GREEN BOOK 4 Alkyl Benzoates - Cosmetic Ingredient Revie · At room temperature and pressure, methyl benzoate, ethyl benzoate, butyl benzoate, and isobutyl benzoate are fragrant,

GREEN BOOK 4

Alkyl Benzoates

CIR EXPERT PANEL MEETING

AUGUST 30-31, 2010

July 30, 2010

RANDUM

MEMO

To: CIR Expert Panel and Liaisons

rom: Lillian C. Becker, M.S.

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

he Cosmetic Ingredient Review (CIR) announced the Scientific Literature Review (SLR) for

12-15 alkyl benzoate is the lead ingredient of this safety assessment. Related alkyl benzoate

comprehensive dossier on the C12-15 alkyl benzoates being ACH program will be completed and provided to CIR in late

r

ed dditional data are required, then the Panel may issue a Tentative Report.

lternatively, the Panel may choose to table the report to await the receipt of the dossier entioned above.

FScientific Analyst and Writer

S Talkyl benzoates in June, 2010. Cingredients are included. CIR has been informed that a

repared for the European REpSeptember or early October. The Panel should review the Draft Report and decide: 1) if it is reasonable to include the othelisted 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 relatingredients. If no aAm

CIR Panel Book Page 1


History of Alkyl Benzoates

June, 2010 – SLR issued. August, 2010 -


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.


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.

© Cosmetic Ingredient Review 1101 17th Street, NW, Suite 412 " Washington, DC 20036-4702 " ph 202.331.0651 " fax 202.331.0088 "

[email protected]


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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


iv

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


1

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


2

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.


3

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 The dermal LD50 of methyl benzoate was ≥ 2000 mg/kg for New Zealand white rabbits (n = 5).28 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 The reported oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats.24,27 Isopropyl Benzoate The reported oral LD50 of isopropyl benzoate was 3730 mg/kg for rats.29 Isobutyl Benzoate The reported oral LD50 of isobutyl benzoate was 3685 mg/kg for rats.18

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.


4

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 incubated to hatching.35 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,


5

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%.


6

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.


7

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

ester of methyl alcohol and benzoic acid that conforms to the formula in Figure 1.

Fragrance ingredient, skin-conditioning agent-emollient, solvent

Benzoic Acid, Methyl Ester;

Methyl Benzenecarboxylate

Methyl benzoate (RIFM)

Morflex Methyl Benzoate

Ethyl Benzoate 93-89-0 Ethyl benzoate is the ester of ethyl alcohol and benzoic acid.

Fragrance Benzoic Acid, Ethyl Ester;

Ethyl benzoate (RIFM)

-

Propyl Benzoate 2315-68-6 Propyl benzoate is the ester of n-propyl alcohol and benzoic acid.

Fragrance ingredient, preservative

Benzoic Acid, n-Propyl Ester;

Propyl benzoate (RIFM)

-

Butyl Benzoate 136-60-7 Butyl benzoate is the ester of butyl alcohol and benzoic acid.

Fragrance ingredient, preservative

Benzoic Acid, n-Butyl Ester;

Butyl benzoate (RIFM)

-

Amyl Benzoate 2049-96-9 Amyl benzoate is the ester of amyl alcohol and benzoic acid that conforms to the formula in Figure 1.

Fragrance ingredient

Benzoic Acid, Pentyl Ester;

Pentyl Benzoate Pentyl benzoate (RIFM)

-

Lauryl/ Myristyl Benzoate

No CAS No. Lauryl/Myristyl benzoate is the organic compound that conforms to the formula in Figure 1.

Skin-conditioning agent-miscellaneous

- Corum 5014

C12-15 Alkyl Benzoate

68411-27-8 C12-15 alkyl benzoate is the mixture of esters of benzoic acid and C12-15 alcohols.

Skin-conditioning agents - emollient

Alkyl (C12-C15) Benzoate;

Benzoic Acid, C12-15 Alkyl Esters;

C12-15 Alcohols Benzoate

AEC C12-15 Alkyl Benzoate;

Botanester AB; Cetiol AB;

Corum 5012; 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 Benzoate

669700-05-2 C16-17 alkyl benzoate is a mixture of esters of C16-17 alcohols and benzoic acid that conforms generally to the formula in Figure 1.

Skin-conditioning agents-emollient, solvent

- Finsolv G-2

Stearyl Benzoate 10578-34-4 Stearyl benzoate is the ester of stearyl alcohol and benzoic acid that conforms to the formula in Figure 1.

Skin-conditioning agent-emollient, solvent

Benzoic Acid, Octadecyl Ester;

Benzoic Acid, Stearyl Ester;

Octadecyl Benzoate

Dub PG; Finsolv 116

Behenyl Benzoate 103403-38-9 Behenyl benzoate is the ester of behenyl alcohol and benzoic acid that conforms to the formula in Figure 1.

Skin-conditioning agent – emollient

Benzoic Acid, Docosyl Ester

Finsolv 137

Branched Alkyl Benzoates Isopropyl Benzoate

939-48-0 Isopropyl benzoate is the ester of isopropyl alcohol and benzoic acid.

Fragrance ingredient

Benzoic Acid, Isopropyl Ester;

Benzoic Acid, 1-Methylethyl Ester; Isopropyl benzoate

(RIFM); 1-Methylethyl Benzoate

-


8

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

ester of isobutyl alcohol and benzoic acid.

Fragrance ingredient, solvent

Benzoic Acid, Isobutyl Ester;

Benzoic Acid, 2-Methylpropyl Ester;

Isobutyl benzoate (RIFM);

2-Methylpropyl Benzoate

-

Isostearyl Benzoate

34364-24-4 Isostearyl benzoate is the ester of isostearyl alcohol and benzoic acid.

Skin-conditioning agent-emollient

Benzoic Acid, Isooctadecyl Ester;

Benzoic Acid, Isostearyl Ester

Finsolv SB

Ethylhexyl Benzoate

5444-75-7 Ethylhexyl benzoate is the ester of 2-ethylhexanol and benzoic acid.

Skin-conditioning agent-emollient, solvent

Benzoic Acid, 2-Ethylhexyl Ester;

2-Ethylhexyl Benzoate Octyl Benzoate

Bernel Ester OB; Finsolv EB

Butyloctyl Benzoate

1888038-97-3 Butyloctyl benzoate is the organic compound that conforms to the formula in Figure 2.

Plasticizer; skin-conditioning agent-emollient, solvent

Benzoic Acid, 2-Butyloctyl Ester

-

Hexyldecyl Benzoate

163883-40-7 Hexyldecyl benzoate is the organic compound that conforms to the formula in Figure 2.

Plasticizer, skin-conditioning agent-emollient, solvent

Benzoic Acid, 2-Hexyldecyl Ester

-

Octyldodecyl Benzoate

108347-89-3 Octyldodecyl benzoate is the ester of octyldodecanol and benzoic acid.

Skin-conditioning agent-emollient

Benzoic Acid, 2-Octyldodecyl Ester

Finsolv BOD


9

Table 2. Physical and Chemical properties of the acetate ingredients.9,9,11,49,49 Methyl

Benzoate Ethyl

Benzoate Propyl

Benzoate Butyl Benzoate Amyl Benzoate Lauryl/Myristal

Benzoate

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

Molecular Weight (g/mol)

136.15 150.17 164.20 178.23 192.25 290.44/318.49

Boiling Point (°C)

198.6 212.9 230.0 247.3 248 225 (Lauryl at 20 mmHg)

Density (g/cm3) 1.09 1.04 1.04 1.00 0.95 0.93(Lauryl)

Vapor pressure (mm Hg @ 20°C)

0.38 0.267 0.136 0.01 0.009 -

Solubility (g/1000g water @ 20°C)

2.1 0.72 0.351 0.059 0.028 -

Log Kow 2.12 2.64 3.01 3.84 4.16 (est.) 7.23 (est. Lauryl) C12-15 Alkyl

Benzoate C16-17 Alkyl

Benzoate Stearyl

Benzoate Behenyl Benzoate

Isopropyl Benzoate Isobutyl Benzoate

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


290.44-332.52 346.55-360.57 374.60 430.71 164.20 178.23

Boiling Point (°C)

363 (est.) - 433 (est.) 518.3 266 237

Density (g/cm3) - - - 0.908 - 1.02


0.00001 (est.)

- 0.00000006 (est.)

0.00000000007 0.161 (est.) 0.0417 (est.)


0.000009 (est.)

- 0.000009 (est.)

0.0000007 0.126 (est.) 0.098 (est.)

