GREEN Decyl Glucoside and Other Alkyl Glucosides CIR EXPERT

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Decyl Glucoside and Other Alkyl Glucosides

CIR EXPERT PANEL MEETING JUNE 27-28, 2011 Administrative

Memorandum

To: CIR Expert Panel Members and Liaisons

From: Monice M. Fiume MMF Senior Scientific Analyst/Writer

Date: June 3, 2011

Subject: Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics – Draft Report

Included is the draft report on the Decyl Glucosides and other Alkyl Glucosides as Used in Cosmetics. This is the first time the Panel is seeing this document. The Scientific Literature Review was issued on February 24, 2011.

Glucoside hydrolases present in human skin may break down these chemicals to their respective starting materials, i.e., glucose and a fatty alcohol. Therefore, summary information from reports on fatty alcohols that have previously been reviewed by the CIR is presented in Table 6. Copies of these reports are available online at http://www.cir-safety.org/jun11.shtml .

The following are included in the data tab: 1. Unpublished information on glucoside ingredients. Memo dated Dec 14, 2010. a. Cognis Care Chemicals. 2010. Data profile PLANTACARE 810 UP (Caprylyl/Capryl Glucoside); b. Cognis Care Chemicals. 2010. Data profile PLANTACARE 818 UP (Coco- Glucoside); c. Cognis Care Chemicals. 2010. Data profile PLANTACARE 1200 UP (Lauryl Glucoside); d. Cognis Care Chemicals. 2010. Data profile PLANTACARE 2000 JP (Decyl Glucoside); e. Consumer Product Testing Company. 1993. Repeat insult patch test of 5% active lauryl glucoside and decyl glucoside; f. Consumer Product Testing Company. 2005. Repeat insult patch test of coco- glucoside (1% active). 2. Concentration of use by FDA product category: glucoside ingredients. Memo dated Jan 7, 2011. 3. Updated concentration of use by FDA product category: Glucoside ingredients. Memo dated Jan 28, 2011.

4. Concentration of use by FDA product category: Hexyldecyl d-Glucoside and Octadecyl d-Glucoside. Memo dated March 15, 2011. 5. In Vitro dermal penetration study: Caprylyl/Capryl Glucoside. Memo dated April 8, 2011 6. Comments on the Scientific Literature Review on Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics. Memo dated March 23, 2011. 7. FDA raw data.

Items 1, 2, and 3 from the above list were included in the SLR.

If there are no additional data needs, the Panel should be prepared to formulate a tentative conclusion with rationale and issue a Tentative Report for public comment. If the data are not complete for making a determination of safety, then an Insufficient Data Announcement should be issued listing the additional data that are needed.

Distributed for Comment Only -- Do Not Cite or Quote

CIR Panel Book Page 1 Distributed for Comment Only -- Do Not Cite or Quote History - Decyl Glucosides and Other Alkyl Glucosides

Scientific Literature Review: February 24, 2011 The following unpublished data were included in the SLR: 1. Unpublished information on glucoside ingredients. Memo dated Dec 14, 2010. a. Cognis Care Chemicals. 2010. Data profile PLANTACARE 810 UP (Caprylyl/Capryl Glucoside); b. Cognis Care Chemicals. 2010. Data profile PLANTACARE 818 UP (Coco-Glucoside); c. Cognis Care Chemicals. 2010. Data profile PLANTACARE 1200 UP (Lauryl Glucoside); d. Cognis Care Chemicals. 2010. Data profile PLANTACARE 2000 JP (Decyl Glucoside); e. Consumer Product Testing Company. 1993. Repeat insult patch test of 5% active lauryl glucoside and decyl glucoside; f. Consumer Product Testing Company. 2005. Repeat insult patch test of coco- glucoside (1% active). 2. Concentration of use by FDA product category: glucoside ingredients. Memo dated Jan 7, 2011. 3. Updated concentration of use by FDA product category: Glucoside ingredients. Memo dated Jan 28, 2011

