xanthan Gum Suntan gels, creams and liquids 0.3-1% Not a spray product % Xanthan Gum Indoor tanning preparations 0.2-1% Xanthan Gum Other suntan preparations 0.4% Dehydroxanthan Gum Shampoos (noncoloring) 0.7% Dehydroxanthan Gum Tonics, dressings and other hair grooming 1% aids Dehydroxanthan Gum Bath soaps and detergents 0.4% Dehydroxanthan Gum Skin cleansing (cold creams, cleansing 0.8% lotions, liquids and pads) Dehydroxanthan Gum Face and neck creams, lotions and 0.1% powders Spray 0.2% Dehydroxanthan Gum Suntan gels, creams and liquids 0.1% Xanthan Gum Crosspolymer Face and neck creams, lotions and 0.03% powders Xanthan Gum Crosspolymer Body and hand creams, lotions and 5% powders Gellan Gum Eye shadow % Gellan Gum Tonics, dressings and other hair grooming 0.5% aids Gellan Gum Face powders % Gellan Gum Foundations % Gellan Gum Lipstick % Gellan Gum Makeup bases 0.3% Gellan Gum Face and neck creams, lotions and % powders Biosaccharide Gum-i Eye liner 0.0 1% Biosaccharide Gum-i Eye shadow 0.01% Biosaccharide Gum-i Eye lotion % Page 4 of 9 CIR Panel Book Page 70

76 Biosaccharide Gum-i Foundations % Biosaccharide Gum-i Lipstick 0.08% Biosaccharide Gum-i Shaving cream (aerosol, brushless and 0.006% lather) Biosaccharide Gum-i Other shaving preparations 0.02% Biosacchande Gum-i Skin cleansing (cold creams, cleansing 5% lotions, liquids and pads) Biosaccharide Gum-i Face and neck creams, lotions and % powders Biosaccharide Gum-i Body and hand creams, lotions and % powders Biosaccharide Gum-i Moisturizing creams, lotions and powders % Biosaccharide Gum-i Night creams, lotions and powders 0.1-1% Biosaccharide Gum-i Paste masks and mud packs % Biosaccharide Gum-i Other skin care preparations % Biosaccharide Gum-i Indoor tanning preparations 0.002% Biosaccharide Gum-2 Face and neck creams, lotions and 1% powders Biosaccharide Gum-2 Night creams, lotions and powders 1% Biosaccharide Gum-4 Eye lotion 0.2% Biosacchande Gum-4 Eye makeup remover % Biosaccharide Gum-4 Foundations % Biosaccharide Gum-4 Makeup fixatives 5% Biosaccharide Gum-4 Skin cleansing (cold creams, cleansing % Lotions, liquids and pads) Biosaccharide Gum-4 Face and neck creams, lotions and % powders Biosaccharide Gum-4 Body and hand creams, lotions and 0.1-1% powders Biosaccharide Gum-4 Moisturizing creams, lotions and powders 0.1% Page 5 of 9 CIR Panel Book Page 71

77 Biosaccharide Gum-4 Indoor tanning preparations 1% Dextran Bath oils, tablets and salts 0.2% Dextran Face and neck creams, lotions and % powders Dextran Night creams, lotions and powders % Dextran Other suntan preparations 0.01% Dextran Sulfate Foundations 0.1% Dextran Sulfate Face and neck creams, lotions and 0.0 1% powders Dextran Sulfate Body and hand creams, lotions and 0.0 1% powders Sodium Dextran Sulfate Eye lotion % Sodium Dextran Sulfate Other eye makeup preparations 0.02% Sodium Dextran Sulfate Other makeup preparations 0.2% Sodium Dextran Sulfate Face and neck creams, lotions and % powders Sodium Dextran Sulfate Body and hand creams, lotions and % powders Sodium Dextran Sulfate Other skin care preparations 0.5% Sclerotium Gum Other bath preparations 0.003% Scierotium Gum Eye lotion % Sclerotium Gum Eye makeup remover 0.6% Sclerotium Gum Mascara % Sclerotium Gum Colognes and toilet waters 0.003% Sclerotium Gum Hair conditioners % Scierotium Gum Shampoos (noncoloring) % Sclerotium Gum Tonics, dressings and other hair grooming % aids Sclerotium Gum Hair dyes and colors (all types requiring 0.8% Page 6 of 9 CIR Panel Book Page 72

78 caution statement and patch test) Sclerotium Gum Blushers (all types) 2% Scierotium Gum Foundations 2% Sclerotium Gum Other manicuring preparations 0.6% Scierotium Gum Other personal cleanliness products 0.5% Sclerotium Gum Shaving cream (aerosol, brushless and 0.02% lather) Sclerotium Gum Skin cleansing (cold creams, cleansing % lotions, liquids and pads) Sclerotium Gum Depilatories 0.5% Sclerotium Gum Face and neck creams, lotions and % powders Sclerotium Gum Body and hand creams, lotions and % powders Sclerotium Gum Moisturizing creams, lotions and powders % Sclerotium Gum Night creams, lotions and powders % Sclerotium Gum Paste masks and mud packs % Sclerotium Gum Other skin care preparations % Hydrolyzed Sclerotium Gum Moisturizing creams, lotions and powders 1% Beta-Glucan Hair conditioners % Beta-Glucan Bath soaps and detergents 0.03% Beta-Glucan Aftershave lotions 0.005% Beta-Glucan Face and neck creams, lotions and 0.002% powders Beta-Glucan Body and hand creams, lotions and % powders Beta-Glucan Moisturizing creams, lotions and powders 0.1% Beta-Glucan Night creams, lotions and powders % Beta-Glucan Paste masks and mud packs 0.001% Page 7 of 9 CIR Panel Book Page 73

79 Beta-Glucan Skin fresheners 0.001% Beta-Glucan Other skin care preparations 0.01% Beta-Glucan Suntan gels, creams and liquids Not a spray product 0.1% Sodium Carboxymethyl Beta- Baby lotions, oils, powders and creams Glucan Powder 0.02% Sodium Carboxymethyl Beta- Other baby products 0.0 1% Glucan Sodium Carboxymethyl Beta- Eye lotion 0.04% Glucan Sodium Carboxymethyl Beta- Hair conditioners 0.04% Glucan Sodium Carboxymethyl Beta- Foundations % Glucan Sodium Carboxymethyl Beta- Vaginal Douches 0.1% Glucan Sodium Carboxymethyl Beta- Aftershave lotions 0.1% Glucan Sodium Carboxymethyl Beta- Skin cleansing (cold creams, cleansing % Glucan lotions liquids and pads) Sodium Carboxymethyl Beta- Face and neck creams, lotions and % Glucan powders Sodium Carboxymethyl Beta- Body and hand creams, lotions and % Glucan powders Sodium Carboxymethyl Beta- Moisturizing creams, lotions and powders % Glucan Sodium Carboxymethyl Beta- Night creams, lotions and powders 0.1% Glucan Sodium Carboxymethyl Beta- Other skin care preparations 0.1% Glucan Pullulan Eye lotion 3% Pullulan Tonics, dressings and other hair grooming 12% Page 8 of 9 CIR Panel Book Page 74

80 aids Pullulan Hair dyes and colors (all types requiring 0.03% caution statement and patch test) Pullulan Other oral hygiene products 17% Pullulan Face and neck creams, lotions and % powders Pullulan Body and hand creams, lotions and 0.9% powders Pullulan Moisturizing creams, lotions and powders 0.9% Hydrolyzed Rhizobian Gum Eye lotion 3% Hydrolyzed Rhizobian Gum Face and neck creams, lotions and 0.4-3% powders Alcaligenes Polysaccharides Face and neck creams, lotions and 0.3% powders Alcaligenes Polysaccharides Paste masks and mud packs 0.005% *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. 0.4% in a rinse-off product Information collected in 2011 Table prepared October 26, 2011 Page 9 of 9 CIR Panel Book Page 75

81 CIR Panel Book Page 76

82 COSMETIC Distributed for Comment Only -- Do Not Cite or Quote Personal Care Products Counci Committed to Safety, ua ity nnovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) Halyna Breslawec, Ph.D. Industry Liaison to the CW Expert Panel DATE: August 22, 2011 SUBJECT: Specifications and Safety data on Sodium Carboxymethyl Beta-Glucan Mibelle Biochemistry CM-Glucan granulate specifications. Kiecak J Summary report CM-Glucan (Sodium Carboxymethyl Betaglucan) in skin care products. RCC Project Therapy and Performance Research Institute (GTLF) Repeated patch test for skin sensitization and photoallergy of CM-Glucan (700-01) in healthy male and female volunteers. Notox B.V Evaluation of the mutagenic activity of CM-Glucan in the Salmonella lyphimurium reverse mutation assay (with independent repeat). Notox Project Life Science Laboratory Primary eye irritation study of CM-Glucan J in rabbits. Test Code No. 99-LXB Life Science Laboratory Primary skin irritation study of CM-Glucan J in rabbits. Test Code No. 99-JXA Life Science Laboratory Primary skin irritation test for CM-Glucan J in human subjects by closed patch test. Test Code No. 99-Xll Life Science Laboratory Reverse mutation study of CM-Glucan J in bacterial. Test Code No. 99-Vil Life Science Laboratory Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method). Test Code No. 99-VIA Life Science Laboratory Single dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg). Test Code No. 99-IA th Street, N.W., Suite 30O Washington, D.C (fax) CIR Panel Book Page 77

83 Eurofins ATS Ocular tolerance test according to NET CAM method: Sodium Carboxymethyl Beta-Glucan. 2 CIR Panel Book Page 78

84 CM-Glucan granulate is a modified water-soluble polysaccharide derived from cell walls of bakers yeast (Saccharomyces cerevisiae). Three out of four glucose-units of the beta 1,3 glucopyranose are carboxymethylated. Composition CM-Glucan 90% Nitrogen (Protein) Glycolic acid Chloroacetic acid volatile matter <0.5% <1.0% <0.05% -4 0% INCI (EU-Declaration / CTFA-Declaration) Sodium Carboxymethyl Betaglucan Physical properties Consistency Odor Color granulate characteristic, isopropyl alcoholic amber, white Characteristics Degree of substitution Preservation 0.75 ± 0.1 without additional preservatives Bacteriology total germ count < 100 CFTJ/g Stability 3 years Packaging Storage 100g. 200g. 500g 25 C, in closed containers at a dark place Specification: CM-Glucan granulate CIR Panel Book Page 79 W rnibeiieiochemiitrv

85 SUMMARY - REPORT Documenting the innocuousness and benefits from the use of CM - GLUCAN (Sodium Carboxymethyl Betaglucan) in skin care products RCC PROJECT FROM MIBELLE AG, COSMETICS BOLIMATTSTRASSE 1 CH BUCHS SWITZERLAND Dr. med. Jiri Kiecak Dermatologist/Dermatotoxicologist Consultant FinaL Version - Birsfelden, 09-March-1998 Date: C i RCC Group CIR Panel Book Page 80

86 RCC PROJECT CM-GLUCAN 2. GENERAL INTRODUCTION Glucans are complex cell-membrane-polysaccharides from the inner wall of sacharomyces cerevisiae, they and show therapeutic potential. Glucan specific immune stimulants. They can modify physiological processes surgical manipulations and (2). and do and its water-soluble derivatives are the to increase host resistance glucose derivatives (1,2). Isolated exhibit various biological activities used as or non course of various physiological infection complications following patho CM-Glucan is from a carboxymethylated water soluble beta-(l- the cell wall of backers yeast s (3). >3) derivative of glucan, isolated CH2OCH 2COO That OH 20H OH 2.1. CHEMICAL CHARACTERISTIC PRODUCT CLASSIFICATION! SPECIFIC USE Cosmetics PHYSICAL FORM/COLOUR PURITY POISONOUS CORROSIVE FLAMMABLE EXPLOSIVE white/brown solid 95% no no no no MOLECULAR WEIGHT approx. IO0 000 SOLUBILITY IN WATER2% ph approx. 7 at a concentration of 2% CIR Panel Book Page 81

87 RCC PROJECT CM-GLUCAN 2.5. MARKET PREPARATIONS from CM-Glucan As intended for marketing and use in cosmetic andjor dermatologic preparations the specifications for those preparations of CM-Glucan are as follows: COMPOSITION CM-Glucan CM-Glucan KF Sodium Carboxymethyl Sodium Carboxymethyl Betaglucan 2% Betaglucan 2% Imidazolidinyl urea 0.5% Ethanol abs. 10% Phenoxyethanol 0.5% Propylene glycol 5% Deionised water ad 100% Deionised water ad 100% PHYSICAL PROPER TIES Consistency liquid, viscous Odour odourless CHARACTERISTICS ph-value Degree of substitution ,1 Preservation 0.5% Imidazolidinyl urea 0.5% Phenoxyethanol Bacteriology total germ count; < 100 germs/g STABILITY stable in storage at 25 C for more than 6 months PACKAGING 10 1 polyethylene containers STORAGE 25 C in closed containers CIR Panel Book Page 82

88 RCC PROJECT CM-GLUCAN 2.6. LD 50 of DIFFERENT INGREDIENTS Sodium Carboxymethyl not determined Betaglucan, Yeast Polysaccharide Imidazolidinyl urea 5.2 g/kg rat, oral Phenoxyethanol > 2000 mg/kg rat, oral Ethanol 12 g/kg rat, intraperitoneal Propylene glycol > 5000 mg/kg rat, oral 2.7. HEAVY METALS in CM-Glucan The examinations showed that heavy metals have not been accumulated in the product CONCENTRATION of CHLOROACETIC ACID in CM-Glucan The concentration of chioroacetic acid has been determined by HPLC analysis and found to be <5 PPM (active material) FORMULATING with CM-Glucan CM-Glucan is supplied as an aqueous solution containing 2% Sodium Carboxymethyl Betaglucan. It can be incorporated directly into most cosmetic and dermatological formulations. It is compatible with most other cosmetic ingredients (i.e. non-ionic and anionic surfactants, alcohol s in concentrations up to 40%). CM-Glucan tolerates normal manufacturing conditions (i.e., ph values from 2-10, high salt concentrations, temperatures as high as 80 C). However, precipitation of CM-Glucan can occur in the presence of positively charged ions and polymers. CIR Panel Book Page 83

89 RCC PROJECT CM-GLUCAN The use levels are between 0.5% - 10% of CM-Glucan (2% solution of Sodium Carboxymethyl Betaglucan), corresponding to a 0.01 to 0.2% concentration of the active ingredient in the final cosmetic or dermatological preparations. The skin tolerance of 2% CM-Glucan final preparations has been carefully proved in healthy volunteers. With the Human Repeated Insult Patch Test for the determination of the irritation, sensitisation, photo-irritation and photo-sensitisation potential, generally a good skin tolerance of the product has been confirmed. CIR Panel Book Page 84

90 G T L F THERAPY ANt) PERFORMANCE RESEARCH INSTITUTE GTLF PROJECT 9142/2 Repeated Patch Test for Skin Sensitization and Photoallergy of CM - GLUCAN (700-01) in Healthy Male and Female Adult Volunteers REPORT Page 1 Out of 24 COPYRIGHT AND PROPERTY All rights strictly reserved. Reproduction or issue to third parties in any form whatever is not permt ted without written authority trom the proprietor. Property of SPONSOR and GTLF. c; SIl.1 ii A Ii l-üo Tl-IEOAPIE- U ND (LI sruxc;sfohscn I NC (Gil.F) M OH MANAC;INC; JJIREC1OR I I{OF. DII. DII. E. FiNK COMPANY IIEGISi1IAlION No JIJEIIIl I, IIAYFIIN Jlnden0iIslrJ0 so is sszo 1:11 naenthlcphow ) JoleJa loulk AcounJ l3v rlsche VcIeinsbjlkErlangcn No BLZ CIR Panel Book Page 85

91 GTLF Project 9142/2 CM Gtucan 2 ol 24 Mibelle AG Cosmetics Report PROJECT STAFF SIGNATURES PRINCIPAL tnvestigatdr/ CLINICAL INVESTIGATOR: Prof. Dr. Dr. E. Fink, GTLF QAU MANAGER: Dr. K. W. Franke 2 STUDY MONITOR: C. Saecker, Mibelle AG Cosmetics PRODUCT SPECIALIST: Dr. F. ZUlu, Mibelle AG Cosmetics date CIR Panel Book Page 86

92 GTLF Project 9142 /2 CM Glucan 3 at 24 Mibetle AG Cosmetics Report SUMMARY AND CONCLUSION The purpose of this study was to evaluate the sensitizing and photoallergic potential of CM - Glucan with the repeated patch test method. CM - Glucan was applied at a concentration of 2 % in distilled water repeatedly during the in duction phase (9 x for sensitization Part A; 6 x for photoallergy, Pad B) for appr. 24 hours in duplicate to non-tanned skin. One duplicate site was irradiated with appr. twice the MED of UV-B (for photoatlergy) within 10 mm. alter patch removal from the test site; the other dupli cafe site remained unexposed (for sensitization). The challenge reaction was provoked by ap plying the test article in an identical manner to a naive test site after a rest period of 10 to 14 days, but by irradiating one duplicate test site with 18 J / cm2 UV-A within 10 mm, after patch removal. Skin reactions of the induction phase were read immediately and 24 hours after patch removal or irradiation in both tests and in addition 48 hours after patch removal for photoallergy. During the challenge phase of both tests skin readings additionally took place 72 hours after patch removal. Reactions were scored in a five-point scale from 0 to 4. The number of individuals, who became sensitized or pholoallergic, is determined by subtract ing the number of individuals with skin reactions 1 in the pretest (GTLF Project 9142/1) from the number of individuals with skin reactions 1 at challenge An overall sensitization rate (OSR) is calculated by dividing the number of volunteers sensi tized by the total number of volunteers clearly negative in the pretest. The photoaltergic potential can be estimated by comparing the OSR of the test article under ir radiation with that without irradiation. 32 subjects, 8 males and 24 females, aging between 18 and 58 years were taken into the study. Demographic data of all participants, their individual MED and skin reactions on the ir radiated and non-irradiated patch areas are given in tables ito 6 of the Appendix. The num ber of reactions and the reaction scores for both areas are summarized under RESULTS. All participants completed the study within the scheduled time. Sensitization (Part A) CM - Glucan did not produce any skin reaction in the pretest (Skin irritation, GTLF Project 9142/1). 2 volunteers showed a doubtful reaction and one volunteer a grade I reaction dur ing induct ion. None volunteer showed any skin reaction after challenge. It can be concluded, that the test article does not have sensitizing properties under conditions applied in this study. Photoaltergy (Part B) CM - Glucan did not produce any skin reaction in the pretest (photo irritation, GTLF Project 9142/1). 31 volunteers showed grade I skin reactions, and one volunteer of grade 2 due to UV-B exposure during induction. One volunteer showed a doubtful reaction after challenge. It can be concluded, that the test article does not have photoallergic properties under conditions applied in this Study. CIR Panel Book Page 87

93 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 /2 CM - 4 of 24 Mibelle AG Cosmetics Report CONTENTS PROJECT STAFF SIGNATURES 2 SUMMARY AND CONCLUSION 3 1. GENERAL Project Staff Schedule Archiving Quality Assurance Statement Statement of Compliance 9 2. RESULTS 10 3, RATIONALEOFTHETRIAL ETHICAL CONSIDERATIONS Risk Assessment Informed Consent Ethics Committee Approval STUDY DESIGN Randomization Concomitant Medication SELECTION OF VOLUNTEERS Numberof Volunteers Inclusion Criteria Exclusion Criteria TEST ARTICLE Test Article Preparation and Dosage Test Article Preparation ,2 Test Article Dosage Accounting for Test Article Supply Test Article Application Site of Application for sensitization for photoallergy Occlusion 16 CIR Panel Book Page 88

94 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - 5 of 24 Mibelle AG Cosmetics Report CONTENTS (cid.) 72.3 Frequency of Application 17 7,2.3.1 for sensitization (pail A) tor photoallergy (part B) for challenge Duration of Experimental Parts Induction Phases for Sensitization (Pail A) and Photoallergy (Pail 8) Treatment Free Period Challenge for Part A and ,2.4,4 Total Minimal Time Duration of Exposure Control Patch Concomitant Exposure SKIN IRRADIATION is 8.1 Light Source Evaluation of the Minimal Erythemal Dose for UV-B Sensitivity Irradiation for Pholoallergy (Pail B) during induction at challenge ASSESSMENT OF SKIN REACTIONS Reading of Skin Reactions Reading of Skin Reactions during the Induction Phase 189 of Sensitization (Part A) and Photoallergy (Part B) Test Reading of Challenge Reactions (Part A and B) Recording and Interpretation of Results RECORDING OF ADVERSE REACTIONS TIMINGANDPRACTICATIQN Control at Compliance Place of Conduction of the Study Amendment Procedures Deviation from the Protocol EVALUATIONOFDATA Premalure terminations Monitoring Control of Adherence to the Protocol Instruction of Staft and Volunteers 21 CIR Panel Book Page 89

95 GTLF Project 9142 / 2 CM - Glucan 6 of 24 Mibelle AG Cosmetics Report CONTENTS (cid.) 13. STATISTICAL EVALUATION INSURANCE LIST OF ABBREVIATIONS DATA COMPILATION REFERENCES DISTRIBUTION 24 APPENDIX: Table 1: List of Participants Table 2: Summary of Individual Skin Reactions Sensitization ( Part A) Table 3: Summary of Individual Skin Reactions Photoaltergy (Part B) Table 4: Reading of Skin Reactions Sensitization (Part A) Table 5: Reading of Skin Reactions Photoatlergy ( Part B) Table 6: Reading of Challenge-Reactions Product Characterization Amendment 1 Ethics Committee Approval Trial Schedule Informed Consent Curriculum Vitae of Principal Investigator Curriculum Vitae of Auditor CIR Panel Book Page 90

96 5033 Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM Gtucan 7 ot 24 Mibetle AG Cosmetics Report 1. GENERAL Title Sponsor Product Specialist Testing Facility Repeated Patch Test for Skin Sensitization and Photoallergy of CM- Glucan (700-01) in healthy male and female adult volunleers. MIBELLE AG COSMETICS Bolimattstr. I CH - Buchs / AG Dr. F. Zulu GTLF - Therapy and Performance Research Institute Ltd., Hair and Skin Care Clinic, Fichtestr. 14, D-8520, Erlangen, Federal Republic of Germany GTLF Project Number 9142 / 2 Test Article CM Glucan (700.01) 1.1 PROJECT STAFF Principal Investigator / Clinical Investigator: Prof. Dr. med. Dr. rer. nat. habit. E. Fink 1.2 SCHEDULE Treatment / Start: November 2, 1992 Clinical part completed: December 19, 1992 Date of Preliminary Report: December 22, 1992 Date of Final Report: July 14, ARCHIVING The GTLF THERAPY AND PERFORMANCE RESEARCH INSTITUTE Ltd., Erlangen, will archive the following data for at least 10 years: protocol, report, duplicate of report, test article reference sample and raw data (CRFs, ICF) CIR Panel Book Page 91