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

Benzoate Ethylhexyl Benzoate

Butyloctyl Benzoate

Hexyldecyl Benzoate

Octyldodecyl Benzoate

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


374.60 234.33 290.44 346.55 402.65

Boiling Point (°C)

426 (est.) 169-170 (at 20 mmHg)

376.9 434.8 449 (est.)

Density (g/cm3) - 0.91 0.939 0.923 -


0.0000001 0.0005 (est.) 0.000007 0.00000009 0.00000002 (est.)


0.00001 0.0011 0.00058 0.000015 0.000001 (est.)

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


10

Table 3. Frequency of use according to duration and exposure.15,16

Use type

Total uses/use

type Uses Concentration

(%) Uses Concentration



(%) 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 ingestion 872 66 3-16 - - - - - -

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 (underarm) 623 6 0.004 - - - - - -

Hair – noncoloring 5687 98 0.3-35 - - - - - -

Hair – coloring 2808 - 0.5-2 Nail 674 2 0.008-10 - - - - - - Mucous Membrane 3732 12 0.01-0.04 2 0.7 - - - -

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 ingestion 872 - - - - - - - -

Inhalation 3447 - 1 - 0.3 - - - - Dermal 26,863 1 - - 0.0005-0.3 - 3-4 1 2 Deodorant (underarm) 623 - - - 0.004 - - - -

Hair – noncoloring 5687 - - - - - - - -

Hair – coloring 2808 Nail 674 - - - - - - - - Mucous Membrane 3732 - - - - - 3 - -

Bath products 745 Infant 357 - - - - - 2 -


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

11


Figure 2. Branched-chain alkyl benzoates: structures, esterase metabolism, and metabolites.

Ethylhexyl benzoateO

O CH3HO CH3

OH

O Benzoic acid **

Ethylhexyl alcohol

Legend

Safe as used

* Not in ICI Dictionary andHandbook, 13th Ed.

Ingredients which are part of thisreview

Result of Esterase metabolism

**Ingredients which are concurrentlyunder review in another report

O CH3

CH3

O

HO CH3

CH3

Isobutyl benzoate

O CH3

OIsopropyl benzoate

CH3

HO CH3

CH3

**Isopropyl alcohol

Isobutyl alcohol

Isostearyl benzoate(one example of the mixture of branched chains)O

O CH3

CH3HO CH3

CH3

Isostearyl alcohol

CH3CH3

*

*

Butyloctyl benzoateO

OHO

Butyloctyl alcohol* CH3

CH3CH3

CH3

Hexyldecyl benzoateO

OHO

Hexyldecyl alcohol* CH3

CH3CH3

CH3

Octyldodecyl benzoateO

OHO

Octyldodecyl alcohol* CH3

CH3CH3

CH3

Figure 3. The synthesis of butyl benzoate.

OH

O

HO CH3H2SO4 O

O

CH3

Δ12


13

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13. Sales specification sheet. Vertellus Specialties. 2010.

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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.

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


http://www.inchem.org/documents/cicads/cicads/cicad64.htm

14

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.

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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.

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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.


15

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.


http://themerckindex.cambridgesoft.com/TheMerckIndex/index.asp

Data

Personal Care Products CouncilCommitted to 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% OctyldodecylBenzoate

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

Personal Care Products CouncilCommitted to Safety,

ua ty nnovation

Memorandum


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

DATE: July9, 2010

SIJEJECT: Studies of C 12-15 Alkyl Benzoate

Cognis. 2007. Cetiol AB (C 12-15 Alkyl Benzoate) data sheet.

Cognis. 2002. UV absorption spectrum of Cetiol AB (C 12-15 Alkyl Benzoate).

Consumer Product Testing Co. 2003. 48 Hour patch test of C-SAT 020093 (C 12-15 Alkyl Benzoate).Experiment Reference Number C02- 1224.01.04.

Consumer Product Testing Co. 2003. Repeated insult patch test of C-SAT 020093 (C 12-15 AlkylBenzoate). 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 caIsCETIOL® AB

Labeling informationINCI name(s)Cl 2-15 Alkyl Benzoate (EU 200612571EC)Cl 2-15 Alkyl Benzoate (CTFA)

RegistrationsIngredient CASR-No. EINECS/ELINCS-No.

6841.1 -27-8 270-112-4

Officially listed in I Quality conforms toJCIC: 012-15 Alkyl Benzoate (Ingredient Code 512004)

Product propertiesAppearanceCetiol® AB is a clear, almost colourless, almost odourless oil.

Example of useCetiol® AB is a traditional emollient for modern skin care applications. Particularly suitable for suncareformulations due to its outstanding solubilizing capacities for crystalline UV filters as well as dispersingproperties for pigments.

Characteristic valuesThe specifications stated in the paragraphs ‘Quality control data and Additional product descriptivedata’ 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 standardOdour conforms to standardAcid value max. 0.5 mg KOH/g ISO 660Saponification value 172- 182 mg KOH/g ISO 3657Colour value (APHA) max. 50 ISO 6271Density (20°C) 0.920 - 0.940 g/ml ISO 2811-3Refractive index (20°C) 1.4830 - 1.4870 ISO 6320Water (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 12058-1Cloud 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 SalesAgreement. COGNIS EXPRESSLY DISCLAIMS ANY RESPONSIBILITY FOR THE SUITABILITY OF THE PRODUCTS FOR ANYSPECIFIC OR PARTICULAR PURPOSES INTENDED BY THE USER. Suggestions for the use and application of the products andguide 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 notassume any liability or risk involved in the use of its products as the conditions of use are beyond its control. The user of theproducts is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectualproperty rights of third parties.

IB.08.2008 CETIOLrABE CIR Panel Book Page 25

S

50,04540-

Cog-02-07141,

Cetiol

AB

C1

AIk

yIiS

2e

353025

—

AB

S

20

——

___

Is105

II

200,0250

300350

400450

500550

600650

700,0

NM


Consumer Product Testing Co.L&E I)75

FINAL REPORT

COLQOO —‘p’CLIENT:

Cognis Deutsehiand GmbH & Co. KG.Henkelstr. 67D-4055 1Duesseldorf, Germany

ATTENTION:Mr. Peter Wierich

TEST: 48 Hour Patch TestProtocol 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

/.Ik1j t3e,.oci+C

EXPERIMENTREFERENCE NUMBER: C02-1224.01-.04

Richard R. Eisenberg, -

Board Certified Dermatologist

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 tiename of these Laboratories nor any member of its staff. may be used in connection with the advertising or sale of any product or processwithout written authorization.

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


Consumer Product Testing Co.[Si. 75

QUALITY ASSURANCE UNIT STATEMENT

Study No.: C02-1224.0l-.04

The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinicallaboratory studies. These studies have been performed with adherence to ICH Guideline E6 for GoodClinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance tostandard operating procedures and applicable protocols. The QAU maintains copies of study protocolsand standard operating procedures and has inspected this study on the date(s) listed below. Thefindings of these inspections have been reported to management and the Study Director. All materialsand 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 14, 2003

Senior personnel involved:

Laura A. Artiles, M.A. - Manager, Quality Assurance

Marie Terlizzese, M.S.Quality Assurance Associate

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

7() New Dutch Lane • Fairfield. New Jersey 07004-2514 • (973) 808-71 1 I • Fax 197:3) 808-7234Clinical • Toxicology • Analytical Chemistry • Microbiology


Cognis Deutscbland GmbH & Co. KG.C02-1224.0l-.04Page 3

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

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

Inclusion Criteria: a. Male and female subjects, age a and over.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


Cognis Deutschland GmbH & Co. KG.C02-1224.0l-.04Page 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 thetest material, or an amount sufficient to cover the contact surface, wasapplied to the 3/4” x 3/4” absorbent pad portion of adhesive dressings*.

When secured to the appropriate treatment site, these dressings formedocclusive 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. Absenceof any visible skin change was assigned a zero value. The test sites werere-evaluated at seventy-two hours.

Evaluation Key: 0 = No visible skin reaction+ = Barely perceptible or spotty erythema1 = Mild erythema covering most of the test site2 = Moderate erythema, possible presence ofmild edema3 = Marked erythema, possible edema4 = 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 testinterval.

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 notindicate a potential for dermal irritation.