Draft Report: June 27-28, 2011 The following were received after the SLR was announced. The data have been incorporated into the report, and the comments have been addressed. 1. Comments on the Scientific Literature Review on Decyl Glucosides and Other Alkyl Glucosides as Used in Cosmetics. Memo dated March 23, 2011. 2. Concentration of use by FDA product category: Hexyldecyl d-Glucoside and Octadecyl d-Glucoside. Memo dated March 15, 2011. 3. In Vitro dermal penetration study: Caprylyl/Capryl Glucoside. Memo dated April 8, 2011

CIR Panel Book Page 2 SciFinder Search – April 7, 2011

History Session began April 7, 2011 at 12:35 PM April 7, 2011 12:36 PM Explore substances by ID: glucoside initiated Explore complete Explore results No answers April 7, 2011 12:48 PM Explore substances by ID: 34625-23-5, 27214-60-4, 65309-84-4, 58846-77-8, 124828-32-6, 148406-76-2, 150679-30-4, 6801-88-3, 6801-92-9, 265312-14-9, 6801-86-1, 41444-56-8 initiated Explore complete Explore results Distributed Answer set 2 created with 6 answers from REGISTRY April 7, 2011 12:57 PM

Explore substances by ID: 30285-48-4, 5391-18-4, 41444-57-9, 29836-26-8, 58846-77-8, 68515-73-1, 141464-42-8, 98283-67-1, 27836-64-2, 110615-47-9, 54549-26-7, for Comment CIR 144982-05-8, 68515-73-1, 110615-47-9, 200413-69-0, 34625-23-5, 27214-60-4, 65309-84-4, 58846-77-8, 124828-32-6, 148406-76-2, 150679-30-4 initiated Explore complete Panel Explore results

Answer set 3 created with 13 answers from REGISTRY Only Book Saved 13 substance answers from Answer set 3 as 'Decyl glucoside 1' --

Page Retrieve reference information in 13 substances of Answer set 3 Do Answer set 4 created with Not 3 3,080 answers from CAPLUS 336 answers from MEDLINE Cite

Refine Answer set 4 by document type or Book, Clinical Trial, Conference, Journal, Letter, Report, Review Quote Answer set 5 created with 1,814 answers from CAPLUS 335 answers from MEDLINE Detailed display from Answer set 5 of Expanding Coverage of the Metabolome for Global Metabolite Profiling Saved 2149 reference answers from Answer set 5 as 'Decyl Gluc 1 Refines by pub type' Refine Answer set 4 by document type Patents only Answer set 6 created with 1,268 answers from CAPLUS April 7, 2011 1:03 PM Explore substances by ID: 6801-88-3, 6801-92-9, 265312-14-9, 6801-86-1, 41444-56-8, C12-18 Alkyl Glucoside, C12-20 Alkyl Glucoside, C12-20 Alkyl Glucoside, C20-22 Alkyl Glucoside, Cetearyl Glucoside, Coco- Glucoside, Octyldodecyl Glycoside initiated Explore complete Explore results Answer set 7 created with 5 answers from REGISTRY Saved 5 substance answers from Answer set 7 as 'decyl gluc 2' Combine the current substance answer set with saved answer sets initiated: decyl gluc 2 OR Decyl glucoside 1 at April 7, 2011 1:04 PM Answer set 8 created with 15 answers from REGISTRY Saved 15 substance answers from Answer set 8 as 'Decyl glucoside All' Retrieve reference information in 15 substances of Answer set 8 Answer set 9 created with 3,080 answers from CAPLUS 336 answers from MEDLINE Saved 3416 reference answers from Answer set 9 as 'Decyl Glucoside All' Refine Answer set 9 by document type Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Answer set 10 created with 1,815 answers from CAPLUS 333 answers from MEDLINE e refined by document type' Saved 2148 reference answers from Answer set 10 as 'Decyl Glucosid Distributed Refine Answer set 10 by research topic sugar surfactant Answer set 11 created with 49 answers from CAPLUS Detailed display from Answer set 11 of Physical properties and inhibitory effects of three-component hybrid liposomes including sugar surfactants for Comment CIR Detailed display from Answer set 11 of Alkylpolyglycoside: carbohydrate based surfactant

Panel Detailed display from Answer set 11 of Adsorption of n-Decyl--D-glucopyranoside and n-Decyl--D-maltopyranoside Mixtures at the Liquid-Vapor Interface Explore results Only Book Answer set 14 created with