97 GTLFProject9l42/2 CM-Glucan 8of 24 Mibelle AG Cosmetics Report 1.4 QUALITY ASSURANCE UNIT STATEMENT STATEMENT PROJECT NUMBER: 9142 / 2 TEST ARTICLE: CM - Glucan (700-01) PRINCIPAL investigator: TITLE: Prof. Dr. Dr. E. Fink Repeated patch test for skin sensitization and photoallergy of CM - Glucan (700-01) in healthy male and female adult volunteers. Based on a quality assurance audit, it is confirmed that this report is a complete and correct reflection of the raw data and that the study was performed in accordance with the EG Note for Guidance Good Clinical Practice for Trials on Medicinal Products in the Europe an Community (111/3976/88-EN, Final of ), Controls on accuracy and correspondence with the protocol were performed on July 7, 1993 and the results reported to the Principal Investigator on July 7, Approved and submitted by: Dr. K. W. Franke Quality Assurance Unit (QAU) 2t 7/3 CIR Panel Book Page 92

98 GTLF Project 9142 / 2 CM - Glucan 9 of 24 Mibelle AG Cosmetics Report 1.5 GOOD CLINICAL PRACTICE STATEMENT OF COMPLIANCE PROJECTNUMBER: 9142/2 TEST ARTICLE: CM - Glucan (700-01) PRINCIPAL INVESTIGATOR: TITLE: Prof. Dr. Dr. F. Fink Repeated patch test for skin sensitization and photoallergy of CM - Glucan (700-01) in healthy male and female adult volunteers. To the best ot my knowledge and belief, the study described in this report was conducted in compliance with the tollowing Good Clinical Practice Standards: Recommended Basis tor the Conduct of Clinical Trials of Medicinal Products in the European Community; EEC/CPMP Working Party on Efficacy of Drugs, (lll/3976/88-en,final of July 11, 1990). DurchfOhrung von klinischen ArzneimittelprUfungen in der Bundesrepublik Deutschland, Merkblatt des SF1. Pharmazeut. md. 50, (1988). Declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. Adopted by the 18th World Medical Assembly, Tokyo, Japan, October 1975, and revised by the World Medical Assembly, Venice, Italy, October 1983, and Hong Kong, September Principal Investigator: Prof. Dr. Dr. F. Fink date tca 6? CIR Panel Book Page 93

99 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 I 2 CM - 10 of 24 Mibelle AG Cosmetics Report 2. RESULTS CM - Gtucan (700-01) Summary of Skin Reactions Snstfzation (Pail A) volunteers positive doubtt. total during induction after challenge PhotoaHergy (Pail B) volunteers positive doubil. total during induction and irradiation with UV-8 after challenge and irradiation with UV-A CIR Panel Book Page 94

100 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project CM - 11 of 24 Mibelle AG Cosmetics Report Evaluation of Sensitization (Part A) Total number ol volunteers at challenge 32 of these were clearly positive at challenge 0 of these were positive already for skin irritation 0 (GTLF Project 9142 / 1) therefore became sensitized during induction 0 out of 32 0 % Evaluation of Photoallergy (Part B) Total number of volunteers at challenge 32 of these were clearly positive at challenge 0 of these were positive already for skin irritation 0 (GTLF Project 9142 / 1) therefore became photoallergic during induction 0 out of 32 = 0 % CIR Panel Book Page 95

101 Glucan Glucan irritant Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - Glucan 12 of 24 Mibelle AG Cosmetics Report 3. RATIONALE OF THE STUDY Because of the intended public use the sensitizing and photoallergic potential of the test article was studied by patch testing iii human adults. Repeated patch tests with or without UV-A and UV-B irradiation are commonly used to predict allergenic and photoatlergic properties of prod ucts intended for public usage. 4. ETHICAL CONSIDERATIONS 4.1 Risk Assessment CM - is the main component of the cell membrane of yeast cells, It is being isolated from Saccharornyces cerevisiae obtained from a yeast producer. The isolated glucan is then derivatized in a second process to water soluble CM (carboxymethyl)- Glucan. CM - is chemically very similar to CMC (carboxymethylcellulose), which is widely used in the cosmetic and pharmaceutical industry. Like that, glucan is a high molecular poly saccharide consisting of glucosidically connected glucose units. A similar modified Glucan (phosphoryl-glucan) intended for treatment of sores was of low tox icily in acute, subacute, and chronic toxicity studies in various animals after intravenous ad ministration up to doses of 2000 mg / kg body weight. The external application of the test article in a concentration of 2% is considered to be of no risk for the volunteers according to the provided data. The investigation of possible irritant and photo - properties of CM - Glucan was required in the general interest of consumer protection. No particular health risk was envisaged for the participating volunteers, considering the chemical nature of the test article and the amounts applied (see also Product Characterization in the Appendix). 4.2 Informed Consent Subjects were taken into the study after having given their written Informed Consent. The signed Informed Consent ncluded the explicit acceptance that the individual data will be known to the Investigator(s), the representatives of the Sponsor and, possibly. the regulatory authorities. CIR Panel Book Page 96

102 GTLF Project 9142 / 2 CM - Glucan 13 of 24 Mibelle AG Cosmetics Report 4.3 Ethics Committee Approval Protocol, CRF and CF were submitted to the Freiburger Ethik-Kommission after their ap proval by Mibelle AG authorities. The Ethics Committee approval was obtained on October 18, STUDY DESIGN The study consisted of two parts: Part A: Testing of sensitizing properties by occlusively applying the test article nine times for 24 hours within three weeks for induction of possible sensitization and proving sensitization by one challenge application (24 hours) after an interval 0110 to 14 days. Part 8: Testing of photoallergic properties by occiusively applying the test article six times tar 24 hours within three weeks and irradiation with UV-B for induction of possible photoallergy and proving of photoallergy after an interval of 10 to 14 days by one challenge application (24 hours) and UV-A irradiation. 5.1 Randomization This was an open controlled trial. Patches with physiological saline figured as negative con trol on the same subject. Randomization was not applicable. 5.2 Concomitant Medication Anhiallergic and/or antiintlammatory drugs were not allowed during the trial. CIR Panel Book Page 97

103 - allergic - inflammatory, - hyperpigmeritalion - moderate Distributed for Comment Only -- Do Not Cite or Quote GTLFProject9l42/2 CM -Glucan Mibeve AG Cosmetics Report 6. SELECTION OF VOLUNTEE}RS 6.1 Number of Volunteers 32 healthy caucasian volunteers, aging between 18 and 58 years, were included into the trial to obtain reliable information about the allergenic potential of the test article. Selection was done by the clinical investigator on basis of a questionnaire and by clinical inspection. 6.2 Inclusion Criteria - Healthy male and female volunteers, aging between 18 and 50 years. - Skin type I and 11 of the scale of BILLHIMER or MED 0.20 Joule /cm Exclusion Criteria Volunteers meeting the inclusion criteria will not be taken into the study, if they give any evi dence of - history of unknown local or systemic allergic reactions. reactions to plasters. - intake of antiallergic or antiintlammatory drugs during the last 2 weeks. eczematous or any other clinical sign of skin alterations on possible test sites. and multiple liver spots (lentigines) on possible test sites. or severe illness within the last 2 weeks before first exposure. - having a so-called very sensitive back. Six volunteers (HAE, HAJ, LAU, MOK, SOC, THG) exceeded the age of 50 years, but were taken into the study because of being regular users of this kind of products and having a very suitable skin. In deviation from the protocol (see 11.4) volunteers up to skin type Ill instead of skin type II of the scale of BILLHIMER were taken into the study, but were excluded from further partici pat ion, if their sensitivity for UV-B exceeded 20 Joule / cm2. CIR Panel Book Page 98

104 GTLF Project 9142 / 2 CM Glucan 15 of 24 Mibelle AG Cosmetics Report 7. TEST ARTICLE Identification: CM - Glucan Description: white powder Batch Number: Purity of test article: Data on file at Mibelle AG Cosmetics Amount: 100 ml Stability of test article (storage Conditions) stable at room temperature for at least 6 months Analytical Certificate: Data on file at Mibelle AG Cosmetics Responsible Manufacturer: Mibelle AG Cosmetics Responsible Analyst: Dr. H. P. Homberger CIR Panel Book Page 99

105 GTLF Project 9142 / 2 CM - Glucan Mibelie AG Cosmetics Report 7.1 Test Article Preparation and Dosage 7.11 Test Article Preparation The test article was provided by the sponsor ready for use in a concentration at 2% in distilled water. The test article was coded on OREs as follows: TA5= CM-Glucan Test ArticLe Dosage The test article preparation (approx. 30 ti]) was tilled to saturation into HAL (HAL Allergie GmbH, Düsseldorf) patch test units Accounting for Test Article Supply The Principal Investigator and the Study Monitor accounted tor all supplies. The Investigator was required to maintain adequate records of the disposition of the test article including dates and times of application (Case Record Form). 7.2 Test Article Application The liquid test article was filled with calibrated pipettes or disposable syringes into HAL (HAL Allergie GmbH, Düsseldorf) patch test units according to instructions regulated by SOP Site of Application HAL patches were tixed in vertical rows (concomitant exposure of other test articles, see be low) on the upper back or arm. lest sites were marked by felt-tipped marking pen and the position recorded on the Case Record Form (CRF). Skin hairs were removed caretully with a disposable shaver, where necessary for sensitization (Part A) Patches were applied on the left back or arm always to the same spot when they were ap plied repeatedly during the induction phase of sensitization. The patch was applied to a naive site on the back or arm at challenge application for photoattergy (Part B) Patches intended br irradiation were put on the right back or arm. Patches were applied to the same spot on the back or arm when they were applied repeatedly during the induction phase of photoallergy. The patch was applied to a naive site on the back or arm at chalienge application. Patch applications for Part A and B were conducted during the same study period Occlusion Complete occlusion was achieved by the construction of the HAL patches. CIR Panel Book Page 100

106 GTLF Project9l42!2 CM -Glucan Mibelle AG Cosmetics Report Frequency of Application tor the induction phase of sensitization (Part A): Test article patches were applied 9 times (3 times weekly on Monday, Wednesday and Friday tar 3 weeks) to the same site on the back or arm during induction phase for the induction phase of photoallergy (Part B): Test article patches were applied 6 times (twice weekly on Monday and Thursday tar 3 weeks) to the same site on the back or arm during induction phase tor challenge (Part A and B): Test article patches were applied once. The patches for challenge of Part A and Part B were applied simultaneously Duration of Experimental Parts: Induction Phases for Sensitization (Part A) and Photoallergy (Part B): 3 weeks Treatment tree period: 2 weeks Challenge for Part A and B: 1 week Total Minimal Time: 6 weeks Duration of Exposure: patches were applied for 24 hours only at each application Control Patch In one HAL unit of the row physiological saline was dispensed (negative control). 7.3 Concomitant Exposures Five further tesl articles were applied on the left and right side. CIR Panel Book Page 101

107 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 /2 CM - 18 of 24 Mibelle AG Cosmetics Report 8. SKIN IRRADIATION 8.1 Light Source The skin site exposed to the test article patches were irradiated with either UV-A or UV-B light produced by the MULTITESTER, Fa. Gerhard Saalmann, Herford. This device allows ir radiation with UV-A and UV-B of defined wave lengths and energy and with very little in frared. The light doses (Ws = J) can be varied by changes of irradiation time The light doses per skin area are kept constant by putting the window of the MULTITESTEP directly on the skin surface. There are special devices on the beam window which allowed an exact adjust ment of the window over the patch sites. While irradiating these sites, care is taken to protect the non-irradiated sites from inadvertent exposure to UV Fight. The irradiation procedure is further regulated by SOP. 8.2 Evaluation of the Minimal Erythemal Dose (MED) tar UV-B Sensitivity The MED was determined by administering a geometric series of 5 UV-8 exposures in which each dose was appr. 20% greater than the previous one, This series could be administered in one exposure using a special filter of the MULTITESTER. The exposures were circular areas with a diameter of appr. 1 cm. About 24 hours later, these sites were examined for the dose which produced minimal but uniform erythema with a distinct border, The MED was de termined individually for each volunteer betore starting the induction phase by irradiating a non-tanned area of the inner fore-arm in the pretest (GTLF Project 9142/ 1). 8.3 Irradiation for photoallergy (Part B) during induction: The patch site on the right back or arm was exposed to about twice the MED of IJV-B within 10 mm. after removal at each induction patch at chaiieng: The patch site on a naive skin area was irradiated with 18 ± 2 J / cm2 of UV- A within 10 mm. after patch removal. This dosage generally produces no erythema, but only slight tanning. CIR Panel Book Page 102

108 GTLF Project 9142 /2 CM - Glucan 19 ot 24 Mibelle AG Cosmetics Report 9. ASSESSMENT CF SKIN REACTION 9.1 Reading of skin reactions Reading of skin reactions was done by the Investigator and/or an experienced staff member of GTLF Readings of skin reactions during the induction phase of sensitization (Part A) and photoallergy test (Part B) were done immediafely and 24 hours (+ 48 hours in photo allergy test) after each pafch removal respectively 24 hours and 48 hours (+72 hours in photoallergy test)) after patch application, but always prior to the next applicat ion Readings of challenge reactions were done immediately, 24, 48, and 72 hours atter patch removal (24, 48, 72 and 96 h after patch application). Volunteers were instructed to present themselves for reexamination, if any reaction sub sequently becomes itchy, uncomfortable or otherwise noticeable. 9,2 Recording and Interpretation of Results Readings were recorded on the CRF and graded as follows: o = negative (no reaction) + = doubtful reaction 1 = weak reaction (well defined erythema) 2 = moderate reaction (erythema with slight-to-moderate edemaj 3 = strong reaction (vesictes or papules) 4 = extreme reaction (bullous, spreading, or other severe reaction) The results obtained tor test article patches were compared with those obtained for negative control patches in the same subject. The test article was considered to show evidence ot irri tation, if the observed dermal reaction was more marked than any dermal reaction seen with the negative control patches on the same subject. If the dermal reaction seen with the test patch was not clearly different from reactions with negative control patches, the results for the test article was classified as questionable. The test article was considered to show no evidence of irritation, it the dermal reaction was identical or less marked and/or less persistent than dermal reactions seen with control pat ches in the same subject. CIR Panel Book Page 103

109 THERAPY Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - Glucan 20 of 24 Mibelle AG Cosmetics Report 10. RECORDING OF ADVERSE REACTIONS Skin reactions on patches were recorded on CRF as described above ( 92) Note was tur ther taken of any untoward sign or symptom spontaneously complained by the volunteer and/or observed by the Investigator. No adverse event was observed during the trial. 11. TIMING AND PRACTICATION The study started on November 2, and ended on December 19, The duration for a single volunteer was 6 weeks Controt ot Compliance Application and removal of patches were supervised by the Investigator and/or an experi enced staff member. Procedures were further regulated by SOP. Volunteers were strictly advised to leave the patches in position, to take care that the patch sites remained under firm occlusion and to take no showers until the patches were removed Place of Conduction of the Study The study took place in the premises of GTLF - AND PERFORMANCE RESEARCH INSTITUTE LTD., Hair and Skin Care Clinic, Fichtestr. 14, D - Federal Republic of Germany Amendment Procedures 8520 Erlangen, One Amendment to the protocol (November 2, 1992) was defined during the study. in addi tion to the lest articles (concomitantly tested) described in the protocol the matrix of one test article was applied (ret.: amendment no. 1) Deviation from the Protocol Because of increasing use of artificial tanning and sun protection devices, volunteers up to skin type Ill instead of skin type II of the scale ot BILLHIMER (being classified on the basis of clinical inspection and personal history of the volunteers) were taken into the study, hut were excluded from further participation, it their sensitivity for UV-B exceeded 20 Joule 1 cm2 at evaluation of the minimal erythemal dose (see 8.2). CIR Panel Book Page 104

110 GTLF Project 9142 / 2 CM - Glucan 21 of 24 Mibelle AG Cosmetics Report 12. EVALUATION OF DATA 12.1 Premature terminations All volunteers taken into the trial completed the trial Monitoring This study was monitored by the Sponsor by means of personal visits and telephone commu nications to ensure that the investigation was conducted in strict accordance with the proto col Control of Adherence to the Protocol Adherence to the protocol by staff and volunteers was controlled by the Investigator and the Monitor Instruction of Staff and Volunteers Volunteers were instructed by CF and verbally through the involved staff members the latter were further instructed by Standard Operating Procedures (SOPs) and verbally through the Principal Investigator. 13, STATISTICAL EVALUATION All results were analysed descriptively and exploratively. Means and standard deviations were calculated where applicable. Main variables were the maximum scores for the skin re actions during challenge after 24h exposure followed by UV-A irradiation or not. No compara tive evaluations were made, because of only one doubtful reaction after challenge on the irra diated site. 14. INSURANCE The Sponsor assumed responsibility for any necessary evaluations of purity, strength and stability of the test article; he was liable tar test article quality and safety. The Principal Investigator was insured and liable for State of the Art conduction of the study. CIR Panel Book Page 105

111 A B Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - Glucan Mibelle AG Cosmetics Report 15. LIST OF ABBREVIATIONS appr. CRF ICF J MED SNR SOP UV - UV - approximately Case Record Form Informed Consent Form Joule (Watt sec.) Minimal Erythemal Dose Subject Number Standard Operating Procedure Ultraviolet light A Ultraviolet light B 16, DATA COMPILATION The following data were recorded on case record forms (CRF) and transcribed for compila lion: Date and hour of test article application and irradiation Date and hour of reading of skin reactions Skin reactions CIR Panel Book Page 106

112 392, 169, Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - Glucan 23 of 24 Mibelle AG Cosmetics Report 17. REFERENCES Information about the lest article was obtained by: 1. Verbal instructions of the Principal Investigator through the representative of the Sponsor. 2. Certificate about tolerance and safety of the test article provided by the Sponsor. The study procedures described in this report are based on fohowing publications: 1. Marzulli, N.F. and Maibach, HI. Predictive testing in humans for contact allergy, in: Fisher, A. (ed), Contact Dermatitis, 3rd Edition, Lea & Febiger, Philadelphia, Schwartz, L. Twenty two years experience in the performance of prophetic patch tests. Southern Medical Journal, 53, 1960, Kligman, A.M. The identification of contact allergens by human assay. I. A critique of standard methods. J. Invest. Dermatol. 47, 1966, Kligman, AM., The identification of contact allergens by human assay. II. Factors influencing the induction and measurement of allergic contact dermatitis. J. Invest. Derrnatol. 47, Kligman, A.M. The identification of contact allergens by human assay. Ill. The maximization test: a procedure for screening and rating contact sensitizers. J. Invest. Dermatol. 47, , Robinson, M.K. Stotts,,J., Dannernan, P.J., Nusair, T.L. A risk assessment process for allergic contact sensitization, Fd. Chern. Toxic. 27, 1989, Henderson, Ch., Riley, E. Certain statistical considerations in patch testing. J. Invest. Derrnatol. 6, 1945, Allen, A.M. Statistics and Experimental Design. Cosmetic Safety. J.H. Whittam (ed.), Cosmetics Science and Technology Series, Vo. 5, Marcel Dekker, New York and Basel, Kligman, A.M. and Epstein, W. Updating the maximization test for identifying contact allergens. Contact Dermatitis 1, Kaidbey, K,H. and Kligman, A.M. Identification of topical photosensitizing agents in humans. J. Invest Dermatol 70, , Kaidbey, K.H. and Kligman, A.M. Photomaximization lest for identifying photoallergic contact sensitizers, Contacl Dermatitis, 6, Billhimer, W.L. Phototoxicity and Photoallergy, in Clinical Safety and Efficacy Testing at Cosmetics, W.C. Waggoner (ed), Marcel Dekker, New York and Easel, 1990, CIR Panel Book Page 107

113 Glucan Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 CM - 24 of 24 Mibelle AG Cosmetics Report 18. DISTRIBUTION 1 Original: Principal Investigator 2 Copies: Sponsor Erlangen Date: July 14, 1993 CIR Panel Book Page 108

114 GTLF Project 9142/2 Mibelle AG Cosmetics Report Appendix to REPORT CIR Panel Book Page 109

115 GTLF Project 9142 / 2 Mibelte AG Cosmetics Report Table 1: LIST CF PARTICIPANTS INIT SNR Age Sex Skin Type MED Joule / cm2 BAN BAD II 0,08 BEA II 0,09 DEA I 0,12 HAE II 014 HAJ II 0,20 HEJ II 0,10 HOP II 0,06 HUF II 0,10 HUN ti 0,12 HUG I 0,20 JOS II 0,08 KEB III 0,10 RLC II 0,08 KRI III 0,10 KRB I 0,05 KRM III 0,09 LAG II 0,06 LAU II 0,07 MAM Ill 0,13 MAB II 0,14 MAS II 0,06 MOK II 0,06 MUC II 0,08 ROB I 0,05 RUB II 0,10 SCU II 0,12 soc I 0,13 THG II 0,15 VOG I 0,06 WAR I 0,10 WAI II 0,15 N=32 mean 36 1 male I n=8 0,10 ±SD 13 2=temale II n= 20 0, n=4 CIR Panel Book Page 110

116 . Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 Mibelle AG Cosmetics Report Table 2: SUMMARY OF INDIVIDUAL SKIN REACTIONS SENSITIZATION (Part A) CM - GL1JCAN (700-01) NIT SNR Max. Reaction Max. Reaction Max. Reaction Irritation during Induction at Challenge (GTLF-Study 9142/1) BAN BAD BEA DEA HAE [ 0 0 HAJ HEJ HOP HUE HUN HUG JOS KEB KLC KRI KRB KIRM LAG LAU MAM I- 0 MAB MAS j MOK MUC ROB RUB SCU SCC THG VOG WAK WAI CIR Panel Book Page 111

117 Irrad.) Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 /2 Mibelle AG Cosmetics Report Table 3: SUMMARY OF INDIVIDUAL SKIN REACTIONS PHOTOALLERGY (Part B) CM - GLUCAN (700-01) NIT SNR Max. Reaction Max. Reaction Max. Reaction Irritation during Induction at Challenge {UV-A Irrad.) (UV-B - (UV-A - Irrad.) (GTLF-Study 9142/1) L BAN BAD BEA DEA HA HAJ 1 0 HEJ HOP HUF 917? HUN p JOS KEB KLC p KRI KRB KRM LAG LAU MAM MAB MAS MOK MUC ROB 9? RUB SCU SCC THG VOG ] 0 1 WAK WAI [ P CIR Panel Book Page 112

