GREEN BOOK 4 Alkyl Benzoates CIR EXPERT PANEL MEETING

Home , Propyl benzoate

GREEN BOOK 4

Alkyl Benzoates

CIR EXPERT PANEL MEETING AUGUST 30-31, 2010

July 30, 2010

MEMORANDUM

To: CIR Expert Panel and Liaisons

From: Lillian C. Becker, M.S. Scientific Analyst and Writer

Subject: Draft Report for C12-15 Alkyl Benzoate and related Alkyl Benzoates

The Cosmetic Ingredient Review (CIR) announced the Scientific Literature Review (SLR) for alkyl benzoates in June, 2010.

C12-15 alkyl benzoate is the lead ingredient of this safety assessment. Related alkyl benzoate ingredients are included.

CIR has been informed that a comprehensive dossier on the C12-15 alkyl benzoates being prepared for the European REACH program will be completed and provided to CIR in late September or early October.

The Panel should review the Draft Report and decide: 1) if it is reasonable to include the other listed ingredients with C12-15 alkyl benzoate in this report and 2) whether any additional data are needed in order to reach a safety conclusion for C12-15 alkyl benzoates and the related ingredients. If no additional data are required, then the Panel may issue a Tentative Report. Alternatively, the Panel may choose to table the report to await the receipt of the dossier mentioned above.

CIR Panel Book Page 1

CIR Panel Book Page 2 History of Alkyl Benzoates

June, 2010 – SLR issued.

August, 2010 -

CIR Panel Book Page 3 Search Strategy for Benzoates

EXPORATORY SEARCH:

PUBMED: “alkyl benzoate” – 7 hits, 1 useful; CAS No. – 0 hits. Internet (Dogpile) – “alkyl benzoate” ‐ 1 MSDS

FULL SEARCH:

PUBMED: “lauryl alcohol” – 53 hits, 6 ordered. Learned that Valerie was doing this ingredient. “tridecyl alcohol” – 0 hits. CAS No. – 0 and 19 hits. 1 useful. “Amyl benzoate” ‐0 hits. CAS no – no hits. “benhyl benzoate” – 0 hits. CAS no – no hits. “Butyl Benzoate” – 9 hits, 1 useful. CAS no. – no hits. “Butyloctyl Benzoate” – 0 hits. No CAS no. “Ethyl Benzoate” – 44 hits, 4 useful. “Ethylhexyl Benzoate” – no hits. “Hexyldecyl Benzoate” – no hits. “Isobutyl Benzoate” – not hits. CAS no. – no hits. “939‐48‐0” OR “34364‐24‐4” OR “Lauryl/Myristyl Benzoate” OR “112‐53‐8” OR “Octyldodecyl Benzoate” OR ”2315‐68‐6” OR “10578‐34‐4” – 224 hits, 19 useful

TOXNET: 68411‐27‐8 – 0 hits; 112‐53‐8 – 253 hits, 47 useful; 112‐70‐9 – 33 hits, 2 useful; 26248‐42‐0 ‐ 10 hits, 4 useful; 629‐76‐5 – 15 hits, 1 useful (already have); 2049‐96‐9 – 6 hits, 0 useful; “amyl alcohol” ‐ 798 hits, 86 + 64 hits so far; 103403‐38‐9 – no hits; 136‐60‐7 ‐ Butyloctyl Benzoate – no hits; butyloctyl alcohol – no hits; C16‐17 Alkyl Benzoate – no hits; palmyl alcohol – no hits; heptadecyl alcohol – no hits; 93‐89‐0 – 56 hits, 64‐17‐5 alcohol – 50000 hits, did not explore yet; Hexyldecyl Benzoate – no hits; Hexyldecyl Benzoate – No hits; 120‐50‐3 – 3 hits, 0 useful; 939‐48‐0 – 2 hits, 0 useful; 34364‐24‐4 – no hits, Isopropyl Benzoate – No hits; Isostearyl Benzoate – no hits; Lauryl/Myristyl Benzoate ‐ no hits; 112‐ 53‐8 [print toxnet] – 248 hits, 112‐53‐8 – 248 hits, 61 useful; 93‐58‐3 – 135 hits, 14 useful; Octyldodecyl Benzoate – no hits; Octyldodecyl Alcohol – no hits; 2315‐68‐6 – 7 hits, 2 useful; 10578‐34‐4 – no hits.

EPA – HPV – One relevant report that included methyl benzoate. Data added to report under original citations.

CIR Panel Book Page 4 Report

Draft Report

Alkyl Benzoates as used in Cosmetics

August 30, 2010

The 2010 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is F. Alan Andersen, Ph.D. This report was prepared by Lillian C. Becker, Scientific Analyst/Writer.

CIR Panel Book Page 5

TABLE OF CONTENTS

TABLE OF CONTENTS ...... iii INTRODUCTION ...... 1 CHEMISTRY ...... 1 Definition and Structure ...... 1 Physical and Chemical Properties ...... 1 Manufacture and Production ...... 1 Impurities ...... 2 Analytical Methods ...... 2 USE ...... 2 Cosmetic ...... 2 Non-Cosmetic ...... 2 GENERAL BIOLOGY ...... 2 Absorption, Distribution, Metabolism, and Excretion ...... 2 Growth Inhibition ...... 3 TOXICOLOGY ...... 3 Acute Toxicity ...... 3 Methyl Benzoate ...... 3 Ethyl Benzoate ...... 3 Isopropyl Benzoate ...... 3 Isobutyl Benzoate ...... 3 Short-Term Toxicity ...... 3 Benzoic Acid and Sodium Benzoate ...... 3 Subchronic Toxicity ...... 3 Benzoic Acid ...... 3 Chronic Toxicity ...... 3 Sodium Benzoate ...... 3 Ocular/Mucosal Irritation ...... 3 Isostearyl Benzoate ...... 3 Dermal Irritation ...... 3 Methyl Benzoate ...... 3 Ethyl Benzoate ...... 4 Propyl Benzoate ...... 4 Butyl Benzoate ...... 4 Dermal Sensitization ...... 4 Reproductive and Developmental Toxicity ...... 4 iii

CIR Panel Book Page 6 Sodium Benzoate ...... 4 Benzoic Acid ...... 4 GENOTOXICITY ...... 4 Methyl Benzoate ...... 4 Benzoic Acid and Sodium Benzoate ...... 4 CARCINOGENICITY ...... 5 Methyl Benzoate ...... 5 Benzoic Acid ...... 5 CLINICAL ASSESSMENT OF SAFETY ...... 5 Absorption, Distribution, Metabolism, and Excretion ...... 5 Toxicity ...... 5 Benzoic Acid ...... 5 Dermal Irritation ...... 5 C12-15 Alkyl Benzoate ...... 5 Benzoic Acid ...... 5 Dermal Sensitization ...... 5 C12-15 Alkyl Benzoate ...... 5 Isostearyl Benzoate ...... 5 Octyldodecyl Benzoate ...... 5 Benzoic Acid ...... 6 Phototoxicity ...... 6 Benzoic Acid ...... 6 SUMMARY ...... 6 TABLES AND FIGURES ...... 7 REFERENCES ...... 13

CIR Panel Book Page 7 INTRODUCTION This is a safety assessment of alkyl benzoate esters that are used in cosmetics. The ingredients included in this literature review are: methyl benzoate, ethyl benzoate, propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl benzoate, C12-15 alkyl benzoate, C16-17 alkyl benzoate, stearyl benzoate, behenyl benzoate, isopropyl benzoate, isobutyl benzoate, isostearyl benzoate, ethylhexyl benzoate, butyloctyl benzoate, hexyldecyl benzoate, and octyldodecyl benzoate. The alkyl benzoate ingredients are esters of benzoic acid and a corresponding alcohol, with the shorter chain alkyl benzoates (methyl, ethyl, propyl, isopropyl, butyl, isobutyl and amyl benzoate) ranging in MW from 136 to 192 and the longer chain alkyl acetates (lauryl/myristyl, C12-15 alkyl, C16-17 alkyl, stearyl, isostearyl, behenyl, ethylhexyl, butyloctyl, hexyldecyl, and octyldodecyl benzoate) ranging in MW from 234 to 431. It is unclear as to whether the alkyl benzoates in this report penetrate the skin. If they do, these compounds may be metabolized in the skin into benzoic acid and the parent alcohol. The conclusions from prior assessments of benzoic acid, sodium benzoate, and the parent alcohols (methyl alcohol, ethyl alcohol, butyl alcohol, myristyl alcohol, behenyl alcohol, isostearyl alcohol) by the Cosmetic Ingredient Review (CIR) Expert Panel are listed below. These conclusions may be relevant to this report if significant penetration and metabolism do occur. Benzoic acid and sodium benzoate - “…safe for use in cosmetic formulations at concentrations up to 5%. The available data are insufficient to support the safety of [benzoic acid and sodium benzoate] in cosmetic products in which a primary route of exposure is inhalation”.1 Methyl alcohol - safe for use as a denaturant in ethyl alcohol for cosmetic products, with qualifications. The Panel has not stated that methyl alcohol is safe or unsafe as a solvent.2 Ethyl alcohol – (as “Alcohol Denat.” with methyl alcohol) - safe in the present practices of use and concentration.3 Butyl alcohol - safe as a cosmetic ingredient in the present practices of use.4 In 2005, the panel looked at new data and the safety conclusion in the report was confirmed. Myristyl alcohol - safe as a cosmetic ingredient in the present practices of use.5 Cetyl alcohol - safe as a cosmetic ingredient in the present practices of use.6 In 2005, the panel looked at new data and the conclusion in the report was confirmed. Stearyl alcohol - safe as currently used in cosmetics.7 In 2006, the panel looked at new data and the conclusion in the report was confirmed. Isostearyl alcohol - safe as cosmetic ingredients in the present practices of use. 6 In 2005, the panel looked at new data and the conclusion in the report was confirmed. Behenyl alcohol - safe as a cosmetic ingredient in the present practices of use.5 In 2005, the panel looked at new data and the conclusion in the current report was confirmed. Propyl alcohol and isopropyl alcohol are concurrently under review by the CIR expert Panel in the report titled Methyl Acetate. Summaries of the data from the benzoic acid and sodium benzoate safety assessment are included in this safety assessment. The potential metabolites of ethylhexyl benzoate, butyloctyl benzoate, hexyldecyl benzoate, isobutyl benzoate, amyl benzoate, pentadecyl benzoate, heptadecyl benzoate, and octyldecyl benzoate are not current cosmetic ingredients in the dictionary, thus have not been reviewed by CIR.

CHEMISTRY Definition and Structure Alkyl benzoates are mostly used as skin-conditioning agents, preservatives, solvents, and plasticizers. The CAS numbers, definitions, functions, as well as technical and trade names of the ingredients under review are presented in Table 1. Structures and potential metabolic pathways of these ingredients are presented in Figures 1 and 2. Physical and Chemical Properties The shorter chain alkyl benzoate esters are colorless liquids. Viscosity generally increases as the molecular mass (chain length) increases.8 The physical and chemical properties of the benzoates are shown in Table 2. At room temperature and pressure, methyl benzoate, ethyl benzoate, butyl benzoate, and isobutyl benzoate are fragrant, colorless oils, and are insoluble in water.9 Manufacture and Production In general, the alkyl benzoates can be produced industrially via esterification of benzoic acid.8 The manufacture of butyl benzoate, for example, is traditionally accomplished via an acid catalyzed (e.g., sulfuric acid) reactive distillation process between benzoic acid and butyl alcohol (Figure 3).10 Methanol and ethanol are normally obtained via fermentation of natural sources. However, some alcohols with chains longer than ethanol are often produced synthetically. An important process for producing C3- C22 industrial alcohols involves a process known as oxo-synthesis (a process for the production of aldehydes which occurs by the reaction of olefins (which can be natural or petroleum sourced) with carbon monoxide, hydrogen and a catalyst [typically cobalt based]), followed by hydrogenation of the aldehyde products, to form the alcohols.11 Recently, a biocatalytic process developed 1

CIR Panel Book Page 8 specifically for the manufacture of esters for use in the formulation of cosmetic and personal care ingredients (i.e. for producing cosmetic grade esters) was developed.12 Impurities The manufacturing processes of the benzoic esters are typically high yielding (>90%) and easily purified (e.g., by distillation). Therefore, the starting materials and water, at least, may be expected to be present in preparations of these esters as the major impurities.8 For example, methyl benzoate is available with a minimum of 99.2% purity, wherein the major contaminants are water (<0.1%) and benzoic acid (<0.02%).13 Analytical Methods The benzoic esters can be analyzed using gas chromatography/mass spectroscopy (GCMS), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectroscopy and infrared (IR) spectroscopy.8,11,14

USE Cosmetic According to the Voluntary Cosmetic Registration Program (VCRP) administered by the Food and Drug Administration (FDA), the total number of uses of C12-15 alkyl benzoate was 971 (858 leave-on and 113 rinse-off products).15 A survey conducted by the Personal Care Products Council (Council) found that C12-15 alkyl benzoate was used at 0.0008% - 35% (tonics, dressings, and other hair grooming aids) in leave-on products and 0.0008% - 50% (paste masks [mud packs]) in rinse-off products.16 There were 2 uses reported of C16-17 alkyl benzoates at 0.7% (bath soaps and detergents). Stearyl benzoate at 2% was reported to have 3 uses (face and neck creams, lotions, and powders). While there were no uses reported by VCRP, the Council reported behenyl benzoate use at 0.04% (lipstick), ethyl benzoate use at 0.0008% - 0.01% (foot powders and sprays), isobutyl benzoate use at 0.01% (perfumes), isostearyl benzoate use at 1% (body and hand creams, lotions, and powders), methyl benzoate use at 0.0005% – 0.3% (perfumes), and octyldodecyl benzoate at 3% - 4% (shaving cream) The uses and concentrations are provided in Table 3. No uses or concentrations of use were reported for propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl benzoate, isopropyl benzoate, ethylhexyl benzoate, butyloctyl benzoate, and hexyldecyl benzoate. These ingredients are used in aerosol/spray products, and effects on the lungs that may be induced by aerosolized products containing these ingredients may be of concern when these ingredients are vaporized. In the EU, methyl benzoate, ethyl benzoate, propyl benzoate and butyl benzoate may be used as preservatives in cosmetics up to 0.5% (acid).17 Non-Cosmetic Alkyl benzoate esters are typically used as solvents in paints, lacquers and coatings, and as intermediates in various chemistry processes.8 Methyl benzoate is used in flavoring and perfumery, and as a solvent in resins.9 Ethyl benzoate is used in flavoring and perfumery, and as a solvent in lacquers and resins.9 Butyl benzoate is used as a solvent for cellulose ether, as a plasticizer, perfume ingredient and for dyeing of textiles.9 Isobutyl benzoate is used in flavoring and perfumery.9

GENERAL BIOLOGY Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to benzyl glucuronide and benzoyl CoA.18 Benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted in the urine. Dermally applied benzoic acid is excreted in the urine within 24 h. Absorption, Distribution, Metabolism, and Excretion There are limited data found on the absorption, distribution, metabolism, and excretion of any of the alkyl benzoates in this safety assessment. However, data on benzyl alcohol show that it is broken down into benzoic acid by simple oxidation.1 Orally consumed benzoic acid is absorbed from the gastrointestinal tract and conjugated with glycine in the liver. The resulting hippuric acid is excreted in the urine (75% - 100% within 6 h). Dermally applied benzoic acid is also excreted in the urine within 24 h. Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to benzyl glucuronide and benzoyl CoA.18 The benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted in the urine. In general, esters can be hydrolyzed to the parent alcohol and acid.8 The rate of this reaction can be increased by raising the temperature and decreased by lowering pH. Secondary and tertiary esters are hydrolyzed more slowly than primary esters. Enzymatic hydrolysis occurs via enzymes called esterases. These enzymes are present in the respiratory tract, skin, blood and gastrointestinal tract.19,20 In general, alcohols can be metabolized by alcohol dehydrogenases to aldehydes or ketones. The aldehydes may be further metabolized by aldehyde dehydrogenases to the corresponding acids. Low molecular weight alkyl esters readily penetrate the skin and mucous membranes. However, benzoic esters are not absorbed through the skin as rapidly as alkyl esters.21 If alkyl benzoates are absorbed and metabolized, the resulting alcohols can be oxidized via alcohol dehydrogenases to produce the corresponding aldehyde or ketone. As noted above, these aldehydes can then be further oxidized via aldehyde dehydrogenases to the corresponding acids.