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


Cognis Deutschland GmbH & Co. KG.C02-1224.01-.04Page 5

Table 1Panel #20020613

Individual Results

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

Subject ObservationsNumber 48 Hours 72 Hours

1 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 0 DNC

10 0 011 0 012 0 013 0 014 0 015 0 016 0 017 0 018 0 019 0 020 0 021 0 022 0 0

DNC = Did not complete study


Cognis Deutschland GmbH & Co. KG.C02-1224.01-.04Page 6


Individual Results



1 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 0 DNC

10 0 011 0 012 0 013 0 014 0 015 0 016 0 017 0 018 0 019 0 020 0 021 0 022 0 0



Cognis Deutsehiand GmbH & Co. KG.C02-1224.01-.04Page 7


Individual Results



1 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 0 DNC

10 0 011 0 012 0 013 0 014 0 015 0 016 0 017 0 018 0 019 0 020 0 021 0 022 0 0



Table 4Panel #200206 13

Individual Results

Cognis Deutschland GmbH & Co. KG.C02-1224.0l-.04Page 8

C-SAT 020093


1 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 0 DNC

10 0 011 0 012 0 013 0 014 0 015 0 016 0 017 0 018 0 019 0 020 0 021 0 022 0 0




Subject Data

Cognis Deutsohiand GmbH & Co. KG.C02-1224.01-.04Page 9

SubjectNumber Initials Age Sex

1 RC 23 F2 BA 46 F3 JE 57 F4 AR 31 M5 WE 63 M6 CV 35 F7 DC 43 F8 LE 47 F9 CD 20 F10 CO 34 F11 AW 73 F12 PR 55 M13 LD 38 F14 EV 34 F15 TV 36 M16 KS 64 F17 SW 36 F18 PM 43 M19 KH 39 F20 BW 63 F21 LB 56 F22 CW 56 F


Substanz-Verwaltung CRTaktuel?er User CNPeter Wierich/OU= DE/OUEMEAIO=Cognis

C-SAT-Nr 020093

IS

Chemische C12/15 AlkylbenzoateBezeich nungTrivial Name Cetiol ABS-GehaIt [%] 100

Losungsmittel(Neben-)KomponentenVerunreinigungen

Identiflzierung der Prufsubstanz

Herstelldatum 01.09.2002Verfallsdatum 01.09.2003

BatchRISS’sCAS

CD2266000351 Zi 200670000

68411 -27-8

lOslichin/suspendierbar nFarbeAggregatzustand belRTpH-Wert

fluchtig bei RT

ParaffinOl, native Ole

farbiosFlussigkeit

Lagerung

Auftraggeber CCC N.MertscheitProduktbetreuerKleber

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

Weitere Informationen zur PrUfsubstanz


C (:92000 -

Consumer Product Testing Co.EST, 1970

CLIENT:

FINAL REPORT

Cognis Deutschland GnibH & Co. KG.Henkelstr. 67D-4055 IDuesseldorf, Germany

ATTENTION: Mr. Peter Wierich

TEST: Repeated Insult Patch TestProtocol No.: 1.01

TEST MATERIAL:

EXPERIMENTREFERENCE NUMBER:

C-SAT 020093

C02-1225.01

Richard R. Eisenberg, MIS.Board Certified Dermatologist

J/Frk, R.N.Study Director

This report is submitted for the exclusive use of the person. partnership, or corporation to whom it is addressed, and neither the report nor thename of these Laboratories nor any member of its staff, may be used in connection with the advertising or sale of any product or processwithout written authorization.

Principal Investigator

70 New Dutch Lane • Fairfield, New Jersey 07004-2514 • (973) 808-71 11 • Fax (973) 808-7234



Study No.: C02-1225.01

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

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

Senior personnel involved:

Richard Hettenbach Senior Director, Regulatory Affairs and Quality Assurance

Marie Terlizzese, M.S.Quality Assurance Associate



CognisDeutschland GmbH & Co. KG.C02- 1225.01Page 3

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

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

Inclusion Criteria: a. Male and female subjects, age 16a and over.b. Absence of any visible skin disease which might be confused with a skin

reaction from the test material.c. Prohibition of use of topical or systemic steroids and/or antihistamines

for at least seven days prior to study initiation.d. Completion of a Medical History form and the understanding and

signing of an Informed Consent form.e. Considered reliable and capable of following directions.

Exclusion Criteria: a. Ill health.b. Under a doctor’s care or taldng medication(s) which could influence the

outcome of the study.c. Females who are pregnant or nursing.d. A history of adverse reactions to cosmetics or other personal care

products.

Test Material: C-SAT 020093

Study Schedule: Panel # Initiation Date Completion Date

20030013 January 13, 2003 February 21, 200320030022 January 20, 2003 February 27, 2003

aWith parental or guardian consent


Cognis Deutschland GmbH & Co. KG.C02-1225.OlPage 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 coverthe contact surface, was applied to the 3/4” x 3/4” absorbent pad portion of anadhesive dressing*. This was then applied to the appropriate treatment site toform an occluded patch.

Induction Phase:

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

With the exception of the first supervised Induction Patch reading, if any testsite exhibited a moderate (2-level) reaction during the Induction Phase,application was moved to an adjacent area. Applications are discontinued forthe remainder of this test phase, if a moderate (2-level) reaction was observedon 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 andThursday removal, and forty-eight hours following each Saturday removal.

Challenge Phase:

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

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


Cognis Deutschland GmbH & Co. KG.C02-1225.OlPage 5

Evaluation Key: 0 No visible skin reaction+ Barely perceptible or spotty erythema1 = Mild erythema covering most of the test site2 = Moderate erythema, possible presence ofmild edema3 = Marked erythema, possible edema4 = Severe erythema, possible edema, vesiculation, bullae and/or

ulceration

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

Observations remained within normal limits throughout the test interval.

Summary: Under the conditions of this study, test material, C-SAT 020093, did notindicate a potential for dermal irritation or allergic contact sensitization.



Cognis Deutsehiand GmbH & Co. KG.C02-1225.01Page 6

Individual Results

C-SAT 020093

Virgin ChallengeSubject — Induction Phase — SiteNumber 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72br

0 0 0

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 08 0 0 0 0 0 0 0 0 0 0 0 09 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 0 013 0 0 0 0 0 0 0 0 0 0 0 0

14 0 -DID NOT COMPLETE STUDY.15 0 0 0 0 016 0 -DID NOT COMPLETE STUDY.17 0 0 —----- —-DID NOT COMPLETE STUDY - ---

18 0 0 0 0 0 0 0 0 0 0 0 019 0 0 0 0 0 0 0 0 0 0 0 020 0 0 0 0 0 0 0 0 0 0 0 021 0 0 0 0. 0 0 0 0 0 0 0 022 0 0 0 0 0 0 0 0 0 0 0 023 0 0 0 0 0 0 0 0 0 0 0 024 0 0 0 0 0 0 0 0 0 0 0 025 0 0 0 0 0 0 0 0 0 0 0 026 0 0 0 0 ----—-----—---DID NOT COMPLETE STUDY27 0 0 0 0 0 0 0 0 0 0 0 028 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0

24 = Supervised removal of l Induction and Challenge Patch-*

= Subject unable to report as scheduled, instructed to remove patch and report on the next test day.


Table 1(continued)

Panel #200300 13

Cognis Deutschland GmbH & Co. KG.C02-1225.01Page 7

Individual Results

C-SAT 020093

Virgin ChallengeSubject ——---Induction Phase —

—-- SiteNumber 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72 hr

293031

32

33

34

35

36373839

40

41 0 0 0 0

42 0 0 0 043 0 0 0 044 0 0 0 045 0 0 0 046 0 0 0 047 0 0 0 048 0 0 0 049 0 0 0 050 0 0 0 051 0 0 0 052 0 0 0 053 0 0 0 054 0 0 0 055 0 0 0 056 0 0 0 0

00

00

0

0

0

00

00

0 0 0 0 0 0

0 0 0 0 0 00 0 0 0 0 0

0 0 0 0 0 00 0 0 0 0 00 0 DID NOT COMPLETE0 0 0 0 0 00 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 00 0 0 0 0 0o o 0 0 0 00 0 0 0 0 0

0 0

0 0

0 00 0

0 0

0 0

0 00 0

0 0

0 00 0

0

0

0

00

STUDY000

00

00

0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 00 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

— -----DID NOT COMPLETE STUDY----0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 00 0 0 0 0 0 0’ 0 0

0

0

0

0

0

000

0

000

DID NOT COMPLETE STUDY---0 0 0 0 0 0 0 00 0 0 0 0 0 0 0

24* = Supervised removal of 1 Induction and Challenge Patch


Table 1(continued)