118 Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 MibelIe AG Cosmetics Report Table 4: SKIN REACTIONS DURING INDUCTION SENSITIZATION (Part A) CM - GLUCAN (70001) Readings# NIT SNR BAN O0.00 BAD :00: :0:0:0.0 0 BEA o:0:o.oo 0 0 0:0000:.0:0 DEA :0 0 0 HAE9912ooo:ooooooo:ooooo :0000.?P2 0: HP : :0.000 UF Pt.: : ; OO 0 0. HUN : :0:0 0 0; HUG 99130:0:0:0: O00 0 o; JOS : : EB 99:3, KRI KRB 91 9,00, : : LAG :0: : LAU oo:oo , : :000 M.S?7 0 0, MOK , ,,,, ROB o : 0, , ::P.LJ?9:49:: ,0, ?P: : A WAI , CIR Panel Book Page 113

119 V VV VVVVVV VV V I (1) o BR BR 1 V 0 U 1 1 (1) Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 Mibelle AG Cosmetics Report Table 5: SKIN REACTIONS DURING INDUCTION PHOTOALLERGY (Part B) CM - GLUCAN (700-01) Readings # INIT SNR i BAN (1) 1 (1) 0 1 (1) (1) (1) (1) BAD BR BR (1) 0 0 (1) 1 (1) 0 1 BEA BR (1) + 0 (1) BR 1 (1) + DEA (1) BR (1) 1 (1) (1) HAE BR (1) (1) BR 0 1 (1) 1 (1) 0 HAJ (1) BR BR 1 (1) 0 (1) BR (1) (1) (1) BR BR (1) BR 0 1 BR 1 (1) (1) VVVtPF.VVVVV V VVHUVVV (1)BR(1) BR 0.0 BR 1 {1)(1) 1 1 1BRBR:1 (1)0.BR 0 1 BR 0 :: : : : : : HUN (1)0BRV(1)0 V()V KEB V (1) BR BR BR BR 0 V BR 0 1 BR 0 BR BR BR (1) VVV.VV BR 1 (1) 0 0 BR 1 (1) (1) VK V (1) (1) V BR (1) 0 1 (1) (1) KRB (1)BR:0 BR 0+ + BR BR KRM BR BR 1 VV.VVV0VVVVVVVVVVV9?292 0 V : (1) V (1) V 0 0 V 1 BR BR (1) 0 0 BR 0 V 0 BRVVt 0 Q () (1) 1 (1) BR. VVVL4V (1)BR;0: (1)0 MAB BR V 1 BR BR 1 V 1 (1) 0 ViVV (1) (1) (1) BR (1) (1) VVMPK V 0 1 V 1 BR BR (1) (1) 1 + BR VV.MVVVV.VV (1) BR BRBRBR011(1) BR VVVOVV (1) (1) BR BR (1) BR 1 V SCU V 0 (1) BR 1 (1) (1) + BR BR BR 0 (1) BR BR BR 0 SCc (1) (1) (1) (1) (1) 1 (1) 0 (1) + 1 (1) (1) VOG (1) (1) (1) + 1 (1) (1) (1) 1 (1) 0 (1) BR 1 (1) (1) V V Q...1 BR = tanned skin CIR Panel Book Page 114

120 .-_ ,... JOS, HUN, MOK, MAB. 99? , 0 Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 / 2 Mibelle AG Cosmetics Report Table 6: Reading ol Challenge Reactions CM - GLUCAN (700-01) Patch non - irradiated irradiated NIT SNR Oh 24h 48h Control after patch removal 0 h 24 h 48 h Control after patch removal and irradiation BAN 99296,,,9 o BAD : 0 0 0,, SEA DEA HAE :0 0:0 0.0: HAJ : 0. 0 HEJ HOP : HUF HUG o :0 0: KES : 0 KLC KRI : 0 0, 0 KRB KRM LAG LAU MAM ,, AS : 0 0 MUG ROB s UB 9..?20 0 0, 0 0,, SOC THG 9?02 : 0, VOG 0, 0 0 0, WAK 99313, : : , , : WAI CIR Panel Book Page 115

121 GTLF Project 9142 I 2 Mibelle AG Cosmetics Report PRODUCT CHARACTERIZATION CM - GLUCAN (700-01) CM - Glucan is a poly-(beta-(1.3)-glucopyranose)-chain; with carboxymethylated glucose units; grade ot substitution ; molecular weight kd. Physica/ characteristics: consistence colour powder white, beige Code Numbers: loose weight 0.54 g / ml ph - valw (2% aqueous solution) 7.0 ± 1.0 viscosity (2% aqueous solution) 150 ± 40 cp Stability Storage conditions stable at room temperature, for at least 6 months <25 C CIR Panel Book Page 116

122 GTLF Project 9142 Protocol Amendment 1 Protocol Amendment 1 Datum: Additional Test Article In addition to the 5 test articles described in the protocol ( 7, pg. 14 to 18) the matrix of test article no. 1 (Lipobelle ST) will be applied as test article no. 6. No additional risks are to be expected by additional application of this test article, and therefore separate approval of the Ethics Committee is considered not to be necessary. Principal Investigator Sponsor Prof. Dr. Dr. E. Fink Mibelle AG Cosmetics, OH Buchs CIR Panel Book Page 117

123 freiburger et[ii ic ko,wission CERTIFICATE Study: Skin Irritation, Skin Sensitization, Phototoxicity and Photoallergy of (120 01) Lipobelle ST, (121 01) BRA - stabilisiertes Lipobelle ST, (130-01) Uano Lipobelle A/E, (131-01) BRA - stabilisiertes Nano - Lipobelle A/E (700-01) CK - Glucan Code; GTLF 9142 Sponsor: Mibelle AG Cosmetics CH Date: 18 October 1992 The Ethics Commission has examined the above study from the ethical, legal and medical science points of view. On behalf of the Ethics Commission there are no objections. there are no objecjenthe detailed recommen dations ltdtuld be met. there are no obje the detailed there are serious listed in objo,es being Hans-Peter Radio 1,., Ph.D. sicist,;e lens.l ele, (iel SchioGheitriq 9 D.7800 Febu1O Tel 0761/ V CIR Panel Book Page 118

124 - Fax Distributed for Comment Only -- Do Not Cite or Quote freiburger eti iiic ko,wission Study: Skin Irritation, Skin Sensitization, Phototoxicity and Photoallergy of (120-01) Lipobelle 57, (121-01) BRA - stabilisiertes Lipobelle 57, (130-01) Nano - Lipobelle A/H, (131-01) BHA - stabilisiertes Nano - Lipobelle AlE (700-01) CM - Glucan Code: GTLF 9142 Sponsor; Mibelle AG Cosmetics - CII Date: 18 October 1992 Me s rese t Pro Dr. Pfei er, Gerd, Karlaruhe Pres dent of the Bundesgerichtshof, rtd. I Chairman PD Dr Common, Herbert, Freiburg lute nist / Hematologist Dr. Herrmann, Manfred, Preiburg Pharmjoist DPhG Dr. Haering, 1audolf, Freiburg Surgeon k Prof. Dr. v n Heymann, Dietrich, Freiburg Theologian A Gra, kcarin-a., rreapurg Medical Layman GxaxhaIIshjIet Di med Di in CIR Panel Book 9 Page 119 ai itins-pdei G:aI ScIiIxfii,xror;no fletiurn in 07 (ii li o33 3

125 GTLF Project 9142 / 2 Mibelle AG Cosmetics Report TRIAL SCHEDULE SENSITIZATION (Part A) INDUCTION PHASE: 3 weeks DAY MON TUE WED THU FRI SAT SUN Patch Application x - x - x - Patch Removal - x - x - x - Reading of Skin Reactionsj (x) x x x (x) = in the second and third week CHALLENGE PHASE: 1 week Patch Application Hours after Patch Removal DAY MON TUE WED THU FRI Patch Application x Patch Removal - x Irradiation (UV - A) - x Reading of Skin Reactions - x x x (x) (x) Readings only if skin reactions 2 or increasing in severity CIR Panel Book Page 120

126 B) x x x - Distributed for Comment Only -- Do Not Cite or Quote GTLF Project 9142 I 2 Mibelle AG Cosmetics Report TRIAL SCHEDULE PHOTOALLERGY (Part B) INDUCTION PHASE: 3 weeks DAY MON TUE WED THU FRI SAT SUN Patch Application x - - x - Patch Removal - x - x - Irradiation (UV x Reading of Skin Reactionsj (x) x x x x (x) = in the second and third week CHALLENGE PHASE: 1 week Patch Application Hours after Patch Removal DAY MON TUE WED THU FRI Patch Application x Patch Removal Irradiation (LV - A) - x - Reading of Skin Reactions - x (x) x (x) = Readings only it skin reactions 2 or increasing in severity CIR Panel Book Page 121

127 zun PROTOKOLL PROBANDENAUFKLARUNG UND EINVERSTANDNISERKLARUNG für Teilnehmer an Prufungen der Hautverlräglichkeit von Produkien zur Haulpilege (APPT) GTLF PROJEKT 9142 Proband(in) r lnita Z1 Herr Prof. Dr. Dr. Ernst Fink leitet die PrUfung der Hautverlräglichkeit von Wirkstoffen zur Hautpflege. Er hat mich heute uberfolgenden Sachverhalt aufgeklärt: vorgesehene SInn unci Zweck der Prufung Prüfung der Hautverträglichkeit von 5 Wirkstolten zur Hautpflege mit und ohne UV-Einwirkung. Diese Wir1stofte sind in ähnlicher Konzentration oder ähnlicher chemischer Struktur schon in Produkten Markies enthalten. Die Produkie werden mit Hilfe eines Pilasterstreifens von 12 x 7 cm, der 10 Naplchen enthält, auf Rücken oder/und Arm aufgeklebt. des DurchfUhrung Die PrUfung beginnt am Sie wird voraussichtlich bis einschliel3lich dauern. Die PrUlung bestehi aus mehreren Abschnhtten 1 Vorprüfung einer möglichen Hautreizung: Die Pt laster werden mir am Montag aufgeklebt und am Dienstag (24 Std. spaler) entfernt, Nach der Abnahme wird die rechie Pflasterstelle 6 Minuten lang mit UV-A belichtet, UV-A machi braun, es entsteht also em brauner Fleck, der ähnlich wie die normale Sonnenbrdune nach einigen Wochen wieder verschwinde(. Die Emptmndlichkeit meiner Haul wird am Montag und Dienstag durch Betichtung mit abgestufter Dosis am Unterarm gepruft. Am Mittwoch, Donnerstag und Freitag mw3 ich dann zur Kant rolle kommen. 2. HauptprUfung einer mglichen Allergisierung: Es werden mir jeweils am Montag, Mittwoch und Fre flag Pilaster immer auf die seibe Stelle links aufgeklebt und am Dienstag, Donnerstag urid Samstag abgenommen. (Am Samstag nehme ich selbst Pilaster ab.) Montag und Donnerstag wird mir auf der rechteri Seite zusätzlich em Pflaster aufgeklebt, am Dienstag und Freitag abgenommen wird. Unmittelbar nach der Abnahme wird die Pflasterstelle für Ca Sekunden mit UV-B bestrahlt; dadurch entsteht don eine Art brand. das das Sonnen Diese Pflasterauftragungen und Bestrahlungen werden 3 Wochen Nach elner Pause von 14 Tagen enlolgt die lang vorgenommen. 3. NachprUfung, die genau wie die VorprUfung abtãuft und 1 Woche dauert. CIR Panel Book Page 122

128 Erlangen, den 1992 Distributed for Comment Only -- Do Not Cite or Quote Solange Ich die Ptlasterstrelfen trage, werde Ich Ubertrlebene Bewegung (sportliche Betätlgung) sowle das Benetzen der Pflaster mit Wasser (Duschen) vermelden. Nebenwrkungen und Herr Prof. Fink hat mir daf3 angesichts der geringen Menge, die aufgebracht wird, Nebenwirkun gen, die durch die Aufnahme cier Wirkstoffe in die Haut bedingt sein konnten, nicflt zu erwarten sind. Solite eine lokale Reizung der Haut auftreten, so äui3ert sich diese durch Brennen, Juckreiz und evtl bis zur Blasenbildung, einern Sonnenbrand. Elne bleibende Schädigung der Haut 1st auch dann nichi zu belurchten; au I3erdem kann ich Pilaster jederzeit nach Verständigung Prüfarztes abnehmen, wenn Brennen und/oder Juckreiz aufireten soitten. Für etwaige battet der Hersteller rn Rahmen der Produkthaftung. RIsken erkirt, hnlicfl das Schaden des Rtung, Datenschutz Alle während der PrOfung erhobenen Daten warden so aulgezeichnet und gespeicherl, dal3 ich anhand der Daten von fremden Dritten nicht idenhifiziert werden kann. Meine Daten (alien unter das Arz tegeheimnis und sind nur dam PrUfarzt und dem Personal zuganglich; ich erkläre jedoch drgcklich, dat) ich damit einverstanden bin, dat) Beauttragte der Herstellertirma und gegebenenfalls auch zuständige Behörden in meine PrQfdaten EinbHck nehmen. rztlichen persnhichen aus Honorar Für die vollständige und zuverlässige Teilnahme an dieser Prüfung erhalte ich em Honorar von DM. Soitten stärkere Hautreakilonen bei der Vorprüfung meine weitere Teilnahme ausschliel3en, erhalte ich für die VorprUtung em Honorar von DM. Erkrung Aufgrund dieser Informationen bin ich bereil, an dieser Prufung teilzunehmen. Meine Teilnahme st frel willig. Herr Prof. Fink hat mir dab ich mein Einvarstandnis jederzeil widerrufen kann. Ich verpfiichte mich, den ärztlichen Anweisungen des Prufungsleiters und PrUfarztes Folge zu leisten und tnsbesondere alles zu unterlassen, was eine vorzeilige Ablösung Ptiasters zur Folge haben konnte, rn Laufe der lung mul3 ich mich insgesamt 25 mat in den der GTLF ainfinden, erkirt, Rumen des Prü Unterschrifl Proband(in) Unterschrift Prüfungsteiter/-arzt CIR Panel Book Page 123

129 - - - Distributed for Comment Only -- Do Not Cite or Quote CURRICULUM VITAE Name Ernst Heinrich F i n k Date of Birth September 19th, 1935 Place of Birth Munich, Germany Marital Status Married; two children (born 1969 and 1972) Scientific Career and Qualifications Studies of Biology, Chemistry and Geography at Maximilian University, Munich, and Friedrich AlexanderUniversity (FAU), Erlangen Nuremberg Studies of Human Medicine at FAU, Erlangen Nuremberg Teachers Degree for Higher Education for the Subjects Chemistry, Biology, and Geography 963 Promotion Dr. rer. flat. 971 Awarded Degree in Medicine 1973 Approbation as Medical Practitioner 1974 Promotion Dr. med Professorial Thesis in Biology (Zoology at FALl Ertangen-Nucemberçj; Dr. rer. nat habil. Parasitology) 1981 Appointment as University Professor (apt.) at FAU Ertangen -Nuremberg CIR Panel Book Page 124

130 today Distributed for Comment Only -- Do Not Cite or Quote Professional Career Head of the Biological Testing Laboratory at the Institute of Pharmacy Food Chemistry, FAU Erlangen - and (Preclinical Testing) at New Drugs Malaria and Lectures on of Bacterial and Parasitic Research Infections and Nurembeig; Development against Chemotherapy Trypanosomiasis; Establishing of a Primate Station; Testing of New Drugs Malaria at the institute of Malaria and Vector Borne Arnani, Tanzania against Diseases, 1972 Establishing of a Station; Preclinical Clinical Testing of New Drugs at the EasI African Organization, Tororo, Uganda and Trypanosomiasis Research Primate against Trypanosomiasis Head of the Project of African Trypanosomiasis of the Agency for International (GTZ) in Kabete, Kenya; Preclinical and Clinical Testing of New Trypanocides Cooperation Chemotherapy German Medical Director of a Middle Sized Company in Germany; for R & D; Clinical on Drugs, Drugs for Respiratory Tract Dermatological Antithrombotics; of Agents, Research Development Responsible Cardiovascular Diseases, Head of the Department of Clinical and Consulting Research Switzerland Pharmaceutical Recombinant Proteinase-Inhibitors Research at RCC, Company, ltingen/base, Managing Director, and Institute Ltd. (GTLF mbh), Erlangen. Research Therapy Performance Erlangen, July 1993 CIR Panel Book Page 125

131 CURRICULUM VITAE me; Dr. med. dent. Karl Wolfgang Franice Further education: Student of Dental Medicine at the Friedrich-Alexander University, Erlangen Graduated (Staatsexamen) from the Friedrich Alexander University, Erlangen. Professional experience and further training: 1957 till 1959 Received doctorate; doctoral thesis on the etiology of cancer of the collum. Assistant doctor at hospital and own practice Medical member of the Field Service of E. Merck, Darmstadt Deputy Head of the Medical Dept. of L. Heuniann & Co. GmbH, Nuremberg Member of the Working Committee Training for Pharmaceutical Representatives of the German Association of the Pharmaceutical Industry (DPI, Bundesverband der pharmazeutischen Industrie) Work as self-employed dental practitioner Member of the Pharmaceutical Coordination and Approval Department at Heumann Pharma GmhH & Co., Nuremberg Head of the Department of Clinical Research at Heumann Pharma GmbH & Co., Nuremberg. since 1990 Head of the Department of Clinical Project Management at Heumann Pharma GmbH & Co., Nuremberg. Nuremberg, October 10, 1991 CIR Panel Book Page 126

132 REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF CM- G LU CAN IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY (WITH INDEPENDENT REPEAT) NOTOX Project NOTOX Substance page 1 of 25 - CIR Panel Book Page 127

133 CM-GLUCAN NOTOX Project STATEMENT OF GLP COMPLIANCE NOTOX B.V., s-hertogenbosch, The Netherlands The study described in this report was conducted in compliance with the most recent edition or: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. Study Director C.M. Verspeek-Rip Date - page 2 CIR Panel Book Page 128 -

134 CM-GLUCAN NOTOX Project QUALITY ASSURANCE STATEMENT NOTOX B.V., s-hertogenbosch, The Netherlands. Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals. This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data. DATES OF QAU INSPECTIONS! REPORTING DATES AUDITS July 17, 1996 July 17, 1996 July 24, 1996 July 24, 1996 August 01, 1996 August 01, 1996 September 18, 1996 September 18, 1996 Quality Assurance Manager C.J. Mitchell 8.Sc. Date page 3 - CIR Panel Book Page 129

135 CMGLUCAN NOTOX Project REPORT APPROVAL STUDY DIRECTOR: C.M. VerspeekRip Date MANAGEMENT: Ing E.J. van de Waart Section Head, Genetic & Eco-Toxicology Date - page 4 - CIR Panel Book Page 130

136 CM-GLUCAN NOTOX Project SUMMARY CM-GLUCAN was four TA100 and TA98) tested in the Salmonella typhimurium reverse mutation assay with of Salmonella typhirnurium (TA1535, TA1537, in two independent experiments. histidine-requiring strains CM-GLUCAN was up to concentrations of 500Q the presence of S9-mix. tested ug/plate in the absence and CM-GLUCAN did not induce a in the number of colonies both in the absence and the presence of S9-niix, in two independently repeated experiments. dose-related increase revertant Based on the of study concluded CM-GLUCAN mutagenic in the Salmonella typhirnurium reverse mutation assay. results this it is that is not - page 5 - CIR Panel Book Page 131

137 CM-GLUCAN NOTOX Project PREFACE Sponsor Mibelle AG Cosmetics Balimattstrasse 1 CH-5033 Buchs Swi tzerl and Study Monitor Dr. F. ZUlu Testing Facility NOTOX B.V. Hambakenwetering DD s-hertogenbosch The Netherlands Study Director C.M. Verspeek-Rip Technical Coordinator G. van Dart Study Plan Start 24 July 1996 Completed : 02 August 1996 TEST SUBSTANCE Identification Description Batch Purity Test substance storage Stability under storage conditions Expiry date Stability in vehicle CM- GLUCAN Beige solid Not indicated by sponsor; treated as 100% pure At room temperature in the dark Stable 01 January 1998 Water :at least 96 h The sponsor is responsible test substance data. for the completeness and GLP Compliance of all VEHICLE The test substance was suspended or dissolved in Milli- water (Millipore Corp., Bedford, Mass., USA). Test substance concentrations were prepared directly prior to use. page 6 - CIR Panel Book Page 132

138 CM-GLUCAN NOTOX Project GUIDELINES The study procedures described in guidelines: this report were based on the following - Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 471: 11 Toxicology: Salmonella typhimurium Reverse Mutation Assay, (adopted May 26, 1983). - European Economic Community (EEC), 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; 8.14: Other Ef f ects-mutagenicity: Salmonella typhimurium - Reverse Mutation Assay. EEC Publication no. L383 (adopted December, 1992). Directive Genetic ARCHIVING NOTOX B.V. will archive the protocol, and raw data for 10 years. at least report, test article reference sample OBJECTIVE Purpose of the study The of study was to evaluate the substance for to induce reverse mutations in a gene of Salmonella typhimurium such mutations would of these micro-organisms (1,2 and 3). ability objective this histidine-independent strains bacterial strains test histidine-requiring result its in Justification for selection of the test system The Salmonella typhimurium reverse mutation assay has shown to be a rapid and adequate for the mutagenic of a wide range of chemical compounds. indicator activity The assay was conducted in the absence and in the presence of system (S9-mix). a metabolic The Salmonella typhimurium TA1535 and TA1537. strains used in this study were TA98, TAIOO, The TA98 and TA1537 are capable of TA100 and TA1535 are capable of mutagens. strains strains detecting detecting base-pair substitution frameshif t mutagens, - page 7 - CIR Panel Book Page 133

139 CM-GLUCAN NOTOX Project MATERIALS AND METHODS TEST SYSTEM Test System Salmonella typhimurium bacteria Rationale Recommended test system in international guidelines (e.g. EPA, OECD, EEC). Source Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (TA1535 and TA1537: 1994, TA98: 1991, TA100: 1993) The characteristics of the individual Salmonella typhimurium strains were as follows: Strain Histidine mutation Mutation type TA1537 hisc3o76 Frameshift TA98 hisd3o52/r*factor* Frameshift TA1535 hisg46 Base-pair substitutions TA100 hisg46/rfactor* Base-pair substitutions *: R-factor = plasmid pkm1o1 (increases error-prone DNA repair) Each tester strain contained the following additional mutations: rfa : deep rough (defective lipopolysaccharide cellcoat) mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrb: loss of the excision repair system (deletion of the ultravioletrepair B gene) The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. Stock cultures of the four strains were stored in liquid nitrogen (-196 C). CELL CULTURE Preparation of bacterial cultures Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37 C, 150 sprn), until the cultures reached an 0.0. of 0.4 at 700 nni (10 cells/ml). Freshly grown cultures of each strain were used for each test. - page 8 - CIR Panel Book Page 134