CIR Panel Book Page 9 Growth Inhibition

In a protein count assay of methyl benzoate, the EC50 (50% of the concentration of maximum effect) was 1506.58 (C.I. 1349.27 - 168.22) mM, the NI50 (the concentration that reduced the uptake of neutral red by 50%) was 683.30 (466.46 – 1000.91) mM in a neutral red uptake assay, and the (the concentration that inhibited growth by 50%) ID50 was 987.19 (605.15-1610.43) mM in a growth inhibition assay using HeLa cells.22 Methyl benzoate (2.5 and 5.0 mg/ml) inhibited mycelia growth and aflatoxin release by Aspergillis flavus and A. parasiticus.23

TOXICOLOGY Acute Toxicity

The oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for rats, and 3000-3330 mg/kg for mice. The oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats. The oral LD50 of isopropyl benzoate was 3730 mg/kg and 3685 mg/kg for isobutyl benzoate for rats. The dermal LD50 of methyl benzoate was ≥ 2000 mg/kg for rabbits. Methyl Benzoate

The reported oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for rats, and 3000-3330 mg/kg for mice.24-27 28 The dermal LD50 of methyl benzoate was ≥ 2000 mg/kg for New Zealand white rabbits (n = 5). There was fecal staining for 3 days after treatment. Irritation was observed at the application site. There was weight loss for 1 – 7 days after treatment. There were no gross findings at necropsy. There were no mortalities. Ethyl Benzoate 24,27 The reported oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats. Isopropyl Benzoate 29 The reported oral LD50 of isopropyl benzoate was 3730 mg/kg for rats. Isobutyl Benzoate 18 The reported oral LD50 of isobutyl benzoate was 3685 mg/kg for rats. Short-Term Toxicity Benzoic acid and sodium benzoate were toxic to rats and mice at oral doses > 1%. Benzoic Acid and Sodium Benzoate In multiple-dose oral toxicity studies on rats and mice, decreased feed consumption, depressed growth, and toxic effects were observed at doses > 1% benzoic acid or sodium benzoate.1 A neurobiological study on rats and mice was negative. Subchronic Toxicity Benzoic acid was toxic to mice at oral doses of 80 mg/kg/d. Benzoic Acid Cross bred white mice (n = 100) were orally administered benzoic acid at 80 mg/kg/d for 3 months.30 The treated group had decreased weight gain. Mortality was increased (68% vs. 60 % in the control group). Chronic Toxicity Sodium benzoate at 880 mg/kg/d incorporated into the feed of rats for 18 – 24 months was not toxic. Sodium Benzoate Sodium benzoate (0, 1%, or 2%; 735 or 880 mg/kg/d) was incorporated into the feed of Fischer 344 rats (n = 102; 50 males, 52 females) for 18 – 24 months.31 There were no differences in mortality between groups. Necropsies were unremarkable. Ocular/Mucosal Irritation Isostearyl benzoate was rated as a slight ocular irritant in two in vitro tests. Isostearyl Benzoate In an ocular irritation assessment of a body lotion containing isostearyl benzoate (0.95%) using neutral red release (NRR) assay, the hen’s egg test on the chorio-allantoic membrane (HET-CAM) assay, and the reconstituted human epithelial culture (REC) assay, the authors rated the lotion as slightly irritating.32 Dermal Irritation Methyl benzoate, ethyl benzoate, propyl benzoate, and butyl benzoate at 100% are dermally irritating to rabbits. Methyl Benzoate Methyl benzoate was applied to the clipped dorsum (100%; 0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6 days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reactions and dermal edema beginning on day 2 followed by dermal hemorrhages, desquamated crust, and thickening of the malpighian stratum beginning on day 5. On the inner ear, there was slight hyperkeratosis at day 6.

CIR Panel Book Page 10 Ethyl Benzoate Ethyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6 days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there was marked cellular changes, edema, desquamated crusts, and thickening of the malpighian stratum beginning on day 1. On the inner ear, there were slight cellular reaction, no edema or hemorrhages, no necrosis, slight to marked desquamated crusts, marked thickening of malpighian stratum, hyperkeratosis, and slight hyperplasia of sebaceous glands beginning day 1. Propyl Benzoate Propyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6 days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reactions, necrosis, thickening of the malpighian stratum beginning on day 1 followed by dermal hemorrhages, desquamated crusts beginning on days 3 or 4.. On the inner ear, there were slight cellular reactions, necrosis, and moderate thickening of the malpighian stratum beginning on day 1 or 3. Butyl Benzoate Butyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6 days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reaction, necrosis, and slight detachment of the dermo-epidermis beginning on day 1 followed by desquamated crusts and thickening of the malpighian stratum beginning on day 3. On the inner ear, there were slight cellular reaction, necrosis beginning on day 1 and moderate desquamed crusts and hyperplasia sebaceous glands on day 2 or 3. Dermal Sensitization There were no animal dermal sensitization data discovered on alkyl benzoates in this safety assessment. Reproductive and Developmental Toxicity Studies on sodium benzoate and benzoic acid did not show reproductive or developmental toxicity. Where effects of the fetus were noted, they occurred at maternally toxic concentrations (> 4% sodium benzoate in rats). Sodium Benzoate Sodium benzoate (0, 1%, 2%, 4%, or 8%; 0, 667, 1333, 1600, or 710 [sic] mg/kg/d) was orally administered in the feed of female Wistar rats (n = 27-30) during gestation.34 On day 20, 20-25 of each group were killed and necropsied. The rest were allowed to live through pregnancy and nurse for 3 or 8 weeks and then killed. Half of the pups were then killed at each of these times and necropsied. The 2 highest dose groups had an increase in the number of dead fetuses and resorbed embryos. The body weights of the viable pups were decreased, there was mild systemic edema observed. The number of fetal abnormalities was increased in a dose-dependent manner. The number of pups born was decreased, the number of perinatal deaths increased to 100%, lactation rate decreased, and survival rate decreased to 0 in the 2 highest dose groups. The effects on the fetus occurred only at maternally toxic concentrations of ≥ 4% sodium benzoate. Sodium benzoate (up to 5mg/egg) was injected twice into the air sac of fertilized chicken eggs at 0 and 96 h and 35 incubated to hatching. Surviving chicks were killed and necropsied. There were no teratogenic effects reported. The LD50 was 4.74 mg/egg. Female Wistar rats (n = 20) were orally administered sodium benzoate (0, 1.75, 8.0, or 175 mg/kg/d) during days 6 – 15 of gestation.36 On day 20 of gestation, the pups were delivered by Caesarean section. There were no differences in the types or amounts of abnormalities observed in any of the treatment groups compared to the control. The fetal and maternal NOAEL was 175 mg/kg. The study above was repeated with mice (n = 20), hamsters (n = 21, 22; gestation days 6 - 10), and rabbits (n = 10). Similar results were reported. Benzoic Acid In oral teratogencity studies, benzoic acid increased the number of resorptions at ≥ 30 mg/kg/d and increased the number of fetal malformations at > 600 mg/kg/d results in hamsters. Results were negative in 2 rat studies up to 500 mg/kg/d.1 Cross bred white mice (n = 25 males, 25 females) were orally administered benzoic acid (40 mg/kg/d) for 8 months before breeding.30 This was continued for 5 generations. The parental and F1 cohorts had increased mortality compared to controls after a 5-day 100% food restriction test. Otherwise, there were no effects on reproduction.

GENOTOXICITY In an Ames test, methyl benzoate was not genotoxic to S. typhimurium or E. coli. Benzoic acid and sodium benzoate were not genotoxic in several assays. Methyl Benzoate In an Ames test using S. typhimurium (TA97, TA98, TA100, TA1535, and TA1537), methyl benzoate (6666 µg/plate) was not mutagenic with or without metabolic activation.37 Methyl benzoate (dose not reported) was not found to be mutagenic using E. coli (Sd-4-73).38 Benzoic Acid and Sodium Benzoate Benzoic acid was negative in several Ames tests using Salmonella typhimurium (including TA98, TA100, TA1535, 4

CIR Panel Book Page 11 TA1537 and TA 1538) with and without metabolic activation.39-43 In one Ames test using S. typhimurium (TA98 and TA100), benzoic acid (0.1 mg/plate) and sodium benzoate (0.1 mg/plate) were genotoxic with activation.42 In a reverse mutation test, benzoic acid (5 mg/disc) was positive for genotoxicity. In a sister chromatid exchange assay using human lymphocytes, benzoic acid (0 – 2.0 mM) was not genotoxic with metabolic activation.40 In a chromosomal aberration test using Chinese hamster fibroblasts, benzoic acid (1.5 mg/ml) and sodium benzoate (2.0 mg/ml) had positive results without metabolic activation.46 Sodium benzoate was positive without metabolic activation and negative with metabolic activation in a reverse mutation assay using Bacillus subtilis.41 Benzoic acid (1.5 mg/ml) was positive in a chromosomal aberration test without metabolic activation. In a sister chromatid exchange assay using hamster lung fibroblasts, sodium benzoate was not clastogenic without metabolic activation.44

CARCINOGENICITY Orally administered methyl benzoate (80 mg/kg/d) to mice increased tumor growth compared to controls. Benzoic acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair dye. Methyl Benzoate In a 1970 review, it was reported that cross bred white mice (n = 100) orally administered methyl benzoate (80 mg/kg/d) had increased tumor growth compared to controls.30 Benzoic Acid Benzoic acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair dye.1

CLINICAL ASSESSMENT OF SAFETY Absorption, Distribution, Metabolism, and Excretion There were no human absorption, distribution, metabolism, and excretion data discovered for alkyl benzoates. Toxicity Benzoic Acid In clinical studies, toxic symptoms (metabolic acidosis, central neural depression, respiratory distress progressing to gasping respiration, hypotension, renal failure, and occasional seizures and intracranial hemorrhages) were observed following doses far exceeding the acceptable daily intake (ADI) established by the World Health Organization.1 Dermal Irritation C12-15 Alkyl benzoate was not irritating at 100%. In multiple clinical studies, the benzoates were recognized to produce non-immunologic contact uriticaria or non-immunologic immediate contact reactions, but it was not clear whether the reactions are histamine or prostaglandin mediated. C12-15 Alkyl Benzoate In an irritation study, C12-15 alkyl benzoate (0, 3%, 10%, 30, and 100% in vegetable oil) was applied to the backs of subjects (n = 21) under occlusion for 48 h.45 No signs of irritation were observed at 48 and 72 h. Benzoic Acid In multiple clinical studies, the benzoates were recognized to produce non-immunologic contact uriticaria or non- immunologic immediate contact reactions, but it was not clear whether the reactions are histamine or prostaglandin mediated.1 Dermal Sensitization In HIRPTs, C12-15 alkyl benzoate at 100%, isostearyl benzoate at 0.95%, and octyldodecyl benzoate at 0.4% were not sensitizing. In 4 studies, test for the sensitization of benzoic acid were negative. C12-15 Alkyl Benzoate A human repeated insult patch test (HRIPT; n = 101) was conducted on C12-15 alkyl benzoate (100%).46 There were no visible reactions to the test substance observed. Isostearyl Benzoate A HRIPT (n = 107) was conducted on a body lotion product containing isostearyl benzoate (0.95%) under semi- occlusion.47 Except for one subject, who also reacted to several other test substances on the shared panel, there were no visible reactions to the product containing isostearyl benzoate at 0.95%. Octyldodecyl Benzoate An HRIPT (n = 105) was conducted on a shaving cream product containing octyldodecyl benzoate (4%) under semi- occlusion.48 The product was diluted to a 10% aqueous solution. There were no visible reactions to the product containing octyldodecyl benzoate at 0.4%. 5

CIR Panel Book Page 12 Benzoic Acid In 4 studies, tests for the sensitization of benzoic acid were negative.1 Phototoxicity Benzoic Acid In several studies, phototoxicity and photosensitivity tests of benzoic acid were negative up to 0.2%.1

SUMMARY This is a safety assessment of alkyl benzoates that are used in cosmetics. Alkyl benzoates are mostly used as skin- conditioning agents, preservatives, solvents, and plasticizers. In general, the alkyl benzoates can be produced industrially via esterification of benzoic acid. The manufacturing processes of the benzoic esters are typically high yielding (>90%) and easily purified (e.g., by distillation). The esters, acids and alcohols can be analyzed using gas chromatography/mass spectroscopy (GCMS), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectroscopy and infrared (IR) spectroscopy. The total number of uses of C12-15 alkyl benzoate was 971 (858 leave-on and 113 rinse-off products) at concentrations up to 35% and 50% in leave-on and rinse-off products, respectively. The highest concentrations of use for C16-17 alkyl benzoates, stearyl benzoate, behenyl benzoate, ethyl benzoate, isobutyl benzoate, isostearyl benzoate, methyl benzoate, and octyldodecyl benzoate were reported to be from 0.01% to 4%. No uses or concentrations of use were reported for propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl benzoate, isopropyl benzoate, ethylhexyl benzoate, butyloctyl benzoate, and hexyldecyl benzoate. Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to benzoyl glucuronide and benzoyl CoA.18 The benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted in the urine. Dermally applied benzoic acid is also excreted in the urine within 24 h. There were no human absorption, distribution, metabolism, and excretion data discovered for alkyl benzoates. The oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for rats, and 3000-3330 mg/kg for mice. The oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats. The oral LD50 of isopropyl benzoate was 3730 mg/kg and 3685 mg/kg for isobutyl benzoate for rats. The dermal LD50 of methyl benzoate was ≥ 2000 mg/kg for rabbits. Benzoic acid and sodium benzoate were toxic to rats and mice at doses > 1% in short-tem oral studies . Benzoic acid was toxic to mice at 80 mg/kg/d in chronic oral studies. Sodium benzoate at 880 mg/kg/d incorporated into the feed of rats for 18 – 24 months was not toxic. Isostearyl benzoate was rated as a slight ocular irritant in two in vitro tests. Methyl benzoate, ethyl benzoate, propyl benzoate, and butyl benzoate at 100% are dermally irritating to rabbits.There were no animal dermal sensitization data discovered on alkyl benzoates in this safety assessment. Studies of sodium benzoic acid and sodium benzoate showed no reproductive or developmental toxicity. Effects noted were at a maternally toxic concentration of > 4% sodium benzoate. Methyl benzoate was not genotoxic to S. typhimurium or E. coli. Benzoic acid and sodium benzoate were not genotoxic in several assays. Orally administered methyl benzoate (80 mg/kg/d) to mice increased tumor growth compared to controls. Benzoic acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair dye. In humans, C12-15 Alkyl benzoate was not irritating at 100%. In multiple clinical studies, the benzoates were recognized to produce non-immunologic contact uriticaria or non-immunologic immediate contact reactions, but it was not clear whether the reactions are histamine or prostaglandin mediated. In HIRPTs, C12-15 alkyl benzoate at 100%, isostearyl benzoate at 0.95%, and octyldodecyl benzoate at 0.4% were not sensitizing. In 4 studies, tests for the sensitization of benzoic acid were negative.

CIR Panel Book Page 13 TABLES AND FIGURES

Table 1. Definitions, functions and structures of alkyl benzoate and alcohol ingredients in this safety assessment. Ingredient CAS No. Definition Function(s) Technical names Trade names Alkyl Benzoates Methyl Benzoate 93-58-3 Methyl benzoate is the Fragrance Benzoic Acid, Methyl Morflex Methyl Benzoate ester of methyl alcohol ingredient, skin- Ester; and benzoic acid that conditioning agent- Methyl conforms to the formula emollient, solvent Benzenecarboxylate in Figure 1. Methyl benzoate (RIFM) Ethyl Benzoate 93-89-0 Ethyl benzoate is the Fragrance Benzoic Acid, Ethyl - ester of ethyl alcohol and Ester; benzoic acid. Ethyl benzoate (RIFM) Propyl Benzoate 2315-68-6 Propyl benzoate is the Fragrance Benzoic Acid, n-Propyl - ester of n-propyl alcohol ingredient, Ester; and benzoic acid. preservative Propyl benzoate (RIFM) Butyl Benzoate 136-60-7 Butyl benzoate is the Fragrance Benzoic Acid, n-Butyl - ester of butyl alcohol and ingredient, Ester; benzoic acid. preservative Butyl benzoate (RIFM) Amyl Benzoate 2049-96-9 Amyl benzoate is the Fragrance Benzoic Acid, Pentyl - ester of amyl alcohol and ingredient Ester; benzoic acid that Pentyl Benzoate conforms to the formula Pentyl benzoate (RIFM) in Figure 1. Lauryl/ Myristyl No CAS No. Lauryl/Myristyl benzoate Skin-conditioning - Corum 5014 Benzoate is the organic compound agent- that conforms to the miscellaneous formula in Figure 1. C12-15 Alkyl 68411-27-8 C12-15 alkyl benzoate is Skin-conditioning Alkyl (C12-C15) AEC C12-15 Alkyl Benzoate the mixture of esters of agents - emollient Benzoate; Benzoate; benzoic acid and C12-15 Benzoic Acid, C12-15 Botanester AB; alcohols. Alkyl Esters; Cetiol AB; C12-15 Alcohols Corum 5012; Benzoate Crodamol AB; Crodamol AB; Dub B1215; Finsolv TN; Hest 25B; Liponate NEB; OriStar AKB; Saboderm AB; Sterol B 125; Tegosoft TN; Tegosoft TN 2 C16-17 Alkyl 669700-05-2 C16-17 alkyl benzoate is Skin-conditioning - Finsolv G-2 Benzoate a mixture of esters of agents-emollient, C16-17 alcohols and solvent benzoic acid that conforms generally to the formula in Figure 1. Stearyl Benzoate 10578-34-4 Stearyl benzoate is the Skin-conditioning Benzoic Acid, Octadecyl Dub PG; ester of stearyl alcohol agent-emollient, Ester; Finsolv 116 and benzoic acid that solvent Benzoic Acid, Stearyl conforms to the formula Ester; in Figure 1. Octadecyl Benzoate Behenyl Benzoate 103403-38-9 Behenyl benzoate is the Skin-conditioning Benzoic Acid, Docosyl Finsolv 137 ester of behenyl alcohol agent – emollient Ester and benzoic acid that conforms to the formula in Figure 1. Branched Alkyl Benzoates Isopropyl 939-48-0 Isopropyl benzoate is the Fragrance Benzoic Acid, Isopropyl - Benzoate ester of isopropyl alcohol ingredient Ester; and benzoic acid. Benzoic Acid, 1- Methylethyl Ester; Isopropyl benzoate (RIFM); 1-Methylethyl Benzoate