Panel #20030022

Cogiiis Deutsohiand GmbH & Co. KG.C02-1225.01Page 8

Individual Results

C-SAT 020093

Virgin ChallengeSubject —-—-—----— Induction Phase--—--— ———

—-—-- SiteNumber 24*hr 1 2 3 4 5 6 7 8 9 24*1w 721w

0 0 + 0 0 0 0 0 Ow 0

2 0 0 0 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0 0 0 0

25 0 0 0 0 0 0 0 0 0 0

26 0 0 0 0 0 0 0 0 0 027 0 0 0 0 0 0 0 0 0 028 0 0 0 0 0 0 0 0 0 0

O 0

0 0

O 0

O 0

0 0

6 0 0 0 07 0 0 0 0

8 0 0 0 0

9 0 0 0 0

10 0 0 0 011 0 0 0 012 0 0 0 013 0 0 0 014 0 0 0 0

15 0 0 0 0

16 0 0 0 0

17 0 0 0 0

18 0 0 0 019 0 0 0 020 0 0 0 021 0 0 0 022 0 0 0 023 - 0 0 024 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 0 0

0 0 0 0 0 0 0 0*

O 0 0 0 0 0 0 0

0 0 0 0 Ow 0 0 0

O 0 o ow o 0 0 oo 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 0 Ow 0 0 0

DID NOT COMPLETE STUDY

O 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

O 0 0 0 0 0 0 0

O 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

o 0 0 0 ow o 0 0

O 0 0 0 Ow 0 0 0

o 0

o oo oO 0

24* Supervised removal t1nduction and Challenge Patch- = Subject not present for supervised patch removal*

= Observation recorded 96 hours post challenge application. Subjectunable to report as scheduled.

W = Inclement weather. Subject unable to report as scheduled and wasinstructed to report on the next scheduled test day.


Table 1(continued)

Panel #20030022

Cognis Deutsehiand GinbH & Co. KG.C02-1 225.01Page 9

Individual Results

C-SAT 020093

Virgin ChallengeSubject —----—---—---—--— Induction Phase--—----— —

—-- SiteNumber 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72 hr

o o 0 0 0 0 0 0 0

o 0 0 0 0 0 0 0 0- DID NOT COMPLETE STUDY

o o 0 0 0 0 0 0 0

o 0 0 0 0 0 0 0o o 0 0 0 0 0 0

o o 0 0 0 0 0 0

0 0 0 0 0 0 0 0

46 0 0 0 0 0 0 0 0 0 0

47 0 0 0 0 0 0 0 0 0 0

49 0 0 0 0 0 0 0 0 0 050 0 0 0 0 0 0 0 0 051 0 0 ------ DID NOT COMPLETE STUDY---52 0 0 0 0 0 0 0 0 0 053 0 0 0 0 0 0 0 0 0 0

54 0 0 0 0 0 0 0 0 0 0

55 0 0 + 0 0 0 0 0 0 056 0 0 0 0 0 0 0 0 0 0

24* = Supervised removal of l Induction and Challenge PatchDNC = Did not complete study

W = Inclement weather. Subject unable to report as scheduled and wasinstructed to report on the next scheduled test day.

0 00 0

0

0 00 0

0 00 0 0

0 00 0

0 0

0 00 0

29

30

3132

33

0 0 0 0 0

0 0 0 0 0

0 0 0 0 00 0 0 0 0

0 0 0 0 0

0 0 0 Ow 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

34 0 0 0 0 0 0 0 0 0 0

35 0 0 0 0 0 0 0 0 0 0

36 0 0 0 0 0

37 0 0 0 0 0

O 0

0 0

0 0O 0

0 00 0

0 0

0 0

0 0O 0 0 0 0

0 0 0 0 0

38 0

39 040 0

4142

4344

45

0 00 0

0 0

O 0

0 0O 00 00 0

48 0 0 0 0 0 0 0 0 0 0 0 0




Subject Data


I SC 54 F2 TJ 33 M3 JS 51 F

4 WF 49 F5 AL 56 F

6 DK 44 F7 DW 46 F8 CC 66 F9 WM 34 F10 CS 41 F11 TM 74 F12 AT 27 F

13 NP 35 M14 RA 36 F15 LS 39 F16 MM 33 M17 SV 33 M18 EM 46 F19 SM 26 F20 MT 58 F21 FP 63 F22 ID 79 F23 CT 34 F24 DE 47 F25 PR 63 F26 PF 77 F27 TM 69 F28 AS 70 F



Table 2(continued)

Panel #20030013

Subiect Data


29 ES ‘39 M

30 SE 50 F31 NE 18 F32 PH 31 F

33 MR 36 F34 DN 21 F

35 RG 45 M36 AT 58 M37 QA 49 F38 RG 55 F39 GG 67 M

40 MG 31 F

41 AA 45 M

42 DM 47 F43 MM 44 F44 KF 45 F45 KP 45 F46 PC 36 F47 DK 19 F48 DT 31 F49 SK 45 F50 SM 55 F51 YL 39 M52 KG 38 F53 JS 62 M54 JP 53 M55 BC 35 M56 JM 66 M



Table 2(continued)

Panel #20030022

Subject Data


1 TC 18 F

2 KF 45 F

3 AK 72 F4 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 F15 JG 44 M

16 BH 25 M

17 PS 28 F18 MV 47 F19 JC 45 F20 JS 44 F21 PS 17 M22 DM 22 M23 DF 69 F24 SB 52 F25 VS 28 F

26 JD 42 M27 LF 36 F28 IC 59 F


Cognis Deutschland GmbH & Co. KG.C02-1225.0lPage 13

Table 2(continued)

Panel #20030022

Subject Data


29 PH 58 M30 RN 74 F31 1V 52 F32 GD 47 F33 VA 48 M34 ES 48 M35 DS 39 F36 RC 63 M37 PS 26 F38 ML .67 F39 EA 41 F40 GA 27 M41 SM 40 F42 SS 51 F43 WB 39 F44 JC 74 F45 DR 36 F46 LR 69 F47 PD 63 F48 PL 73 F49 TB 53 M50 CA 37 F51 CS 31 F52 EH 68 M53 EA 63 F54 CC 55 F55 AS 34 F56 SW 52 F


Substanz-Verwaltung CRTaktueller User CN=Peter Wierich!OU=DE/OU=EMEAIO=Cognis

C-SAT-Nr 020093

Chemische C12115 AlkylbenzoateBezeichnungrrivial Name Cetiol ABS-Gehalt [%) 100LOsungsmittel(Neben-)KomponentenVerunreinigungen

Identifizierung der Prufsubstanz

Herstelldatum 01.09.2002Verfallsdatum 01.09.2003

BatchRISsLsCAS

CD2266000351 ZI 200670000

68411 -27-8

lslich ParaffinOl, native Oleinlsuspendierbar inFarbe farblosAggregatzustand bei FlOssigkeitRTpH-Wert

flochtig bei RT I

Lagerung

Auftraggeber CCC N.MertscheitProduktbetreuerKleber

Gefahrenhinweise (z.B. giftig/reizend!Lzend/enUndIich/expIosiv)

Weitere Informationen zur PrUfsubstanz


Personal Care Products CouncilCommitted to 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 lotioncontaining 0.95% Isostearyl Benzoate) through alternative methods to the Draize test. Reportreference: CTOXJO5 161.

1101 17th Street, N.W., Suite 30O Washington, D.C. 20036-4702 202.331.1770 I 202.331.1969 (fax) www.personakarecouncil.org CIR Panel Book Page 51

Consumer Product Testing Co.I-sr

FINAL RE PORT

CLIENT:-

ew Jersey 07950-2451

ATTENTION: James FlanaganSr. Cherr4ist Microbiology

TEST: Repeated Insult Patch TestProtocoiNo.: 1.01

TEST MATERIAL: i Body Lotion TL45-.24-2

• 9ç irJ

i3irnzc’c1-cEXPERIMENT C05-072.01REFERENCE NUMBER:

Richard R. Eisenberg, M.D.Board Cprtified Dermatologist

P-ank,(‘xeLitive Vice President, Clinical Evaluations

Report Date: ‘O// 472

This report is submitted Jor the exetusive use of the person, parfnershi4, or corporation to whom it ‘a addressed, and neither the report nor thenrrrne of these Laboratories 001 any membor of its staff, may be use in corinsciron with the edverltsin ot safe of any pcoduct or processwittiest written authorization

() N(v IhItcIl Lant • Iiirfirki New ,Jers t45 14 t$73) O-7 II Iax (97;lf 8O-7234


Consumer Product Testing Co.