140 CM-GLUCAN NOTOX Project Agar plates Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid. code L28) in Vogel-Bonner Medium E, 10 g glucose, 0.5 mg biotin and 0.6 mg histidine. Top agar Vogel -Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 mm at 121 C. Environmental conditions All incubations were carried out in the dark at 37 C. The temperature was monitored during the experiment. REFERENCE SUBSTANCES Negative control: The vehicle of the test article, being Milli-Q water. Positive controls: Without metabolic activation (-S9-mix): Strain Chemical Concentration/plate Solvent TA1535 sodium azide (SA) (Fluka) 1 pg Saline TA aminoacridine (9AC) 60 ug Saline (Janssen Chimica) TA98 daunomycine (OM) (Sigma) 4 pg Saline TA100 methylmnethanesulf onate (MMS) 650 g OMSO (Merck) With metabolic activation (.i-s9-rnix): Strain Chemical Concentration/plate Solvent TA1535 and TA aniinoanthracene (2AA) 5 pg DMSO TA98 and (Sigma) TA100 2-aminoanthracene (2AA) 0.5 pg OMSO (Sigma) Solvents for reference substances Saline = physiological saline (Fresenius AG, Bad Homburg, Germany) DMSO = dimethylsulphoxide of spectroscopic quality (Merck). - page 9 - CIR Panel Book Page 135

141 CM-GLUCAN NOTOX Project METABOLIC ACTIVATION SYSTEM Preparation of S9-homogenate: Rat liver microsonial enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP s. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0 C) sterile 0.1 H sodium phosphate buffer (ph 7.4) containing 0.1 mm Na 2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 mm at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 C) and identified by the day of preparation. Preparation of S9-mix: 59-mix was prepared immediately before use and kept on Ice. 59-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 H sodium phosphate buffer ph 7.4; 1 ml 0.08 M MgC1 solution; 1 ml 0.33 H KC1 solution. The above solution was filter ( m)-sterilized. 0.5 ml S9-fraction (batch 96-3) was added to 9.5 ml S9-mix components (5% (v/v) S9-fraction). EXPERIMENTAL PROCEDURE Dose range finding test Selection of an adequate range of doses was based on a dose range finding test with strain TA100, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the experiment of the mutagenesis assay. The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate. - page 10 - CIR Panel Book Page 136

142 CM-GLUCAN NOTOX Project Mutation assay Five different doses (with approximately half-log steps) substance were tested in triplicate in each strain. of the test The test substance each strain, in Top was two agar in top agar tubes tested bath in the absence and presence of S9-mix in independent experiments. was solutions were successively added to fresh bacterial culture (i0 various volumes of either 0.5 ml a molten and heated to 45 C. 3 ml The molten top agar: 0.1 following of 9 cells/ml) of one of the tester strains, dilution of the test substance in Milli-Q water and S9-mix (in case phosphate buffer (in case mixed on a of of activation assays) or 0.5 non-activation assays). Vortex and the content of the top agar tube The was ml ml 0.1 N ingredients were a poured onto selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 C revertant (histidine independent) colonies were counted. for 48 h. After this period a Colony counting The with revertant colonies (histidine independent) were counted automatically a Protas model colonies per plate were present colony counter or manually, if less than 40 - page 11 - CIR Panel Book Page 137

143 CM-GLUCAN NOTOX Project ACCEPTABILITY OF ASSAY A Salmonella typhimurium reverse mutation assay considered acceptable meets the following a) The negative control data (number of spontaneous per should reasonably be within the background range for each b) The control chemicals should produce responses in which also are reasonably within the laboratory range documented for each control substance. Furthermore, the mean count should be two times the concurrent control group mean. C) The dose range should include a toxic concentration as demonstrated by the dose range finding with TA100 or should extend to 5 mg/plate. it strains positive selected tester strain. vehicle criteria: plate positive laboratory test is at least clearly revertants strain historical plate) all tester historical if DATA EVALUATION AND STATISTICAL PROCEDURES No formal hypothesis was done. A substance considered negative (not mutagenic) in the a) The number of in any at any concentration not than two times the control value, with or without metabolic b) The negative response should be reproducible in one independently repeated experiment. A a) test is test total greater is testing revertants activation. solvent tester strain at least test if: substance considered (mutagenic) in the induces a dose increase in the number of with respect to the number induced by the solvent control in any of the with or without metabolic However, any mean count of than 20 considered to be not It revertants significant. is at least 2f old, positive tester strains, either plate less related b) The response should be reproducible in independently repeated experiment. positive is at least test if: one activation. The preceding enter into the criteria final were not absolute and other modifying evaluation decision. factors might - page 12 - CIR Panel Book Page 138

144 CM-GLUCAN NOTOX Project RESULTS DOSE RANGE FINDING TEST (Table 1, Appendix 2) CM-GLUCAN was tested in strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000.igIplate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test (Table 1). The individual data are presented in Appendix 2. Precipi tate CM-GLUCAN did not precipitate in the top agar. Precipitation of CM-GLUCAN was not observed on the plates at the start or at the end of the incubation period in tester strain TA100. Toxicity No reduction of the bacterial background lawn and no decrease in the number of revertants was observed. Based on these data, CM-GLUCAN was tested in the mutation assay up to concentration of 5000 iigfplate in the absence and the presence of S9-mix. MUTATION ASSAY (Table I and 2, Appendix 2) Precipitate CM-GLUCAN did not precipitate in the top agar. Precipitation of CM-GLUCAN was not observed on the plates at the start or at the end of the incubation period in all tester strains. Toxicity of the test substance The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. Number of revertants All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. CONCLUSION Based on the results of this study it is concluded that CH-GLUCAN is not mutagenic in the Salmonella typhimurium reverse mutation assay. - page 13 - CIR Panel Book Page 139

145 CM-GLUCAN NOTOX Project REFERENCES 1) Ames, 8.N., McCann, 3. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res,, 31, ) Maron, D.M. and Ames, 8,N., 1983, Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, Erratum, 1983, Mutation Res., 113, ) Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coli: partial purification and some properties. 3. Biol. Chem., 218, page 14 - CIR Panel Book Page 140

146 CM-GLUCAN NOTOX Project TABLE 1 MUTAGENIC RESPONSE OF CM-GLUCAN IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY Experiment 1 Dose Mean number of revertant (His) colonies! (iig/plate) 3 replicate plates (± S.D.) with different strains of Salmonella typhimurium TA1535 TA1537 TA98 TA100 Without S9-mix 3 129± ± ± ± 2 9 ± 1 16 ± ± ± 2 8 ± 2 19 ± ± ± 3 10 ± 4 26 ± ± ± 3 8 ± 3 26 ± ± ± 2 6 ± 2 27 ± ± 7 Solvent control 10 ± 1 9 ± 3 21 ± ± 6 Positive control 234 ± ± ± ± 107 With 59-mix 3 117± ± ± ± 1 7 ± 1 33 ± ; ± 5 7 ± 1 30 ± ± ± 2 7 ± 1 31 ± ± ± 3 7 ± 2 24 ± ± ± 4 7 ± 4 27 ± ± 6 Solvent control 11 ± 4 9 ± 1 30 ± ± 5 Positive control 297 ± ± ± ± ml Milli-Q water 2 One plate infected - page 15 - CIR Panel Book Page 141

147 CM-GLUCAN NOTOX Project TABLE 2 MUTAGENIC RESPONSE OF CM-GLUCAN IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY Experiment 2 Dose Mean number of revertant (His) colonies! (iig/plate) 3 replicate plates (± S.D.) with different strains of Salmonella typhimurium TA1535 TA1537 TA98 TA100 Without S9-rnix ± 5 8 ± 3 24 ± ± ± 2 9 ± 5 21 ± 5 99 ± ± 3 6 ± 2 20 ± ± ± 3 12 ± 1 21 ± 1 97 ± ± 3 12 ± 2 23 ± 5 98 ± 9 Solvent control 11 ± 2 9 ± 2 21 ± ± 11 Positive control 201 ± ± ± ± 8 With S9-mix ± 2 9 ± 5 30 ± ± ± 4 12 ± 2 25 ± 3 99 ± ± 4 11 ± 4 24 ± ± ± 2 9 ± 2 24 ± ± ± 1 11 ± 2 26 ± ± 18 Solvent control 12 ± 3 11 ± 3 29 ± ± 8 Positive control 112 ± ± ± ± ml Milli-Q water - page 16 - CIR Panel Book Page 142

148 CM- GLUCAN NOTOX Project APPENDIX 1 Bacterial background The condition of the both macroscopically (results are normal lawn evaluation bacterial background and microscopically lawn by using unless indicated in tables) is evaluated (if indicated), a dissecting microscope Definition Characteristics Normal Slightly reduced Moderately reduced Extremely reduced Absent Distinguished Distinguished by a by a healthy microcolony lawn. noticeable thinning of the ruicrocolony lawn and an increase in the size of the microcolonies compared to the solvent control plate. Distinguished by a marked thinning of the microcolony and an increase in the size of the microcolonies compared to the solvent control plate. Distinguished lawn and an by an extreme thinning of Increase in the size of the compared to the solvent control plate. Distinguished background lawn. by a complete lack of any the microcolony microcolonies microcolony lawn Precipitation evaluation Evidence of test article precipitate the following precipitation definition. on the plates is recorded by addition of Definition Characteristics Slight Precipitate Distinguished Mode rate by noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate. Precipitate Distinguished Heavy by a requiring the plate to Precipitate Distinguished by a marked amount of precipitate hand counted. be large amount of precipitate making the required hand count difficult. on on the plate, the plate, page 17 CIR Panel Book Page 143

149 CM- C LU CANNOTOX Project APPENDIX 2 Individual plate counts; (following pages) Strain TA1535 Experi merit 1 WITHOUT S9-MIX plate MEAN SD Dose(.ig/plate) ± 2 11 ± 2 10 ± 3 14 ± 3 11 ± 2 Solvent control Positive control ± ± 19 WITH S9-MIX plate MEAN SD Oose(i.tglpl ate) ± 1 10 ± 5 10 ± 2 14 ± 3 11 ± 4 Solvent control Positive control ± ± 68 - page 18 CIR Panel Book Page 144

150 CM-GLUCAN NOTOX Project Strain TA1537 Experiment 1 WITHOUT S9MIX plate MEAN SO Dose(pg/pl ate) ± ± ± ± ±2 Solvent control ± 3 Positive control ± 19 WITH S9-MIX plate MEAN SO Dose(ig/plate) ± ± ± ± ± 4 Solvent control ± 1 Positive control ± 74 - page 19 - CIR Panel Book Page 145

151 CM- C LU CAN NOTOX Project Strain Experi rnent TA98 1 WITHOUT S9-MIX plate MEAN SO Dose(iig/pl ate) ± 3 19 ± 4 26 * 1 26 ± 2 27 ± 5 Solvent control Positive control ± I 511 ± 24 WITH S9-MIX plate MEAN SO Dose(iig/plate) ± 3 30 ± 8 31 ± 2 24 ± 5 27 ± 7 Solvent control Positive control ± ± 82 - page 20 - CIR Panel Book Page 146

152 CM- GLUCAN NOTOX Project Strain Experi ment TA100 1 WITHOUT S9-MIX plate MEAN SD Dose(pg/plate) ± 107 ± 107 ± 122 ± 118 ± 116 ± 123 ± 114 ± Solvent control Positive control ± ± 107 WITH S9-MIX plate MEAN SD Dose(pg/pl ate) INF ± ± ± ; ± ± ± ± 6 Solvent control Positive control ± ± 32 INF: Plate infected - page 21 - CIR Panel Book Page 147

153 CM GLUCAN NOTOX Project Strain Experi ment TA WITHOUT S9-MIX plate MEAN SO Dose(pg/pl ate) ± 5 10 ± 2 13 ± 3 12 ± 3 16 ± 3 Solvent control Positive control ± ± 12 WITH S9-MIX plate MEAN SD Oose(iig/pl ate) ± 2 11 ± 4 13 ± 4 14 ± 2 14 ± 1 Solvent control Positive control ± ± 26 - page 22 - CIR Panel Book Page 148

154 CM - GLUCAN NOTOX Project Strain Experiment TA WITHOUT S9-MIX plate MEAN SD Ocse(pg/p late) ±3 9±5 6±2 12 ± 1 12 ± 2 Solvent control Positive control ±2 479 ± 85 WITH S9-MIX plate MEAN SO Dose(itg/pl ate) ±5 12 ± 2 11 ± 4 9±2 11 ± 2 Solvent control Positive control ± ± 67 - page 23 - CIR Panel Book Page 149

155 CM- GLUCAN NOTOX Project Strain Experi nent TA98 2 WITHOUT S9-MIX plate MEAN SD Dose(ig/pl ate) ± 5 21 ± S 20 ± 2 21 ± 1 23 ± 5 Solvent control Positive control ± ± 6 WITH S9-MIX plate MEAN SD Dose(iiglplate) ± 9 25 ± 3 24 ± 1 24 ± 1 26 ± 6 Solvent control Positive control ± ± 26 - page 24 - CIR Panel Book Page 150

156 CM-GLUCAN NOTOX Project Strain Experiment TA100 2 WITHOUT S9411X plate MEAN SD Dose(ug/pl ate) ± ± ± 3 98 ± 9 Solvent control Positive control ± ± 8 WITH S9-MIX plate MEAN SD Dose(pg/pl ate) ± 7 99 ± ± ± ± 18 Solvent control Positive control ± ± 49 - page 25 - CIR Panel Book Page 151

157 ALiFE SC ENCE LABORATORY chome, Nishihonmachi. Nishi-ku, OSAKA. JAPAN. TeI &ftv*i2 T 5 19 TeLQ (fk) Test Code No. 99-IXB STUDY REPORT Primary eye irritation study of CM-Glucan J in rabbits Report issued: November 29, 1999 English translation issued: November 29, 1999 amoto, M.D., Ph. D Director Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan CIR Panel Book Page 152

158 LIFE SCIENCE LABORATORY N5AN TeL T5 19 Tel (fe) Title Primary eye irritation study of CM-Glucan J in rabbits Purpose As a part of safety evaluation for CM-Glucan J, primary eye irritation study was performed using rabbits to examine whether or not the test substance has the irritant effects on the eyes after application of a single dose. This study was conducted by reference to the methods for toxicity studies in Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.) 3 and in accordance with the SOP of our Laboratory. Sponsor C. Holstein Co., Ltd. 3-7 Doshoinachi 1-chome, Chuo-ku, Osaka , Japan Contract Laboratory Head Office Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan Testing Facility The Second Chihaya-akasaka Laboratory, Life Science Laboratory 900, Aza-Chihayadani, Chihaya-akasaka-mura, Minamikawachi-gun, Osaka , Japan Testing Period From October 21 to November 29, 1999 Test Substance 2 CIR Panel Book Page 153

159 LIFE SCIENCE LABORATORY 5-19, 2-chomo, Nshhonmachi. Nishi-ku, OSAKA. JAPAN. Te I *T2T5l 9 TeLO () Name: CM-Glucan J (Sodium Carboxymethyl Betaglucan) Lot No.: Structure: Aqueous carboxymethiated 3 1,3-glucan derived from the cell walls of yeast (Saccharomyces cerevisiae), with molecular weight of about 100,000 Purity: More than 90% CM-Glucan 3 Appearance: White granular powders with a tinge of amber color and with alcoholic odor S olubility: Water soluble Stability: Stable for more than 12 months at 25CC Storage conthtion: Kept in a tightly closed container at room temperature Supplied by: C. Holstein Co., Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Preparation of the Test Substance The test substance was used for the study undiluted as delivered by the sponsor. Animals Eight male Japanese white rabbits (conventional), weighing 2.05 to 2.35 kg, were purchased from Hokusetsu Sangyo Co. Ltd. (Settsu, Osaka) on November 1, After 8 days of acclimation, six healthy animals without eye irritation, ocular defects or pre-existing corneal injury were used for the study. 3 CIR Panel Book Page 154

160 LIFE SCIENCE LABORATORY 5192-chorneMishihonmachi, fr2 T s 9 TeL Tel W) Animals were identified individually by marking of temporary animal numbers on right ears with black felt pen at arrival and of experimental animal numbers on left ears with red felt pen at grouping, and also by cageand rack-labeling. On the day before starting the test, both eyes of each animal were examined for confirming no corneal injury by application of 0.1 ml of 2% sodium fluorescein aqueous solution to the eyes, immediate washing out with 20 ml of distilled water for injection, and use of a slit lamp (Neitz Instruments Co., Ltd., Tokyo). No pre-existing corneal injury was found in any eyes of the animals selected for the study. Environmental Conditions Rabbits were housed individually in aluminum cages (320W X 550D X 350H mm) and maintained in an animal facility at 20 to 25 C and 50 to 70 % relative humidity with 10 changes or more of room air per hour on an 8-hour light cycle(0900-l700 hour). Every day the room and cage racks were cleaned and the floor was disinfected with Neochior Clean (Shikoku Chemicals Corporation), Standard laboratory chow (RC4, Oriental Yeast Co., Ltd., Tokyo) and tap water (from Chihaya-akasaka-mura water supply) by automatic water supplier were provided adlibitun2. Methods Treatment: On the day of starting the test, six animals weighing 2.30 to 2.50 kg were divided randomly into two groups of three animals each, no eye-washing group (animal Nos. 1 to 3) and eye-washing group (animal Nos. 4 to 6). The test substance was applied to the conjunctival sac of one eye (right eye) of each animal (animal Nos. 1 to 6) at a dose of 0.1 g after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one minute in order to spread the test substance over the whole area of the eyeball. The other eye (left eye), which remained untreated, served as a control. In the no eye-washing group the treated eyes remained unwashed 4 CIR Panel Book Page 155

161 LIFE SCIENCE LABORATORY chome, Nishihonmachi, 1IK*WT2T 5 19 Nishi-ku, OSAKA. JAPAN. TeI TeI () through the observation period, while in the eye-washing group the treated ones were washed out with 30 ml of distilled water for injection one minute after the test substance treatment. Animals were restricted with stainless steel cangue for one hour. Observation and measurement: Animals were observed for general conditions every day during the observation period. Their body weights were measured on 0 (the day of treatment) and 7 days (at the end of the observation period) after treatment. According to the evaluation criteria of primary eye irritation by Draize (shown in Attached Table), the reactions of the treated eyes (right eyes) to the test substance were examined both in no-eye washing and eye-washing groups and evaluated at 1, 24, 48 and 72 hours, and further 4 and 7 days The observation of ocular responses included the conjuntivae (chemosis, discharge and redness), cornea (opacity and area of cornea involved) and iris (values). after treatment in comparison with the untreated eyes (left eyes). Photographs for the eyes of each animal both in no-eye washing and eyewashing groups were taken at, 24 and 72 hours, and 7 days after treatment. Results 1) General conditions There were no changes in general conditions of all animals except the ocular responses during the observation period. 2) Body weights (Table 1) Body weights at the end of the observation period (7 days after treatment) were increased uneventfully in all animals. 3) Observation of ocular responses (Tables 2 to 7, Table 8, Photo) No eye-washing group (animal Nos. 1 to 3): 5 CIR Panel Book Page 156

162 LIFE SCIENCE LABORATORY 51 9, 2-chome. Nishihonmachi, Nishi-ku, OSAKA. JAPAN. TeI TeI () For conjunctivae, a slight redness was observed in the treated eyes (right eyes) of all three animals at 1 hour after the test substance treatment, but these signs disappeared in two animals at 24 hours and in the remaining one within 48 hours. For cornea and iris, on the other hand, no irritation was found in the treated eyes of all animals during the observation period. The mean total scores of primary eye irritation showed 2.0 at 1 hour after the test substance treatment, 0.7 at 24 hours and 0 at 48 hours and thereafter. Eye-washing group (animal Nos. 4 to 6): For conjunctivae, a slight redness was seen in the treated eyes (right eyes) of all three animals at 1 hour after the test substance treatment, but these signs disappeared in two animals at 24 hours and in the remaining one within 48 hours. No abnormal changes were found in cornea and iris of the treated eyes of all animals during the observation period. The mean total scores of primary eye irritation were 2.0 at 1 hour after the test substance treatment, 0.7 at 24 hours and 0 at 48 hours and thereafter. Discussion and Conclusion CM-Olucan J was applied to the conjunctival sacs of right eyes of six rabbits at a dose of 0.1 g. The eyes of the three animals were washed out with distilled water for injection one minute after treatment, while those of the other three animals remained unwashed. The reactions of the treated eyes to the test substance were examined for 7 days in comparison with the untreated left eyes as control, according to the evaluation criteria of primary eye irritation by Draize. For conjunctivae, a slight redness was observed in all animals both in the no eye-washing and eye-washing groups at 1 hour after the test substance treatment, but these signs disappeared within 48 hours. For cornea and iris, no irritation was seen in all animals both in the no eye-washing and eyewashing groups. There were no differences in the severity of primary eye irritation between no-eye washing and eye-washing groups. The irritation 6 CIR Panel Book Page 157

163 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi. Nishi ku, OSAKA, JAPAN. TeI Rft *$2T51 9 TeI.OG (fe) was considered to be mainly due to the physical property of the test substance which is a granular powder. CM-Glucan J was evaluated as practically non-irritating for eyes, judging from the classification on eye irritation properties by the method of Kay and Calandra 2. Archives The protocol, raw data, documentation records and the study report are retained in the archival room specified by Life Science Laboratory (Room No. 126, 2F1. in Okazakibashi Bldg., 9-11 Utsubohonmachi 2-chome, Nishi-ku, Osaka , Janan). References 1) J. H. Draize, 0. Woodard and H. 0. Calvery: Methods for the study of irritation and toxicity of substances topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. Z, (1944) 2) J. H. Kay and J. C. Calandra: Interpretation of eye irritation test. J. Soc. Cosmet. Chem. 13., (1962) 3) Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.), Supervised by Evaluation and Registration Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Edited by Japanese Association for Standards, Yakuji Nippo Sha, Tokyo (1996) (in Japanese) 7 CIR Panel Book Page 158

164 LIFE SCIENCE LABORATORY 4Jpn chornNtshihonmach, *2T51 9 Tel O Tel ({-) Attached Table Evaluation of primary eye irritation (by Draize) Ocular lesions Grade A Conjunctivae (a) Chemosis No swelling 0 Any swelling above normal (includes nictitating membrane) 1 Obvious swelling with partial eversion of the lids 2 Swelling with lids about half closed 3 Swelling with lids about half closed to completely closed 4 (b) Discharge No discharge 0 Any amount different from normal (does not include small amount observed in inner canthus of normal animals) 1 Discharge with moistening of the lids and hairs just adjacent to the lids 2 Discharge with moistening of the lids and considerable area around the eye 3 (c) Redness (refers to palpebral and bulbar conjunctivae) Vessels normal 0 Vessels definitely injected above normal 1 More diffuse, deeper crimson red, individual Vessels not easily discernible 2 Diffuse beefy red 3 8 CIR Panel Book Page 159