16 answers from CAPLUS -- Page 131 answers from MEDLINE Do

Detailed display from Answer set 14 of Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 105 patients Not 4

Full text accessed for Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 105 patients from Dermatitis : contact, Cite atopic, occupational, drug : official journal of the American Contact Dermatitis Society, North American Contact Dermatitis Group Volume: 21 Issue: 5 Pages: 262-8 2010 or April 7, 2011 1:22 PM Quote Explore references by research topic: decyl glucoside initiated, resulting in 2 candidates Explore complete Candidates Selected 413 references were found containing "decyl glucoside" as entered. Automatically removed 6 duplicate MEDLINE answer(s) Explore results Answer set 15 created with 399 answers from CAPLUS 8 answers from MEDLINE

April 7, 2011 1:23 PM Explore references by research topic: decyl glucoside initiated, resulting in 2 candidates Explore complete Candidates Selected 413 references were found containing "decyl glucoside" as entered. Automatically removed 8 duplicate MEDLINE answer(s) 929 references were found containing the concept "decyl glucoside". Automatically removed 8 duplicate MEDLINE answer(s) Explore results Answer set 18 created with 915 answers from CAPLUS 6 answers from MEDLINE

Explore references by research topic: glucoside initiated, resulting in 2 candidates April 7, 2011 1:26 PM Explore references by research topic: glucoside initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review, Conference April 7, 2011 1:27 PM Distributed Explore references by research topic: 30285-48-4 initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review, Conference Explore complete for Comment CIR Candidates Selected

Panel 59 references were found containing "30285-48-4" as entered. Automatically removed 3 duplicate MEDLINE answer(s) Only Book Explore results

Answer set 21 created with -- Page 48 answers from CAPLUS Do

8 answers from MEDLINE Not 5

April 7, 2011 1:29 PM Cite Explore references by research topic: 30285-48-4 5391-18-4 41444-57-9 29836-26-8 58846-77-8 68515-73-1 141464-42-8 98283-67-1 27836-64-2 110615-47-9 54549-26-7 or 144982-05-8 68515-73-1 110615-47-9 initiated, resulting in 0 candidate

Quote April 7, 2011 1:31 PM Explore references by research topic: irritancy of glucosides initiated, resulting in 4 candidates Explore complete Candidates Selected 25 references were found where the two concepts "irritancy" and "glucosides" were present anywhere in the reference. Explore results Answer set 22 created with 21 answers from CAPLUS 4 answers from MEDLINE Detailed display from Answer set 22 of Non-irritating cosmetic and pharmaceutical compositions for irritating agents retinoids and - and -hydroxy acids Detailed display from Answer set 22 of Application of surfactants. 51. Alkyl polyglucoside surfactants. 1 Detailed display from Answer set 22 of Lactobionic acid in a natural alkylpolyglucoside-based vehicle: assessing safety and efficacy aspects in comparison to glycolic acid April 7, 2011 1:34 PM Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates Explore complete Candidates Selected 145 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 23 duplicate MEDLINE answer(s) Explore results Answer set 23 created with 121 answers from CAPLUS 1 answer from MEDLINE April 7, 2011 1:37 PM Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Distributed Candidates Selected 112 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 22 duplicate MEDLINE answer(s) Explore results for Comment CIR Answer set 25 created with

Panel 89 answers from CAPLUS 1 answer from MEDLINE Only Book Full text accessed for Comparative testing for the identification of skin- of nonionic sugar lipid from Regulatory Toxicology and Pharmacology Volume: 58 Issue: 2

Pages: 301-307 2010 -- Page Full text accessed for Irritant and of eight commonly used in skin cleansers: an evaluation of 105 patients from Dermatitis : contact, atopic, occupational, drug : official Do

journal of the American Contact Dermatitis Society, North American Contact Dermatitis Group Volume: 21 Issue: 5 Pages: 262-8 2010 Not 6

Detailed display from Answer set 25 of Zinc and its derivatives: their applications in cosmetic Cite April 7, 2011 1:42 PM or