Study No.: C05-0729.01

The objective of the Quality Assurance Unit (QkU) is to monitor the conduct and reporting of clinicallaboratory studies. These studies have been performed with adherence to the applicable ICH GuidelineE6 for Good Clinical Practice and requiremenuts provided for in 21 CFR parts 50 and 56 and inaccordance to standard operating procedures and applicable protocols. The QAU maintains copies ofstudy protocols and standard operating procedures and has inspected this study. All data pertinent to thisstudy will be stored in the Consumer Product Testing Company archive, unless speciiied otherwise, inwriting by the Sponsor.

Quality Assurance personnel involved:

)cdkô /o/i4Qu ty Assurance Date


70 New Duth Lane FirfieIc1, New Jersw O704-2 14 973) 8Q-7 I 11 • Fax ($73) 8O87234(linicI • Toxicology \fliI\tiCI Chcmiry Microbiology


Page 3

Objective: To determine by repetitive epidermal contact the potential of a test materialto induce primary or cumulative irritation and!or allergic contactsensitization.

Participants: One hundred fifteen (115) qualified subjects, male and female, ranging in agefrom 16 to 78 years. rere selected for this evaluation. One hundred seven(107) subjects completed this study. The remaining subjects discontinuedtheir participation for various reasons, none of which were related to theapplication of the test material.

Inclusion Criteria: a. Male and female subjects, age 16a and over.b. Absence of any viibIe skin disease which might be confused with a skin

reaction from the test material.Prohibition of use of topical or systemic steroids andlor antihistaminesfor at least seven dkys prior to study initiation.

d. Completion of a Medical History form and the understanding andsigning of an Informed Consent form.

e. Considered reliable and capable of following directions.

Exclusion Criteria: a. 01 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 advçrse reactions to cosmetics or other personal care

products.

Test Material: Body Lotion TL45-24-2

Study Schethile: Panel # 1nitiaton Date Completion Date

20050393 Augut 15. 2005 September 23, 200520050401 August 17, 2005 September 29, 2005

‘With parental or guardian consent


Page 4

Methodology: The upper back between the scapulae served as the treatment area.Approximately 0.2 nIl of the test material, or an amount sufficient to coverthe contact surface, vas applied to the 1’ x I’ absorbent pad portion of aclear adhesive dressijg*. This was then applied to the appropriate treatmentsite to form a semi-o4elusive patch.

Induction Phase:

Patches were applied three (3) times per week (e.g., Monday, Wednesday,and Friday) for a totai of nine (9) applications. The site was marked to ensurethe continuity of patch application. Following supervised removal andscoring of the first Induction patch, participants were instructed to remove allsubsequent lnductioa patches at home, twenty-thur hours after application.The evaluation of this site was made again just prior to re-application. If aparticipant was unabl to report for an assigned test day, one (1) makeup daywas permitted. This day was added to the Induction period. It was noted thatdue to a holiday weekend, which occurred during the Induction Phase,subjects who requited a makeup day experienced a delay betweenapplications.

With the exception of the first supervised Induction Patch reading, if any testsite exhibited a moerate (2-level) reaction during the Induction Phase,application was moved to an adjacent area. Applications are discontinued forthe remainder of this test phase, if a moderate (2-level) reaction was observedon 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 andThursday removal, and forty-eight hours following each Saturday removal.

Chaliene Phase:

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

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


Page 5

Evaluatkrn Key: 0 = No visible skin eaction+ = Barely perceptible or spotty eiythema

= Mild erythema covering most of the test site2 Moderate erythena, possible presence of mild edema3 = Marked erythema, possible edema4 = Severe erythemà, possible edema, vesiculation, bullae and/or

ulceration

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

With one exception, observations remained negative throughout the testinterval.

it was noted that Subject #25, Panel #20050393, exhibited numerousreactions during the Induction Phase and moderate (2) to barely perceptible(+) responses post-challenge application. This subject also reacted to anumber of test materials on this shared panel. Although his medical profiledoes not reflect a histoly of allergies, it is the Laboratory’s opinion that he isa reactive individual. He will be prohibited from participation in future patchtests.

Suimuary: Under the conditions f this study, test material, Body LotionTL45-24-2, did not irdicate a clinically significant potential for dermalirritation or allergic contact sensitization.


Page 6


1ndiviIua1 Results

1. Body Lotion TL45-24-2

Virgin ChallengeSubject —-———-—------—-------hdueticPhase--—-—--—--—-—----—-—---- SiteNumber 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72br

1 0 0 0 0 0 Q 0 0 0 0 0 02 (1 0 0 0 0 4 0 0 0 0 0 03 0 0 0 0 0 0 0 0 0 0 0 04 0 0 0 0 0 0 0 0 0 0 0 05 0 0 0 0 0 0 0 0 0 0 0 06 0 0 0 0 0 0 0 0 0 0 07 0 0 0 0 0 0 0 0 0 0 0 08 0 0 0 0 0 0 0 0 0 0 0 09 0 0 0 0 0 0 0 0 0 0 010 0 0 0 0 0 0 0 0 0 0 0ii 0 0 0 0 0 0 0 0 0 0 0 012 0 0 0 0 0 q 0 0 0 0 0 013 0 0 0 0 0 0 0 0 0 0 0 014 0 0 0 0 0 0 0 0 0 0 0 015 0 0 0 0 0 0 0 0 0 0 016 0 0 0 0 0 O. 0 0 0 0 0 017 0 0 0 0 0 0 0 0 0 0 0 018 0 0 0 0 0 ol 0 o 0 0 0 019 0 0 0 0 0 0 0 0 0 0 0 020 0 0 0 0 --—-.-—4———DID NOT COMPLETE STUDY-------—----——21 0 0 0 0 0 0 0 0 0 0 0 022 0 0 0 0 0 0 0 0 0 0 0 023 0 0 0 0 0 0 0 0 0 0 0 024 0 0 0 0 0 0 0 0 0 0 0 025 0 0 1 1 1 1 + 0 0 0 2 1 ÷**

26 0 0 0 0 0 0’ 0 0 0 0 0 027 0 0 0 0 0 0 0 0 0 0 0 028 0 0 0 0 0 0 0 0 0 0 0 029 0 0 0 0 0 0 0 0 0 0 0 0

24* Supervised removal of l Induction and Challenge Patch96 hour follow-up evaluation


Page 7

30 0 031 0 032 0 033 0 034 0 035 0 036 0 037 0 038 0 039 0 040 0 041 0 042 0 043 0 044 0 045 0 046 0 047 0 048 0 049 0 050 0 051 0 052 0 053 0 054 0 0

Tb1e I(cotinued)

Panel #20050393

individual Results

ody Lotion TL45-24-2

0 0

0 00 00 00 00 0

0 00 0

0 00 0

0 00 00 00 00 0

0 00 0

0 00 00 00 00 00 00 0

0 00 0

Virgin ChallengeSubject -----——---------——-------Inductiot Phase —-——----—-- SiteNumber 24*hrL 1_ 3 4 6 7 8 9 24*hr72 hr

0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 ol 0 0 0 00 0 0 Oj 0 0 0 00 0 0 01 0 0 0 00 0 0 01 o 0 0 00 0 0 0 0 0 0 0

---—-—-—---P NOT COMPLETE STUDY-0 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 01 0 0 0 00 0 0 0 0 0 0 00 o 0 ol 0 0 0 00 0 0 o 0 0 0 00 0 0 0 0 0 0 00 0 0 ol o 0 0 00 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 ol 0 o 0 0

55 0 0 0 0 0 OI o 0 0 056 0 0 0 0 0 01 0 0 0 0

24* Supervised removal of 1 Induction and Challenge Patch


Page 8

o 0 0 0

o 0 0 0o 0 0 0o 0 0 0o o 0 0o 0 0 0o 0 0 0o 0 0 0o 0 0 0o o 0 0o 0 0 0o o 0 0o 0 0 0o o 0 0

o 0 0 0o 0 0 0o 0 0 0o 0 0 0o o 0 0o o 0 0o o 0 0o 0 0 0o 0 0 0o 0 0 0o o 0 01 0 0 0

o o 0 0 0—DTD NOT COMPLETE STUDY-o o• 0 0 0o o 0 0 0o o 0 0 0o o 0 C) 0o o 0 C) 0o o 0 0 0o o 0 0 0o o 0 0 0o o 0 0 0o o 0 0 0o o 0 0 0o o 0 0 0o o 0 0 0-DID NOT COMPLETE STUDY•-DID NOT COMPLETE STUDY-o 0 0 0 0o 0 0 0 0o 0 0 0 0o O 0 0 0o o 0 0 0o 0 0 0 0o 0 0 0 0o O 0 0 0o 0. 0 0 0o 0 0 0 0o 0 0 0 0o 0 0 0 0