CIR Panel Book Page 14 Table 1. Definitions, functions and structures of alkyl benzoate and alcohol ingredients in this safety assessment. Ingredient CAS No. Definition Function(s) Technical names Trade names Isobutyl Benzoate 120-50-3 Isobutyl benzoate is the Fragrance Benzoic Acid, Isobutyl - ester of isobutyl alcohol ingredient, solvent Ester; and benzoic acid. Benzoic Acid, 2- Methylpropyl Ester; Isobutyl benzoate (RIFM); 2-Methylpropyl Benzoate Isostearyl 34364-24-4 Isostearyl benzoate is the Skin-conditioning Benzoic Acid, Finsolv SB Benzoate ester of isostearyl alcohol agent-emollient Isooctadecyl Ester; and benzoic acid. Benzoic Acid, Isostearyl Ester Ethylhexyl 5444-75-7 Ethylhexyl benzoate is Skin-conditioning Benzoic Acid, 2- Bernel Ester OB; Benzoate the ester of 2- agent-emollient, Ethylhexyl Ester; Finsolv EB ethylhexanol and benzoic solvent 2-Ethylhexyl Benzoate acid. Octyl Benzoate Butyloctyl 1888038-97-3 Butyloctyl benzoate is the Plasticizer; skin- Benzoic Acid, 2- - Benzoate organic compound that conditioning agent- Butyloctyl Ester conforms to the formula emollient, solvent in Figure 2. Hexyldecyl 163883-40-7 Hexyldecyl benzoate is Plasticizer, skin- Benzoic Acid, 2- - Benzoate the organic compound conditioning agent- Hexyldecyl Ester that conforms to the emollient, solvent formula in Figure 2. Octyldodecyl 108347-89-3 Octyldodecyl benzoate is Skin-conditioning Benzoic Acid, 2- Finsolv BOD Benzoate the ester of agent-emollient Octyldodecyl Ester octyldodecanol and benzoic acid.

CIR Panel Book Page 15 Table 2. Physical and Chemical properties of the acetate ingredients.9,9,11,49,49 Methyl Ethyl Propyl Butyl Benzoate Amyl Benzoate Lauryl/Myristal Benzoate Benzoate Benzoate Benzoate

CAS No. 93-58-3 93-89-0 2315-68-6 136-60-7 2049-96-9 -

Molecular 136.15 150.17 164.20 178.23 192.25 290.44/318.49 Weight (g/mol) Boiling Point 198.6 212.9 230.0 247.3 248 225 (Lauryl at 20 mmHg) (°C) Density (g/cm3) 1.09 1.04 1.04 1.00 0.95 0.93(Lauryl)

Vapor pressure 0.38 0.267 0.136 0.01 0.009 - (mm Hg @ 20°C) Solubility 2.1 0.72 0.351 0.059 0.028 - (g/1000g water @ 20°C) Log Kow 2.12 2.64 3.01 3.84 4.16 (est.) 7.23 (est. Lauryl)

C12-15 Alkyl C16-17 Alkyl Stearyl Behenyl Isopropyl Benzoate Isobutyl Benzoate Benzoate Benzoate Benzoate Benzoate

CAS No. 68411-27-8 667900-05-2 10578-34-4 103403-38-9 939-48-0 120-50-3

Molecular 290.44-332.52 346.55-360.57 374.60 430.71 164.20 178.23 Weight (g/mol) Boiling Point 363 (est.) - 433 (est.) 518.3 266 237 (°C) Density (g/cm3) - - - 0.908 - 1.02

Vapor pressure 0.00001 - 0.00000006 0.00000000007 0.161 (est.) 0.0417 (est.) (mm Hg @ (est.) (est.) 20°C) Solubility 0.000009 - 0.000009 0.0000007 0.126 (est.) 0.098 (est.) (g/1000g water (est.) (est.) @ 20°C)

Log Kow 7.23 (est.) - 10.18 (est.) 13.35 3.18 3.23 (est.)

Isostearyl Ethylhexyl Butyloctyl Hexyldecyl Octyldodecyl Benzoate Benzoate Benzoate Benzoate Benzoate

CAS No. 34364-24-4 5444-75-7 188038-97-3 163883-40-7 108347-89-3

Molecular 374.60 234.33 290.44 346.55 402.65 Weight (g/mol)

Boiling Point 426 (est.) 169-170 (at 20 376.9 434.8 449 (est.) (°C) mmHg)

Density (g/cm3) - 0.91 0.939 0.923 -

Vapor pressure 0.0000001 0.0005 (est.) 0.000007 0.00000009 0.00000002 (est.) (mm Hg @ 20°C) Solubility 0.00001 0.0011 0.00058 0.000015 0.000001 (est.) (g/1000g water @ 25°C)

Log Kow 10.10 (est.) 5.7 (est.) 7.857 9.982 11.09 (est.) est.= Values were estimated using the EPI Suite, Version 4.0 program or Advanced Chemistry Development (ACD/Labs) Software V11.02. - Not found

CIR Panel Book Page 16 Table 3. Frequency of use according to duration and exposure.15,16 Total uses/use Concentration Concentration Concentration Concentration Use type type Uses (%) Uses (%) Uses (%) Uses (%) C12-15 Alkyl benzoate C16-17 Alkyl benzoate Ethyl benzoate Isobutyl benzoate Total/range 36,808 971 0.0008-50 2 0.7 - 0.0008-0.01 - 0.01 Duration of use Leave-on 23,788 858 0.0008-35 - - - 0.0008-0.01 - 0.01 Rinse-off 13,020 113 0.3-50 2 0.7 - - - - Exposure type Eye area 3663 69 0.0008-11 ------Possible 872 66 3-16 ------ingestion Inhalation 3447 25 0.3-12 - - - 0.003-0.01 - 0.01 Dermal 26,863 870 0.0008-50 2 0.7 - 0.0008-0.01 - 0.01 Deodorant 623 6 0.004 ------(underarm) Hair – 5687 98 0.3-35 ------noncoloring Hair – coloring 2808 - 0.5-2 Nail 674 2 0.008-10 ------Mucous 3732 12 0.01-0.04 2 0.7 - - - - Membrane Bath products 745 ------Infant 357 9 10 - - - - -

Isostearyl benzoate Methyl benzoate Octyldodecyl benzoate Stearyl benzoate Total/range 36,808 1 1 - 0.0005-0.3 - 3-4 3 2 Duration of use Leave-on 23,788 1 - - - - - 3 2 Rinse-off 13,020 - - - 0.007-0.3 - 3-4 - - Exposure type Eye area 3663 - - - 0.0005-0.3 - - - - Possible 872 ------ingestion Inhalation 3447 - 1 - 0.3 - - - - Dermal 26,863 1 - - 0.0005-0.3 - 3-4 1 2 Deodorant 623 - - - 0.004 - - - - (underarm) Hair – 5687 ------noncoloring Hair – coloring 2808 Nail 674 ------Mucous 3732 - - - - - 3 - - Membrane Bath products 745 Infant 357 - - - - - 2 -

CIR Panel Book Page 17

Figure 1. Straight chain alkyl benzoates: structures, esterase metabolism, and metabolites.

CIR Panel Book Page 18 Figure 2. Branched-chain alkyl benzoates: structures, esterase metabolism, and metabolites.

Legend Ingredients which are part of this review

Safe as used

* Not in ICI Dictionary and Handbook, 13th Ed. **Ingredients which are concurrently under review in another report

Result of Esterase metabolism

O Benzoic acid ** OH

Isopropyl benzoate **Isopropyl alcohol O CH3 CH3 O CH3 HO CH3 O Isobutyl benzoate Isobutyl alcohol CH O 3 CH3 HO * CH3 CH3 Isostearyl benzoate Isostearyl alcohol O (one example of the mixture of branched chains) CH CH O 3 HO 3 CH3 CH3

O Ethylhexyl alcohol Ethylhexyl benzoate OCH3 HO CH3* CH3 CH3

O Butyloctyl alcohol Butyloctyl benzoate O CH3 HO CH3* CH3 CH3

O Hexyldecyl alcohol Hexyldecyl benzoate O CH3 HO CH3* CH3 CH3

Octyldodecyl alcohol O Octyldodecyl benzoate * O CH3 HO CH3 CH3

CH3

Figure 3. The synthesis of butyl benzoate.

O O HSO OH HO CH 2 4 3 O CH3 Δ 12

CIR Panel Book Page 19 REFERENCES 1. Andersen FA. Final report on the safety assessment of benzyl alcohol, benzoic acid, and sodium benzoate. International Journal of Toxicology. 2001;20(Suppl 3):23-50.

2. Andersen, F. A. Final Report on the Safety Assessment of Methyl Alcohol. International Journal of Toxicology. 2001;20((Suppl. 1)):57-85.

3. Andersen, F. A. Alcohol Denat., including SD Alcohol 3-A, SD Alcohol 30, SD Alcohol 39, SD Alcohol 39-B, SD Alcohol 39-C, SD Alcohol 40, SD Alcohl 40-B, and SD Alcohol 40-C, and the Denatonium Bezoate, Quassin, and Brucine Sulfate/Brucine. 2008 CIR Compedium. 2005;9-12.

4. Andersen, F. A. n-Butyl Alcohol Amended Report. 2008 CIR Compedium. 2005;49-51.

5. Elder RL. Final report on the safety assessment of cetearyl alcohol, cetyl alcohol, isostearyl alcohol, myristyl alcohol, and behenyl alcohol. Journal of the American College of Toxicology. 1988;7(3):359-413.

6. Elder, R. L. Final Report on the Safety Assessment of Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl Alcohol. Journal of the American College of Toxicology. 1988;7(3):359-413.

7. Elder, R. L. and El. Final Report on the Safety Assessment of Stearyl Alcohol, Oleyl Alcohol, and Octyl Dodecanol. Journal of the American College of Toxicology. 1985;4(5):1-29.

8. Riemenschneider, W. Organic Esters. 2002. 6th:(12): pp.305-328. New York: Wiley-VCH.

9. Richard J Lewis Sr. Hawley's Condensed Chemical Dictionary. 2007.

10. Robert S.Huss, Fengrong Chen, Michael F.Malone, and Michael F.Doherty. Reactive Distillation for Methyl Acetate Production. Computers and Chemical Engineering. 2003;271855-1866.

11. Falbe, J., Bahrmann, H., Lipps, W., and Mayer, D. Aliphatic Alcohols. 2002. 6:(2): pp.19-46. New York: Wiley-VCH.

12. T.Veit. Biocatalysis for the Production of Cosmetic Ingredients. Engineering in Life Sciences. 11-2-2004. 4:(6): pp.508-511.

13. Sales specification sheet. Vertellus Specialties. 2010.

14. International Programme on Chemical Safety.Butyl Acetates. http://www.inchem.org/documents/cicads/cicads/cicad64.htm. Accessed 11- 20-2009.

15. Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database. 2010. Washington, DC: FDA.

16. Personal Care Products Council. 7-8-2010. Concentration of use C12-15 alkyl benzoate, amyl benzoate, behenyl benzoate, butyl benzoate, butyloctyl benzoate, C16-17 benzoate, ethyl benzoate, ethylhexyl benzoate, hexydecyl benzoate, isobutyl benzoate, isopropyl benzoate, isostearyl benzoate, lauryl/myristyl benzoate, methyl benzoate, octyldodecyl benzoate, propyl benzoate and stearylbenzoate.

17. Commission of the European Comunities. COMMISSION DIRECTIVE 2007/17/EC of 22 March 2007 amending Council Directive 76/768/EEC, concerning cosmetic products, for the purposes of adapting Annexes III and VI thereto to technical progress . 2007. Commision Directive 2007/17/EC:

18. Adams TB, Cohen SM, Doull J, Feron VJ, Goodman JI, Marnett LJ, Munro IC, Portoghese PS, Smith RL, Waddell WJ, and Wagner BM. The FEMA GRAS assessment of benzyl derivatives used as flavor ingredients. Food and Chemical Toxicology. 2005;431207-1240.

19. Dahl, A. R., Miller, S. C., and Petridou-Fischer, J. Carboxylesterases in the respiratory tracts of rabbits, rats and Syrian hamsters. Toxicol Lett. 1987;36(2):129-136.

20. Longland, R. C., Shilling, W. H., and Gangolli, S. D. The hydrolysis of flavouring esters by artificial gastrointestinal juices and rat tissue preparations. Toxicology. 1977;8(2):197-204.

21. Kirk-Othmer Concise Encyclopedia of Chemical Technology. 4 ed. New York, NY: Wiley, 2001.

22. Shen, Y. and West, C. Toxicity of aromatic aerobic biotransformation products of toluene to hela cells. Bulletin.of Environmental.Contamination.and Toxicology. 1998;60(2):177-184.

23. Chipley, J. R. and Uraih, N. Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid. Appl.Environ.Microbiol.%1980., Aug. 40(2):352-7.(2:352-7):Applied.

24. Graham BE and Kuizenga MH. Toxicity studies on benzyl benzoate and related benzyl compounds. Journal of Pharmacology and Experimental Therapeutics. 1945;84358-362. 13

CIR Panel Book Page 20 25. Jenner PM, Hagan EC, Taylor JM, Cook EL, and Fitzhugh OG. Food flavorings and compounds of related structure I. Acute oral toxicity. Food Cosmet.Toxicol. 1964;2327-343.

26. Kravets-Bekker AA and Ivanova OP. Sanitary-toxicological characteristics of methyl benzoate and potassium benzoate. Farmacevtski Vestnik. 1970;2125-129.

27. Smyth Jr.HF, Carpenter CP, and Weil CS. Range finding toxicity data: List V. Archives of Industrial Hygiene and Occupational Medicine. 1954;4119-122.

28. Merriman TN. An acute dermal toxicity study in rabbits with methyl benzoate (C-2000) Final report. Springborn Laboratories, Inc. 1995. Report No. 3206.347.

29. Smyth Jr.HF, Carpenter CP, and Weil CS. Range finding toxicity data: List IV. Archives of Industrial Hygiene and Occupational Medicine. 1951;4119-122.

30. Shtenberg AJ and Ignat'ev AD. Toxicological evaluation of some combinations of food preservatives. Food Cosmet.Toxicol. 1970;8369-380.

31. Sodemoto Y and Enomoto M. Report of carcinogenesis bioassay of sodium benzoate in rats: absence of carcinogenicity of sodium benzoate in rats. Journal of Environmental Pathology and Toxicology. 1980;487-95.

32. Skin Research Dpt. 2005. Assessment of the eye irritating potential of a cosmetic product (body lotion containing 0.95% isostearyl benzoate) through alternative methods to the Draize test.

33. Branca, M., Garcovich, A., Linfante, L. D., Macrì, A, Mantovani, A., Olivetti, G., and Salvatore, G. Macro- and microscopic alterations in 2 rabbit skin regions following topically repeated applications of benzoic acid n-alkyl esters. Contact Dermatitis.%1988., Nov. 1988;19(5):320-34.(5:320-34):Contact.

34. Onodera H, Ogiu T, Matsuoka C, Furuta K, Takeuchi M, Oono Y Kubota T, Miyahara M, Maekawa A, and Odashima S. Studies on effects of sodium benzoate on fetuses and offspring of Wistar rats. Bull Nat Inst Hyg Sci. 1978;9647-55.

35. Verrett MJ, Scott WF, Reynaldo EF, Alterman EK, and Thomas CA. Toxicity and teratogencity of food additive chemicals in the developing chicken embryo. Toxicol Appl Pharmacol. 1980;56265-273.

36. Morgareidge K. Teratologic evaluation of FDA 71-37 (sodium benzoate). U.S. Food and Drug Administration. 1972. Report No. PB-221 777.

37. Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ.Mol.Mutagen.%1992.;19.Suppl 21:2-141. 1992.

38. Szybalski W. Special microbial systems. II. Observations on chemical mutagenesis in microorganisms. Annals of the New York Academy of Sciences. 1958;475-489.

39. Andersen FA. Final report on the safety assessment of benzyl alcohol, banzoic acid, and sodium benzoate. International Journal of Toxicology. 2001;20(Suppl. 3):23-50.

40. Jansson T, Curvall M, Hedin A, and Enzell CR. In vitro studies of biological effects of cigarette smake condensate. II. Induction of sister- chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents. Mutation Research. 1986;169129-139.