Explore references by research topic: sensitization potential of surfactants initiated, resulting in 4 candidates Quote Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 112 references were found containing the two concepts "sensitization potential" and "surfactants" closely associated with one another. Automatically removed 22 duplicate MEDLINE answer(s) Explore results Answer set 26 created with 89 answers from CAPLUS 1 answer from MEDLINE Keep Me Posted profile 'sensitization potential of surfactants' created from Answer set 26 Refine Answer set 26 by research topic cosmetics Answer set 27 created with 3 answers from CAPLUS 1 answer from MEDLINE April 7, 2011 1:44 PM Explore references by research topic: irritation of surfactants initiated, resulting in 5 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Explore complete Candidates Selected 668 references were found containing the two concepts "irritation" and "surfactants" closely associated with one another. Automatically removed 169 duplicate MEDLINE answer(s) Explore results Answer set 28 created with 442 answers from CAPLUS Distributed 57 answers from MEDLINE Keep Me Posted profile 'irritation of surfactants' created from Answer set 28 Refine Answer set 28 by research topic cosmetics for Comment CIR Answer set 29 created with

Panel 152 answers from CAPLUS 7 answers from MEDLINE Only Book Detailed display from Answer set 29 of Ocular Irritection: in vitro method for testing ocular irritancy

Refine Answer set 29 by research topic -- Page glucoside Do

Answer set 30 created with Not 7

1 answer from CAPLUS Cite 1 answer from MEDLINE or Detailed display from Answer set 30 of Photostability of naturally occurring whitening agents in cosmetic microemulsions Quote Refine Answer set 28 by research topic glucoside Answer set 31 created with 3 answers from CAPLUS 1 answer from MEDLINE Detailed display from Answer set 31 of Evaluation of irritation potential of surfactant mixtures Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_04_07_2011_134818.ris" Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_04_07_2011_134835.ris" Exported Evaluation of irritation potential of surfactant mixtures in 'RIS' format as "Reference_04_07_2011_134854.ris" Session began April 7, 2011 at 1:50 PM April 7, 2011 1:50 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 32 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE April 7, 2011 1:54 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 34 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE Answer set 35 created with 4 answers from CAPLUS Detailed display from Answer set 35 of Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants Exported Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants in 'RIS' format as "Cho.ris" Exported Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants in 'RIS' format as "Cho.ris" April 7, 2011 1:56 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 36 created with 1,815 reference answers from CAPLUS 333 reference answers from MEDLINE Distributed April 7, 2011 1:57 PM Saved answer set 'Decyl Glucoside refined by document type' opened Answer set 37 created with 1,815 reference answers from CAPLUS for Comment CIR 333 reference answers from MEDLINE

Panel April 7, 2011 1:57 PM Saved answer set 'Decyl Glucoside All' opened Only Book Answer set 38 created with

3,080 reference answers from CAPLUS -- Page 336 reference answers from MEDLINE Do

Refine Answer set 38 by document type Not 8

Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Cite Answer set 39 created with or 1,815 answers from CAPLUS Quote 333 answers from MEDLINE April 7, 2011 1:58 PM Saved answer set 'Decyl glucoside All' opened Answer set 40 created with 15 substance answers from REGISTRY Retrieve reference information in 15 substances of Answer set 40 Answer set 41 created with 3,080 answers from CAPLUS 336 answers from MEDLINE Refine Answer set 41 by document type Book, Clinical Trial, Conference, Journal, Preprint, Report, Review Answer set 42 created with 1,815 answers from CAPLUS 333 answers from MEDLINE Keep Me Posted profile 'Glucosides Refined by Document Type' created from Answer set 42 April 7, 2011 2:01 PM Explore references by research topic: alkyl polyglucosides initiated, resulting in 2 candidates Explore complete Candidates Selected 2649 references were found containing the concept "alkyl polyglucosides". Automatically removed 37 duplicate MEDLINE answer(s) Explore results Answer set 43 created with 2,610 answers from CAPLUS 2 answers from MEDLINE April 7, 2011 2:02 PM Explore references by research topic: alkyl polyglucosides initiated, resulting in 2 candidates Limiters Book, Preprint, Clinical Trial, Report, Journal, Review English Distributed Explore complete Candidates Selected 359 references were found containing the concept "alkyl polyglucosides". Automatically removed 34 duplicate MEDLINE answer(s) for Comment CIR Explore results