0 0 0

o oo 0o 0o 0

o oo 0o oo oo 0o 0o oo 0o 0

o oo 0o oo oo 0o oo 0o o() 0o oo 0

o 0

Table I(continued)

Panel #20050401

Individual Results

Body Lotion TL45-24-2

Virgin ChallengeSubiect -— — Induction Phase—----—--—-—-— — SiteNumber 24*hr 1 2 3 4 5 — 6 7 8 9 24*hr 72 hr

34

67

$910

1112

131415

1617

18

1920

21

222324

26

27

28

29

000

0

0

0

00

00

00

0

0

0

0

0

00

000

00

0

24* Supervised removal of 1e Induction and Challengq Patch


Page 9

Table 1(cohtinued)

Panel #20050401

Individual Results

Body Lotion TL45-24-2

SubjectNumber 24*hr I

30 0 0 031 0 0 032 0 0 033 0 0 034 0 0 035 0 0 036 0 0 037 0 0 038 0 0 039 0 0 040 0 0 041 0 0 042 0 0 043 0 0 044 0 0 045 0 0 046 0 0 047 0 0 048 0 0 049 0 0 050 0 0 051 0 0 052 0 0 053 0 0 054 0 0 055 0 0 056 0 0 057 0 0 058 0 0 059 0 C) 0

00000 q

Virgin ChallengeSite

24*hr 72 hr

0 0o 0

0 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 0-DID NOT COMPLETE STUDY-0 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 0o 0 0 00 0 0 00 0 0 0

-----——---------------------Inductio Phase— —-——

2 3 4 6 7 8 9

0 0 0 00 0 0 0

-1311) NOT COMPLETE TUDY0 0 0 00 0 0 0 00 0 Ô 0 00 0 0 00 0 01 0 00 0 01 0 00 0 01 0 00 o 0! 0 00 0 o! 0 00 0 0 0 00 0 0 00 0 0 0 00 0 0 0 00 0! 0 00 0 01 0 o0 0 0 0 0o 0 0 0 00 o o! o o0 0 0! 0 00 0 0 0 00 0 o 00 0 0 0 00 0 0 0 00 0 0 0 00 0 0 0 0

—--—i DID NOT COMPLETE STUDY-—-—-——0 0 0 0 0 0 0 0 0

24 Supervised removal of 1 Induction and Chal1eng Patch


Page 10

liable 2Panel 20050393

Subject Data

SubjectNumber Initials

LG 64 M2 AK 45 F3 RM 43 F4 VT 39 F5 KL 40 F6 ND 36 M7 BA 39 F8 SF 53 F9 MM 58 M10 EG 55 F11 AG 22 M12 VV 16 M13 HG 72 M14 MA 16 F15 VA 51 M16 GD 49 F17 CD 25 F18 JO 54 F19 MV 42 F20 JM 24 F21 CN 63 F22 AB 62 M23 3M 68 F24 IV 72 F25 AD 55 M26 RD 70 M27 LZ 43 F28 FS 21 M29 AD 53 F


Table 2(coitinued)

Panel #20050393

Subject Data

Coty, Inc.C05-0729.0 1Page 11

SubjectNumber Initials —

30 GS 76 F31 JD 21 M32 VD 73 F33 AD 69 M34 RW 49 M35 AL 77 F36 JM 44 M37 RC 20 M38 MG 66 F39 LS 40 F40 JD 59 F41 LV 72 F42 GN 36 F43 EM 29 F44 AM 60 F45 RF 35 F46 HP 75 F47 iF 70 F48 TM 52 F49 DC 64 F50 MC 30 F51 DC 18 F52 MC 39 F53 AC 57 F54 EF 76 F55 EV 65 F56 CW 75 F


Page 12

‘Fable 2(continued)

Panel #20050401

Subjct Data

SubjectNumber Initials - Age Sex

I JV 66 F2 LP 38 F3 JP 34 F4 LM 48 F5 DD 49 F6 CM 53 F7 AS 58 F8 PR 66 F9 LM 35 F10 CD 65 F11 RT 21 F12 LS 42 F13 AP 57 F14 JH 47 F15 OS 26 F16 SB 30 F17 RB 57 F18 CL 54 M19 JC 31 F20 RV 66 F21 EM 42 F22 BL 43 F23 LD 35 F24 JA 37 M25 52 M26 MA 51 F27 MP 42 F28 AL 46 F29 AO 46 F


Page 13

Täbie2(continued)

Panel #20050401

Subject Data


30 AS 57 M31 JS 55 F32 RM 35 F33 SM 65 M34 YV 51 • F35 RV 26 F36 CB 21 F37 RF 40 M38 JR 30 F39 RR 55 F40 VR 60 M41 PF 37 M42 DB 28 F43 TP 64 F44 MW 71 F45 JS 54 M46 FF 51 M47 ED 45 F48 AL 78 M49 LL 76 F50 DS 67 F51 AD 41 M52 BR 34 F53 JS 39 M54 JY 47 M55 YC 41 F56 HM 77 F57 JB 36 F58 GZ 41 F59 EH 58 F


Assessment of the Eye Irritating Potentialof a Cosmetic Product through

Alternative Methodsto the Draize Test

Test Product: Body LotionReference: 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 weconsider that the estimated Draize classification of the test product might beslightly irritant with Draize score which might range from 0 to 15

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

According to the estimated Draize score, the following warning maybe 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

TABLE CONTENT

1- INTRODUCTION

2- TECHNICAL INFORMATIONS

21 Product characteristics

2-2 Testing facilities

2-3 Data storage

2-4 Authentication of the study

3- ALTERNATIVE METHODS USED

3-1 The Neutral Red Release (NRR) assay

3-2 The Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM)

3-3 The Reconstituted human Epithelial Culture (REC) assay

4- RESULTS AND CONCLUSIONS

4-1 NRR assay

4-2 HET-CAM

4-3 REC assay

5- FINAL ASSESSMENT

6- REFERENCES

Skin Research Opt Cell Toxicology lab. p. 21 26 CIR Panel Book Page 66

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 vitro or ex vivo assays have been proposed worldwideas alternatives to the Draize test. Despite the tremendous efforts concentrated eitherby the Cosmetic Toiletries and Fragrances Association (CTFA) or the EuropeanCommunity (EC) and British Home Office (BHO), none of these alternative methodshave been successfully validated.

However, in some particular fields suct as eye irritation, it is clear that under theincreasing pressure of consumer associations, regulatory agencies tend to be moreand 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-AllantoIcMembrane (HET-CAM) and the Neutral Red Release (NRR) assay as official testmethods for determining the irritating potential of cosmetic products (Journal Officielde Ia République Française, 26112196, Annexe IV; 30/12199, Annexe VI).

The aim of this study was to predict the eye irritation potential of formulated products.For that purpose, we developed a particular in vitro>> approach, which combinesseveral alternative methods. Indeed, many international studies have clearlydemonstrated the interest of combining at least 2 or 3 alternative methods whenassessing eye irritation through in vitro tests.

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(<relevant>) alternative methods the 3 following in vitro tests:

- 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 ofdifferent end-points and thus explores various types of mechanisms (cytotoxicity,acute vascular effect, toxicokinetic, transepithelial absorption,...) which are generallyconsidered as taking part in the eye irritation phenomena (Rougier et al., 1994).

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

Skin Research Dpt — Cell Toxicology lab. 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 sponsorMorris Plains (USA) on the 16 August 2005.The test product, identified as a skin care froduct, is a white milk, having a pH of 7.2at 22.2°C.

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

All the data relative to the study will be stored in the premises of the Skin ResearchDpt for a period of 10 years.

Skin Research Opt Cell Toxicølogy lab, 5 1 26 CIR Panel Book Page 69

2-4 Authentication of the study

I, the undersigned Olivier DOUCET, Director of the Study, certify that this study has

been carried out in the oremises of the - — —

standard protocol under the responsibility of Myléne LANVIN and Technical

Investigators of the Cell Toxicology Laboratory.

Olivie OUCET Pharim D.Toxicolog UROTOX)Head of Skin esearch Opt

I, the undersigned Mylène LANVIN, Reponsible for the Study, certify that this study

has been performed under my supervising in accordance with our internal standardprotocol.