41. Results of recent studies on the relevance of various short-term screening tests in carcinogenicity evaluation. 1980. The Predictive Value of Short-Term Screening Tests in Carcinogencity Evaluation. Williams GM, Kroes R, Waaijers HW, and van de Pol KW.

42. Kuboyama N and Fujii A. Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal species. J Nihon Univ Sch Dent. 1992;34(3):183-195.

43. McCAnn J, Choi E, Yamasaki E, and Ames BN. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proceedings of the National Academy of Sciences USA. 1975;72(12):5135-5139.

44. Cooperative programe on short-term assays for carcinogencity in Japan. 1980. Molecular and Cellular Aspect of Carcinogen Screening Tests. Montesano R, Bartsch H, and Tomatis L. Lyon, France: International Agency for Research on Cancer Scientific Publications.

45. Consumer Product Testing Co. 48 Hour patch test of C-SAT 020093 (C12-15 Alkyl benzoate). 2003. Report No. C02-1224.01.04. pp. 1-10.

46. Consumer Product Testing Co. Repeated insult patch test of C-SAT 020093 (C12-15 alkyl benzoate). 2003. Report No. C02-1224.01.04. pp. 1-14.

47. Consumer Product Testing Co. 2005. Repeated insult patch test of a body lotion containing 0.95% isostearyl benzoate. Experiment reference number C05-0728.01. 14

CIR Panel Book Page 21 48. Personal Care Products Council. 2010. Summary of an HRIPT of a Shaving Cream Product containig 4% octyldodecyl benzoate.

49. The Merck Index. http://themerckindex.cambridgesoft.com/TheMerckIndex/index.asp. Accessed 10-20-2009.

CIR Panel Book Page 22 Data PersonalCare ProductsCouncil Committedto Safety, Quality&Innovation

Memorandum

TO: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR)

FROM: John Bailey, Ph.D. Industry Liaison to the Cifi Expert Panel

DATE: July 9, 2010

SUBJECT: Summary of an HRIPT of a Shaving Cream Product containing 4% Octyldodecyl Benzoate

A shaving cream product, containing 4% Octyldodecyl Benzoate, was tested in an NRIPT by a 3rd party testing facility. The test included 105 panelists, was semi-occlusive, and the product was diluted to a 10% aqueous solution before application. The results of HR]PT “did not demonstrate a potential for eliciting dermal irritation or sensitization.” More specifically, for all 105 panelist no visible skin reaction was observed over the testing period.

11011 7th Street, N.W., Suite 300 Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personalcarecouncil.org

CIR Panel Book Page 23 PersonalCare ProductsCouncil Committedto Safety, ua ty nnovation Memorandum

TO: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR)

-‘FROM: ‘JohnBai1ey,Ph.D Industry Liaison to the CIR Expert Panel

DATE: July9, 2010

SIJEJECT: Studies of C 12-15 Alkyl Benzoate

Cognis. 2007. Cetiol AB (C12-15 Alkyl Benzoate) data sheet.

Cognis. 2002. UV absorption spectrum of Cetiol AB (C12-15 Alkyl Benzoate).

Consumer Product Testing Co. 2003. 48 Hour patch test of C-SAT 020093 (C12-15 Alkyl Benzoate). Experiment Reference Number C02- 1224.01.04.

Consumer Product Testing Co. 2003. Repeated insult patch test of C-SAT 020093 (C12-15 Alkyl Benzoate). Experiment Reference Number C02- 1225.01.

11011 7th Street, N.W., Suite 3O0 Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personalcarecouncil.org

CIR Panel Book Page 24 cgnisUom caIs CETIOL® AB Labeling information INCI name(s) Cl 2-15 AlkylBenzoate (EU 200612571EC) Cl 2-15 AlkylBenzoate (CTFA) Registrations Ingredient CASR-No. EINECS/ELINCS-No. 6841.1-27-8 270-112-4

Officially listed in I Quality conforms to JCIC: 012-15 Alkyl Benzoate (Ingredient Code 512004) Product properties Appearance Cetiol® AB is a clear, almost colourless, almost odourless oil.

Example of use Cetiol® AB is a traditional emollient for modern skin care applications. Particularly suitable for suncare formulations due to its outstanding solubilizing capacities for crystalline UV filters as well as dispersing properties for pigments. Characteristic values The specifications stated in the paragraphs ‘Quality control data and Additional product descriptive data’ finally and conclusively describe the properties of the Product. Provisional quality control data (Data which is used for quality release and is certified for each batch.) Appearance conforms to standard Odour conforms to standard Acid value max. 0.5 mg KOH/g ISO 660 Saponification value 172- 182 mg KOH/g ISO 3657 Colour value (APHA) max. 50 ISO 6271 Density (20°C) 0.920 - 0.940 g/ml ISO 2811-3 Refractive index (20°C) 1.4830 - 1.4870 ISO 6320 Water (Karl Fischer) max. 0.3 ISO 4317 Provisional additional product descriptive data (Data which is proven statistically but not determined regularly.) Viscosity (20°C) max. 30 mPas ISO 12058-1 Cloud point max. - 5°C ISO 3015

Revision-No. 2-08.2007

All products in the text marked with an ® are trademarks of the Cognis group. The information on product specifications provided herein is only binding to the extent confirmed by Cognis in a written Sales Agreement. COGNIS EXPRESSLY DISCLAIMS ANY RESPONSIBILITY FOR THE SUITABILITY OF THE PRODUCTS FOR ANY SPECIFIC OR PARTICULAR PURPOSES INTENDED BY THE USER. Suggestions for the use and application of the products and guide formulations are given for information purposes only and without commitment. Such suggestions do not release cognis’ customers from testing the products as to their suitability for the customer’s intended processes and purposes. Cognis does not assume any liability or risk involved in the use of its products as the conditions of use are beyond its control. The user of the products is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties.

IB.08.2008 CETIOLrABE

CIR Panel Book Page 25

200,0

250

300

350

400

450

500

550

600

650

700,0

I I

10 Is CIR

Panel

—— ___

Book

ABS

Page 25 —

Cog-02-07141,

Cetiol

C 1

A k iS

I 2 e

50,0 S Consumer Product Testing Co. L&E I)75 FINAL REPORT COLQOO —‘p’ CLIENT: Cognis Deutsehiand GmbH & Co. KG. Henkelstr. 67

D-4055 1 Duesseldorf, Germany ATTENTION: Mr. Peter Wierich

TEST: 48 Hour Patch Test Protocol No.: CZKH-OOl

TEST MATERIALS: .01 C-SAT 020093 (diluted 3% in vegetable oil) .02 C-SAT 020093 (diluted 10% in vegetable oil) .03 C-SAT 020093 (diluted 30% in vegetable oil) .04 C-SAT 020093 1/.Ik t3e,.oci+C EXPERIMENT j REFERENCE NUMBER: C02-1224.01-.04

Richard R. Eisenberg, - BoardCertifiedDermatologist

Robert W. Shanahan, Ph.D. Principal Investigator

4yFfank, R.N. ‘study Director

This report is submitted for the exclusive use at the person, partnership, or corporation to whom it is addressed, and neither the report nor tie name of these Laboratories nor any member of its staff. may be used in connection with the advertising or sale of any product or process without written authorization.

7() Ntv 1)uth I,rlll(’ • IfirfiC1(I. Ncv ,Jcrsc\’ 07004-2514 • (973) 808-7111 • 1x (973) 808-7234

CIR Panel Book Page 27 [Si. The laboratory Clinical New Study and standard and findings regarding Date(s) in The Laura Senior 7() accordance 75 data objective representative standard Jersey, New A. No.: personnel of of Practice operating pertinent Artiles, Consumer such studies. inspection: Dutch these C02-1224.0l-.04 07004, of operating with procedures the inspections M.A. and Lane involved: to procedures signature standard These unless Quality this QUALITY Clinical requirements • procedures study Fairfield. studies and specified operating Assurance of have - Quality Marie and January • protocols. the will Toxicology have been applicable Quality New ASSURANCE be Terlizzese, otherwise, provided and Assurance procedures 14, been stored Unit reported Manager, Jersey has Product CIR 2003 Assurance performed • (QAU) protocols. inspected in Panel for Analytical in M.S. the Associate to 07004-2514 writing and Quality in Book management Archive is 21 study Unit UNIT to with Page this The CFR by monitor Chemistry Assurance signifies protocol the 28 adherence Facility study QAU STATEMENT parts • Sponsor. (973) and the maintains Testing on that 50 as the • at conduct 808-71 Microbiology the well to 70 and this Study ICH New date(s) as study 56 copies and 1 government Director. Guideline I Dutch and • reporting has listed Fax of in been Lane, study accordance 197:3) All below. E6 regulations Co. performed of protocols for Fairfield, materials 808-7234 clinical Good The to Cognis Deutscbland GmbH & Co. KG. C02-1224.0l-.04 Page 3

Objective: To determine by epidermal contact the primary irritation potential of a test material.

Participants: Twenty-two (22) subjects, male and female, ranging in age from 20 to 73 years, who qualified were selected for this evaluation. Twenty-one (21) subjects completed this study. The remaining subject discontinued her participation for personal reasons unrelated to the use of the test materials.

Inclusion Criteria: a. Male and female subjects, age a andover. b. Absence of any visible skin disease which might be confused with a skin reaction from the test materials. c. Prohibition of use of topical or systemic steroids andlor antihistamines for at least seven days prior to study initiation. d. Completion of a Medical History form and the understanding and signing of an Informed Consent form. e. Considered reliable and capable of following directions.

Exclusion Criteria: a. Ill health. b. Under a doctor’s care or taking medication(s) which could influence the outcome of the study. c. Females who are pregnant or nursing. d. A history of adverse reactions to cosmetics or other personal care products.

Test Materials: .01 C-SAT 020093 (diluted 3% in vegetable oil) .02 C-SAT 020093 (diluted 10% in vegetable oil) .03 C-SAT 020093 (diluted 30% in vegetable oil) .04 C-SAT 020093

Study Schedule: Panel # Initiation Date Completion Date

20020613 December 31, 2002 January 3, 2003

aWjth parental or guardian consent

CIR Panel Book Page 29 Cognis Deutschland GmbH & Co. KG. C02-1224.0l-.04 Page 4

Methodology: Prior to the initiation of this study, the test material was prepared as 3%, 10%, and 30% dilutions, using vegetable oil.

The upper back between the scapulae served as the treatment area. Approximately 0.2 ml of the diluted, as well as an undiluted sample of the test material, or an amount sufficient to cover the contact surface, was applied to the 3/4” x 3/4” absorbent pad portion of adhesive dressings*. When secured to the appropriate treatment site, these dressings formed occlusive patches.

The test materials remained in contact with the skin for a total of forty- eight hours. These sites were then evaluated for gross changes. Absence of any visible skin change was assigned a zero value. The test sites were re-evaluated at seventy-two hours.

Evaluation Key: 0 = No visible skin reaction + = Barely perceptible or spotty erythema

1 = Mild erythemacovering most of the test site 2 = Moderate erythema,possible presence of mild edema 3 = Marked erythema, possible edema 4 = Severe erythema, possible edema, vesiculation, bullae andlor ulceration

Results: The results of each participant are appended (Tables 1-4).

Observations of all treated areas remained negative throughout the test interval.

Summary: Under the conditions of this study, the test material, C-SAT 020093, applied neat and at 3%, 10%, and 30% dilutions in vegetable oil, did not indicate a potential for dermal irritation.

*Manufactured by TruMed Technologies, Inc., Bumsville, MN

CIR Panel Book Page 30 Cognis Deutschland GmbH & Co. KG. C02-1224.01-.04 Page 5

Table 1 Panel #20020613

Individual Results

C-SAT 020093 (diluted 3% in vegetable oil)

Subject Observations Number 48 Hours 72 Hours

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 DNC 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 21 0 0 22 0 0

DNC = Did not complete study

CIR Panel Book Page 31 Cognis Deutschland GmbH & Co. KG. C02-1224.01-.04 Page 6

Table 2 Panel #20020613

Individual Results

C-SAT 020093 (diluted 10% in vegetable oil)

Subject Observations Number 48 Hours 72 Hours

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 DNC 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 21 0 0 22 0 0

DNC = Did not complete study

CIR Panel Book Page 32 Cognis Deutsehiand GmbH & Co. KG. C02-1224.01-.04 Page 7

Table 3 Panel #20020613

Individual Results

C-SAT 020093 (diluted 30% in vegetable oil)

Subject Observations Number 48 Hours 72 Hours

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 DNC 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 21 0 0 22 0 0

DNC = Did not complete study

CIR Panel Book Page 33 Cognis Deutschland GmbH & Co. KG. C02-1224.0l-.04 Page 8

Table 4 Panel #200206 13

Individual Results

C-SAT 020093

Subject Observations Number 48 Hours 72 Hours

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 DNC 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 21 0 0 22 0 0

DNC = Did not complete study

CIR Panel Book Page 34 Cognis Deutsohiand GmbH & Co. KG. C02-1224.01-.04 Page 9

Table 5 Panel #20020613

Subject Data

Subject Number Initials Age Sex

1 RC 23 F 2 BA 46 F 3 JE 57 F 4 AR 31 M 5 WE 63 M 6 CV 35 F 7 DC 43 F 8 LE 47 F 9 CD 20 F 10 CO 34 F 11 AW 73 F 12 PR 55 M 13 LD 38 F 14 EV 34 F 15 TV 36 M 16 KS 64 F 17 SW 36 F 18 PM 43 M 19 KH 39 F 20 BW 63 F 21 LB 56 F 22 CW 56 F

CIR Panel Book Page 35 Substanz-Verwaltung CRT aktuel?er User CNPeter Wierich/OU= DE/OUEMEAIO=Cognis IS

C-SAT-Nr 020093

Chemische C12/15 Alkylbenzoate Bezeich nung Trivial Name Cetiol AB S-GehaIt [%] 100 Losungsmittel (Neben-) Komponenten Verunreinigungen Identiflzierung der Prufsubstanz

Herstelldatum 01.09.2002 Verfallsdatum 01.09.2003

Batch CD22660003 RIS 51Zi 200670000 S’s CAS 68411 -27-8

lOslich ParaffinOl,native Ole in/suspendierbar n Farbe farbios Aggregatzustand bel Flussigkeit RT pH-Wert fluchtig bei RT

Lagerung

Auftraggeber CCC N.Mertscheit ProduktbetreuerKleber

Gefah renhinweise (z.B. qiftiqlreizend/tzend/entzundIichIexpIosiv)

Weitere Informationen zur PrUfsubstanz

CIR Panel Book Page 36 EST, 70 This without name

1970

TEST

ATTENTION:

EXPERIMENT

TEST:

REFERENCE CLIENT: New report of written these is

Dutch Consumer

submitted MATERIAL: authorization. Laboratories Lane

for NUMBER: the nor •

exclusive C any

Fairfield, (:92000 member use

of FINAL of New the its person. staff,

Jersey

J/Frk,

may Board

Principal

Study Richard

Protocol

Repeated C-SAT

Mr. D-4055 Henkelstr. C02-1225.01

Duesseldorf, Cognis CIR

partnership, Product

be Peter

07004-2514 Panel REPORT

used

Director

Certified

Deutschland

020093

R. No.:

in Investigator Book

Insult

Wierich

R.N. 67 -

connection Eisenberg,

corporation Germany

Page

1.01

Dermatologist Patch • 37 (973) with

to GnibH

whom

Test MIS. the 808-71 advertising

it Testing

is &

addressed, Co. 11

or KG. sale • and Fax of neither any (973) product the 808-7234 report or

process Co. nor the QUALITY ASSURANCE UNIT STATEMENT

Study No.: C02-1225.01

The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical laboratory studies. These studies have been performed with adherence to ICH Guideline E6 for Good Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to standard operating procedures and applicable protocols. The QAU maintains copies of study protocols and standard operating procedures and has inspected this study on the date(s) listed below. The findings of these inspections have been reported to management and the Study Director. All materials and data pertinent to this study will be stored in the Archive Facility at 70 New Dutch Lane, Fairfield, New Jersey, 07004, unless specified otherwise, in writing by the Sponsor.

Date(s) of inspection: January 20, 2003 February 26, 2003 February 14, 2003 March 10,2003 March 11, 2003

Senior personnel involved:

Richard Hettenbach Senior Director, Regulatory Affairs and Quality Assurance

Marie Terlizzese, M.S. Quality Assurance Associate

The representative signature of the Quality Assurance Unit signifies that this study has been performed in accordance with standard operating procedures and study protocol as well as government regulations regarding such procedures and protocols.

CIR Panel Book Page 38 CognisDeutschland GmbH & Co. KG. C02-1225.01 Page 3

Objective: To determine by repetitive epidermal contact the potential of a test material to induce primary or cumulative irritation and/or allergic contact sensitization.

Participants: One hundered and twelve (112) qualified subjects, male and female, ranging in age from 16 to 79 years, were selected for this evaluation. One hundered and one (101) subjects completed this study. The remaining subjects discontinued their participation for various reasons, none of which were related to the application of the test material.