Panel Answer set 44 created with 323 answers from CAPLUS Only Book 2 answers from MEDLINE

Keep Me Posted profile 'Alkyl Polygolucosides' created from Answer set 44 -- Page Detailed display from Answer set 44 of Profile of irritant patch testing with detergents: sodium lauryl sulfate, sodium laureth sulfate and alkyl polyglucoside Do

Detailed display from Answer set 44 of Toxicology of alkyl polyglycosides Not 9

Detailed display from Answer set 44 of Alkyl polyglycosides Cite Detailed display from Answer set 44 of Alkyl polyglycosides or Full text accessed for from Surfactant Science Series Volume: 98 Issue: Detergency of Specialty Surfactants Pages: 1-69 2001 Quote Detailed display from Answer set 44 of Nonionic Surfactants: Alkyl Polyglucosides. [In: Surfactant Sci. Ser., 2000; 91] Exported 3 reference answers from Answer set 44 in 'RIS' format as "APGs.ris" Detailed display from Answer set 44 of Dermatological properties of alkyl polyglycosides Full text accessed for Dermatological properties of from Alkyl Polyglycosides Pages: 169-176 1997 Detailed display from Answer set 44 of Alkyl polyglycosides in personal care products Full text accessed for in personal care products from Alkyl Polyglycosides Pages: 71-98 1997 Detailed display from Answer set 44 of Using alkyl polyglycosides in personal-care products Detailed display from Answer set 44 of Alkylpolyglycosides - a new cosmetic concept for mildness and care Full text accessed for - a new cosmetic concept for mildness and care from Agro-Food-Industry Hi-Tech Volume: 5 Issue: 5 Pages: 20-8 1994 Detailed display from Answer set 44 of Alkyl polyglycosides: properties and applications Exported 6 reference answers from Answer set 44 in 'RIS' format as "APGs2.ris" April 7, 2011 2:18 PM

Keep Me Posted searches checked weekly

NLM STN Intl EU FDA ChemPortal conc Toxline- Misc Regis- ECE- SIDS-HPV-IUCL # uses NTIS RTECS Merck EU SCCS IARC NTP EAFUS GRAS OTC NIOSH Misc data Pubmed NLM try TOC ID date searched 1-28-11 11-19-10 11-23 Oct2010 1-12-11 1-12 2-08 2-08 2-08 2-08 2-08 2-08 1-12 Decyl Glucoside x 2 0 x x no no no no no no no no no x Arachidyl Glucoside x 0 x x no no no no no no no no no no Butyl Glucoside no 1 0 x x no no no no no no no no no x C10-16 Alkyl Glucoside no 0 x x no no no no no no no no no x C12-18 Alkyl Glucoside no 0 x no no no no no no no no no no C12-20 Alkyl Glucoside x 0 x no no no no no no no no no no C20-22 Alkyl Glucoside no 0 no no no no no no no no no no Caprylyl/Capryl Glucoside x 3 x x no no no no no no no no no x Caprylyl Glucoside x 0 x x x no no no no no no no no no x 352 Cetearyl Glucoside x 0 x no no no no no no no no no no Distributed Coco-Glucoside x 0 x no no no no no no no no no no Ethyl Glucoside x 2 0 x x no no no no no no no no no x Isostearyl Glucoside no 0 x x no no no no no no no no no no for Lauryl Glucoside x 2 0 x x no no no no no no no no no x CIR Comment Myristyl Glucoside x 1 0 x x no no no no no no no no no x Panel Octyldodecyl Glucoside no 0 x no no no no no no no no no no Palm Kernel/Coco Glucoside no 0 x no no no (deleted from report) no Only Book Undecyl Glucoside no 1 0 x x no no no no no no no no no x Tradenames Search 639 0 -- Page

In VCRP/but not INCI Not 10 Hexadecyl D-Glucoside x no no no no no x Cite (54549-27-8)

Octadecyl D-Glucoside or

x no no no no no x Quote (27836-65-3)

References Ordered Whit – 25 papers (11-23-10) NTIS – 3 documents (11-23-10)