165 LIFE SCIENCE LABORATORY 519 2chom Nishthonrnach *T2 T 51 9 TeI TeI (ft Attached Table (Continued) B Cornea (a) Opacity-Degree of density (area which is most dense is taken for reading) No opacity 0 Scattered or diffuse area, details of iris clearly visible 1 Easily discernible translucent areas, details of iris slightly obscured 2 Opalescent areas, no details of iris visible, size of pupil barely discernible 3 Opaque, iris invisible 4 (b) Area of cornea involved One quarter (or less) but not zero 1 Greater than one quarter less than one-half 2 Greater than one-half less than three quarters 3 Greater than three quarters up to whole area 4 C Iris (a) Values Normal 0 Folds above normal, congestion, swelling, circumcorneal injection (any one or all of these or combination of any thereof), iris still reacting to light (sluggish reaction is positive) 1 No reaction to light, hemorrhage, gross Destruction (any one or all of these) 2 A Score for conjunctivae: (a+b+c)x2 B Score for cornea: axbx5 C Score for iris: a X 5 9 CIR Panel Book Page 160

166 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nishi-ku. OSAKA. JAPAN. Tel *&$IZ*T2T519 TeI (ft) Persons engaged in the study Animal care Atsunobu Matsuo November 29, 1999 )_ Administration and observation Hitoshi Yamamoto November29, 1999 /224Lt 4 NobukaIatsumura November 29, CIR Panel Book Page 161

167 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nish-ku, OSAKA, JAPAN. Tel *T2T519- TeI () Study director Hitoshi Yarnamoto November 29, 1999 Management Shtgeru Morita, Ph. D. November 29, CIR Panel Book Page 162

168 LIFE SCIENCE LABORATORY chornNshihonmachi, T5 1 9 Tel TeI () Table 1 Body weight changes in rabbits during the observation period after a single application of CM-Glucan J to the right eye Animal Body weight after application (kg) No. 0 day 7 days Mean ±SD ±0.07 ± CIR Panel Book Page 163

169 LIFE SCIENCE LABORATORY 5-19, 2-chome. Nshihonmachi, Nshi-ku. OSAKA. JAPAN. Te I TeI (44) Table 2 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 1: No eye-washing) Obervation Scores after application (right eye) item 1 h 24 h 48 h 72 h 4 d 7 d A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b+c)x B Cornea (a) Opacity (b) Area involved ScoreaXbX C Iris (a) Value Scoreax Total scores A+B+C No abnormal findings were found in the left eye (untreated control). h: hours, d: days 13 CIR Panel Book Page 164

170 IJFE SCIENCE LABORATORY chome. Nishihonmach, Nishi-ku, OSAKA, JAPAN. Tel *$T2T519 Tel (It) Table 3 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 2: No eye-washing) Obervation item A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b±c)x2 Total scores Scores after application (right eye) lh 24h 48h 72h 4d 7d o o o o B Cornea (a) Opacity (b) Area involved Score axbx C Iris (a) Value Scoreax A+B+C No abnormal findings were found in the left eye (untreated control). h: hours, d: days 14 CIR Panel Book Page 165

171 LIFE SCIENCE LA8ORATORY 5-19, 2-chome, NishhonmachL Nshi-ku, OSAKA, JAPAN. TeL *T2T519 TeL () Table 4 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 3: No eye-washing) Obervation Scores after application (right eye) item lh 24h 48h 72h 4d 7d A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b+c)x B Cornea (a) Opacity (b) Area involved ScoreaXbX C Iris (a) Value ScoreaX Total scores A+B+C No abnormal findings were found in the h: hours, d: days left eye (untreated control). 15 CIR Panel Book Page 166

172 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nishi-ku, OSAKA, JAPAN. Tel. O *J2T519 TeI () Table 5 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 4: Eye-washing) Obervation Scores after application (right eye) item 1 h 24 h 48 h 72 h 4 d 7 d A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b+c)x B Cornea (a) Opacity (b) Area involved Score axbx C Iris (a) Value ScoreaX Total scores A+B+C No abnormal findings were found in the left eye (untreated control). h: hours, days: days [(3 CIR Panel Book Page 167

173 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nshihonmachi, Nishi-ku, OSAKA, JAPAN. Tel *J2T5i9 Tel ( hi) Table 6 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 5: Eye-washing) Obervation Scores after application (right eye) item lh 24h 48h 72h 4d 7d A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b+c)x B Cornea (a) Opacity (b) Area involved Scoreaxbx C Iris (a) Value Score ax Total scores A+B+C No abnormal findings h: hours, days: days were found in the left eye (untreated control). 17 CIR Panel Book Page 168

174 LIFE SCIENCE LABORATOF1Y 5-19, 2-chome, Nishhormachi, Nishi-ku, OSAKA. JAPAN. Te I *fli*t2t 5i 9 TeI (fe) Table 7 Primary eye irritation scores for individual rabbits after a single application of CM-Glucan J to the right eye (Animal No. 6: Eye-washing) Obervation item A Conjunctivae (a) chemosis (b) discharge (c) redness Score (a+b+c)x2 B Cornea (a) Opacity 0 (b) Area involved 0 Score axbx5 0 Scores after application (right eye) lh 24h 48h 72h 4d 7d o o o o o o o C Iris (a) Value ScoreaX Total scores A+B+C No abnormal findings were found in the left eye (untreated control). h: hours, days: days 18 CIR Panel Book Page 169

175 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nishi-ku, OSAKA, JAPAN. Tot Tel (i) Table 8 Total scores of primary eye irritation in primary eye irritation study of CM-Glucan J in rabbits Group Animal Total scores after application (right eye) No. lh 24h 48h 72h 4d 7d No eye-washing IVIean Eye-washing Mean No abnormal findings were observed in the left eye (untreated control). h: hours, d: days 19 CIR Panel Book Page 170

176 Photo Changes in primary eye i. tation for individual rabbits after a si le application of CM-Glucan J to the right eye Distributed for Comment Only -- Do Not Cite or Quote CIR Panel Book Page 171

177 LIFE SCIENCE LABORATORY chome, Nishihonmachi, Nishi-Icu, OSAKA. JAPAN. Tel *rn*r2t51 9 Tel (i) Test Code No. 99-IXA STUDY REPORT Primary skin irritation study of CM-Glucan J in rabbits Report issued: November 29, 1999 English translation issued: November 29, 1999 oyao,i.dep.d. Director Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan I CIR Panel Book Page 172

178 LIFE SCIENCE LABORATORY 4JIPfl 519 2chorne. Nishihonrnach iz*nt2 T 5 i 9 TeIO TeI (ft) Title Primary skin irritation study of CM-Glucan J in rabbits Purpose As a part of safety evaluation for CM-Glucan J, primary skin irritation study was performed using rabbits to examine whether or not the test substance has the irritant effects on the skin after application of a single dose. This study was conducted by reference to the methods for toxicity studies in Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.), Supervised by Evaluation and Registration Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Edited by Japanese Association for Standards, Yakuji Nippo Sha, Tokyo (1996) (in Japanese) and in accordance with the SOP of our Laboratory. Sponsor C. Holstein Co., Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Contract Laboratory Head office: Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan Testing Facility: The Second Chihaya-akasaka Laboratory, Life Science Laboratory 900, Aza-Chihayadani, Chihaya-akasaka-mura, Minamikawachi-gun, Osaka , Japan Testing Period From October 21 to November 29, CIR Panel Book Page 173

179 LIFE SCIENCE LABORATORY chome, Nishihonmachi, &VT2T51 9* Nishi-ku. OSAKA, JAPAN. TeI TeI.O (lij Test Substance Name: CM-Glucan J (Sodium Carboxymethyl Betaglucan) Lot No.: Structure: Aqueous carboxymethylated 13-1,3-glucan derived from the cell walls of yeast (Saceharornyces cei evisiae, with molecular weight of about 100,000 Purity: More than 90% CM-Glucan J Appearance: odor White granular powders with a tinge of amber color and with alcoholic Sobubility: Water soluble Stability: Stable for more than 12 months at 25 C Storage condition: Kept in a tightly closed container at room temperature Supplied by: C. Holstein Co., Ltd. 3-7 Doshomachi, Chuo-ku, Osaka , Japan Preparation of the Test Substance The test substance was used for the study undilute as delivered by the sponsor. Animals Eight male Japanese white rabbits (conventional), weighing 2.15 to 2.40 kg, were purchased from Hokusetsu Sangyo Co. (Settsu, Osaka) on CIR Panel Book Page 174

180 $1 LIFE SCIENCE LABORATORY chomejshthonrnachi. Tel *2T51 9 Tel (it) November 1, After 7 days of acclimation, 6 animals with healthy intact skin were used for the study. Animals were identified individually by marking of temporary animal numbers on right ears with black felt pen at arrival and of experimental animal numbers on left ears with red felt pen at grouping, and also with cage- and rack-labeling. Environmental Conditions Rabbits were housed individually in aluminum bracket cages (320W X 550D )< 350H mm) and maintained in an animal room at a temperature of 20 to 25 C and a relative humidity of 50 to 70% with 10 changes or more of room air per hour on an 8-hour light cycle (0900 to 1700 hour). Every day the room and cage racks were cleaned and the floor was disinfected with Neochlor Clean (Shikoku Chemicals Corporation). Standard laboratory chow (RC4, Oriental Yeast Co., Ltd., Tokyo) in feed baskets and tap water (from Chihaya-akasaka-mura water supply) by automatic water system were freely available. Methods Six rabbits were used for the study. Fur was removed carefully from the dorsal area of the trunk of each animal by clipping with an electric clipper on the day of the test substance application. As shown in Chart, four sites of the dorsal area were designated for the position of the test substance application. The two were abraded by making four epidermal incisions (two perpendicular to two others like *) with a needle of l8g not so as to injure dermis (abraded skin) and the other two remained intact (intact skin). A dose of 0.5 g of the test substance moistened with 0.2 ml of distilled water was loaded on the cloth disc (2.5 cm in diameter) of adhesive plaster for patch test and applied to the two sites (abraded skin and intact skin) of each animal by closed patch for 24 hours. As control, oniy adhesive plaster (nothing was loaded on the cloth disc) was applied to the remaining two sites 4 CIR Panel Book Page 175

181 LIFE SCIENCE LABORATORY 5-192chorneMishhonmachi 4T2T TeLOS TeI, () in a similar way. The application sites were covered with hypo-allergenic cloth tape (Dermicel Tape). _J The applied site No. 1 (intact skin): The test substance 0.5 g 7 c The applied site No. 2 (abraded skin): 4 / Jff \ The test substance 0.5 g i \ The applied site No. 3 (intact skin): 11 [ 2 Adhesive plaster only ) The applied site No. 4 (abraded skin): Adhesive plaster only Animals were observed for general conditions every day during the observation period and weighed on 0 (the day of application) and 5 days (at the end of the observation period) after application. The patches were removed from the application sites 24 hours after application. Skin reactions including erythema/eschar formation and edema formation were examined at 24, 48 and 72 hours, and further 4 and 5 days after application. The evaluation criteria of skin reactions were according to Draize (shown below). Photographs of the skins at the applied sites of the test substance and only adhesive plaster were taken at 24 and 72 hours, and further 5 days after application. A Evaluation of skin reactions (by Draize) Skin reactions Grade Erythema and eschar formation No erythema 0 Very slight erythema (barely perceptible) 1 Well defined erythema 2 Moderate to severe erytherna 3 Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4 5 CIR Panel Book Page 176

182 LIFE SCIENCE LABORATORY 5-19, 2-chome. Nishhonrnachi, T Nishi-ku, OSAKA. JAPAN. Te1.O TeLO (fe) B Edema formation No edema 0 Very slight edema (barely perceptible) 1 Slight edema (edges of area well defined by definite raising) 2 Moderate edema (area raised approximately 1 mm) 3 Severe edema (raised more than 1 mm and extending beyond area of exposure) 4 The skin reactions were evaluated by primary irritation index (P.1.1., shown below), namely, the combined average of the averages of the 24- and 72-hour scores (erythema/eschar formation and edema formation) for the intact and for the abraded skin. Primary irritation index Evaluation only mildly irritating 3 5 moderate irritant severe irritant Results 1) General conditions and body weights The body weights on the day of application and at the end of the observation period are shown in Table 1. No abnormal findings were observed in the general conditions of all rabbits except the skin reactions and their body weights were increased satisfactorily during 5 days of the observation period. 2) Skin reactions The scores of skin reactions in the application sites of each animal during the observation period are shown in Tables 2 to 5. The application site No. 1 (intact skin): CM-Glucan J (Table 2, Photo 1) (3 CIR Panel Book Page 177

183 L)FE SCIENCE LABORATORY 5-19,2-chorne.Nis[-honmachi, *NT2T51 9 Tel Tel ({ ) No skin irritations such as erythema!esch.ar and edema were found in all animals through 5 days of the observation period. The application site No. 2 (abraded skin): CM-Glucan J (Table 3, Photo 2) Very slight erythema was observed in all six animals at 24 hours after application, but disappeared in one animal at 48 hours, in three at 72 hours and in the remaining two at 4 days. No eschar and edema were seen in any animals during the observation period. The application site No. 3 (intact skin): adhesive plaster only (Table 4, Photo 3) No skin irritations such as erythema/eschar and edema were found in all animals through 5 days of the observation period. The application site No. 4 (abraded skin): adhesive plaster only (Table 5, Photo 4) Very slight erythema was observed in all six animals at 24 hours after application, but disappeared in one animal at 48 hours, in four at 72 hours and in the remaining one at 4 days. No eschar and edema were found in all animals during the observation period. 3) Evaluation on the basis of P. I. I. (Table 6) The average P. I. I. was 0.33 for the test substance and 0.29 for the control (only adhesive plaster). Therefore, the test substance was evaluated as non-irritating to only mildly irritating, but the results suggesting that only adhesive plaster also produced a similar degree of skin reactions to the test substance. Discussion and Conclusion When applied to the intact skin, CM-Glucan J as well as only adhesive plaster did not cause skin irritations in any rabbits during the observation period (5 days). CIR Panel Book Page 178

184 LJFE SCIENCE LABORATORY chomNisMionmachi, 1T2 T S1 9- TeLO TeLO () When the test substance was applied to the abraded skin, very slight erythema, but not eschar and edema, was observed in all animals at 24 hours after application, which was reduced gradually and disappeared in all animals within 4 days. Only adhesive plaster also showed a similar degree of skin irritations to the test substance when applied to the abraded skin. Both the test substance and only adhesive plaster produced somewhat a greater skin irritation in the abraded skin than in the intact skin. P. I. I. was 0.33 for CM-Glucan J and 0.29 for only adhesive plaster. From these results, CM-Glucan J was evaluated as non-irritating to only mildly irritating according to the criteria of Draize, but it was considered to be non-irritating because only adhesive plaster also caused a similar degree of skin irritations to the test substance. Archives The protocol, raw data, documentation records and the study report are retained in the archival room specified by Life Science Laboratory (Room No. 126, 2 Fl. in Okazakibashi Bldg., 9-11 Utsubohonmachi 2-chome, Nishi-ku, Osaka , Japan) after the completion of the study. References 1) J. H. Draize: Dermal toxicity, In Appraisal of the safety of chemicals in food, Drugs and cosmetics, Association of Food and Drug Officials of the United States, Texas State Department of Health, Austin (1959) 8 CIR Panel Book Page 179

185 / Distributed for Comment Only -- Do Not Cite or Quote LIFE SCIENCE LABORATORY 519, 2-chome, Nishihonmach, Msh-ku. OSAKA. JAPAN. Tel *T2 T 5 li9 TeI, () Persons engaged in the study Animal care: - \ - iij Atsunobu Matsuo November 29, 1999 Administration and observation )A/ Hitoshi Yamamoto November 29, 1999 Nobukalu Matsumura November 29, CIR Panel Book Page 180

186 LIFE SCIENCE LABORATORY chome, N1shihonmachi, Nishi-ku. OSAKA. JAPAN. Tel &4zT2T51 9 Tel (ø) Study director /;;4nas Hitoshi Yamarnoto November 29, 1999 Management Shigeru Morita, Ph. D. November 29, CIR Panel Book Page 181

187 LIFE SCIENCE LABORATORY chome, Nishihonmachi, Nishi-ku, OSAKA, JAPAN. TeI *J2T5i 9 TeI (fe) Table 1 Body weight changes in rabbits during the observation period after application of CM-Glucan J to the skin for 24 hours Animal Body weight after appjicatien (kg) No. 0 day 5 days Mean ± S D ±0.05 ±0.06 Ii CIR Panel Book Page 182

188 LIFE SCIENCE LABORATORY 6-19, 2-chome, Nfshjhonmachi, Nish-ku, OSAKA. JAPAN. Tel *WT2T5l 9 TeI ({t) Table 2 Scores for primary skin irritation in individual rabbits after the application of the test substance to the intact skin The application site No. 1 (intact skin): CM-Glucan J Animal Erythema/eschar formation No. 24h 48h 72h 4d Sd Total h: hours d: days Edema formation 24h 48h 72h 4d 5d Table 3 Scores for primary skin irritation in individual rabbits after the application of the test substance to the abraded skin The application site No. 2 (abraded skin): CM-Glucan J Animal No Total Erythema/eschar formation 24h 48h 72h 4d h: hours d: days Sd Edema formation 24h 48h 72h 4d Sd CIR Panel Book Page 183

189 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nshihonrnachi, Nishi-ku. OSAKA, JAPAN. Tel J2T5i 9 TeI ({) Table 4 Scores for primary skin irritation in individual rabbits after the application of only adhesive plaster to the intact skin The application site No. 3 (intact skin): Adhesive plaster only Animal Erythema/eschar formation No. 24h 48h 72h 4d 5d Total h: hours d: days Edema formation 24h 48h 72h 4d 5d Table 5 Scores for primary skin irritation in individual rabbits after the application of only adhesive plaster to the abraded skin The application site No. 4 (abraded skin): Adhesive plaster only Animal Erythema/eschar formation No. 24h 48h 72h 4d Sd Total h: hours d: days Edema formation 24h 48h 72h 4d Sd CIR Panel Book Page 184

190 LIFE SCIENCE LABORATORY chome. Nishihorimechi, Nishi-ku, OSAKA. JAPAN, Te I *T2T5i9 TeI (1) Table 6 Primary irritation index (P. I. I.) of CM-Glucan J when applied to the rabbit skin Animal Test substance Control No. CM-Glucan J Only adhesive plaster Mean CIR Panel Book Page 185

191 ,. - Distributed for Comment Only -- Do Not Cite or Quote Photo 1 Skin reactions in individual rabbits after application of CM-Glucan J to the intact skin The application site No. 1 An in al After 24 hours After 72 hours After 5 days 1 F r? r:., l.r.., tip I II &. 5 6 :. i---. CIR Panel Book Page 186

192 Photo 2 Skin reactions in individual rabbits after application of CM-Glucan J to the abraded skin The application site No. 2 Animal After 24 hours After 72 hours After 5 days : 2 E: 4 3 I * 4 ue ----q 5 : 6 * 1 1 CIR Panel Book Page 187

193 Photo 3 Skin reactions in inthvidual rabbits after application of adhesive plaster only to the intact skin The application site No. 3 Animal No. After 24 hours After 72 hours After 5 days I I o. - I 5 hr 6.O..r. hr p - CIR Panel Book Page 188

194 Photo 4 Skin reactions in inthvidual rabbits after application of adhesive plaster only to the abraded skin The application site No. 4 Animal No, After 24 hours After 72 hours After 5 days I. 2 4 ; 1.. *4.. L -,l1 4 :4.,:,,,,,,-..Iir 5 ::..,..,l,,! -- J U,S.,., $k..4o I... I 4 CIR Panel Book Page 189

195 LIFE SCIENCE LABORATORY chornNisIhonmachi *$T2 T Tel Tel () Test Code No. 99-XII STUDY REPORT Primary skin irritation test for CM-Glucan J in human subjects by closed patch Report issued: December 6, 1999 English translation issued: December 6, 1999 YO1rer:Ph.D. Life Science Laboratory 5-19, 2 -Chome, Nishihonmachi, Nishi-ku, Osaka , Japan CIR Panel Book Page 190

196 LIFE SCIENCE LABORATORY chornNIshihonmachi, 1*T2 T 51 9 TeI TeI fi) Title Primary skin irritation test for CM-Glucan J in human subjects by closed patch Summary CM-Glucan J was found to have no primary skin irritation potential by a 24-hour closed patch test when applied to 40 human subjects. Purpose As a part of safety evaluation for CM-Glucan J, a 24-hour closed patch test was performed in 40 human volunteers to examine whether or not the test material has a primary skin irritation potential. Sponsor C. Holstein Co., Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Contract Laboratory Life Science Laboratory 5-19, 2-Chome, Nishi-ku, Osaka , Japan Testing Facilities Life Science Laboratory Head office: 5-19, 2-Chome, Nishi-ku, Osaka , Japan Chihaya-akasaka Laboratory: 1082, Aza-Chihaya, Chihaya-akasaka-mura, Minamikawachi-gun, Osaka , Japan 2 CIR Panel Book Page 191

197 LIFE SCIENCE LABORATORY cnornNishihonmachi, 9 Tel Tel () Testing Period From November 18 to December 6, 1999 Test Material Name: CM Glucan J (Sodium Carboxymethyl Betaglucan) Lot No.: Structure: Aqueous carboxymethylated -1,3-glucan derived from the cell walls of yeast (Saccharomyces cerevi.iae, with molecular weight of about 100,000 Purity: More than 90% CM-Glucan J Appearance: White granular powders with a tinge of amber co or and with alcoholic odor S olubility: Water soluble Stability: Stable for more than 12 months at 25 C Storage condition: Kept in a tightly closed container at room temperature Supplied by: C. Holstein Co., Ltd. 3-7 Doshomachi l-chome, Chuo-ku, Osaka , Japan Received on: October 20, 1999 Preparation of the Test Material It was used for the study undiluted as delivered by the sponsor. 3 CIR Panel Book Page 192

198 LIFE SCIENCE LABORATORY 519, 2-chorn Nishonm ach isi2 T 5 Tel Tel (i) Control (Blank) Cloth disc of adhesive plaster for patch test (Libatape Pharmaceutical Co., Ltd.). Test Methods women) participated in this study. Before starting the study, the purpose, nature and potential risks of the test were explained to the subjects and their signed consent forms (in Japanese) were obtained. A blank format of its English translation is attached as Appendix. A 0.1 g of CM-Glucan J was loaded on one of the two cloth discs of the adhesive plaster for patch test using a small amount of vaseline for adhesion, while nothing was loaded on the other. The paired discs, loaded and not loaded, were placed on the interior surface of either upper arm of each subject under the adhesive patch. After 24 hours the patches were removed Forty volunteers (27 men and 13 and the skin reactions of the applied sites were observed macroscopically and evaluated by the medical doctor (study director) according to the score of the Japan Patch Test Research Group (the standard criteria of Japan). The photographs of the applied sites were taken from each subject. Evaluation of skin reactions (Score employed by the Japan Patch Test Research Group) 0 H) No change 0.5 (±) Slight erythema 1 (+) Erythema 2 (++) Erythema+Edema 3 (+ + +) Erythema+Edema+Papule, Serous Papule, Vesicle 4 (++++) Bulla Results 4 CIR Panel Book Page 193