Inclusion Criteria: a. Male and female subjects, age a16 and over. b. Absence of any visible skin disease which might be confused with a skin reaction from the test material. c. Prohibition of use of topical or systemic steroids and/or antihistamines for at least seven days prior to study initiation. d. Completion of a Medical History form and the understanding and signing of an Informed Consent form. e. Considered reliable and capable of following directions.

Exclusion Criteria: a. Ill health. b. Under a doctor’s care or taldng medication(s) which could influence the outcome of the study. c. Females who are pregnantor nursing. d. A history of adverse reactions to cosmetics or other personal care products.

Test Material: C-SAT 020093

Study Schedule: Panel # Initiation Date Completion Date

20030013 January 13, 2003 February 21, 2003 20030022 January 20, 2003 February 27, 2003

aWith parental or guardianconsent

CIR Panel Book Page 39 Cognis Deutschland GmbH & Co. KG. C02-1225.Ol Page 4

Methodology The upper back between the scapulae served as the treatment area. Approximately 0.2 ml of the test material, or an amount sufficient to cover the contact surface, was applied to the 3/4” x 3/4” absorbent pad portion of an adhesive dressing*. This was then applied to the appropriate treatment site to form an occluded patch.

Induction Phase:

Patches were applied three (3) times per week (e.g., Monday, Wednesday, and Friday) for a total of nine (9) applications. The site was marked to ensure the continuity of patch application. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent Induction patches at home, twenty-four hours after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assignedtest day, one (1) makeup day was permitted. This day was added to the Induction period.

With the exception of the first supervised Induction Patch reading, if any test site exhibited a moderate (2-level) reaction during the Induction Phase, application was moved to an adjacent area. Applications are discontinued for the remainder of this test phase, if a moderate (2-level) reaction was observed on this new test site. Applications would also be discontinued if marked (3- level) or severe (4-level) reactivity was noted.

Rest periods consisted of twenty-four hours following each Tuesday and Thursday removal, and forty-eighthours following each Saturday removal.

Challenge Phase:

Approximately two (2) weeks after the final Induction patch application, a Challenge patch was applied to a virgin test site adjacent to the original Induction patch site, following the same procedure described for Induction. The patch was removed and the site scored at the clinic twenty-four and seventy-two hours post-application

*Manufactured by TruMed Technologies, Inc., Burnsville, MN

CIR Panel Book Page 40 Cognis Deutschland GmbH & Co. KG. C02-1225.Ol Page 5

Evaluation Key: 0 No visible skin reaction + Barely perceptible or spotty erythema

1 = Mild erythemacoveringmost of the test site 2 = Moderate erythema,possible presence of mild edema 3 = Marked erythema,possible edema 4 = Severe erythema,possible edema, vesiculation, bullae and/or ulceration

Results: The results of each participant are appended (Table 1).

Observations remained within normallimits throughout the test interval.

Summary: Under the conditions of this study, test material, C-SAT 020093, did not indicate a potential for dermal irritation or allergic contact sensitization.

CIR Panel Book Page 41 Cognis Deutsehiand GmbH & Co. KG. C02-1225.01 Page 6

Table 1 Panel #20030013

Individual Results

C-SAT 020093

Virgin Challenge Subject — Induction Phase — Site

Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72br

-* 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 14 0 -DID NOT COMPLETE STUDY. 15 0 0 0 0 0 0 0 0 0 0 0 0 16 0 -DID NOTCOMPLETESTUDY. 17 0 0 —----- —-DID NOT COMPLETE STUDY - --- 18 0 0 0 0 0 0 0 0 0 0 0 0 19 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 21 0 0 0 0. 0 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 0 0 26 0 0 0 0 ----—-----—---DIDNOT COMPLETE STUDY 27 0 0 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0

24 = Supervisedremovalof l Inductionand ChallengePatch -* = Subjectunableto report as scheduled,instructedto removepatchand report on thenexttest day.

CIR Panel Book Page 42 Cognis Deutschland GmbH & Co. KG. C02-1225.01 Page 7

Table 1 (continued) Panel #200300 13

Individual Results

C-SAT 020093

Virgin Challenge Subject ——---InductionPhase — —-- Site Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72 hr

29 0 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 0

35 — -----DID NOT COMPLETE STUDY---- 36 0 0 0 0 0 0 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 38 0 0 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0’ 0 0 0 0 0 41 0 0 0 0 0 0 0 0 0 0 0 0 42 0 0 0 0 0 0 0 0 0 0 0 0 43 0 0 0 0 0 0 0 0 0 0 0 0 44 0 0 0 0 0 0 0 0 0 0 0 0 45 0 0 0 0 0 0 0 0 0 0 0 0 46 0 0 0 0 0 0 DID NOT COMPLETE STUDY 47 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 o o 0 0 0 0 0 0 53 0 0 0 0 0 0 0 0 0 0 0 0 54 0 0 0 0 DID NOT COMPLETE STUDY--- 55 0 0 0 0 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremoval of 1 Inductionand ChallengePatch

CIR Panel Book Page 43 Cogiiis Deutsohiand GmbH & Co. KG. C02-1225.01 Page 8

Table 1 (continued) Panel #20030022

Individual Results

C-SAT 020093

Virgin Challenge Subject —-—-—----— Induction Phase--—--— ——— —-—-- Site 24*1w Number 24*hr 1 2 3 4 5 6 7 8 9 721w

Ow 0 0 + 0 0 0 0 0 0 O 0 2 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 O 0 4 0 0 0 0 0 0 0 0 0 0 O 0 5 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0*

10 0 0 0 0 O 0 0 0 0 0 0 0 Ow 11 0 0 0 0 0 0 0 0 0 0 0 ow 12 0 0 0 0 O 0 o o 0 0 o 13 0 0 0 0 o 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 Ow 16 0 0 0 0 0 0 0 0 0 0 0 17 0 0 0 0 DID NOT COMPLETE STUDY

18 0 0 0 0 O 0 0 0 0 0 0 0 19 0 0 0 0 0 0 0 0 0 0 0 0

20 0 0 0 0 O 0 0 0 0 0 0 0

21 0 0 0 0 O 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 0 0 ow 23 - 0 0 0 o 0 0 0 o 0 0 Ow 24 0 0 0 0 O 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 o 0 26 0 0 0 0 0 0 0 0 0 0 o o 27 0 0 0 0 0 0 0 0 0 0 o o 28 0 0 0 0 0 0 0 0 0 0 O 0

24* Supervisedremoval t1nduction and ChallengePatch - = Subjectnot presentfor supervisedpatchremoval * = Observationrecorded96 hourspost challengeapplication.Subject unableto report as scheduled. W = Inclementweather.Subjectunableto report as scheduledand was instructedto report on the nextscheduledtestday.

CIR Panel Book Page 44 Cognis Deutsehiand GinbH & Co. KG. C02-1 225.01 Page 9

Table 1 (continued) Panel #20030022

Individual Results

C-SAT 020093

Virgin Challenge Subject —----—---—---—--— InductionPhase--—----— — —-- Site Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72 hr

29 0 0 0 0 0 0 0 0 0 0 O 0 Ow 30 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 O 0 33 0 0 0 0 0 0 0 0 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 O 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 38 0 o o 0 0 0 0 0 0 0 0 0 39 0 o 0 0 0 0 0 0 0 0 0 0 40 0 - DID NOT COMPLETE STUDY 41 o o 0 0 0 0 0 0 0 0 42 o 0 0 0 0 0 0 0 0 0 0 0 43 o o 0 0 0 0 0 0 0 0 O 0 44 o o 0 0 0 0 0 0 0 0 0 0 45 0 0 0 0 0 0 0 0 O 0 0 0 46 0 0 0 0 0 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 51 0 0 ------DID NOT COMPLETE STUDY--- 52 0 0 0 0 0 0 0 0 0 0 0 0 53 0 0 0 0 0 0 0 0 0 0 0 0 54 0 0 0 0 0 0 0 0 0 0 0 0 55 0 0 + 0 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof l InductionandChallengePatch DNC = Didnot completestudy W = Inclementweather.Subjectunableto reportasscheduledandwas instructedto report on thenextscheduledtestday.

CIR Panel Book Page 45 Cognis Deutschland GmbH & Co. KG. C02-1225.01 Page 10

Table 2 Panel #20030013

Subject Data

Subject Number Initials Age Sex

I SC 54 F 2 TJ 33 M 3 JS 51 F 4 WF 49 F 5 AL 56 F 6 DK 44 F 7 DW 46 F 8 CC 66 F 9 WM 34 F 10 CS 41 F 11 TM 74 F 12 AT 27 F 13 NP 35 M 14 RA 36 F 15 LS 39 F 16 MM 33 M 17 SV 33 M 18 EM 46 F 19 SM 26 F 20 MT 58 F 21 FP 63 F 22 ID 79 F 23 CT 34 F 24 DE 47 F 25 PR 63 F 26 PF 77 F 27 TM 69 F 28 AS 70 F

CIR Panel Book Page 46 Cognis Deutschland GmbH & Co. KG. C02-1225.01 Page 11

Table 2 (continued) Panel #20030013

Subiect Data

Subject Number Initials Age Sex

29 ES ‘39 M 30 SE 50 F 31 NE 18 F 32 PH 31 F 33 MR 36 F 34 DN 21 F 35 RG 45 M 36 AT 58 M 37 QA 49 F 38 RG 55 F 39 GG 67 M 40 MG 31 F 41 AA 45 M 42 DM 47 F 43 MM 44 F 44 KF 45 F 45 KP 45 F 46 PC 36 F 47 DK 19 F 48 DT 31 F 49 SK 45 F 50 SM 55 F 51 YL 39 M 52 KG 38 F 53 JS 62 M 54 JP 53 M 55 BC 35 M 56 JM 66 M

CIR Panel Book Page 47 Cognis Deutschland GmbH & Co. KG. C02-1225.01 Page 12

Table 2 (continued) Panel #20030022

Subject Data

Subject Number Initials Age Sex

1 TC 18 F 2 KF 45 F 3 AK 72 F 4 EH 51 M 5 KC 23 F 6 AC 27 M 7 DD 47 F 8 FD 53 M 9 BT 46 F 10 LC 32 F 11 GT 40 F 12 MW 48 F 13 LG 40 F 14 JB 16 F 15 JG 44 M 16 BH 25 M 17 PS 28 F 18 MV 47 F 19 JC 45 F 20 JS 44 F 21 PS 17 M 22 DM 22 M 23 DF 69 F 24 SB 52 F 25 VS 28 F 26 JD 42 M 27 LF 36 F 28 IC 59 F

CIR Panel Book Page 48 Cognis Deutschland GmbH & Co. KG. C02-1225.0l Page 13

Table 2 (continued) Panel #20030022

Subject Data

Subject Number Initials Age Sex

29 PH 58 M 30 RN 74 F 31 1V 52 F 32 GD 47 F 33 VA 48 M 34 ES 48 M 35 DS 39 F 36 RC 63 M 37 PS 26 F 38 ML .67 F 39 EA 41 F 40 GA 27 M 41 SM 40 F 42 SS 51 F 43 WB 39 F 44 JC 74 F 45 DR 36 F 46 LR 69 F 47 PD 63 F 48 PL 73 F 49 TB 53 M 50 CA 37 F 51 CS 31 F 52 EH 68 M 53 EA 63 F 54 CC 55 F 55 AS 34 F 56 SW 52 F

CIR Panel Book Page 49 aktueller

C-SAT-Nr Substanz-Verwaltung Identifizierung LOsungsmittel Chemische Herstelldatum Verunreinigungen Bezeichnung Verfallsdatum flochtig Komponenten (Neben-) Auftraggeber Lagerung S-Ge rrivial Gefahrenhinweise Weitere Batch sLs RIS Aggregatzustand CAS lslich ProduktbetreuerKleber inlsuspendierbar Farbe RT pH-Wert Name halt bei Informationen User

[%) RT der in bei (z.B. CN=Peter Prufsubstanz zur 01.09.2003

020093 C12115 51 CD22660003 68411 farblos ParaffinOl, giftig/reizend!Lzend/enUndIich/expIosiv) FlOssigkeit 01.09.2002 I CCC Cetiol 100 ZI PrUfsubstanz 200670000 N.Mertscheit -27-8 AB Alkylbenzoate Wierich!OU=DE/OU=EMEAIO=Cognis native Ole CIR Panel Book

CRT Page 50 PersonalCare ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

TO: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CW)

FROM: John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel

DATE: July 9, 2010

SUBJECT: Studies on a Body Lotion Containing 0.95% Isostearyl Benzoate

Consumer Product Testing Co. 2005. Repeated insult patch test of a body lotion containing 0.95% Isostearyl Benzoate. Experiment Reference Number C05-0728 .01.

Skin Research Dpt. 2005. Assessment of the eye irritating potential of a cosmetic product (body lotion containing 0.95% Isostearyl Benzoate) through alternative methods to the Draize test. Report reference: CTOXJO5161.

1101 17th Street, N.W., Suite 30O Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personakarecouncil.org

I CIR Panel Book Page 51

Report

I-sr

() wittiest This

nrrrne

EXPERIMENT

REFERENCE

TEST

TEST:

ATTENTION:

N(v

CLIENT:

report

Date:

written

these

IhItcIl

Consumer

submitted

MATERIAL:

authorization

Laboratories

‘O//

Lant

Jor

472

the

NUMBER:

001

•

exetusive

any

Iiirfirki

membor

use

New

FINAL

the

its

person,

staff,

,Jers

(‘xeLitive

C05-072.01

ProtocoiNo.:

Repeated

James

Sr.

may

Board

Richard parfnershi4,

CIR

Product Cherr4ist

P-ank, t45

Panel

use

Flanagan

Cprtified

Insult

in or

Book

Vice

corinsciron

Eisenberg,

corporation

PORT Microbiology

Body

14 1.01 ew

Page

Patch

President,

Dermatologist

Lotion

t$73)

with to

52 Jersey

whom

Test

M.D.

the

O-7

edverltsin

TL45-.24-2

Clinical

07950-2451

‘a

Testing

addressed,

•

9ç

Evaluations

safe

and

Iax

neither

any

(97;lf

pcoduct

the

irJ

i3irnzc’c1-c

8O-7234

report

process

nor

Co. the

The

regarding

Quality

writing E6

study

accordance The

study

laboratory Study

New

accordance

for

representative

objective

protocols

will No.:

Duth

Good

Assurance

such

the

Consumer

studies.

C05-0729.01

stored with

Lane

Sponsor.

procedures

standard

Clinical

and the

(linicI

signature

standard

These

standard

personnel

Quality

FirfieIc1,

QUALITY

the

Practice

operating

and

studies )cdkô

•

Consumer

operating of

Toxicology

operating

Assurance

protocols.

involved: the

New

and

have

procedures

Quality

ASSURANCE

Assurance

Jersw

requiremenuts

Product

procedures

been Unit

CIR

Product Assurance \fliI\tiCI

Panel performed

O704-2

(QkU)

and

Testing and

Book

and

applicable

provided is

Chcmiry

study

Unit has

Page

Company

UNIT to

with

monitor inspected

signifies protocol

973)

adherence

protocols.

for

STATEMENT

Date

archive,

/o/i4 the Microbiology

8Q-7

Testing

this

that

conduct

well

study.

CFR

The this

unless the

study

QAU applicable

•

parts

and

All government

Fax

speciiied

data

has

reporting maintains

($73)

been

pertinent

and

ICH otherwise,

8O87234

regulations

Co.

performed

Guideline

copies

clinical

and

this

in in

‘With Study Test

Exclusion

Inclusion

Participants:

Objective:

Material:

parental

Schethile:

Criteria:

guardian

consent

Panel

20050393

20050401

d. b.

application

their

(107)

from

One

sensitization.

determine

products.

outcome

Under

Females

Completion

Considered

Absence signing

for

Male induce Prohibition

reaction

hundred

participation

subjects

health.

history

and

least

Body

doctor’s

who

from

primary

fifteen

the

female

years.

seven

completed of the

CIR

reliable

any

August

Augut 1nitiaton

test are

repetitive Informed

the Lotion

advçrse

for use

Panel

study.

viibIe

(115)

pregnant

dkys

subjects,

care

material.

test

rere

various

Medical

of and

Book

15.

17,

TL45-24-2

material.

topical

Date or

prior

qualified

this

Consent

selected cumulative

skin

reactions

epidermal

2005

capable taking

Page

age

study.

reasons,

disease

History nursing.

study

16 a

medication(s)

subjects,

form.

for

systemic

and

contact

following

The

Completion

September

this

which

initiation.

none

irritation

cosmetics

form

over.

remaining

evaluation.

male

steroids

the

might

and

directions.

which

potential

and

29,

23,

and!or

Date

the

2005

female,

Page

andlor

subjects

other

confused

were

could

One

understanding

allergic

hundred

antihistamines

personal

ranging

influence

discontinued

test

with

material

contact

seven

care

skin

and

the

age the

*Manufacred

Methodology:

TruMed

seventy-two

The

Technologies, Induction

Challenge

Thursday

Approximately

Chaliene

Rest

level) the

application

site

With

applications.

was

due

The

subjects participant

the

subsequent

scoring

Patches

and

Induction

clear

site

Approximately

the

The

this

remainder

Friday) patch

permitted.

contact

exhibited

continuity

evaluation

periods

the

adhesive

upper

form

new

severe

were

exception

removal,

patch

who

patch

holiday

the Phase:

was

Phase:

was

hours

lnductioa

test

for

surface,

back

consisted

Inc., semi-o4elusive

CIR

first

applied

dressijg*.

two

(4-level)

moved

removed unabl

This

of of

requited

site,

0.2

site.

this

was.

totai

moerate post-application.