Distributed for Comment Only -- Do Not Cite or Quote Search Terms

((DECYL OR ETHYL OR BUTYL OR CAPRYLYL OR UNDECYL OR LAURYL OR MYRISTYL OR ARACHIDYL OR CAPRYL OR ALKYL OR CETEARYL OR COCO OR (PALM AND KERNEL) OR ISOSTEARYL OR ISOOCTADECYL OR OCTYLDODECYL OR OCTYL OR DODECYL OR TETRADECYL OR EICOSYL) AND GLUCOSIDE) OR ((ETHYL OR BUTYL OR OCTYL OR DECYL OR UNDECYL OR DODECYL OR LAURYL OR MYRISTYL OR TETRADECYL OR ARACHIDYL OR EICOSYL OR ISOSTEARYL OR ISOOCTADECYL) AND GLUCOPYRANOSIDE) OR 30285-48-4 OR 5391-18-4 OR 41444-57-9 OR 29836-26-8 OR 58846-77-8 OR 68515-73-1 OR 141464-42-8 OR 98283-67-1 OR 27836-64-2 OR 110615-47-9 OR 54549-26-7 OR 144982-05-8 OR 68515-73-1 OR 110615-47-9 OR 200413-69-0 OR 34625-23-5 OR 27214-60-4 OR 65309-84-4 OR 58846-77-8 OR 124828-32-6 OR 148406-76-2 OR 150679-30-4 OR 6801-88-3 OR 6801-92-9 OR 265312-14-9 OR 6801-86-1 OR 41444-56-8

ORAMIX OR PLANTACARE OR PLANTAREN OR ORAMIX OR SUCRAPH OR AVANEL OR ORISTAR OR DESULF OR (TRITON AND SURFACTANT) OR SEPISOFT OR (TEGO AND CARE)

SCiFinder Seach Terms – 4-7-11

30285-48-4 5391-18-4 41444-57-9 29836-26-8 58846-77-8 68515-73-1 141464-42-8 98283-67-1 27836-64-2 110615-47-9 54549-26-7 144982-05-8 68515-73-1 110615-47-9 200413-69-0 34625-23-5 27214-60-4 65309-84-4 58846-77-8 124828-32-6 148406-76-2 150679-30-4

6801-88-3 6801-92-9 265312-14-9 6801-86-1 41444-56-8 C12-18 Alkyl Glucoside C12-20 Alkyl Glucoside C12-20 Alkyl Glucoside C20-22 Alkyl Glucoside Cetearyl Glucoside Coco- Glucoside Octyldodecyl Glycoside Alkyl Glucoside

CIR Panel Book Page 11 Decyl Glucosides and Other Alkyl Glucosides Data Profile* – June 2011 – Writer, Monice Fiume – (updated 5-15-2011) Inhalaiton Inhalaiton Repro/Dev Tox Genotox Carcinogeni- city Dermal Irr/Sens Ocular Irritaton Reported Use Method of Manufacture Toxicokinetic Data Tox –Animal Acute, Derrmal Tox –Animal Acute, Oral Tox, Animal Acute, Inhalation Tox –Animal Rptd Dose, Dermal Tox, Animal Rptd Dose, Oral Tox –Animal Rptd Dose, Decyl Glucoside X X X Arachidyl Glucoside X C10-16 Alkyl Glucoside X X C12-18 Alkyl Glucoside C12-20 Alkyl Glucoside X C20-22 Alkyl Glucoside Caprylyl/Capryl Glucoside X X X X X X X X Caprylyl Glucoside X X X Distributed Cetearyl Glucoside X Coco-Glucoside X X X Ethyl Glucoside X X X

Isostearyl Glucoside for CIR Lauryl Glucoside X X X X Comment

Panel Myristyl Glucoside X Octyldodecyl Glucoside Only Book Undecyl Glucoside Alkyl Polyglucosides (APGs), General Info X X X X -- Page Previously Reviewed Fatty Alcohols Yr. Rev. Do n-Butyl Alcohol 2008 X X X X X X X X Not 12 Cetearyl Alcohol 1988 X X Cetyl Alcohol 1988 X X X X X X X X Cite

Coconut Alcohol 2008 or

Isostearyl Alcohol 1988 X X X Quote Myristyl Alcohol 1988 X X X X Octyl Dodecancol 1985 X X X X Stearyl Alcohol 1985 X X X X X X X

*“X” indicates that data were available in a category for the ingredient

Report

Draft Report

Decyl Glucoside and Other Alkyl Glucosides as Used in Cosmetics

June 27, 2011

The 2011 Cosmetic Ingredient Review Expert Panel members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is F. Alan Andersen, Ph.D. This report was prepared by Monice M. Fiume, Senior Scientific Analyst/Writer, and Bart A. Heldreth, Ph.D., Chemist, CIR.