R arch AssistantHead of Cell Toxcoiogy Dpt

We, the undersigned Carine LINOSSIER, Connne THILLQU, Vincent COMTE and

Boris MERLIN, Technical Investigators, certify that all the observations and numerical

data presented in this document are an accurate reflection of the results obtainedduring this study.

cr4ER CorirN1OUTèehfàal Investigator Tec4&I)rrestigatorCell Toxicology Dpt C5.e1foIogy Dpi

Vincent COMZ Boris MERLINTechnical Igvator Technical Inve torCell Cell

Skin Research Opt Cell Toxicology lab. p. 61 26 CIR Panel Book Page 70

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 testsystem 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 releasetheir neutral red dye. The neutral red contained in surviving cells was extract with arevelation solution and spectrophotometrically measured. The test productconcentration that gives rise to the release of 50% NR dye (NR50) is used asendpoint to reflect the cytotoxicity of the test product.Two stages can be necessary to asses$ the NR50 of a test product. The first Stageallows the estimation of the NR50 wheéas the second stage permit to accuratelyassess the final score.

Materials

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

Rabbit cornea SIRC cells: Rabbit cornea fibroblasts SIRC (ATCC n°CCL6O) werebought in the United States at ATCC (American Type Culture Collection Rockville,Maryland, USA) through a French supplier (CERDIC, Sophia Antipolis, France). Cellswere cultured according to the internal procedures of our laboratory for freezing,unfreezing and subculturing. Briefly, cells were maintained in medium MEMsupplemented with 2% antibiotics and 1% MEM Non Essential Amino Acids. Thiscompleted medium was extemporaneously supplemented with 10% of ‘rnodified”fcatal calf serum. Cells were incubated in humidified atmosphere at 37°C, 5% CO2.

Experimental procedure

Cell seeding: For treatment, cells were seeded in all the wells of 24-well plates. Theplates were incubated for 24 hours at 37°C, 5% CO2.

Skin Research Dpt Cell Toxicology b p. 7126 CIR Panel Book Page 71

Application of the neutral red dye: The NRR assay was carried out according to the

principle of the colorimetric test described by Borenfreund and Puerner (1984, 1985).

After incubation, I ml of neutral red solution test, centrifuged before using, was

added to each well of the plates. The plates were incubated for 3 hours at 37°C, 5%

Co2.

Test product dilutions: According to the physico-chemical characteristics of the testproduct, the dilutions ware performed extemporaneously in an hydrophilic (wtv) or alipophilic substance (w/w). During the first stage, the product was tested diluted at0%; 5%; 15%; 25%; 35% and 50%.According to this preliminary results, some dilutions were selected for the secondstage. The principle is given in the following Table 1.

Table 1: Dilutions to be selected for the second stage

1 Stage n°1 Stagen°2NR5O (%) [iIutions to be tested %)

<4 0.1 . 1 54and6 1 5 10

>6and<13 5 10 15

— 10 5>17 and <23 15 20 — 2523arid27 20 25 30>27and<33 25 30 3533and37 30 35>37anci<46 351 4 —____ 50

46 and 50 40 50 - 60> 50 Slight cytbtoxIcity I Unnecessary Stag2

Test product application: After incubation for 3 hours, the neutral red solution wasremoved and I ml of complete culture medium was added in each well of the plate.The plate was maintained at room temperature for 30 minutes. Then each well wasrinsed with 2 ml of PBS and 500 p1 of ech test product dilution were added in thesame time in two wells of the plate. Aftera 55-second contact (or 25 seconds for thepositive control), each well was rinsed with PBS. The plate was gently stirringthroughout contact time.

Skin Research Dpt Cell Toxicology lab. p. 8126 CIR Panel Book Page 72

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 NR50 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 NR50 according to the scale

presented in Table 2.

Table 2: Cytotoxicity scale from the NR endpoint

% of deathNR50 (%) obSerVed at the Clssiflcation COTY Conclusion

dilution_50%

> 5020 Negligible cytotoxicity (PNI)

Slightly cytotoxic (SI)> 20 and < 50 Not very important cytotoxicity (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 NR50 of the positive control ranged from

0.127% to 0.185%.

Skin Research Opt Cell Toxicology lab. p. 9126 CIR Panel Book Page 73

3-2 The Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM)


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).


Upon their arrival, all the defective eggs and eggs which weight is not ranged from 50to 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 atemperature 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 a5 ml saline solution (NaCl 0.9% with distilled water) application and the removal ofthe inner shell membrane, the vascular chorioallantoic membrane (CAM) wasexposed 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 of37.5°C, the test product was applied onto the surface of the CAM. After a 20-secondcontact, the membrane was gently rinsed off by using 5 ml (10 ml or more ifnecessary) of saline solution kept at 37.5°C.

Investigator observations: Observations were achieved by using a specific lampKL1 500 electronic (SCHOTT, France) emitting a cold and white light. Blood vesselsand albumen were continuously observed by a trained person for a 5-minute period.Irritant effects, such as hyperhaemia, haemorrhage and coagulation (opacity and/orthrombosis), were scored according to their occurrence within the test period.

Skin Research Dpi Cell Toxicology 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

If the 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) presentedin Table 3.

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

Vascular Time (t)

effect Q<t3O. 30s.<t2min. 2min.<t5min

Hyperhaemia 5 3 1

Haemorrhage 7 5 3

Coagulation 9 7 5

For each parameter (Hyperhaemia, Haemorrhage, Coagulation) the individual scoresobtained from the 4 eggs were averaged. The sum of these 3 values gave the socalled “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 calculatedaccording to the classification developed by Luepke (1985, 1986) and described inthe 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 irritantI . 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 internalprocedure, the study complied if the Het-Cam score for the positive control rangedfrom 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


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 obtainedby culturing transformed human keratinocytes (TR146 cell line) derived from

squamous carcinoma (Regnier at al., 1987; Rupniak et al., 1985).


Test product application: The product wa tested neat or diluted according to the typeof product. Test sample was directly applied onto the apical surface of the epithelialculture. Product was gently spread with a brush. Cultures were treated with the testproduct 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, 5%C02 /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) wasperformed. After incubation in MiT reagent, the formazari crystals were extracted byisopropariol. Optical densities were read at 540 nm, by using a spectrophotometer(isopropanol served as blank”).

Skin Research Dpt- Cell Toxicology lab.

—p. 12126


with:

SMCI =

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

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 thenegative control. The time course of toxicity for the applied product was expressed asa cumulative simplified mean cytetoxicity index (SMCI) calculated over 24 hours, asfollows:

3

%cyt. lh = % of cytDtoxicity of 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 classificationpresented 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 theinternal procedure, the study compIle only when the SMCI of the positive control lieswithin the confidence internal range.

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

Skin Research Dpt Cell Tocology lab. p. 13/26 CIR Panel Book Page 77

Tab

le6

:T

able

of

concord

ance

betw

een

the

Invi

tro

sco

res

and

the

est

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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 NR5 supehor to 50% was obtained fo

the product Body Lotion, ref. TL45-24-2. From this result. the testproduct was considered slightly cytotoic.

4-1-2 Results

Stage 1Th firct f thic iirv wc inititd in the oremises of the

19 September2005 and was completed on the 20 Septernber2uu.

The test product Body Lotiorli was 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 productand the positive control are presented in Tables 7 and 8 respectie’. The graphicassessment of the NR for the test product and the positive control were presentedin Fig. 1 and 2 respectively.

Skin Research Dp Cell Toxipoogy lab. p. 15126


Table 7: Optical densities (O.D.) and cytotoxicity (%) obtained for the test product

and the negative control (representing the 0% dilution) during the stage n°1

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

—

Product dilution (%) 0 5 15 25 35 50

Well 1 0.951 0.962 0.964 0.962 0,913 0.744

Well 2 0.944 0.99 1.012 0.992 0.931 0.743

We113 0.951 - I - - - -

Well 4 0.962 - - - - -

Mean 0.952 - 0,97 0.988 0.977 0.922 0.744

SD 0.007 0.021 0.034 0.021 0.013 0.001

Cytotoxicity (%) 0.00 0.00 0.00 0.00 3.15 21.90

100

80

>60,:x40:>

C.)

20

0

.•‘._ -

0 10 20 30 40 50

Product dilutions (%)

Fig. 1: Assessment of the NR50 for the test product

NR>50%

Skin Research Dpt Cell Toxicology lab. p. 16/26 CIR Panel Book Page 80

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

C.)x

0

0

Product dilutions (%)

Fig. 2: Assessment of the NR50 for the positive control

I NRO.160%

(O.D.) and cytotoxicity (%) obtained for the positive controland 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 - - -

Well 4 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

180 i

60

40

20

0

0 0.05 0.1 0.15 0,2 0.25

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, itwas not necessary to perform the stage 2

4-1-3 Conclusion

Under the experimental conditions use, the NR50 of the productBody Lotion was superior to 50%. Frm this result, the test product may beconsidered 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 tothe same category.