Panel

this

and

Burnsville,

Induction

patch

weekend,

between

(2)

nIl

day

test

following

vas

Applications

applied

the

patches

three

forty-eight to

Book

site

weeks

reactivity

was

phase,

nine

and

report

first

applied

application.

the

This

twenty-four was

Page

(2-level)

(3)

adjacent

patch,

makeup

patch.

added

the

(9)

which

the

MN to

test

supervised

after

the

was

made for

times

applications.

home, a

hours

site

was

scapulae

material,

would

same

moderate

virgin

participants

the

then

the

occurred

area.

reaction

again

the

noted.

scored

assigned

per

day

following

hours

final

Following

twenty-thur

procedure

1’

applied

Induction

also

Induction

test

week

Applications

just

experienced

served

(2-level)

Induction

following

I’

The

during

during site

were

test

prior

the

(e.g.,

absorbent

discontinued

each

amount

site

Patch

supervised

adjacent

period. described

the

day,

hours

clinic

instructed

reaction

the

Monday, Page

the

Saturday

was

appropriate

patch

the

are

one

re-application.

each

reading,

sufficient

Induction

after

twenty-four

marked

discontinued It

pad

delay

(1)

treatment

was for

application,

was

Tuesday

removal

the

removal.

makeup

portion

Wednesday,

application.

marked

Induction.

remove

noted

observed

treatment

between

original

any

Phase,

ensure

cover

area. and

and

that

test

day

and

for

(3-

all

a a

Suimuary:

Results:

Evaluatkrn

Key:

0 TL45-24-2,

+ irritation

Under

tests.

a number

does

reactions

it (+)

interval.

With

The

reactive

was =

responses

results

not

one

ulceration

Marked the

Severe

Moderate

Barely

Mild

noted

reflect

during

individual.

visible

conditions

test

exception,

allergic

did

erythema

perceptible

erythemà,

each

post-challenge

erythema,

materials that

CIR

not

erythena, the

histoly skin

Panel

participant

contact

Subject

Induction

irdicate

covering

eaction

observations possible

Book

this

will

possible

sensitization.

possible

allergies,

spotty this

Page

study,

#25,

are

application.

Phase

most

prohibited

shared

clinically edema,

edema

appended

eiythema

Panel

presence

test

remained

and

the

panel.

material,

vesiculation,

the

moderate

#20050393,

test

significant

from

(Table

This

Laboratory’s

site

negative

Although

mild

participation

subject

1).

(2)

edema

bullae

potential

Page

exhibited

throughout

his

also

barely

opinion

and/or

medical

Body

reacted

future

for

perceptible

numerous

that

the

dermal

Lotion

profile

patch

test

is a

24*

22 20

ii Number

Subject

8 5

Supervised

hour

24*hr 0

0 0

follow-up

removal

0 0

—-———-—------—------hdueticPhase--—-—--—--—-—----—-—----

evaluation

1 0

0 0

Induction

1 0 0

0 0

and

0 0

--—-.-—4———DID

0 0

4 Challenge

CIR

1ndiviIua1

Panel 1.

Panel

Body

0’

0 0

Table

0 0 0

#20050393 Patch

Book

Lotion

Results

1 0

0 0

Page 0

0 0

6 TL45-24-2

NOT

0 0

COMPLETE

0 0

STUDY------—----——

Page

Virgin

0 0

24*hr

Site

Challenge

0 0

72br

0 ÷**

24*

Number

49 46

56 48 47 45 44 Subject

55 43 40

54 42

53 50 41

52 51

35 32

36 34 33

Supervised

24*hr

0 0

0 0 0

0 0 0 0 0 0

0 0

0 0 0

removal

0 0

0 0 0 0 0 0

0 0

0 0 0

0 0 0 0

0 0 0

-----——------——------Inductiot

0 0 0

0 0

0 0 0

0 0

0 0 0

0 0

Induction

0 0 0

0 0

0 0 o

0 0

0 0 0 0

0 0

3 ---—-—-—---P

and

0 0 0

0 0

0 0 0

0 0

4 Challenge

CIR

individual Panel

Panel

(cotinued)

ody

Tb1e

01 OI

0 0 0

0 o

ol ol

01 0

0 0 01

0 Oj

#20050393

Phase Patch

Book

Lotion

Results

NOT

0 0

o 0 0

0 o

0 0 0 0 0

Page 0 0 0 o

0 0

0 0 0

COMPLETE TL45-24-2

0 0

o 0 0

0 0

0 0 0 0

0 7

—-——----—--

0 0 0

0 0

0 0 0

0 0

STUDY-

0 0 0 0 0 0

0 0 0

0 0

Page

Virgin

0 0

24*hr72 0

0 0

0 0 0

Site

Challenge

0 0 0

0 0 0 0

0 0

0 hr 24* 29 28 21 Number 27 26 23 20 22 24 Subiect 17 16 19 18 12 14 11 10 13 15 6 9 $ 4 7 3 Supervised

24*hr

o o

o o o

o o 1 o

o o

o o o

removal o

0 0

0 o

0 o o 0 0 0

o 0 o 0

o 0 0 o 0 0 0 0 0 0 -— 1 of 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 — 1 e Induction 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 and •-DID -DID

—DTD

o o

o o o o

o o o

o o

o o 4 Induction Challengq Individual Panel CIR NOT NOT NOT (continued) Body Panel Table 0 0 O

0 0 0 0. o 0

0 0

O o

o o

o o o

o• o 5 #20050401 Phase—----—--—-—-— Patch — COMPLETE COMPLETE COMPLETE Book Lotion Results I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 Page TL45-24-2 59 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C) 0 C) 0 0 0 7 STUDY- STUDY STUDY- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 — 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 Page 8

Virgin

o 24*hr

o o

() o

o o

o o 0 Site Challenge

0 o

0 0

0 o 0

o o 72

o 0 o

0 0

0 o

o 0 o

0 o 0

0 0 o 0 hr

58 56

57 53

55 51 49

52 50 48

47 45 41

46 44

42 40 Number

Subject

38 36

34 32

33 31

Supervised

0 0

24*hr 0

0 0

removal

0 0

0 0 0

0 0

-----——------Inductio

0 0

0 0 0

0 0

Induction

0 0

o 0

0 0 0

0 0

000

and

0 0

0 o

0 0

4 Chal1eng

CIR

Individual

—--—i Panel

Panel

(cohtinued)

Body

0 0!

01 0

o! 0

0 0

01 0!

Table

#20050401

Patch Phase—

Book

Lotion

-1311)

DID

Results

0 0

Page

NOT NOT TL45-24-2

0 0

o 0

0 0

COMPLETE 0 0

COMPLETE 0

0 7

—-——

0 0

-DID

STUDY-—-—-——

TUDY

NOT

0 0

COMPLETE

Page

Virgin

0 0

0 24*hr

Site

STUDY-

Challenge

0 0

0 0 72

0 hr Page 10

liable 2 Panel 20050393

SubjectData

Subject Number Initials

LG 64 M 2 AK 45 F 3 RM 43 F 4 VT 39 F 5 KL 40 F 6 ND 36 M 7 BA 39 F 8 SF 53 F 9 MM 58 M 10 EG 55 F 11 AG 22 M 12 VV 16 M 13 HG 72 M 14 MA 16 F 15 VA 51 M 16 GD 49 F 17 CD 25 F 18 JO 54 F 19 MV 42 F 20 JM 24 F 21 CN 63 F 22 AB 62 M 23 3M 68 F 24 IV 72 F 25 AD 55 M 26 RD 70 M 27 LZ 43 F 28 FS 21 M 29 AD 53 F

CIR Panel Book Page 61 Coty, Inc.

C05-0729.0 1 Page 11

Table 2 (coitinued) Panel #20050393

Subject Data

Subject Number Initials —

30 GS 76 F 31 JD 21 M 32 VD 73 F 33 AD 69 M 34 RW 49 M 35 AL 77 F 36 JM 44 M 37 RC 20 M 38 MG 66 F 39 LS 40 F 40 JD 59 F 41 LV 72 F 42 GN 36 F 43 EM 29 F 44 AM 60 F 45 RF 35 F 46 HP 75 F 47 iF 70 F 48 TM 52 F 49 DC 64 F 50 MC 30 F 51 DC 18 F 52 MC 39 F 53 AC 57 F 54 EF 76 F 55 EV 65 F 56 CW 75 F

CIR Panel Book Page 62 Page 12

‘Fable2 (continued) Panel #20050401

Subjct Data

Subject Number Initials - Age Sex

I JV 66 F 2 LP 38 F 3 JP 34 F 4 LM 48 F 5 DD 49 F 6 CM 53 F 7 AS 58 F 8 PR 66 F 9 LM 35 F 10 CD 65 F 11 RT 21 F 12 LS 42 F 13 AP 57 F 14 JH 47 F 15 OS 26 F 16 SB 30 F 17 RB 57 F 18 CL 54 M 19 JC 31 F 20 RV 66 F 21 EM 42 F 22 BL 43 F 23 LD 35 F 24 JA 37 M 25 52 M 26 MA 51 F 27 MP 42 F 28 AL 46 F 29 AO 46 F

CIR Panel Book Page 63

Number

Subject

59 58

55 49

56 53

47 46

54 51 48 45

44 41

52 43 40

50 42

39 37

38 35

Initials

BR DS

RM ED

SM LL

JB JY CB

JS PF

JR JS

CIR

Panel

Subject (continued)

Panel

Täbie2 #20050401

Book Data

Page

Age

36 47

58 77

34 76 45

64 60

54 28

55 21

30 26

55 57 64

•

Sex

M M

M F

F F

F M

F F

Page 13 Assessment of the Eye Irritating Potential of a Cosmetic Product through Alternative Methods to the Draize Test

Test Product: Body Lotion Reference: TL45-24-2 10

Report Date: 27 September 2005 S° S o Report Ref: CTOXJO5161

CONCLUSION:

Taking into account the responses of the 3 alternative methods used we consider that the estimated Draize classification of the test product might be slightly irritant with Draize score which might range from 0 to 15

According to our experience and with respect to the type of product tested (skin care product), we consider that this product is as well tolerated as products belonging to the same category.

According to the estimated Draize score, the following warning may be proposed:

“No statement”

Olivie OUCET Pharm. ., D. Toxicotog UROTOX) Head of Skin esearch Opt

Skin Research Opt Cell Toxicology lab. p. 1126 CIR Panel Book Page 65

1- Skin 5-

TECHNICAL ALTERNATIVE

INTRODUCTION

RESULTS

FINAL REFERENCES

Research

3-2

3-1

2-2

2-4

2-3

4-3

4-2 3-3

4-1 ASSESSMENT

Opt

Testing

Product

NRR

The

Authentication

The

Data AND

HET-CAM

REC

INFORMATIONS

Neutral

Hens

Reconstituted

METHODS

assay

storage

assay

CONCLUSIONS

facilities

characteristics

Egg

Red

Test

USED

Release

the

TABLE human

CIR on

Cell

study the Panel

Toxicology

(NRR)

Epithelial

CONTENT Chorio-Allantoic

Book assay Page

lab. Culture

Membrane

(REC)

assay (HET-CAM) p. 21 26 1-INTRODUCTION

There is a need to evaluate the eye irritation potential of cosmetic products for purposes of consumer safety and reguatory requirements. For the time being, the Draize rabbit eye test is practically the nly method for determining ocular irritation, which is acceptable to various regulatory groups.

For the last few years, a clear desire, based on both ethical and scientific grounds. has been arising to replace the use of nimals, in cosmetic product testing. In that respect, a wide number of in vitroor ex vivoassays have been proposed worldwide as alternatives to the Draize test. Despite the tremendous efforts concentrated either by the Cosmetic Toiletries and Fragrances Association (CTFA) or the European Community (EC) and British Home Office (BHO), none of these alternative methods have been successfully validated. However, in some particular fields suct as eye irritation, it is clear that under the increasing pressure of consumer associations, regulatory agencies tend to be more and more favorable to the use of these methods for safety assessment. For instance, the French government recently registered the Hen’s Egg Test on Chorio-AllantoIc Membrane (HET-CAM) and the Neutral Red Release (NRR) assay as official test methods for determining the irritating potential of cosmetic products (Journal Officiel de Ia République Française, 26112196,Annexe IV;30/12199, Annexe VI).

The aim of this study was to predict the eye irritationpotential of formulated products. For that purpose, we developed a particular in vitro>>approach, which combines several alternative methods. Indeed, many international studies have clearly demonstrated the interest of combining at least 2 or 3 alternative methods when assessing eye irritation through in vitrotests.

Skin Research Dpi Cell Tox,cology lab. P. 3/26

CIR Panel Book Page 67 Taking into account both the results obtained during the last international validation studies (Balls et a!., 1995; Gettings et aL, 1990; Gettings et at. 1994; Gettings et aL, 1996) and the recent advances in te use of in vitro models we selected as ()alternative methods the 3 following in vitrotests:

- the Neutral Red Release (NRR) assay • the Hens Egg Test on the ChorioAllaritoic Membrane (HET-CAM) - the Reconstituted Human Epithelial Culture (REC) assay

The combination of these different in vitro methods allows the assessment of different end-points and thus explores various types of mechanisms (cytotoxicity, acute vascular effect, toxicokinetic, transepithelial absorption,...) which are generally considered as taking part in the eye irritation phenomena (Rougier et al., 1994).

The conclusion of the study results from a global assessment, systematically based on the responses of the 3 methods used, since none single alternative method can predict eye irritation with a sufficient level of safety.

Skin Research Dpt — CellToxicologylab. p4/26

CIR Panel Book Page 68 2- TECHNICAL INFORMATIONS

2-1 Product characteristics

The product Body Lotion, ref. TL45-24-2, was received from the sponsor Morris Plains (USA) on the 16 August 2005. The test product, identified as a skin care froduct, is a white milk, having a pH of 7.2 at 22.2°C.

Upon receipt, it was stored at room temperature in the Cell Toxicology Laboratory. An aliquot of the test product was stored mla specific room of the Skin Research Dpt. According to the internal Skin Research. procedures, it will be kept there for a minimum period of 3 years.

2-2 Testing facilities

The test were performed in the Cell Toxicology Laboratory of Skin Research Dpt -

2-3 Data storage

Allthe data relative to the study will be stored in the premises of the Skin Research Dpt for a period of 10 years.

Skin Research Opt Cell Toxicølogy lab, 5 126 CIR Panel Book Page 69

Skin

during

data

We,

Boris

has

protocol.

standard

Investigators been 2-4

the the

Research

been

Authentication

the

presented

MERLIN,

undersigned undersigned

carried

this undersigned

performed

protocol study.

Opt

Tèehfàal

out

cr4ER

Cell

Technical Vincent

Cell

Technical

the

this

Toxicology

Mylène

Olivier

Cell

under

the

under

Carine

COMZ

document

Toxicology

Investigator

oremises

Igvator

Investigators,

DOUCET,

the

LANVIN,

the

LINOSSIER,

Dpt

study

supervising responsibility CIR

Cell

are

Laboratory. Reponsible

Panel Director

Toxicology

the

certify accurate

Book

Connne in

that accordance Page

lab. of

—

the

for

Tec4&I)rrestigator

Technical

Boris

CorirN1OU

Cell

C5.e1foIogy

Olivie Head

Toxicolog

Head

all Myléne

reflection

THILLQU,

Study,

the

arch

MERLIN

Study, observations

—

Cell

OUCET

with

Skin

certify

LANVIN

Assistant

Inve

Vincent

Toxcoiogy

our

certify

the

UROTOX)

esearch

Dpi

that

Pharim

internal

results

and

tor

and

that

this

COMTE

Opt

Dpt

numerical

study

this

Technical

obtained standard

p. study

and has

26 D. 3- ALTERNATPIE METHODS USED

3-1 The Neutral Red Release (NRR) assay

Principle of the method The NRR assay with rabbit cornea cells (SIRC) is a short-term monolayer culture test system in which cells are first exposed to Neutral Red dye (NR) then to the test material. According to the toxicity of the product, the cells are damaged and release their neutral red dye. The neutral red contained in surviving cells was extract with a revelation solution and spectrophotometrically measured. The test product concentration that gives rise to the release of 50% NR dye 50(NR is used as endpoint to reflect the cytotoxicity of the test product. ) Two stages can be necessary to asses$ the 50NR of a test product. The first Stage allows the estimation of the 50NR wheéas the second stage permit to accurately assess the final score.