199 LIFE SCIENCE LABORATORY 5-19,2-chornNishonrnachi, T2T51 9 TeI TeI () The results obtained at 24 hours after closed patch of CM-Glucan J to human skins are shown in Table and Photographs. No primary skin irritation was observed in all 40 subjects tested after the patches were removed. Conclusion CI vi-glucan J was considered to have no primary skin irritation potential for humans in the 24-hour closed patch test under our test conditions. Person Engaged in the Study and Study Diector Yukio Yanagimoto, M.D., Ph.D. Archives The protocol, all records obtained during the testing period, raw data and the study report are retained in the archival locker specified by Life Science Laboratory. References 1) T. Kawamura, S. Sasagawa, T. Masuda, S. Honda, M. Kinoshita, S. Harada, T. Ishizaki, R. Nagai, K. Hirokawa, T. Anzai, K. Anekoji, A. Hidano, T. Kawano, I. Ikegami, S. -Sato and T. Aoyama: Basic studies on the standardization of patch test. Nihon Hifuka Gakkai Zasshi 80, (1970) (in Japanese) 5 CIR Panel Book Page 194

200 LIFE SCIENCE LABORATORY 5-19, 2 -chome. Nishihonmachi, &t1a*r2t Nishi-ku, OSAKA. JAPAN. TeI (4k) Tel Study Director Yuk a g December 6, 1999 Management Seru Morita, Ph.D. December 6, CIR Panel Book Page 195

201 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihormachi, Nishi-ku. OSAKA, JAPAN. Tel, **T2T519 TeLO ( t) Appendix: English translation of the original blank format of Consent Form in Japanese To Yukio Yanagimoto Director of Life Science Laboratory Date: Address of subject: Name of subject: Date of birth: Consent Form I hereby consent as a subject to participate in the primary skin irritation test (24-hour closed patch test) for the test material given below and observe the following matters that demand special attention. Test material: The matters that demand special attention: 1) If you feel an irritation and/or a pain in your skin at the applied site of the test material, remove the patch immediately and wash the skin fully with water, though a preliminary skin irritation test has been performed for its safety evaluation in experimental animals. 2) Do not wet the applied site of the test material during the test period. 3) Refrain from hard exercises or works causing perspiration during the test period. 4) Do not scratch the applied site. Observation and evaluation: rpweflty,.fotr hours after closed patch of the test material 7 CIR Panel Book Page 196

202 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishhonmachi. Nishi-ku, OSAKA, JAPAN. Tel *$*r2t51 9* TeI () Table Evaluation of the skin reaction to CM-Glucan J Subject Evaluation Score and Subject (Sex, Age) Photo No. Test material Control (Blank) 1 KR. M SM. M HY. M S.M.F T.N. M A.]\ LM R.H. F MK.M RN. M HK. M TK. F KU. F N.0F H.NF Y.K F HO. M A.W. lvi KS. M HA. M SH. F YT. M S.N. F N.T. F YH. M Y.K. M CIR Panel Book Page 197

203 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmach, Nishiku, OSAKA, JAPAN. Te I *T2T5i 9 TeI, (f) 26 M.O.M N.N.F T.A. F M.O.M H.M.M A.S. M T.S. M S.M. M S.HF M.G.M Y.K. M E.S. M A.M.M N.M.M H.Y. M CIR Panel Book Page 198

204 in -. L. V... (O V)... - O - Photographs of the applied sites material in human subjects Test material: CM-Glucan J Distributed for Comment Only -- Do Not Cite or Quote at 24 hours after closed patch of the test Test material Test material Test material Test material V. jw4-4 cuo p.c 4.. co )Z c,z.. 4:. Q) 3) (1) E V. C 3O (O : 2 2. c 3 2 I,. : ,2cq I ci D U0 _.:q,n r.. -.,:.r. :._, rnz - -. CZ o,-.. V co ciz j U csiz 3, 3) c p PC%4 lo rjz., r ,. i !.-.io i 1) V a. a V - -. V.. COZ , 30 g j 40 h CIR Panel Book Page 199

205 Photographs ol the applied. sites at 4 hours alter ciosea. patcn 01 tue test material in human subjects Test material: Cloth disc of adhesive plaster (control) Sob. No. Control No. Control b Control Control 4-I 00 2 i 3t ) QZ H - 21 J..) c $ ; DO QZ 22 C_, I a. gg I.4 _) I J C 0 QZ 32 C4 4.. C I )Jc c 00 QZ.4 4J , 5 6 4I 00 I 00 jz C QZ 1 c.) ; , Jco.) 00 Qz z. 00 ciz 7 ) I DO QZ 0 I 4 1,r% 17 r4j : ) 8 4-Ico 00 Qz i ) C c.)z QZ 19 Ho QZ QZ 30 I ) C I F QZ C 00 QZ CIR Panel Book Page 200

206 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nishi-ku, OSAKA, JAPAN. Tel Distributed for Comment Only -- Do Not Cite or Quote *T2T5l9 TeI (fe) 7 Test Code No. 99-Vu STUDY REPORT Reverse mutation study of CM-Glucan J in bacteria Report issued: November 29, 1999 English translation issued: November 29, 1999 Yukio imoto, M.D., Ph. D. Director Life Science Laboratory 5-19, 2- Chome, Nishihonmachi, Nishi-ku, Osaka , Japan 1 CIR Panel Book Page 201

207 LIFE SCIENCE LABORATORY 519 2chorne Nis[uhonrnach, *T2 T 51 9* TeI TeI (t) Title Reverse mutation study of CM-Glucan J in bacteria Purpose As a part of safety evaluation for CM-Glucan 3, reverse mutation study using bacteria was performed to examine whether or not the test substance has a mutagenic potential. This study was conducted by reference to the methods for toxicity studies in Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.) and in accordance with the SOP of our Laboratory. Sponsor C. Holstein Co., Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Contract Laboratory Head Office: Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan Testing Facility: The First Chihaya-akasaka Laboratory, Life Science Laboratory 1082, Aza-Chihaya, Chihaya-akasaka-mura, Minamikawachi-gun, Osaka , Japan Testing Period From October 21 to November 29, 1999 Test Substance Name: 2 CIR Panel Book Page 202

208 LIFE SCIENCE LABORATORY 519, 2-chomo, NishihormachL *T2 T5 1 9 Nshi-ku. OSAKA. JAPAN TeI (ft) Tel CM-Glucan 3 (Sodium Carboxymethyl Betaglucan) Lot No.: Structure: Aqueous carboxymethylated 13-1,3-glucan derived from the cell walls of yeast (Saccharoznyces cerevisiae), with molecular weight of about 100,000 Purity: More than 90% CM-Glucan J Appearance: White granular powders with a tinge of amber color and with alcoholic odor Solubility: Water soluble Stability: Stable for more than 12 months at 25CC Storage condition: Kept in a tightly closed container at room temperature Supplied by: C. Holstein Co., Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Positive Controls N-Ethyl-N -nitro-n-nitrosoguanidine (ENNG) 2-Nitrofluorene (2-NF) 9 -Aminoacridine hydrochloride (9-AA) 2-Arninoanthracene (2-AA) Sodium azide (NaN) Vehicle Control (Negative Control) Purified water :3 CIR Panel Book Page 203

209 LIFE SCIENCE LABORATORY chomo. Nishihonmchi. &IT2T Nishi-ku, OSAKA. JAPAN. Tef (ft) TeI Reagents 59 S9 (Lot ) prepared by the following procedure was purchased from Oriental Yeast Co., Ltd. (Osaka) on October 29, 1999 and stored at 80( until used. Preparation of 59: Male Sprague-Dawley rats (7 weeks old, 205.9±9,6 g) were intraperitoneally treated with phenobarbital (PB) at 30 mg/kg on day 0 and at 60 mg/kg on days 1 to 3 repeatedly and also with 5,6-benzoflavone BF) at 80 mg/kg on day 2. Animals were sacrificed on day 4 and their livers were removed to prepare 9000 X g supernatant (59) by a routine procedure. Nutrient broth (Lot JD, Difco Laboratories) Agar powder (Lot XD, Difco Laboratories) Other reagents Commercially available reagents of special grade were used. Test System Bacterial tester strains used: Salmonella typhimurinin TA100, TA98, TA1535 and TA1537 Escherichia coil WP2 uvra Procurement and storage: Salmonella strains and Escherichia coil strain were obtained from Institute for Fermentation, Osaka on June 13, In our Laboratory, the bacterial tester strains are stored in sterile small screw-capped plastic vials at 80 C after freezing on dry ice a fresh nutrient broth culture (0.8 ml of bacterial cell suspension with 0.07 ml of DMSO). The tester strains are subcultured at intervals within a year and their characteristics are examined at intervals of 3 months to confirm the genetic stability. 4 CIR Panel Book Page 204

210 LIFE SCIENCE LABORATORY 5-19, 2-chome. Nishihonmach, $*r2 T si 9- Nishi-ku. OSAKA, JAPAN. TeI TeI (ft> Preparation of Test Materials Test substance: The test substance was dissolved in purified water in the following five different concentrations, for the preliminary dose-finding test, 195, 781, 3125, and ji g/ml, and for the subsequent main test, 3125, 6250, 12500, and ii g/ml (based on the results of the dose-finding test). The test substance was autoclaved (at 121 C for 15 minutes) according to the indication of the sponsor and used for the test, because growth of bacteria was observed in the test substance solutions in the sterility test beforehand performed. Positive controls: Strain-specific positive controls were selected on the basis of the types of bacterial tester strains and dissolved in DM50. The positive controls used and the dose levels for the corresponding tester strains are shown below. Tester strain Without S9 Mix Positive control( i g/plate) With 59 Mix Positive control( p. g/plate) TA100 NaN AA 1 TA98 2-NF 1 2-AA 0.5 TA1535 NaN AA 2 TA AA AA 2 WP2 uvra ENNG 2 2 AA 20 S9 Mix: S9 Mix was prepared with a sterile technique when used. S9 Mix contains per ml: 0.1 ml of S9, 0.02 ml of 0.4 M magnesium chloride (8 p mol), 0.02 ml of 1.65 M potassium chloride (33 j2 mol), ml of 1 M glucose-6-phosphate (5 p mol), 0.04 ml of 0.1 M NADPI1 (4p mol), 0.04 ml of 0.1 M NADH (4 p mol), 0.5 ml of 0.2 M sodium phosphate buffer (100 p. mol), 0 CIR Panel Book Page 205

211 LIFE SCIENCE LABORATORY 4 )ffi chome. Nishihonmachi. *T2T5 1 S Njshi-ku. OSAKA. JAPAN - - TeLO Tel () and purified water for balance. Nutrient liquid broth: Nutrient broth 0.8 g Sodium chloride 0.5 g Purified water 100 ml Ten ml of this medium was poured into each L-shaped tube and autoclaved. Minimal glucose agar plate medium: Minimal Vogel-Bonner medium E (undiluted, 10-times concentrated) containing the following constituents was prepared at first. Magnesium sulfate heptahydrate 2 g Citric acid monohydrate 20 g Potassium phosphate, dibasic, anhydrate 100 g Ammonium sodium phosphate tetrahydrate 35 g These compounds were dissolved in purified water, in order, to make a total volume of 1000 ml (ph 7). Then, minimal glucose agar medium containing the following constituents was prepared. Minimal Vogel-Bonner medium E (undiluted) 80 ml Glucose 16 gin 100 ml of purified water Agar powder 12 g of 620 inl of purified water After individually autoclaved and cooled to about 60 C, these were mixed together. Thirty ml of the mixture was poured into each gamma-raysterilized petri dish (90 mm diameter, Ina-Optika Co., Ltd. Osaka) and allowed to solidify for preparation of minimal glucose agar plate medium (hard agar). Top agar: Aqueous solution containing 0.6% agar and 0.5% sodium chloride was autoclaved and kept at about 45 C in a molten state until used. When used, amino acid solution I (aqueous solution containing 0.5 mm L-histithne and 0.5 mm D-biotin for Salmonella tester strains) or II (aqueous solution 13 CIR Panel Book Page 206

212 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi. Nish-ku, OSAKA, JAPAN. Tel T519 Tel (It) containing- 0.5 mm L-tryptophan for Escherichia coil tester strain) was added to this molten agar at a volume ratio of 1:10, and mixed thoroughly for preparation of top agar. These amino acid solutions I and II were beforehand sterilized by using a vacuuni-driven disposable filtration system (0.22 p m pore size, Iwaki Glass Co., Ltd., Tokyo). Methods The study was carried out by the preincubation method with minor modification of Ames eta]. Dose finding and main tests: In the preliminary dose-finding test, the frozen stock cell suspension of each bacterial tester strain was thawed at room temperature. Twenty p L of the cell suspension was inoculated into 10 ml of nutrient liquid broth in a L-shaped tube and incubated with shaking (65 times/minute) at 37 C for 12 hours in a darkroom. A 0.1 ml aliquot of the test substance solution in purified water at 195, 781, 3125, or p g/ml (19.5, 78.1, 312.5, 1250 or 5000 p g/plate) was mixed with 0.5 ml of S9 Mix for the test with metabolic activation [S9 Mix(+)] or 0.1 M sodium phosphate buffer (ph 7.4) for the test without metabolic activation [S9 Mix( )] in a sterile test tube and then 0.1 ml of the tester strain culture was added. The mixture was preincubated with shaking at 37 C for 20 minutes. After addition of 2 ml of top agar containing the amino acid solution I for Salmonella tester strains or the amino acid solution II for Eschei ichla coil tester strain, the reaction mixture was vortexed and poured over the surface of minimal glucose agar plate medium in the petri dishes. The dishes were placed in an incubator at 37 C for 48 hours. Revertant colonies were counted macroscopically and growth inhibition was examined using a stereoscopic microscope. Duplicate plates were used for each dose level. The subsequent main test was performed in a similar way to the dose finding test for confirming the reproducibility. The dose levels of the test substance were 3125, 6250, 12500, and p g/ml (312.5, 625, 1250, 2500 and 5000 p g/plate) for each bacterial tester strain, which were 7 CIR Panel Book Page 207

213 LIFE SCIENCE LABORATORY 5 l 9, 2-chome, Nishhonmachi, *11rz*r2T Nish-ku, OSAKA. JAPAN. TeI TeI tt) determined on the basis of the results obtained from the dose-finding test. The test for vehicle control (negative control) was carried out in a similar way to the dose.fmding test, using 0.1 ml of purified water in place of the test substance solution in purified water. The test for positive controls was carried out in a similar way to the dosefinding test, using 0.1 ml of the specified positive control solution in DM50 for each bacterial tester strain at the indicated concentration in place of the test substance solution in purified water. Sterility test for 59 Mix and the test substance solutions in purified water: A 0.1 ml aliquot of S9 Mix or the test substance solutions in purified water with different concentrations (after sterilization by autoclave) was spread over the surface of standard agar plate (20 ml, ph 7.2, Eiken Chemical Co., Ltd., Tokyo). After 48-hour incubation of the plates at 37 C, sterility was examined for confirming no growth of bacteria. 0.1 M sodium phosphate buffer and two kinds of top agars were also subjected to the sterility test. Evaluation of the results: The mean number of revertant colonies per plate in duplicate was calculated in the test substance groups, and the positive and negative (vehicle) control groups. The test substance was evaluated to be positive for mutagenic activity when it induced two-fold or more increase in the number of revertants compared with that of the spontaneous ones (negative control) and when this effect was dose-dependent and reproducible. Results The results obtained are shown in Tables 1 and 2 and Figs. 1 and 2. With and without metabolic activation[s9 Mix (+) and S9 Mix ( )j, all positive controls induced more than twice increase in the number of revertants compared with that of spontaneous ones (negative control) in all bacterial tester strains (Tables 1 and 2). S CIR Panel Book Page 208

214 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi *1IZ*T2 T 5 l 9 Nishi-ku, OSAKA. JAPAN. Tel Tel, () In the sterility test, no growth of bacteria was observed in any samples tested. The dose-finding test was performed at 5 dose levels of 19.5, 78.1, 312.5, 1250 and 5000 t g/plate (doses increasing in a geometric progression by a factor of 4) for all bacterial tester strains (Table 1). Eveii at 5000 g/plate, the test substance caused neither growth inhibition nor twice or more increase in the number of revertants compared with the negative control in any bacterial tester strains with or without S9 Mix. Therefore, the main test was performed at 5 dose levels of 312.5, 625, 1250, 2500 and 5000 p g/plate (doses increasing in a geometric progression by a factor of 2) for all bacterial tester strains (Table 2 and Figs. 1 and 2). Even at 5000 ji g/plate, neither growth inhibition nor twice or more increase in the number of revertants compared with the negative control was observed in any bacterial tester strains with or without S9 Mix. Conclusion Both in the dose-finding test and subsequent main test, the test substance induced neither growth inhibition nor twice or more increase in the number of revertants compared with the negative control in any bacterial tester strains tested with or without S9 Mix even at the highest dose level (5000 t g/plate. The similar tendency and reproducibility were observed in these results obtained from the two separate experiments. In conclusion, the test substance is considered to be negative for mutagenic activity in Salmonella typhimuriun2 TA100, TA1535, TA98 TA1537 and Escherichia coil WP2 uvra, judging from the SOP of our Laboratory. Archives The protocol, all records obtained during the testing period, raw data and the study report are retained for 10 years in the archival room specified by Life Science Laboratory. 9 CIR Panel Book Page 209

215 LIFE SCIENCE LABORATORY 519 2chorn Nish0,onrnach T2 T 51 9 Tel Tel () Study director: Tanehiko Azuma Person engaged in the study: Yutaka Kageyama Management: Shigeru Morita, Ph. D. References 1) Ames, B. N., Durston, W. E., Yamasaki, E. and Lee, F. D. : Proc. Nat. Acad. Sci. USA1., (1973) 2) McCann, J., Spingarn, N.E., Kobori, J. and Ames, B. N. : Proc. Nat. Acad. Sci. USA.72, (1975) 3) Ames, B. N., McCann, J., and Yamasaki, E. : Mutation Res. j, (1975) 4) Yahagi, T., Degawa, M., Seino, Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto, Y. : Cancer Lett. 1, (1975) 5) De Serres, F. J. and Shelby, M. D. : Science 203, (1979) 6) Brusick, D. J., Simmon, V. F., Rosenkranz, H. S., Ray, V. A. and Stafford, R. S. : Mutation Res..7, (1980) 7) OECD guideline for testing of chemicals, Supervised by Office of Environmental Chemical Safety, Environmental Health Bureau, Ministry of Health and Welfare, Yakugyo Jiho Sha, Tokyo (1991) (in Japanese) 8) Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.), Supervised by Evaluation and Registration Division, Pharmaceutical Affairs Bureau, Ministry of Health and \,Teffare, Edited by Japanese Association for Standards, Yakuji Nippo Sha, Tokyo (1996) (in Japanese) 10 CIR Panel Book Page 210

216 LIFE SCIENCE LABORATORY 5-19, 2-chome. Nishihonmachi, Nishi-ku, OSAKA. JAPAN Tel *NT2T519 TeI () Study director /,i. Tanehiko Azuma November29, 1999 Person engaged in the study Yutaka Kageyama November 29, 1999 anagement igeru Morita, Ph. D. November 29, CIR Panel Book Page 211

217 Table 1 Reverse mutation test with bacteria of CM Glucan J (Dose finding test) With (+) or Test substance Number of revertants (number of colonies/plate) without ( ) dose Base oair substitution type Frameshift type S9 Mix (pg/plate) TA100 TA1535 WP2uvrA TA9B TA1537 Negative control 154 (153) 12 (13) 23 (23) 44 (41) 6 (7) (134) 13 (14) 19 (16) 33 (39) 7 (5) SSMix( ) 149 (154) 15 (16) 19 (21) 30 (34) 6 (5) (131) 11 (11) 15 (16) 27 (34) 5 (5) (151) 17 (14) 14 (14) 33 (35) 5 (5) (141) 18 (17) 18 (19) 47 (43) 2 (3) Negative control 151 (147) 9 (10) 23 (20) 44 (42) 5 (7) (129) 14 (14) 17 (22) 45 (41) 8 (11) SSMix(+) 121 (123) 7 (8) 17 (20) 38 (49) 6 (6) (152) 8 (11) 22 (21) 43 (46) 6 (6) (140) 12 (12) 28 (26) 32 (36) 8 (6) (140) 9 (10) 16 (16) 39 (47) 5 (7) Positive Name NaN 3 NaN 3 ENNG 2 NF control not Dose requiring ( p g/plate) S9 Mix Number of colonies/plate 529 (470) 320 (314) 692 (644) 270 (288> 280 (290) Positive Name 2 AA 2 AA 2 AA 2 AA 2 AA control Dose requiring ( p g/plate) S9Mix Number of colonies/plate 818 (799) 157 (178) 726 (717) 231 (221) 95 (102) 1 When inhibition is found against growth of tho bacteria, mark the applicable value with an asterisk, 2.Fill the average number of colonies in each dose in the( ). 3. Number of revertants Fill in the observed value and average value in order beginning with low doses of the test substance. 4.When the precipitate is observed on the plate, mark the applicable dose of the test substance with +. 5,ENNG: N ethyl N nitro N nitrosoguanidine, 2 NF: 2 nitrofluorene 9 AA: 9 aminoacridine hydrochloride. 2 AA: 2 arninoanthracene, f1an3 sodium azido CIR Panel Book Page 212