Skin Research Dpi 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 ofimmediate effects following application of:the test substance onto the surface of thechorloallantoic membrane of 10-day-old feitile hen’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 theFrench 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 thechorioallantoic 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 appliedneat to the hen’s egg chorioallantoIc membrane.

4-2-2 Results

This study was initiated in the oremises of the

completedon the 13 September 2005.

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

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

Skin Research Opt Cet TxcØlogy lab, p 19126 CIR Panel Book Page 83

Negative cor,troi: saline solution

Table 11: Individual egg score

Test product: Body Lotion ref. TL4S-24-2

Table 9: Individual egg scores obtained for the test product

Mean Score = 7.0

Remarks: A very slight haemorrhage re

egg whereas slight ones were observed t

00

o0

Classification: Moderate’y irritant

ction was observed to tre second treated

the others treated eggs

Table 10: Individual egg obtained for the positive control

Mean Score = 16.0

obtained for the negative control

Mean 0 0

Mean Score 0

Skin Research Opt Cell To

a

cology lab, p. 20126


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.


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

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:

Batch n°:

14 September 2005 Date of expiry:

05022B0902 Age of the culture:

23 September 2005

7 days

The REC score, expressed as a simplified mean cytotoxicity

for the product, tested neat is presented irilTable 12.

index (SMCI), obtained

-r

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


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.


product), we consider that this product is as well tolerated as products belonging tothe 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 productsby using a particular <<in vitro>> approach, which combines several alternativemethods:

- 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 resultsand classifications were obtained:

NRR assay: NR50 > 50% slightly cytotoxic

HET-CAM: Score = 7.0 moderately irritantREC assay: SMCI 1.87 slightly cytotoxic

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

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

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

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

ECIHO International 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 vitro alternatives 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 vitro alternatives to the Draize

primary eye irritation test (phase II) p11/water emulsions. Food and ChemicalToxicology. 32, 943-976

- GETTINGS, S. D., LORDO R. A., HINTZE K. L., et al. (1996). The CTFA evaluationof alternatives program: an evaluation df in vitro alternatives to the Draize primary

eye irritation test (phase ill) surfactant-based formulations. Food and ChemicalToxicology. 34, 79-117.

Skin Research Dpt Cell Thxiology lab.* p. 25! 26


- 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 cytotoxiceffects of chemicals and formulations. ATLA 17. 28-33.

- REGNIER, M., DESBAS, C., BAILLY, C., and DARMON, M. (1987). Differentiationof normal and tumoral human keratinocytes cultured on dermis. Reconstruction ofeither 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 vitromethods 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 fournew human cell lines derived from squamous cell carcinomas of the head and neck.J. NatI. Cancer Inst. 75, 621-635.

Skin Research Dpt Cell Toxicology lab, p. 26)26 CIR Panel Book Page 90

C12 15 ALKYL BENZOATE 08B ti fteners 1

VCRP Data for Alkyl BenzoatesIngredient Use Category No. of UsesC12-15 ALKYL BENZOATE 01B - Baby Lotions, Oils, Powders, and Creams 9C12-15 ALKYL BENZOATE 03A - Eyebrow Pencil 1C12-15 ALKYL BENZOATE 03C - Eye Shadow 28C12-15 ALKYL BENZOATE 03D - Eye Lotion 20C12-15 ALKYL BENZOATE 03E - Eye Makeup Remover 1C12-15 ALKYL BENZOATE 03F - Mascara 1C12-15 ALKYL BENZOATE 03G - Other Eye Makeup Preparations 18C12-15 ALKYL BENZOATE 04B - Perfumes 1C12-15 ALKYL BENZOATE 04E - Other Fragrance Preparation 13C12-15 ALKYL BENZOATE 05A - Hair Conditioner 12C12-15 ALKYL BENZOATE 05B - Hair Spray (aerosol fixatives) 6C12-15 ALKYL BENZOATE 05C - Hair Straighteners 3C12-15 ALKYL BENZOATE 05F - Shampoos (non-coloring) 2C12-15 ALKYL BENZOATE 05G - Tonics, Dressings, and Other Hair Grooming Aids 42C12-15 ALKYL BENZOATE 05I - Other Hair Preparations 33C12-15 ALKYL BENZOATE 07A - Blushers (all types) 16C12-15 ALKYL BENZOATE 07B - Face Powders 26C12-15 ALKYL BENZOATE 07C - Foundations 42C12-15 ALKYL BENZOATE 07D - Leg and Body Paints 1C12-15 ALKYL BENZOATE 07E - Lipstick 66C12-15 ALKYL BENZOATE 07F - Makeup Bases 7C12-15 ALKYL BENZOATE 07G - Rouges 1C12-15 ALKYL BENZOATE 07I - Other Makeup Preparations 17C12 15 ALKYL BENZOATE- 08B Cuticle Softeners - Cu cle So 1C12-15 ALKYL BENZOATE 08C - Nail Creams and Lotions 1C12-15 ALKYL BENZOATE 10A - Bath Soaps and Detergents 11C12-15 ALKYL BENZOATE 10B - Deodorants (underarm) 6C12-15 ALKYL BENZOATE 10E - Other Personal Cleanliness Products 1C12-15 ALKYL BENZOATE 11A - Aftershave Lotion 15C12-15 ALKYL BENZOATE 11D - Preshave Lotions (all types) 1C12-15 ALKYL BENZOATE 11E - Shaving Cream 5C12-15 ALKYL BENZOATE 11G - Other Shaving Preparation Products 3C12-15 ALKYL BENZOATE 12A - Cleansing 30C12-15 ALKYL BENZOATE 12C - Face and Neck (exc shave) 91C12-15 ALKYL BENZOATE 12D - Body and Hand (exc shave) 102C12-15 ALKYL BENZOATE 12E - Foot Powders and Sprays 5C12-15 ALKYL BENZOATE 12F - Moisturizing 174C12-15 ALKYL BENZOATE 12G - Night 35C12-15 ALKYL BENZOATE 12H - Paste Masks (mud packs) 11C12-15 ALKYL BENZOATE 12I - Skin Fresheners 3C12-15 ALKYL BENZOATE 12J - Other Skin Care Preps 48C12-15 ALKYL BENZOATE 13A - Suntan Gels, Creams, and Liquids 17C12-15 ALKYL BENZOATE 13B - Indoor Tanning Preparations 36C12-15 ALKYL BENZOATE 13C - Other Suntan Preparations 9


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 2STEARYL BENZOATE 07C - Foundations 1


Personal Care’ Products CouncilCommitted to Safety,Quality & Innovation

Memorandum


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 thissubmission (found at http ://www .epa. gov!chemrtklpubs/summaries/benzylde/c 1 3450tc.htm)was reviewed for relevant information.

Benzaldehydep-Methoxybenzaldehydem-Methoxy-p-hydroxybenzaldehydeBenzyl acetateBenzyl benzoateMethyl benzoateMethyl p-methylbenzoateMethyl 2-hydroxybenzoatePentyl 2-hydroxybenzoateBenzyl 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 werealso included in the text. Rather than stating that no uses were reported for the rest of theingredients, it would be helpful if the names of the ingredients for which no uses were reportedwere stated.

p.2 - The statement about the permitted uses of methanol in the EU suggests that the EU regulationswere checked for all the ingredients in the report. Please see EU Annex VI entry la forpermitted uses of Methyl Benzoate, Ethyl Benzoate, Propyl Benzoate and Butyl Benzoate aspreservatives.

p.2 - As the alcohols are not included in the report. The information about alcohols can be deletedfrom the Non-Cosmetic Use section. If this information is left in the report, a reference shouldbe 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 onbenzoates.

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 ofbenzoic esters, the primary references should be obtained and cited.

p.3 Please define NI50.p.3 - How many rabbits were used in the dermal LD50 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. Pleasestate whether or not histopathological examinations of major organs were completed in thisstudy.

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”? Howlong was this test?

p.5 - Reference 33 is a carcinogenesis bioassay. Please provide the results concerning carcinogenicityin 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?

2


GREEN BOOK 4 Alkyl Benzoates - Cosmetic Ingredient Revie · At room temperature and pressure, methyl benzoate, ethyl benzoate, butyl benzoate, and isobutyl benzoate are fragrant,

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