Materials Chemicals: Sodium Dodecyl Sulfate (SDS) and Sodium Chloride (NaCI) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Neutral red dye was supplied by Fluka AG (Buchs, CH). Modified Eagle’s Medium (MEM), fcetal calf serum, antibiotics (penicillin/streptomycin 5000Ul/5000jgIml and fungizon amphotericin B 250jglmI), MEM Non Essential Amino Acids (NEAA) and Phosphate- Buffered Saline (PBS) were supplied by lnvitrogen (Cergy-Pontoise, France). Before using, ftal calf serum was maintained in a bain-marie at 56°C during 30 minutes in order to obtain a modified” fcatal calf serUm.

Rabbit cornea SIRC cells: Rabbit cornea fibroblasts SIRC (ATCC n°CCL6O) were bought in the United States at ATCC (American Type Culture Collection Rockville, Maryland, USA) through a French supplier (CERDIC, Sophia Antipolis, France). Cells were cultured according to the internal procedures of our laboratory for freezing, unfreezing and subculturing. Briefly, cells were maintained in medium MEM supplemented with 2% antibiotics and 1% MEM Non Essential Amino Acids. This completed medium was extemporaneously supplemented with 10% of ‘rnodified” fcatal calf serum. Cells were incubated in humidified atmosphere at 37°C, 5% .2CO Experimental procedure Cell seeding: For treatment, cells were seeded in all the wells of 24-well plates. The plates were incubated for 24 hours at 37°C, 5% .2CO

Skin Research Dpt Cell Toxicologyb p. 7126

CIR Panel Book Page 71 1

Skin

throughout

same The

Test

positive rinsed

removed

According

stage.

0%;

Test

product,

After

lipophilic Co 2 .

added Application

principle

>37anci<46

33and37

>27and<33

23arid27 >17

Research >6and<13

plate

4and6

5%;

Stage

product

NR5O

product

time

incubation,

with

The to

and

control),

the

15%;

of and

substance

each

was

contact

principle

2 (%)

n°1

the of

Dpt

<23 dilutions

two

application:

dilutions:

this

the

25%;

maintained

well

colorimetric

Table

wells

each

preliminary

neutral

time.

PBS

35%

ml (w/w).

—

complete

ware

given

the

According

well

Dilutions

and

the

After

and

red

plates.

neutral

test

performed

During

was

room

500

plate.

50%.

40 0.1

351 20

dye:

results,

the

incubation

culture Slight

described The CIR

Cell

following

rinsed

temperature

The

the red

Aftera

of the Panel

Toxicology

cytbtoxIcity

plates

extemporaneously

some

medium

first

ech

selected

solution

[iIutions NRR

. physico-chemical with

Book

for

55-second

stage,

Table

were

test

dilutions

Borenfreund

assay 3 Page

lab.

PBS.

was

Stagen°2

for

test,

hours,

product to

Unnecessary

incubated the

25 20

35 30

the

was

added 1

The

centrifuged

contact

were

minutes.

product

second

the

tested

carried

dilution

and

plate

characteristics in

- neutral

—

selected

for

each

(or

Puerner

stage

was

hydrophilic

Then Stag2

—____

out

was

were

before

hours

red

well

tested

according

seconds

for

each

gently

(1984,

added

solution

15 the

using,

the

(wtv)

37°C,

diluted

well the

second

stirring

for

1985).

plate.

8126

was

test

the

at a Revelation of the cytotoxicity: An ethanol/acetic acid/distilled water solution was then added to each well. The plates were gently stirring during about 15 minutes until having an homogeneous coloration. 200 .iI of the resulting solutions were put, in dupilcate, in the wells of a 96-well plate.

Reading: The optical densities (O.D.) were read at 540 nm by using a multi-well spectrophotometer. The ethanol/acetic acid/distilled water solution served as ‘blank’,

Control solution application: The positive control (Sodium Dodecyl Sulfate = SDS) was tested diluted at 0%; 0.01%: 0.05%; 0.2% and 0.25%. The dilutions were performed in 0.9% sodium chloride sokition. The dilution 0% which represents the negative control was applied to the cells during 55 seconds whereas the dilutions 0.01%; 0.05%; 0.2% and 0.25% were applied during only 25 seconds.

Test scoring: Data were expressed as a percentage of cytotoxicity, compared to the negative control (dilution 0%). The 50NR was calculated by interpolation from the curve representing the percentage of viability versus the concentration of test product.

The cytotoxicity of the product was obtained from the 50NR according to the scale presented in Table 2.

Table 2: Cytotoxicity scale from the NR endpoint

% of death 50NR (%) obSerVed at the Clssiflcation COTY Conclusion dilution_50% 20 Negligible cytotoxicity (PNI) > 50 Slightly cytotoxic (SI) > 20 and < 50 Not very importantcytotoxicity (SI) ‘ 25 and 50 Moderate cytotoxicity (MI) Moderately cytotoxic (MI) 25 Important Cytotoxicity (I) Cytotoxic (I)

The conforrrnty of the study was checked bY using a positive control. According to the internal procedure, this study complied if the 50NR of the positive control ranged from 0.127% to 0.185%.

Skin Research Opt CellToxicologylab. p. 9126

CIR Panel Book Page 73 3-2 The Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM)

Principle of the method The Het-Cam is an in vitro method used to evaluate the irritant potential of a test material (J.O.R.F., 26/12196, Annexe IV). The test procedure is based on the assessment by a trained person of the immediate effects following application of test product to the chorioallantoic membraneof 10-day-old fertile eggs. The determination of the Net-Cam score, according to the scale described by Luepke (1985, 1986) allows the assessment of the irritating potential of the test product.

Materials Chemicals: Sodium Dodecyl Sulfate (SDS) and Sodium Chloride (NaCl) were purchased from Sigma Chemical Co. :(St Louis, MO, USA). Sterilized water for injections (Wi)was purchased from Aguettant Laboratory (Lyon, France).

Hen’s eggs: Fresh fertile White Legom hen’s eggs, weighing 50 -65 g, were supplied by INRA (Tours, France).

Experimental procedure Upon their arrival, all the defective eggs and eggs which weight is not ranged from 50 to 65 g, were eliminated. The hen’s eggs were incubated at 15°C during at least 48 hours. Then, they were placed, on their long axis, in a rotating incubator under a temperature of 37.5°C ± 1°C; 60% ± 5% relative humidity (Union FrancoSuisse, Evreux, France) for 10 days. The eggshell was removed around the airspace. After a 5 ml saline solution (NaCl 0.9% with distilled water) application and the removal of the inner shell membrane, the vascular chorioallantoic membrane (CAM) was exposed to the air.

Test product application: Four eggs are treated with 0.3 ml of the product, tested neat or diluted according to the type of product. Previously maintained at a temperature of 37.5°C, the test product was applied onto the surface of the CAM. After a 20-second contact, the membrane was gently rinsed off by using 5 ml (10 ml or more if necessary) of saline solution kept at 37.5°C.

Investigator observations: Observations were achieved by using a specific lamp KL1500 electronic (SCHOTT, France) emitting a cold and white light. Blood vessels and albumen were continuously observed by a trained person for a 5-minute period. Irritant effects, such as hyperhaemia, haemorrhage and coagulation (opacity and/or thrombosis), were scored according to their occurrence within the test period.

Skin Research Dpi CellToxicology lab. p. 10126

CIR Panel Book Page 74 Control solutions application: Two eggs treated with a sodium dodecyl sulfate solution in sterilized water for injections (SDS solution) served as positive control while at least 2 eggs treated with saline solution were used as negative controL Ifthe test product was diluted in mineral oil, 2 eggs were treated with this lipophilic diluant to check its Het-Cam score.

The irritating effect of the test product (if any) was quantified according to the scoring system described in the French regulaticn (J.O.R.F., 26(12196, Annexe IV)presented in Table 3.

Table 3: Hat-Cam scoring syøtem according to Luepke’s scale

Vascular Time (t) effect Q

Hyperhaemia 5 3 1

Haemorrhage 7 5 3 Coagulation 9 7 5

For each parameter (Hyperhaemia, Haemorrhage, Coagulation) the individual scores obtained from the 4 eggs were averaged. The sum of these 3 values gave the so called “Het-Cam score” of the test product on a scale ranging from 0 to 21.

The magnitude of the eye irritating potential of the test product was then calculated according to the classification developed by Luepke (1985, 1986) and described in the French regulations (J.O.R.F., 26/12/90, Annexe IV),see Table 4.

Table 4: Test product classification

Hot-Cam score CIasslIcatIofl COW Conclusion

Score < 1 Practicallynon irritant . I Slightly irntant (SI) 1 Score < 5 SlIghtly imtant 5 Score < 9 Moderately irritant Moderately irritant (Ml) Score 9 Irritant Irritant (I)

The conformity of the study is checked by using controls. According to the internal procedure, the study complied if the Het-Cam score for the positive control ranged from 15 to 18 and the Hat-Cam score for the negative control ranged from 0 to 1.

Skin Research Dpi Cell Toxology lab. 11/ 26 . CIR Panel Book Page 75 3-3 The Reconstituted human Epithellal Culture (REC) assay

Principle of the method The REC assay is a cytotoxicity test based on a time course approach. The formulated product is applied onto three-dimensional reconstituted human epithelial cultures, having the feature of the epithelial part of the cornea. The quantification of the test product cytotoxicity is performed through a colorimetric assay: the MIT test (Mosmann, 1983). The determination of a simplified mean cytotoxicity index (SMCI) is used to quantify the time course toxicity for the applied substance, according to the procedure described by Doucet et al. (198).

Materials Chemicals: Sodium Dodecyl Sulfate (SDS) and 3-(4, 5-dimethylthiazol-2-yl) 2, 5- diphenyltetrazolium bromide (MiT) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Isopropanol was supplied by Carlo Erba (Milan, Italy) and Phosphate—Buffered Saline (PBS) by Invitrogen (Cergy-Pontoise, France). Saline solution and modified culture medium (MCDB 153) were supplied by SkinEthic Laboratories (Nice, France).

Reconstituted human Epithelial Cultures (REC): Reconstituted human epithelial cultures were supplied by SkinEthic Laboratories (Nice, France). They were obtained by culturing transformed human keratinocytes (TR146 cell line) derived from squamous carcinoma (Regnier at al., 1987; Rupniak et al., 1985).

Experimental procedure Test product application: The product wa tested neat or diluted according to the type of product. Test sample was directly applied onto the apical surface of the epithelial culture. Product was gently spread with a brush. Cultures were treated with the test product in duplicate. The cultures were transferred to a 24-well culture dish, each well containing fresh medium MCDB 15, They were incubated at 37°C, 25%C0 / 95% air atmosphere for 1 hour, 3 hours and 24 hours. After each exposure time, cultures were washed with PBS, and the MiT assay (Mosmann, 1983) was performed. After incubation in MiT reagent, the formazari crystals were extracted by isopropariol. Optical densities were read at 540 nm, by using a spectrophotometer (isopropanol served as blank”).

Skin Research Dpt - Cell Toxicology lab. p. 12126 — CIR Panel Book Page 76 Control solutions application: For each time, 2 cultures treated with a sodium dodecyl sulfate solution in saline (SDS solution) served as positive control while 2 cultures treated with saline solution were used as negative control. If the test product was diluted in mineral oil, 2 cultures treated with this lipophilic diluant were used also as negative control.

The results were expressed as a percentage of cytotoxicity compared with the negative control. The time course of toxicity for the applied product was expressed as a cumulative simplified mean cytetoxicity index (SMCI) calculated over 24 hours, as follows:

(%cyt.lh) + (%cyt.3h/3) + (%cyt. 24hl24) SMCI = 3

with: %cyt. lh = % of cytDtoxicityof the test product after 1 hour. %cyt. 3h % of cytotoxicity of the test product after 3 hours. %cyt. 24h = % of cytotoxicity of the test product after 24 hours.

The cytotoxicity of the product was determined according to the classification presented in Table 5.

Table 5: Cytotoxicity scale from the SMCI endpoint

The conformity of the study is checked y using a positive control, According to the internal procedure, the study compIle only when the SMCI of the positive control lies within the confidence internal range.

The product classification in terms of eye irritation results from a global assessment based on the responses of the 3 in vitroitiethods used. The following Table 6 reflects this multi-technical approach and gives information about the proposed safety classification of the test product. This latter is extrapolated from the results of the 3 alternative methods and presented as an estimated Draize classification. Based on this, an attempt is made for issuing somespecific US warnings,

Skin Research Dpt Cell Tocology lab. p. 13/26

CIR Panel Book Page 77 Table 6 : Table of concordance between the In vitro scores and the estimated Draize classification for care 3 skin products, sun care products, alcoholic products and make-up products.

Test Method In Vitro Threshold Estimated European Draize US Warning Classification

REC Assay Score <7.5 Slightly lrritant CIR Hot-Cam Score <9 (Draize score :0- 15) No statement NRR Assay Score >50 Panel o x -______—.-----__-—.------_------—--—- Book 8 REC Assay 7.5 <= Score < 15 Moderately Irritant Avoid contact with eyes. Page

Hot-Cam Score < 9 (Draize score: 15.1 - 30) If contact occurs, flush with water. NRR Assay 25< Score <= 50 78

REC Assay 15 < Score < 24.5 Irritant This product may cause irritation. Avoid contact with eyes. Hot-Cam Score >= 9 (Draize score : 30.1 50) If contact occurs, flush with plenty of water. NRR Assay Score <= 25 It irritation persists, consult a physician.

REC Assay Score >= 24.5 Strongly Irritant Determination of appropriate warning statements.

Hot-Cam Score > 9 (Draize score : 50.1 - 110) Suitability to market the product has to be considered. NRR Assay Score <= 25 4- RESULTS AND CONCLUSIONØ

4-1 NRR assay

4-1-1 Summary

The Neutral Red Release (NRR) assab’ conducted on rabbit cornea fibrobiasts SiRC is an in vitro method currently used to assess the cytotoxicfty of a test product after a short contact time of the test sibstance with the cells by measuring the neutral red release from pre-loaded cells (Brantom at aL, 1997; Reader et aL, 1989). The cytotocity is revealed by the cncentration of test product (NR) which inhibited of 50% the cell survival and grov.th. n this study, the procedure used was adpted from the protocol described in French regulation as official method for the ssessment of the irritating potential of formulated cosmetic products (JO.RR. 30112199,Annexe VI). Under the experimental conditions used,an 5NR supehor to 50% was obtained fo the product Body Lotion, ref. TL45-24-2. From this result. the test product was considered slightly cytotoic.

4-1-2 Results

Stage 1 Th firct f thic iirv wc inititd in the oremises of the 19 September 2005 and was completed on the 20 Septernber2uu.

The test product Body Lotiorliwas tested diluted at 0%: 5%: 5%: 25%: 35% and 50% in saline solution.

The optical densities and the percentags of viability obtained for the test product and the positive control are presented in Tables 7 and 8 respectie’. The graphic assessment of the NR for the test product and the positive control were presented in Fig. 1 and 2 respectively.

Skin ResearchDp CellToxipoogy lab. p. 15126

CIR Panel Book Page 79

Skin

Table

Product

Research

Cytotoxicity

and

C.)

: 40:

>60,

the

Well

We113

Well

Mean

100

Optical

dilution

Dpt (O.D.)

negative

(%)

Fig.

densities

(%)

and

control

cytotoxicity Assessment

—

0.007

0.952

0.962

0.951

0.944

0.951

. 0.00

(O.D.)

and

(representing Product -

CIR

the

Cell

and

0.021

0,97 0.99

0.962

0.00

NR>50%

of (%)

Panel

Toxicology

5 negative -

cytotoxicity

the

dilutions obtained

Book

NR 50

the

0.034

0.988

0.964

1.012 0.00

Page

lab.

0% control -

for

(%)

(%) for

dilution) the

•‘._

the

obtained

test

0.021

0.977

0.962

0.992

0.00 25 -

test

during

product product

for

0.013

0.922

0,913

0.931

3.15

the

35 the -

stage

test

product

0.001

21.90 0.744

0.744

0.743

n°1

p. 50 -

- 16/26 Table 8: Optical densities (O.D.) and cytotoxicity (%)obtained for the positive control and the negative control (representing the 0% dilution) during the stage n91

(O.D.) and cytotoxicity (%)obtained for the positive control and the negative control

Product dilution (%) 0 O.01 0.05 0.2 0.25

Well 1 1.058 i.083 0.861 0.449 0.246 Wel12 1.075 1.082 0.770 0.410 0.229

We113 1.059 - - -

Well4 1.052 - - - - Mean 1.061 1.083 0.816 0.430 0.238 SD 0.010 0.001 0.064 0.028 0.012

Cytotoxicity (%) 0.00 0.00 23.14 59.52 77.62

100

80 i

60 C.) x 0 40 0 20

0 0 0.05 0.1 0.15 0,2 0.25 Product dilutions(%)

Fig. 2: Assessment of the 50NR for the positive control

I NRO.160%

Skin Research Opt Cell Toxicology lab. p. 17/26

CIR Panel Book Page 81 Stage 2

According to table 2 and to the results obtained during the first stage of this study, it was not necessary to perform the stage 2

4-1-3 Conclusion

Under the experimental conditions use, the 50NR of the product Body Lotion was superior to 50%. Frm this result, the test product may be considered slightly cytotoxic.