218 Table 2 Reverse mutation with bacteria of CM Glucan J (Main Test) With (+) or Test substance Number of revertants (number of colonies/plate) without ( ) close Base pair substitution type Frameshift type S9 Mix (pg/plate) TAIOO TA1535 WP2uvrA TA98 TA1 537 Negative control 128 (125) 8 (9) 17 (16) 46 (53) 8 (9) ( (8) 18 (18) 60 (51) 7 (7) S9Mix( ) (133) 10 (10) 13 (16) 56 (59) 5 (6) (148) 12 (10) 18 (16) 63 (62) 6 (7) (144) 8 (13) 14 (17) 71 (70) 7 (7) (128) 13 (16) 14 (14) 47 (52) 9 (9) Negative control 111 (111) 12 (10) 13 (17) 40 (42) 11 (14) (109) 12 (14) 20 (21) 56 (53) 7 (9) S9Mix(+) (126) 12 (11) 25 (22) 66 (59) 9 (9) (124) 9 (9) 13 (17) 44 (43) 14 (11) (111) 14 (10) 20 (20) 56 (50) 12 (10) (110) 11 (13) 16 (19) 39 (45) 11 (10) Positive Name NaN 3 NaN 3 ENNG 2 NF 9-M control not Dose requiring (ii g/plate) S9 Mix Number of colonies/plate 420 (416) 210 (208) 682 (643) 323 (318) 390 (384> Positive Name 2 AA 2-4A 2 AA 2 A\ 2 AA control Dose requiring ( j.t g/plate) S9 Mix Number of colonies/plate 844 (856) 144 (149) 948 (846) 227 (231) 94 (103) 1 When inhibition is found against growth of the bacteria, mark the applicable value with an asterisk. 2.Fill the average number of colonies in each dose in the ( ). 3. Number of revertants Fill in the observed value and average value in order beginning with ow doses of the test substance. 4.When the precipitate is observed on the plate, mark the applicable dose of the test substance with + 5.ENNG: N-ethyl N nitro--n--nitrosoguanidine, 2 NF: 2-nitrofluorene 9 Ak 9 aminoacridine hydrochloride, 2 M: 2 aminoanthracene NaN3: sodium azide CIR Panel Book Page 213

219 250 Styph/murium TM 00 a) 0 a) i Z o S9Mix(-).- S9M1x(+) p Test substance concentration( jig/plate) 30 S.typhimurium TA1535 a> a. 20 o- S9MIx( ) O I S9MIx(+) Test substance concentration( jig/plate) 30 Ecoli WP2uvrA a) o > S9Mix( ). S9Mix(+) Test substance concentration(p g/plate) Figi Dose response curves of mutation of base pair substitution type Stypht murium TA100, TA1 535 and Ecoli WP2uvrA by CM Glucan J (Main test) CIR Panel Book Page 214

220 80 S.typhimur,um TA > 60 0, i :: 0 -- S9Mix(+) Test substance concentration( LI g/plate) 20 S.typhimurwm TA ) 0 0, 0) C ) -o E 2:...: Test substance concentration( LI g/plate) Fig.2 Dose response curves of mutation of frameshift type S.typhimuriurn TA98 and TA1537 by CM Glucan J (Main test) CIR Panel Book Page 215

221 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nshihonmachi, Nishi-ku, OSAKA. JAPAN. Tel, TeI, () Test Code No. 99 VIA STUDY REPORT Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method) Report issued: December 17, 1999 English translation issued: December 17, 1999 Director Life Science Laboratory D.,Ph.D. 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan 1 CIR Panel Book Page 216

222 LIFE SCIENCE LABORATORY NN TeI *J2 T 51 TeLO (fe) 9 Title Skin sensitization study of CM-Glucan J in guinea pigs (by maximization test method) Purpose As a part of safety evaluation for CM-Glucan J, skin sensitization study was performed using guinea pigs to examine whether or not the test substance has a skin sensitization potential. This study was conducted by reference to the methods for toxicity studies in Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.) and in accordance with the SOP of our Laboratory Sponsor C. Holstein Company, Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , Japan Contract Laboratory Head office: Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan Testing facility: The Second Chihaya-akasaka Laboratory, Life Science Laboratory 900, Aza-Chihayadani, Chihaya- akas aka-mura, IVlinamikawachi-gun, Osaka , Japan Testing Period From October 21 to December 17, 1999 Test Substance Name: CM-Glucan J (Sodium carboxymethyl betaglucan) Lot No.: 2 CIR Panel Book Page 217

223 LIFE SCIENCE LABORATORY 5-19, 2-chonie, Nishihonmachi, Nishi-ku, OSAKA. JAPAN. Te I **r2t51s TeIO (4) Structure: Aqueous carboxymethylated 1-1,3-glucan derived from cell walls of yeast (Saccharornyces cerevisiae), about 100,000 of molecular weight Purity: More 90% CM-Glucan Appearance: White granular powders with a tinge of amber color and with alcoholic odor Solubility: Water soluble Stability: Stable for more than 12 months 25 C Storage condition: Kept in a tightly closed container room temperature Supplied by: C. Holstein Company, Ltd. 3-7 Doshomachi 1-chome, Chuo-ku, Osaka , than J at at the Japan Positive Control Substance Name: 2,4-Dinitro- 1-chlorobenzene (DNCB) Lot No.: TPQ272 1 Appearance: Pale-yellow crystals Stability: Stable at room temperature (confirmed by the manufacturer) Storage condition: Stored at room Manufactured by: Wako Pure Chemical Industries, Ltd. 1-2 Doshomachi 3-chome, Osaka , temperature Chuoku, Japan Preparation of the Test Substance Solution and Positive Control Substance 3 CIR Panel Book Page 218

224 LIFE SCIENCE LABORATORY Tel *1T2 T51 9- Tel (-) Solution In order to determine the highest dose concentration of the test substance that does not cause the primary skin irritation, the test substance was applied to the skin of three guinea pigs at concentrations of 50, 10 and 2(wlv)% for 24 hours by closed patch. As the results, the primary skin irritation was observed at a concentration of 50(w/v)%. In this study, therefore, the test substance was used at a dose level of lo(w/v)% for the first and second induction and the challenge. The test substance was dissolved in distified water when used. The positive control (DNCB) was dissolved in olive oil at a concentration of 0.l(wlv)% when used. Animals Used Thirty female albino guinea pigs of Hartley strain, four weeks old, were purchased from Japan SLC, Inc. ( Koto-cho, Hamamatsu, Shizuoka ) on November 4, Their body weights were 264 to 298g at arrival. After 12 days of acclimation including quarantine, animals with healthy intact skin were used for the experiments. At grouping 24 animals were assigned to four groups of six animals each such that mean body weights were nearly equivalent in the groups. Their body weights were 299 to 345g at grouping. Animals were identified individually by marking of temporary animal numbers on right ears with black felt pen at arrival and of experimental animal numbers on left ears with red felt pen at grouping, and also by cageand rack-labeling. Environmental Conditions Animals were housed individually in stainless steel wire-meshed cages (185W >< 260D X 1 75H mm) and maintained in an animal facility at 20 to 25 C and 50 to 70% relative humidity with more than 10 changes of room air per hour on 8-hour light cycle ( hour). Every day the room and cage racks were cleaned and the floor was thsinfected with Neochlor Clean Shikoku Chemicals Corporation). Standard laboratory chow (RC4, Oriental Yeast Co., Ltd.) in feed baskets and tap water (Chihaya-akasaka mura water supply) in polycarbonate bottles were freely available during 4 CIR Panel Book Page 219

225 LIFE SCIENCE LA8ORATOY chornelshionmacni. Tel Tel *T2T51 9* () the experiment. Methods Skin sensitization study was performed according to the maximization test method using guinea because the test substance could be dissolved in water. Guinea pigs were divided into four groups of six animals each: the test substance-sensitized group, the substance-non-sensitized group, the positive control-sensitized group and the positive control-non-sensitized group. The dose volumes of the substance (l0(w/v)% solution) and the positive control DNCB, 0.l(w/v)% solution) were 0.1, 0.2 and 0.1 ml for the first induction (by injection), for the second induction (by topical application) and for the challenge, respectively. pigs, test intradermal test The substance-sensitized group: On the first day of experiment (the day of injection, day 0), the of each animal was removed from a 4 >< 6 cm on the shoulder by clipping short with an electric clipper and by shaving closely with an electric razor. As shown in chart, three of symmetrical injection (0.05 ml each) were given into this area (2 x 4 cm) simultaneously for first induction: 0.1 ml of equi-volume mixture of distified water and Freund s complete adjuvant (FCA), 0.1 ml of the test substance solution and 0.1 ml of equi-volume mixture of the substance and FCA. Six days after injection (day 6), the on the same area was clipped and shaved in a similar way. 0.5 g of lo(w/w)% sodium lauryl sulfate (SLS)-vaseline ointment was applied to the injection site by open patch to enhance sensitization by provoking a mild inflammatory reaction. After 24 hours (day 7), the ointment was removed with 70% ethanol. Seven days after injection (day 7), 0.2 ml of the test substance solution was topically applied to the same site for the second induction. The area was covered with a 2 X 4 cm paper and impermeable adhesive plaster, and secured with surgical tape (Dei micel, Johnson & Johnson Medical Inc.) for 48 hours. test hair pairs intradermal intradermal test intradermal area intradermal hair intradermal ifiter 5 CIR Panel Book Page 220

226 LIFE SCIENCE LABORATORY chome, Nishhonmachi, Nishi-ku, OSAKA, JAPAN, Tel Tel ({) *1ITr *&$1*cT2T 51 9 (A) The site of induction The first induction (by intradermal injection) 0.05 ml of equi-volume mixture of distified water and FCA 0.05 ml of the test substance solution 0.05 ml of equi-volume mixture of the test substance and FCA The second induction (by topical application) 0.2 ml of the test substance solution 24 hours after pretreatment with l0(w!w)% SLS (P: the test substance) (B) The site of challenge (by topical application) 0,1 ml of the test substance solution (Ch: the test substance) Twenty days after intradermal injection (day 20), the hair of each animal was removed from a 5 X 5 cm area on the flank by clipping and shaving. Twenty-one days after intradermal injection (day 21), 0.1 ml of the test substance solution was topically applied to the shaved site for the challenge. The area was covered with an adhesive plaster for patch (2.5 cm in diameter, Libatape Pharmaceutical Co., Ltd.) and secured with Dermicel for 24 hours. The positive control-sensitized group: Animals were treated with the same procedures as the test substancesensitized group using DNCB solution in place of the test substance solution. The test substance-non-sensitized and the positive control-non-sensitized groups: In the first and second induction, animals were treated with the same procedures as the corresponding sensitized groups using vehicle (distilled water or olive oil) in place of the test substance solution or the positive control solution, respectively. In the challenge, animals were treated with the same procedures as the corresponding sensitized groups using the test substance solution or the positive control solution. 6 CIR Panel Book Page 221

227 LIFE SCIENCE LABORATORY chornNishonmach, 9 Tel Tel (fe) Observation and evaluation: The skin reactions were evaluated at 24 and 48 hours after the 24-hour challenge according to the following evaluation eriteria 2. General conditions of animals were observed every day during the experiment. Their body weights were measured on the day of arrival, days (the day of second induction), 21 (the day of challenge) and 24 (the last day of O (the first day of experiment, the day of first induction), experiment) after intradermal injection. Photographs of the applied sites of the test substance were taken on the days of evaluation of skin reactions. 7 Evaluation of skin reaction (Scale for scoring) (1) Erythema formation (2) No erythema Very slight erythema Well-defined erythema Moderate to severe erythema Severe erythema (crimson red) to slight eschar formation injuries in depth) 4 Edema formation No edema Slight edema Moderate edema Severe edema, raised more than beyond area of exposure 1 mm and extending Sensitization rate Number of animals with positive reaction (%) = X 100 Number of tested animals Mean response = (1) + (2)] Number of tested animals Statistical analysis: Data on skin sensitization were analyzed among the groups by Wilcoxon s CIR Panel Book Page 222

228 LIFE SCIENCE LABORATORY chome, Nishhonmachi, Nishi-ku, OSAKA, JAPAN. Tel, Tel (4) 1 9 rank-sum test (Mann-Whitney s U-test). Results Skin sensitization study of CM-Glucan J was performed according to the maximization test method using guinea pigs. The following results were obtained. 1) General conditions No abnormal general conditions were observed in any animals of the groups except the skin reactions during the experimental period. 2) Body weight Individual and mean body weight changes of the groups during the experimental period are shown in Table 1 The mean body weight increase was almost similar among the groups during the experimental period. 3) Skin reactions Individual skin reactions and mean skin response (sensitization rate and mean response) of the groups after 24-hour challenge with the test substance or DNCB are shown in Tables 2 and 3. Summing-up of the mean skin response of the groups are shown in Table 4. Photographs of skin reactions in individual animals are shown in Photos 1 and 2. CM-Glucan J-sensitized and non-sensitized groups (Tables 2 and 4, Photo 1): Both in the test substance-sensitized and non-sensitized groups at 24 and 48 hours after 24-hour challenge with the test substance, no abnormal skin reactions were found in any animals. Therefore, the sensitization rate and mean response were 0% and 0, respectively, in these groups. Positive control DNCB)-sensitized and non-sensitized groups (Tables 3 and 4, Photo 2): In the DNCB-sensitized group at 24 hours after 24-hour challenge with DNCB, well-defined erythema was observed in three of six animals, moderate erythema in two, and severe erythema (with eschar formation) in one, and also slight edema was seen in five of six animals and moderate 8 CIR Panel Book Page 223

229 LIFE SCIENCE LABORATORY S l 9, 2-chome, Nshhonmachi, Nih-ku, OSAKA, JAPAN. TeI TeI () 5fRfi edema in one. At 48 hours after 24-hour challenge, well-defined erythema was observed in five of six animals and severe erythema (with eschar formation) in one, and also slight edema was seen in all animals. Moreover, scale was found in four of six animals. The sensitization rate was 100% at 24 and 48 hours after 24-hour challenge. The mean response was 3.83 and 3.33 at 24 and 48 hours after 24-hour challenge, respectively. Skin reactions in the DNCB-sensitized group were significantly different from those in the DNCB-non-sensitized group, the test substance-sensitized and non-sensitized groups P<0.05). In the DNCB-non-sensitized group at 24 and 48 hours after 24-hour challenge with DNCB, on the other hand, there were no changes observed in skin reactions in all animals. Thus, the sensitization rate and mean response were 0% and 0, respectively. Discussion and Conclusion In a preliminary study, primary skin irritation was examined by a topical application of CM-Glucan J to the skin of three guinea pigs at concentrations of 50, 10 and 2(wlv)%. The test substance caused a slight skin irritation at 50(wlv)%. Therefore, according to the guinea pig maximization test method, the skin sensitization study on CM-Glucan J was performed by application to six animals per group at a concentration of 10(w/v)% for the first and second induction and the challenge. There were no abnormal general conditions observed in all animals except the skin reactions during the experimental period. The mean body weight increase was almost similar among the groups during the experimental period. As for the skin reactions at 24 and 48 hours after 24-hour challenge with CM-Glucan J, no abnormal findings such as erythema and edema were found in the test substance-sensitized and non-sensitized groups. As for the skin reactions at 24 and 48 hours after 24-hour challenge with DNCB, the positive control, no abnormal findings were observed in the DNCB-non-sensitized group. I-Iowever, well-defined to severe erythema and slight to moderate edema were seen in all animals of the DNCB sensitized group, suggesting that DNCB causes an apparent skin sensitization. The observation shows that this test system for detecting the skin sensitization potential of the test substance is suitable. 9 CIR Panel Book Page 224

230 UFE SCIENCE LABORATORY chornNishthonmachi, TeI *T2T519 TeLO (4) In conclusion, CM-Glucan J is considered to have no skin sensitization potential in guinea pigs under our experimental conditions. Archives The protocol, raw data, documentation records and study report of this study are retained in the archival room of Life Science Laboratory after the completion of the study. References 1> B. Magnusson and A. M. Kligman: The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 2, (1969) 2) Y. Sato, Y. Katsumura, H. Ichikawa, T. Kobayashi and K. Nakajima: Development of contact photosensitization test in guinea pigs Adjuvant-strip method. Nishi-nippon Hifuka 42, (1980) (in Japanese) 3) Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.), Supervised by Evaluation and Registration Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Edited by Japanese Association for Standards, Yakuji Nippo Sha, Tokyo (1996) (in Japanese) 10 CIR Panel Book Page 225

231 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nshhonmachi, t Jishi-ku. OSAKA, JAPAN. Tel *T2 T TeI ]-1881 W) 519 Persons engaged in the study Animal care: Atsunobu Matsuo December 17, 1999 Administration and observation: cv Eiji Sakurai December 17, 1999 Atsunobu Matsuo December 17, 1999 ii CIR Panel Book Page 226

232 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishhonmachi, Nishi-ku. OSAKA. JAPAN. Tel a*T2T519 TeI (fk) Study director Atsunobu Matsuo December 17, 1999 Management Shigeru Morita, Ph. D. December 17, CIR Panel Book Page 227

233 LIFE SCIENCE LABORATORY 9. Nishi ku. OSAKA. JAPAN. Tel chome, Nshihonmachi, Table 1 TeI (fe) Body weight changes in guinea pigs during the experimental period in the skin_sensitization study of CM-Glucan J 9 Group Animal Body weight g) No. 0 day 7 days 21 days 24 days The test substance-sensitized group 1 (first day) (2nd induction) (challenge (last day) Mean ±SD ±16.1 ±27.7 ±15,5 ±16,2 The test substance-non-sensitized group Mean ±SD ±13.9 ±18.6 ±29.0 ±30,1 The positive control-sensitized group Mean ±SD ±11.9 ±26.0 ±29.8 ±33.0 The positive control-non-sensitized group Mean SD: Standard (leviation ±SD ±12.0 ±31.1 ±40.5 ±43,0 CIR Panel Book Page 228

234 LIFE SCIENCE LABORATORY chome, NishihonmachL Nishi-ku, OSAKA. JAPAN. Tel TeI *T2T519 (fe> Table 2 Skin reactions at 24 and 48 hours after 24-hour challenge with CM-Glucan J in the skin sensitization study of CM-Clucan J Animal Sensitized group Animal Non-sensitized group Nc). 24 hours 48 hours No. 24 hours 48 hours 1 Ery. Ecle. Ery. Ede. Ery. Ede. Ery. Edo Ii SR(%) MR Ery.: erythema, F...de.: edema SR: sensitization rate, ME,: mean response CIR Panel Book Page 229

235 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmach, Nishi-ku, OSAKA. JAPAN. Tel **T2T19 TeI (fit) Table 3 Skin reactions at 24 and 48 hours after 24-hour challenge with DNCB in the skin sensitization study of CM-Glucan J Animal Sensitized group Animal Non-sensitized group No. 24 hours 48 hours No. 24 hours 48 hours Ery. Ede. Ery. Edo. Ery. Ede. Ery. Edo # 1. 4# SR(%) MR 3.83* 333* 0 0 Ery.: erythema, Ede.: edema SR: sensitization rate, ME.: mean response #: with eschar, +: with scale *: Significantly different from the corresponding non-sensitized group at P < 0.05 CIR Panel Book Page 230

236 Distributed for Comment Only -- Do Not Cite or Quote LIFE SCIENCE LABORATORY chome, Nishihonmachi. Nishi-ku, OSAKA, JAPAN. TeI TeI () 9 Table 4 Summing-up of the sensitization rate and mean response at 24 and 48 hours after 24-hour chaflenge with CM-Glucan J or.dncb in the skin sensitization study of CM-Glucan J Group Sensitization rate (%) Mean response CM-Glucan 3 DNCB *: 24 hours 48 hours 24 hours 48 hours Sensitized group Non-sensitized group Sensitized group * 333* Non-sensitized group 0 0 Significantly different from the corresponding other groups at P< CIR Panel Book Page 231

237 Photo 1 Skin reactions at 24 and 48 hours after 24-hour challenge with CM-Glucan J in the skin sensitization study of CM-Glucan J in guinea pigs z At 24 hours At 48 hours, 4- : : At 24 hours. At 48 hours Test substance-sensitized group Test substance-non-sensitized group A 9.. r - I IS C, w 1 : 4. I. C k.:12. CIR Panel Book Page 232

238 ._. At At. - - Distributed for Comment Only -- Do Not Cite or Quote Photo 2 I. Skin reactions at 24 and 48 hours after 24-hour challenge with DNCB, the positive control, in the skin sensitization study of CM-Glucan in guinea pigs J Positive control-sensitized group 24 hours I o Positive control-non-sensitized group At 48 hours At 24 hours - r hours :;lt r, li -. llt.l. 20 c I a k Abt. IIW. 4 j lit.. e a a. m b. d a r 1b 4! a it. * 24-7 It- -ii 1.-,..,4l p. -r -- r.. i;. --- IL it: e. v -. : -,,a.a - I CIR Panel Book Page 233

239 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishilionmachi. Nishi-ku, OSAKA, JAPAN. Tel **T2T5*1 9 TeI (fe) Test Code No. 99-IA STUDY REPORT Single oral dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg) Report issued: November 25, 1999 English translation issued: November 25, 1999 Director M. D., Ph. D. Life Science Laboratory 5-19, 2-Chome, Nishthonmachi, Nishi-ku, Osaka , Japan 1 CIR Panel Book Page 234

240 LIFE SCIENCE LABORATORY 5-19, 2 -chome, Nishihonmachi, *&$I*WJ2 T Nishi-ku, OSAKA, JAPAN. Tet TeI (fe) Title Single oral dose toxicity study of CM-Glucan J in mice (at a dose level of 2000 mg/kg) Purpose As a part of safety evaluation for CM-Glucan, single oral dose toxicity study was performed to examine whether or not the test substance has adverse effects on mice by observing the animals for general conditions, behavior and signs of toxicity for 14 days after dosing by gavage. This study was conducted by reference to the methods for toxicity studies in Guidebook for applications for approval to manufacture cosmetics and quasi-drugs (3rd ed.), Supervised by Evaluation and Registration Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Edited by Japanese Association for Standards, Yakuji Nippo Sha, Tokyo (1996) (in Japanese) and in accordance with the SOP of our Laboratory. Sponsor C. Holstein Co., Ltd. 3-7 Doshomachi l-chome, Chuo-ku, Osaka , Japan Contract Laboratory Head office: Life Science Laboratory 5-19, 2-Chome, Nishihonmachi, Nishi-ku, Osaka , Japan Testing facility: The First Chihaya-akasaka Laboratory, Life Science Laboratory 1082, Aza-Chihaya, Chihaya-akasaka-rnura, Minamikawachi-gun, Osaka , Japan Testing Period 2 CIR Panel Book Page 235

241 LIFE SCIENCE LABORATORY 519, 2chorne. Nshihonmachi, TeI *FRJ2 T519 TeiO (i) From October 21 to November 25, 1999 Test Subsatance Name: CM-Glucan J (Sodium Carboxymethyl Betaglucan) Lot No.: Structure: Aqueous carboxymethylated /3-1,3-glucan derived from the cell walls of yeast (Saceharomyces cerevisiae, with molecular weight of about 100,000 Purity: More than 90% CM-Glucan J Appearance: White granular powders with a tinge of amber color and with alcoholic odor Solubiity: Water soluble Stability: Stable for more than 12 months at 25CC Storage condition: Kept in a tightly closed container at room temperature Supplied by: C. Holstein Co., Ltd. 3-7 Doshomachi l-chome, Chuo-ku, Osaka , Japan Vehicle Used and Preparation of the Test Substance Solution The test substance was dissolved in purified water to make a 20(w/v)% solution when used. Animals Four-week old 10 male and 10 female of ddy strain were purchased from 3 CIR Panel Book Page 236