According to our experience and with respect to the type of product tested (skin care product), we consider that this product is as well tolerated as products belonging to the same category.

SkinResearchDpi cell Toxióology lab. p. 18126 CIR Panel Book Page 82 4-2 HET-CAM

4-2-1 Summary

The Hen’s Egg Test-Chorioallantoic Melnbrane (Het-Cam) is an in vitro method currently used to assess the eye irritating potential of a test product (Balls et aL, 1995; Gettings et al., 1994). The test procedure is based on the evaluation of immediate effects following application of:the test substance onto the surface of the chorloallantoic membrane of 10-day-old feitilehen’s eggs. In this study, the protocol used was adapted from Luepke (Luepke, 1985; Luepke and Kemper, 1986) and was performed according to the method described by the French regulation (JD.R.F., 26112196,Annexe IV).The Het-Cam score of the product Body Lotion, ref. TL45-24-2, was determined after application to the chorioallantoic membrane of 0.3 ml of neat test material. Under the experimental conditions used, the Het-Cam score of the test product was 7.0. Consequently, this product may be classified as moderately irritant when applied neat to the hen’s egg chorioallantoIc membrane.

4-2-2 Results

This study was initiated in the oremises of the completed on the 13 September 2005.

Date of arrival of the eggs: 30 August 2005 Date of incubation at 15°C: 30 August 2005 Date of incubation at 37,5°C: 01-02 September 2005

The individual results obtained for the test product, tested neat, the positive and the negative controls are respectively presented in Tables 9, 10 and 11.

Skin Research Opt Cet TxcØlogy lab, p 19126 CIR Panel Book Page 83 Test product: Body Lotion ref. TL4S-24-2

Table 9: Individual egg scores obtained for the test product

0 0 o 0

Mean Score = 7.0 Classification: Moderate’y irritant

Remarks: A very slight haemorrhage re ction was observed to tre second treated egg whereas slight ones were observed t the others treated eggs

Table 10: Individual egg obtained for the positive control

Mean Score = 16.0

Negative cor,troi: saline solution

Table 11: Individual egg score obtained for the negative control

Mean 0 0 a

Mean Score 0

Skin Research Opt Cell To cologylab, p. 20126

CIR Panel Book Page 84 4-23 Conclusion

Under the experimental conditions usd, the Het-Cam score of the product Body Lotion was 7.0. Frcn this result, the test product may be considered moderately irritant when app!ied neat to the hen’s egg CAM.

According to our experience and with respect to the type of product tested (skin care product), we consider that this product is as well tolerated as products belonging to the same category.

Skin Research Opt C&I Thxiogy lab p21/26 CIR Panel Book Page 85 -r

4-3 REC assay

4-3-1 Summary

The Reconstituted human Epitheliat Culture (REC) assay is an in vitro method used to assess the cytotoxicity of a test product through a three-dimensional epithelial modal. After application of the test product, the cytotoxicity is evaluated by a rapid colorimetric test: the MU test, according to the protocol described by Mosmann (1983). In this study, the protocol used was adapted from the test procedure described by Doucet at al. (1998). The time course of toxicity for the applied product, expressed as a cumulative simplified mean cytotoxicity index (SMCI) was used as endpoint. Under the experimental conditions used, a SMCI of 1.87 was calculated. From this result, the product ody Lotion. ref. TL45-24-2, was considered slightly cytotoxic when applied neat onto the reconstituted epithelial cultures.

4-3-2 Results

This study w inititd in th nrmiscs nf the I

on the 15 September 2005.

Cell cultures Date of arrival: 14 September 2005 Date of expiry: 23 September 2005 Batch n°: 05022B0902 Age of the culture: 7 days

The REC score, expressed as a simplified mean cytotoxicity index (SMCI), obtained for the product, tested neat is presented irilTable 12.

Table 12: REC score (SMCI) and cytotoxic potential obtained for the test product

CIR Panel Book Page 86 4-33 Conclusion

Under the experimental conditions used, he SMCI of the test product Body Lotion was 1.87. From this result, the test product may be considered slightly cytotoxic when applied neat onto reconstiti ted human epithelial cultures.

According to our experience and with respect to the type of product tested (skin care product), we consider that this product is as well tolerated as products belonging to the same category.

Skin Research Opt Cell ToxiIogy lab. p. 231 2 CIR Panel Book Page 87 5- FINAL ASSESSMENT

The aim of this study was to predict the ee irritation potential of formulated products by using a particular <>approach, which combines several alternative methods:

- the Neutral Red Release (NRR) assay the Hen’s Egg Test on the Chorio.AUantoic Membrane (HET-CAM) the Reconstituted Human Epithelial Culture (REC) assay

For the tested product, Body Lotion, ref. TL45-24-2, the following results and classifications were obtained:

NRR assay: 50NR > 50% slightly cytotoxic HET-CAM: Score = 7.0 moderately irritant REC assay: SMCI 1.87 slightly cytotoxic

Taking into account the responses of these 3 methods, we consider that the estimated Draize classification of the test product might be slightly irritant with Draize score, which might range from 0 to 15.

According to our experience and with respect to the type of product tested (skin care product), we consider that this product is as well tolerated as products belonging to the same category.

According to the estimated Draize score, the following iS warning may be proposed:

No sta1ement”

Skin Research Dpt CeKThxicØogy lab. CIR Panel Book Page 88 6- REFERENCES

- BALLS, M, BOTHAM, P.A., BRUNER, L.H, and SPIELMANN, H. (1995). The ECIHOInternational Validation Study on Alternatives to the Draize Eye Irritation Test. Toxicology In Vitro. 9, 871-929.

- BORENFRE1JND, E. and PUERNER, J.A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Methods. 9, 7-9.

- BORENFREUND, E. and PUERNER, J.A. (1985). Toxicity determined in vitro by morphological alteration and neutral red absorption. Toxicol Letters. 24, 119-124.

- BRANTOM P.G., BRUNER L.H., CH4\MBERLAIN M. et a), (1997). A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test (1997). Toxicology in vilo 11, 141-179.

- DOUCET, 0., LANVIN, M. and ZASIROW, L. (1998). A new in vitro human epithelial model for assessing the eye. irritating potential of formulated cosmetic products. In Vitro & Molecular Toxicology 11, 273-283.

- GETTINGS, S. D, DIPASQUALE, L. C., BAGLEY, 0. M., et al. (1990). The CTFA evaluation of alternatives program: an evaluation of in vitroalternatives to the Draize primary eye irritation test (phase I) hdro-alcoholic formulations: a preliminary communication. In Vitro Toxicology. 3, 293-302.

- GETTINGS, S. D., DIPASQUALE, L. Ci., BAGLEY, D. M., et al. (1994). The CTFA evaluation of alternatives program: an evaluation of in vitroalternatives to the Draize primary eye irritation test (phase II) p11/water emulsions. Food and Chemical Toxicology. 32, 943-976

- GETTINGS, S. D., LORDO R. A., HINTZE K. L., et al. (1996). The CTFA evaluation of alternatives program: an evaluation df in vitroalternatives to the Draize primary eye irritation test (phase ill) surfactant-based formulations. Food and Chemical Toxicology. 34, 79-117.

Skin Research Dpt CellThxiology lab. * p. 25! 26

CIR Panel Book Page 89 - JOURNAL OFFICIEL DE LA REPUBLIQUE FRANcAISE. Arrêté du 29 Novembre 1996, J.O. du 26 Décembre 1996. Annexe IV. Méthode Officielle d’Evaluation dii Potential Irritant par Application sur Ia Membrane Chorio-AllantoIdienne de l’oeuf de poule.

- JOURNAL OFFICIEL DE LA REPUBL1UE FRANçAIsE. Arrêté dii 27 Décembre 1999. J.O.R.F. du 30 Décembre 1999. Annexe VI. Méthode officielle devaluation dii potentiel irritant par application directe sur monocouche do fibroblastes de cornée de lapin par Ia méthode do relargage dii rouge neutre.

- LUEPKE, NP. (1985). Hen’s egg chorioallantoic membrane test for irritation potential. Food and Chemical Toxicology 23, 287-291.

- LUEPKE, N.P. and KEMPER, RH. (1986). Het-Cam test: an alternative to the Draize eye test. Food and Chemical Toxicology. 24, 495-496.

- MOSMANN, T. (1983). Rapid colorirnetric assay for cellular growth survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, 55-63.

- READER S.J., BLACKWELL V., OHARA R., CLOTHIER R., GRIFFIN G. and BALLS M. (1989). A vital dye release method for assaying the short-term cytotoxic effects of chemicals and formulations. ATLA 17. 28-33.

- REGNIER, M., DESBAS, C., BAILLY,C., and DARMON, M. (1987). Differentiation of normal and tumoral human keratinocytes cultured on dermis. Reconstruction of either normal or tumoral architecture. In Vitro Cell Dev, Bio. 24, 625-632.

- ROUGIER, A., COTTIN, M., DE SILVA, 0., et al. (1994). The use of in vitro methods in the ocular irritation assessment of cosmetic products. Toxicology In Vitro. 8, 893-905.

- RUPNIAK, H.T., ROWLATT, C., LANE, E.B., et at. (1985). Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck. J. NatI. Cancer Inst. 75, 621-635.

Skin Dpt Research CellToxicologylab, p. 26)26

CIR Panel Book Page 90 VCRP Data for Alkyl Benzoates Ingredient Use Category No. of Uses C12-15 ALKYL BENZOATE 01B - Baby Lotions, Oils, Powders, and Creams 9 C12-15 ALKYL BENZOATE 03A - Eyebrow Pencil 1 C12-15 ALKYL BENZOATE 03C - Eye Shadow 28 C12-15 ALKYL BENZOATE 03D - Eye Lotion 20 C12-15 ALKYL BENZOATE 03E - Eye Makeup Remover 1 C12-15 ALKYL BENZOATE 03F - Mascara 1 C12-15 ALKYL BENZOATE 03G - Other Eye Makeup Preparations 18 C12-15 ALKYL BENZOATE 04B - Perfumes 1 C12-15 ALKYL BENZOATE 04E - Other Fragrance Preparation 13 C12-15 ALKYL BENZOATE 05A - Hair Conditioner 12 C12-15 ALKYL BENZOATE 05B - Hair Spray (aerosol fixatives) 6 C12-15 ALKYL BENZOATE 05C - Hair Straighteners 3 C12-15 ALKYL BENZOATE 05F - Shampoos (non-coloring) 2 C12-15 ALKYL BENZOATE 05G - Tonics, Dressings, and Other Hair Grooming Aids 42 C12-15 ALKYL BENZOATE 05I - Other Hair Preparations 33 C12-15 ALKYL BENZOATE 07A - Blushers (all types) 16 C12-15 ALKYL BENZOATE 07B - Face Powders 26 C12-15 ALKYL BENZOATE 07C - Foundations 42 C12-15 ALKYL BENZOATE 07D - Leg and Body Paints 1 C12-15 ALKYL BENZOATE 07E - Lipstick 66 C12-15 ALKYL BENZOATE 07F - Makeup Bases 7 C12-15 ALKYL BENZOATE 07G - Rouges 1 C12-15 ALKYL BENZOATE 07I - Other Makeup Preparations 17 C12-15 ALKYL BENZOATE 08B - CuticleCuti cle SoftenersSofteners 1 C12-15 ALKYL BENZOATE 08C - Nail Creams and Lotions 1 C12-15 ALKYL BENZOATE 10A - Bath Soaps and Detergents 11 C12-15 ALKYL BENZOATE 10B - Deodorants (underarm) 6 C12-15 ALKYL BENZOATE 10E - Other Personal Cleanliness Products 1 C12-15 ALKYL BENZOATE 11A - Aftershave Lotion 15 C12-15 ALKYL BENZOATE 11D - Preshave Lotions (all types) 1 C12-15 ALKYL BENZOATE 11E - Shaving Cream 5 C12-15 ALKYL BENZOATE 11G - Other Shaving Preparation Products 3 C12-15 ALKYL BENZOATE 12A - Cleansing 30 C12-15 ALKYL BENZOATE 12C - Face and Neck (exc shave) 91 C12-15 ALKYL BENZOATE 12D - Body and Hand (exc shave) 102 C12-15 ALKYL BENZOATE 12E - Foot Powders and Sprays 5 C12-15 ALKYL BENZOATE 12F - Moisturizing 174 C12-15 ALKYL BENZOATE 12G - Night 35 C12-15 ALKYL BENZOATE 12H - Paste Masks (mud packs) 11 C12-15 ALKYL BENZOATE 12I - Skin Fresheners 3 C12-15 ALKYL BENZOATE 12J - Other Skin Care Preps 48 C12-15 ALKYL BENZOATE 13A - Suntan Gels, Creams, and Liquids 17 C12-15 ALKYL BENZOATE 13B - Indoor Tanning Preparations 36 C12-15 ALKYL BENZOATE 13C - Other Suntan Preparations 9

CIR Panel Book Page 91 C16-17 ALKYL BENZOATE 10A - Bath Soaps and Detergents 2

ISOSTEARYL BENZOATE 12C - Face and Neck (exc shave) 1

STEARYL BENZOATE 01B - Baby Lotions, Oils, Powders, and Creams 2 STEARYL BENZOATE 07C - Foundations 1

CIR Panel Book Page 92 PersonalCare’ ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

TO: F. Alan Andersen, Ph.D. Director - COSMETIC INGREDIENT REVIEW (CIR)

FROM: John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel

DATE: July 26, 2010

SUBJECT: Comments on the Scientific Literature Review on Alkyl Benzoates

The following 10 substances were included in an HPV submission to EPA.. It is not clear if this submission (found at http ://www .epa.gov!chemrtklpubs/summaries/benzylde/c 13450tc.htm) was reviewed for relevant information. Benzaldehyde p-Methoxybenzaldehyde m-Methoxy-p-hydroxybenzaldehyde Benzyl acetate Benzyl benzoate Methyl benzoate Methyl p-methylbenzoate Methyl 2-hydroxybenzoate Pentyl 2-hydroxybenzoate Benzyl 2-hydroxybenzoate p.2 - As reference 12 was published in 2004, the meaning of “began a couple of years ago” is not clear, and will become less clear after the CIR report is published. p.2 - It would be helpful if the product categories in which use was reported in the Council survey were also included in the text. Rather than stating that no uses were reported for the rest of the ingredients, it would be helpful if the names of the ingredients for which no uses were reported were stated. p.2 - The statement about the permitted uses of methanol in the EU suggests that the EU regulations were checked for all the ingredients in the report. Please see EU Annex VI entry la for permitted uses of Methyl Benzoate, Ethyl Benzoate, Propyl Benzoate and Butyl Benzoate as preservatives. p.2 - As the alcohols are not included in the report. The information about alcohols can be deleted from the Non-Cosmetic Use section. If this information is left in the report, a reference should be added for the use of isopropyl alcohol as a topical antimicrobial. p.3 - It is not clear why the discussion regarding metabolism of acetates is included in this report on benzoates.

11011 7th Street, N.W., Suite 3O0 Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personalcarecouncil.org

CIR Panel Book Page 93 p.3 - If Kirk-Othmer (reference 23) cited any primary references regarding the dermal penetration of benzoic esters, the primary references should be obtained and cited. p.3 Please define 50NI p.3 - How many rabbits. were used in the dermal 50LD study of Methyl Benzoate. p.3 - Please provide the species used in the Benzoic Acid and Sodium Benzoate studies. p.3 - How many rats were used in the chronic study of Methyl Benzoate. Although this study is under the Methyl Benzoate subheading, and the text says “Methyl benzoate”, the title of the study (reference 33) in the reference section indicates the study was about Sodium Benzoate. Please state whether or not histopathological examinations of major organs were completed in this study. p.4 - What was the concentration of Methyl Benzoate used in the dermal irritation study in rabbits. p.4 - Please correct the spelling of “strume”, “hyperkeatosis”, “straatmu”, “hyperkertosis” p.4 - The information on Benzyl Benzoate, needs to be removed from this report and added to Benzoic Acid, Benzyl Benzoate et al. report. p.4 - Did reference 35 report any maternal effects? p.4 - Please provide the number of rats per treatment group used in reference 37. pA - Please provide the number of mice, hamsters and rabbits per group used in reference 37. On what gestation days were these species treated? p.5 - Please provide the gestation days on which the hamsters were treated with Benzoic Acid. p.5 - It is not clear what 50.5% vs 12.5% represents, mortality of the parental and Fl cohorts combined of controls compared to treated mice? What is meant by a “100% food restriction test”? How long was this test? p.5 - Reference 33 is a carcinogenesis bioassay. Please provide the results concerning carcinogenicity in the Carcinogenicity section. p.S - Please define ADI. What is the value of the ADI for Benzoic Acid? p.S - Were the studies of Benzoic Acid really clinical studies, or were they case reports? p.5 - What concentrations of Benzoic Acid were negative for sensitization, phototoxicity and photosensitivity? Where is the Summary for this report?

CIR Panel Book Page 94