242 LIFE SCIENCE LABORATORY 5-19,2 chornejsihonmachi. TeI *T2 T 519 TeI () Japan SLC, Inc. (their body weights at arrival: 19.9 to 21.1 g for males and 16.9 to 19.6 g for females). After 7 days of acclimation, 6 male and 6 female mice in good general conditions were used for the test at 5 weeks of age. Their body weights were 28.3 to 29.7 g for males and 24.6 to 25.9 g for females on the day of administration (after fasting). Animals were identified individually by tail-marking of temporary animal numbers with black felt pen at arrival and of experimental animal numbers with red felt pen at grouping, and also by cage- and rack-labeling. Environmental Conditions Mice were housed 6 animals per cage in stainless steel wire mesh cages (185W x 260D x 175H mm) and maintained in a barrier system animal room at a temperature of 22± 3cC and a relative humidity of 55 ± 10% with 16 changes or more of room air (all fresh air) per hour on a 12-hour light cycle (0700 to 1900 hour). Every day the room and cage racks were cleaned and the floor was thsinfected with Neochior Clean (Shikoku Chemicals Corporation). Animals had free access to standard laboratory chow (NMF, Oriental Yeast Co., Ltd.) in feed baskets and tap water from Chihaya-akasaka-mura water supply) in polycarbonate bottles except for food deprivation before and after dosing the test substance. Methods After 4 hours of food deprivation, mice (6 males and 6 females) were given the test substance by gavage at a single dose of 2000 mglkg as indicated by the sponsor to confirm the safety of the test substance by oral exposure route. Animals were examined for general conditions, behavior and signs of toxicity for 14 days after dosing. Their body weights were measured on 0 (the day of dosing), 1, 4, 7 and 14 days after dosing. All surviving animals were sacrificed under ether anesthesia at the end of the observation period and necropsied for a gross pathological examination of 4 CIR Panel Book Page 237

243 LIFE SCIENCE LABORATORY ceh,machi. TeIO TeLOS () 519 major organs/tissues. Results 1) Body weight changes Individual and mean body weight changes of the groups during the observation period are shown in Table 1. Mean body weights were increased uneventfully in males and females through 14 days of the observation period. 2) IViortality and general conditions Mortality of the groups during the observation period is shown in Table 2. Neither deaths nor abnormal findings in general conditions were found in males and females during the observation period. 3) Necropsy All surviving animals were sacrificed under ether anesthesia at the end of the observation period and their organs/tissues were examined inacroscopically. No gross abnormalities were observed in any animals. Discussion and Conclusion CM-Glucan J was given to male and female mice by gavage at a single dose of 2000 mg/kg as indicated by the sponsor. All animals of both sexes survived and did not show any abnormal general conditions and signs of toxicity through 14 days of the observation period. Their body weights were increased uneventfully during the observation period. In necropsy at the end of the observation period, no gross pathological changes were found in any animals. Oral LD5O value for CM-Glucan J was more than 2000 mg/kg for both male and female mice. Archives 5 CIR Panel Book Page 238

244 LIFE SCIENCE LABORATORY chome. Nishihonmach. T2T519 Nihi-ku, OSAKA, JAPAN. TeI B81 TI (fe) The protocol, raw data, documentation records and study report of this study are retained in the archival room of Life Science Laboratory (Room No. 126, 2 Fl. in Okazakibathi Bldg., 9-11 Utsubohonmachi 2-chorne, Nishi-ku, Osaka , Japan) after the completion of the study. 6 CIR Panel Book Page 239

245 LIFE SCIENCE LABORATORY chome, Nishihonmachi. Nishi-ku. OSAKA. JAPAN. Tel &IitX*T2 T51 9* TeLQ6-653l-1881 (ft) Persons engaged in the study Animal care: Atsunobu Matsuo November 24, 1999 Administration and observation: _i 7zc Eiji Sakurai November 24, 1999 Atsunobu Matsuo November 24, CIR Panel Book Page 240

246 LIFE SCIENCE LABORATORY 5-19, 2-chome, Nishihonmachi, Nishi-ku, OSAKA, JAPAN. Tel *T2T519 TeI () Study director Atsunobu Matsuo November 25, 1999 Management Shigeru Morita, Ph. D. November 25, CIR Panel Book Page 241

247 sl. Distributed for Comment Only -- Do Not Cite or Quote LIFE SCIENCE LABORATORY chome, Nshihonmachi, Nishi-ku, OSAKA, JAPAN. Te I TeI, (44) Table 1 Body weight changes in male and female mice of ddy strain during the observation period after a single oral dose of CM-Glucan J by gavage Sex Dose Animal Body weights after dosing (g) (mg/kg) Male 2000 Female 2000 SD: standard deviation N 0 day 1 day 4 days 7 days 14 days Ml M M M M M Mean ±SD ±0.51 ±0.88 ±1.11 ±1.08 ±1.63 Fl F F F , F FG Mean ±SD ±0.49 ±0.74 ±0.77 ±1.05 ±1.29 Table 2 Mortality of male and female mice during the observation period after a single oral dose of CM-Glucan J by gavage Sex Dose Cumulative mortality after dosing/animals used (mg/kg) 0 day 14 days IViale /6 > > 0/6 Female /6. 0/6 9 CIR Panel Book Page 242

248 JORF Actimart 1140, FAX eurofins ATS P IiI Distributed for Comment Only -- Do Not Cite or Quote Test report Provisoire Page n : du : 30/11/07 1/1 TEST REPORT These results concern only the sample tested in the laboratory which is defined here after. Except specific case, the sample will be kept in our premises during 2 months from the date above mentioned. MIBELLE AG COSMETICS BOLIMATTSTRASSE 5033 BUCHS/AG SUISSE I The hereby report is not aimed to gore a conformity to the present legislation. It only refers to qualitative and quantitative criteria which allow to declare the conforrnityto specihcations of the reference file when this one has been provided by the customer The copy of this report is only authorized by unabridged edition. ATS internal code: SUBJECT: OCULAR TOLERANCE TEST-OCULAR TOLERANCE TEST ACCORDING HET CAM METHOD Client: MIBELLE AG COSMETICS Quotation: DESCRIPTION OF THE SAMPLE TO BE TESTED: Sample n : Denomination: CM-GLUCAN P Brand: ECHANTILLON SocJJrn Ccivbzer.y ii - Iii.yil 3 el-cr Batch n : Pick up: Start of the analysis MIBELLE AG COSMETICS Pick up Date :31/ Registration date ELECTRONIC CHECKING OF THE REPORT MADE BY: Technical controller: Rapport non validé YOUR TECHN1CAL CONTACT: CECILE COLOMBAN - CeciIeCoIomban@Eurofins.com RESULTS TESTS Specifications Results In vitro Het Cam tolerance (5 to 9 products) - - A.29/1 1/96 Study code HC/025 Date of the study 27)11/2007 Concentration of the stock Irritation result Classification solution % 5% 1,3 Weakly irritant L (o Jo CM G ICci Eurofins ATS -POle dactivité daix-en-provence - - Rue Ampere - Aix-en-Provence Cedex 3- France TEL ) (0) W SIRET: Code APE 743 B CIR Panel Book Page 243

249 XANTHAN GUM 01A - Baby Shampoos 1 29 XANTHAN GUM 01B - Baby Lotions, Oils, Powder 11 5 XANTHAN GUM 01C - Other Baby Products 17 XANTHAN GUM 02A - Bath Oils, Tablets, and Salt XANTHAN GUM 02D - Other Bath Preparations 4 XANTHAN GUM 03A - Eyebrow Pencil XANTHAN GUM 03B - Eyeliner 59 XANTHAN GUM 03C - Eye Shadow 23 2,749 XANTHAN GUM 03D - Eye Lotion 53 XANTHAN GUM 03E - Eye Makeup Remover 3 XANTHAN GUM 03F - Mascara 54 XANTHAN GUM 03G - Other Eye Makeup Prepara 49 XANTHAN GUM 04C - Powders (dusting and talcu 4 XANTHAN GUM 04E - Other Fragrance Preparatio XANTHAN GUM 05A - Hair Conditioner XANTHAN GUM 05C - Hair Straighteners 22 XANTHAN GUM 05E - Rinses (non-coloring) 3 XANTHAN GUM 05F - Shampoos (non-coloring) 18 XANTHAN GUM 05G - Tonics, Dressings, and Oth 20 XANTHAN GUM 05I - Other Hair Preparations 21 XANTHAN GUM 06A - Hair Dyes and Colors (all ty XANTHAN GUM 06F - Hair Lighteners with Color 1 XANTHAN GUM 06H - Other Hair Coloring Prepar 1 XANTHAN GUM 07A - Blushers (all types) 8 XANTHAN GUM 07B - Face Powders 1 XANTHAN GUM 07C - Foundations 63 XANTHAN GUM 07D - Leg and Body Paints 7 XANTHAN GUM 07E - Lipstick 6 37 XANTHAN GUM 07F - Makeup Bases 24 XANTHAN GUM 07G - Rouges 2 XANTHAN GUM 07H - Makeup Fixatives 1 XANTHAN GUM 07I - Other Makeup Preparations 30 XANTHAN GUM 08B - Cuticle Softeners 2 8 XANTHAN GUM 08C - Nail Creams and Lotions 2 XANTHAN GUM 08E - Nail Polish and Enamel 1 XANTHAN GUM 08G - Other Manicuring Preparat 3 XANTHAN GUM 09A - Dentifrices 19 XANTHAN GUM 09B - Mouthwashes and Breath F 6 XANTHAN GUM 09C - Other Oral Hygiene Produc 6 XANTHAN GUM 10A - Bath Soaps and Detergents 61 XANTHAN GUM 10B - Deodorants (underarm) 1 XANTHAN GUM 10E - Other Personal Cleanliness 55 XANTHAN GUM 11A - Aftershave Lotion 35 XANTHAN GUM 11E - Shaving Cream 11 XANTHAN GUM 11G - Other Shaving Preparation 4 XANTHAN GUM 12A - Cleansing 204 XANTHAN GUM 12B - Depilatories 1 XANTHAN GUM 12C - Face and Neck (exc shave 441 XANTHAN GUM 12D - Body and Hand (exc shave 293 XANTHAN GUM 12E - Foot Powders and Sprays 7 XANTHAN GUM 12F - Moisturizing 706 XANTHAN GUM 12G - Night 102 XANTHAN GUM 12H - Paste Masks (mud packs) 103 XANTHAN GUM 12I - Skin Fresheners 14 2,409 XANTHAN GUM 12J - Other Skin Care Preps XANTHAN GUM 13A - Suntan Gels, Creams, and 21 7 XANTHAN GUM 13B - Indoor Tanning Preparation 65 XANTHAN GUM 13C - Other Suntan Preparations 11 2,964 DEHYDROXANTHAN GUM 03G - Other Eye Makeup Prepara 1 DEHYDROXANTHAN GUM 05F - Shampoos (non-coloring) 3 DEHYDROXANTHAN GUM 10A - Bath Soaps and Detergents 1 DEHYDROXANTHAN GUM 12A - Cleansing 5 DEHYDROXANTHAN GUM 12F - Moisturizing 1 CIR Panel Book Page 244

250 DEHYDROXANTHAN GUM 12G - Night 1 6 DEHYDROXANTHAN GUM 12J - Other Skin Care Preps 2 DEHYDROXANTHAN GUM 13B - Indoor Tanning Preparation 1 15 GELLAN GUM 03C - Eye Shadow 4 GELLAN GUM 03F - Mascara 1 GELLAN GUM 07A - Blushers (all types) 2 GELLAN GUM 07B - Face Powders 6 GELLAN GUM 07C - Foundations 12 GELLAN GUM 07E - Lipstick 1 GELLAN GUM 07F - Makeup Bases 2 GELLAN GUM 12A - Cleansing 1 GELLAN GUM 12D - Body and Hand (exc shave 1 GELLAN GUM 12F - Moisturizing 3 GELLAN GUM 12J - Other Skin Care Preps 1 34 SCLEROTIUM GUM 01B - Baby Lotions, Oils, Powder 4 SCLEROTIUM GUM 02D - Other Bath Preparations 1 SCLEROTIUM GUM 03D - Eye Lotion 9 25 SCLEROTIUM GUM 03E - Eye Makeup Remover SCLEROTIUM GUM 03F - Mascara 5 SCLEROTIUM GUM 03G - Other Eye Makeup Prepara 10 SCLEROTIUM GUM 04E - Other Fragrance Preparatio 9 13 SCLEROTIUM GUM 05A - Hair Conditioner 1 17 SCLEROTIUM GUM 05F - Shampoos (non-coloring) 2 SCLEROTIUM GUM 05G - Tonics, Dressings, and Oth 2 SCLEROTIUM GUM 05H - Wave Sets 1 SCLEROTIUM GUM 05I - Other Hair Preparations 11 SCLEROTIUM GUM 07C - Foundations 4 SCLEROTIUM GUM 07F - Makeup Bases 1 SCLEROTIUM GUM 08B - Cuticle Softeners 1 SCLEROTIUM GUM 10E - Other Personal Cleanliness 11 SCLEROTIUM GUM 11A - Aftershave Lotion 1 SCLEROTIUM GUM 11E - Shaving Cream 1 SCLEROTIUM GUM 11G - Other Shaving Preparation 1 SCLEROTIUM GUM 12A - Cleansing 7 SCLEROTIUM GUM 12C - Face and Neck (exc shave 23 SCLEROTIUM GUM 12D - Body and Hand (exc shave 5 SCLEROTIUM GUM 12F - Moisturizing 43 SCLEROTIUM GUM 12G - Night 8 SCLEROTIUM GUM 12H - Paste Masks (mud packs) 6 SCLEROTIUM GUM 12J - Other Skin Care Preps 10 SCLEROTIUM GUM 13A - Suntan Gels, Creams, and SCLEROTIUM GUM 13B - Indoor Tanning Preparation 2 31 SCLEROTIUM GUM 13C - Other Suntan Preparations RHIZOBIAN GUM 03D - Eye Lotion 1 RHIZOBIAN GUM 03G - Other Eye Makeup Prepara 1 RHIZOBIAN GUM 12C - Face and Neck (exc shave 1 RHIZOBIAN GUM 12I - Skin Fresheners 1 HYDROLYZED RHIZOBIAN GUM 12A - Cleansing 1 BIOSACCHARIDE GUM-1 02A - Bath Oils, Tablets, and Salt 1 BIOSACCHARIDE GUM-1 02B - Bubble Baths 1 BIOSACCHARIDE GUM-1 03D - Eye Lotion 8 25 BIOSACCHARIDE GUM-1 03E - Eye Makeup Remover 1 BIOSACCHARIDE GUM-1 03G - Other Eye Makeup Prepara BIOSACCHARIDE GUM-1 04E - Other Fragrance Preparatio 1 3 BIOSACCHARIDE GUM-1 05A - Hair Conditioner 8 18 CIR Panel Book Page 245

251 BIOSACCHARIDE GUM-1 05F - Shampoos (non-coloring) 4 BIOSACCHARIDE GUM-1 05G - Tonics, Dressings, and Oth 5 BIOSACCHARIDE GUM-1 05I - Other Hair Preparations 1 BIOSACCHARIDE GUM-1 06D - Hair Shampoos (coloring) 1 BIOSACCHARIDE GUM-1 07C - Foundations 8 BIOSACCHARIDE GUM-1 07H - Makeup Fixatives 2 BIOSACCHARIDE GUM-1 07I - Other Makeup Preparations 2 BIOSACCHARIDE GUM-1 10A - Bath Soaps and Detergents 1 BIOSACCHARIDE GUM-1 10E - Other Personal Cleanliness 1 BIOSACCHARIDE GUM-1 11A - Aftershave Lotion 2 BIOSACCHARIDE GUM-1 11G - Other Shaving Preparation 6 BIOSACCHARIDE GUM-1 12A - Cleansing 6 BIOSACCHARIDE GUM-1 12C - Face and Neck (exc shave 81 BIOSACCHARIDE GUM-1 12D - Body and Hand (exc shave 24 BIOSACCHARIDE GUM-1 12E - Foot Powders and Sprays 1 BIOSACCHARIDE GUM-1 12F - Moisturizing 79 BIOSACCHARIDE GUM-1 12G - Night 10 BIOSACCHARIDE GUM-1 12H - Paste Masks (mud packs) 8 BIOSACCHARIDE GUM-1 12I - Skin Fresheners 3 BIOSACCHARIDE GUM-1 12J - Other Skin Care Preps BIOSACCHARIDE GUM-1 13B - Indoor Tanning Preparation 1 36 BIOSACCHARIDE GUM-1 13C - Other Suntan Preparations BIOSACCHARIDE GUM-2 03G - Other Eye Makeup Prepara 1 BIOSACCHARIDE GUM-2 12A - Cleansing 1 BIOSACCHARIDE GUM-2 12C - Face and Neck (exc shave 5 BIOSACCHARIDE GUM-2 12D - Body and Hand (exc shave 1 BIOSACCHARIDE GUM-2 12F - Moisturizing 1 BIOSACCHARIDE GUM-2 12H - Paste Masks (mud packs) 3 12 BIOSACCHARIDE GUM-4 03C - Eye Shadow 1 BIOSACCHARIDE GUM-4 03D - Eye Lotion 2 BIOSACCHARIDE GUM-4 03G - Other Eye Makeup Prepara 4 BIOSACCHARIDE GUM-4 07C - Foundations 2 BIOSACCHARIDE GUM-4 12A - Cleansing 2 BIOSACCHARIDE GUM-4 12C - Face and Neck (exc shave 19 BIOSACCHARIDE GUM-4 12D - Body and Hand (exc shave 5 BIOSACCHARIDE GUM-4 12F - Moisturizing 4 BIOSACCHARIDE GUM-4 12G - Night 1 BIOSACCHARIDE GUM-4 12H - Paste Masks (mud packs) 2 BIOSACCHARIDE GUM-4 12J - Other Skin Care Preps 2 44 DEXTRAN 03E - Eye Makeup Remover 1 DEXTRAN 03G - Other Eye Makeup Prepara 1 DEXTRAN 12A - Cleansing 1 DEXTRAN 12C - Face and Neck (exc shave 16 DEXTRAN 12F - Moisturizing 1 DEXTRAN 12G - Night 7 DEXTRAN 12J - Other Skin Care Preps 3 DEXTRAN 13C - Other Suntan Preparations 1 31 SODIUM CARBOXYMETHYL DEXTRAN 12C - Face and Neck (exc shave 1 SODIUM CARBOXYMETHYL DEXTRAN 12D - Body and Hand (exc shave 1 SODIUM CARBOXYMETHYL DEXTRAN 12F - Moisturizing 1 SODIUM CARBOXYMETHYL DEXTRAN 12G - Night 1 DEXTRAN SULFATE 03D - Eye Lotion 2 DEXTRAN SULFATE 03G - Other Eye Makeup Prepara 6 DEXTRAN SULFATE 07I - Other Makeup Preparations 2 CIR Panel Book Page 246

252 SODIUM DEXTRAN SULFATE 03D - Eye Lotion 1 SODIUM DEXTRAN SULFATE 12C - Face and Neck (exc shave 2 SODIUM DEXTRAN SULFATE 12J - Other Skin Care Preps 2 BETA-GLUCAN 01A - Baby Shampoos 2 BETA-GLUCAN 01B - Baby Lotions, Oils, Powder BETA-GLUCAN 01C - Other Baby Products 1 BETA-GLUCAN 03C - Eye Shadow 1 8 BETA-GLUCAN 03D - Eye Lotion 4 BETA-GLUCAN 03E - Eye Makeup Remover 1 BETA-GLUCAN 03G - Other Eye Makeup Prepara 2 BETA-GLUCAN 05A - Hair Conditioner 4 BETA-GLUCAN 05F - Shampoos (non-coloring) 6 BETA-GLUCAN 07B - Face Powders 1 BETA-GLUCAN 07C - Foundations 1 BETA-GLUCAN 07E - Lipstick 1 BETA-GLUCAN 07F - Makeup Bases 2 BETA-GLUCAN 09A - Dentifrices 1 BETA-GLUCAN 10A - Bath Soaps and Detergents 6 9 BETA-GLUCAN 10E - Other Personal Cleanliness 1 BETA-GLUCAN 11E - Shaving Cream 3 BETA-GLUCAN 12A - Cleansing 12 BETA-GLUCAN 12C - Face and Neck (exc shave 20 BETA-GLUCAN 12D - Body and Hand (exc shave 3 BETA-GLUCAN 12F - Moisturizing 28 BETA-GLUCAN 12G - Night 3 BETA-GLUCAN 12H - Paste Masks (mud packs) 2 78 BETA-GLUCAN 12I - Skin Fresheners 2 38 BETA-GLUCAN 12J - Other Skin Care Preps SODIUM CARBOXYMETHYL BETA-GLUCA03F - Mascara 1 SODIUM CARBOXYMETHYL BETA-GLUCA03G - Other Eye Makeup Prepara 1 SODIUM CARBOXYMETHYL BETA-GLUCA12A - Cleansing 7 SODIUM CARBOXYMETHYL BETA-GLUCA12C - Face and Neck (exc shave 28 SODIUM CARBOXYMETHYL BETA-GLUCA12D - Body and Hand (exc shave 2 SODIUM CARBOXYMETHYL BETA-GLUCA12F - Moisturizing 10 SODIUM CARBOXYMETHYL BETA-GLUCA12G - Night 1 SODIUM CARBOXYMETHYL BETA-GLUCA12H - Paste Masks (mud packs) 4 SODIUM CARBOXYMETHYL BETA-GLUCA12J - Other Skin Care Preps 2 56 PULLULAN 03D - Eye Lotion 1 PULLULAN 03G - Other Eye Makeup Prepara 6 PULLULAN 05G - Tonics, Dressings, and Oth 3 PULLULAN 07F - Makeup Bases 1 PULLULAN 09B - Mouthwashes and Breath F 4 PULLULAN 09C - Other Oral Hygiene Produc 1 PULLULAN 12C - Face and Neck (exc shave 8 PULLULAN 12D - Body and Hand (exc shave 5 PULLULAN 12F - Moisturizing 6 PULLULAN 12H - Paste Masks (mud packs) 1 PULLULAN 12J - Other Skin Care Preps 4 40 SCHIZOPHYLLAN 12C - Face and Neck (exc shave 2 SCHIZOPHYLLAN 12F - Moisturizing 1 SCHIZOPHYLLAN 13A - Suntan Gels, Creams, and 1 ALCALIGENES POLYSACCHARIDES 03D - Eye Lotion 2 ALCALIGENES POLYSACCHARIDES 07F - Makeup Bases 1 ALCALIGENES POLYSACCHARIDES 12A - Cleansing 2 CIR Panel Book Page 247

253 ALCALIGENES POLYSACCHARIDES 12C - Face and Neck (exc shave 1 ALCALIGENES POLYSACCHARIDES 12F - Moisturizing 4 ALCALIGENES POLYSACCHARIDES 12J - Other Skin Care Preps 1 CIR Panel Book Page 248

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