TABLE OF CONTENTS

Introduction ...... 1 Chemistry ...... 1 Definition and Structure...... 1 Impurities, Constituents, and Physical and Chemical Properties...... 2 Method of Manufacture ...... 2 Use ...... 2 Cosmetic ...... 2 Non-Cosmetic ...... 3 Toxicokinetics ...... 3 Absorption, Distribution, Metabolism, and Excretion ...... 3 Dermal ...... 3 Oral ...... 4 Absorption Enhancement ...... 4 Toxicological studies ...... 4 Single Dose (Acute) Toxicity ...... 5 Dermal ...... 5 Oral ...... 5 Repeated Dose Toxicity ...... 5 Dermal ...... 5 Oral ...... 7 Reproductive and Developmental Toxicity ...... 7 Oral ...... 7 In Vitro Estrogenicity Assays ...... 8 Genotoxicity ...... 8 Carcinogenicity ...... 8 Irritation and Sensitization ...... 8 Skin Irritation...... 9 Non-Human...... 9 Human ...... 9 Sensitization ...... 9 Non-Human...... 9 Human ...... 9 Mucosal Irritation ...... 9 Case Studies ...... 10 Summary ...... 10 Tables ...... 12 Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment...... 12 Table 2. Chemical and physical properties ...... 15 Table 3a. Frequency and concentration of use according to duration and type of exposure ...... 17 Table 3b. Ingredients Not Reported to be Used ...... 18 Table 4. Skin irritation and sensitization studies ...... 19 Table 5. Mucosal irritation studies ...... 23 Table 6. Summaries of information on fatty alcohols from previous CIR reports ...... 25 References ...... 28

CIR Panel Book Page 14 Distributed for Comment Only -- Do Not Cite or Quote

INTRODUCTION This document is a review of studies relevant to the safety of 17 alkyl glucoside ingredients as used in cosmetic formulations. Most of these ingredients function in cosmetics as surfactants. A few do not function as surfactants, but are used as skin conditioning agents, hair conditioning agents, or emulsion stabilizers. The ingredients included in this review are obtained by the condensation of an alcohol with a cyclic form of glucose (D- glucopyranose). The ingredients included in this group are: Decyl Glucoside Cetearyl Glucoside Arachidyl Glucoside Coco-Glucoside Butyl Glucoside Ethyl Glucoside C10-16 Alkyl Glucoside Isostearyl Glucoside C12-18 Alkyl Glucoside Lauryl Glucoside C12-20 Alkyl Glucoside Myristyl Glucoside C20-22 Alkyl Glucoside Octyldodecyl Glucoside Caprylyl/Capryl Glucoside Undecyl Glucoside Caprylyl Glucoside

While the names of these ingredients imply that they are mono-glucosides, these ingredients, however, are not limited to mono-glucosides, but may involve products that are the result of a number of condensed glucose repeat units. Glucoside hydrolases in human skin are likely to break down these chemicals to release their respective fatty alcohols and glucose. Therefore, summary information on the appropriate fatty alcohols that have previously been reviewed by the Cosmetic Ingredient Review (CIR) is presented at the end of this report in the last table (Table 6).

CHEMISTRY Definition and Structure This group of ingredients consists of anomerically-alkyl-substituted D-glycopyranosides. Specifically, the alkyl substituents range from 2 to 22 carbons in length, and the D-glycopyranosides consist of glucose-type mono-, di-, tri-, oligo-, or poly-saccharides (e.g., mono = glucose (i.e. D-glucopyranoside) and di = maltose (i.e. D-maltopyranoside)). Regardless of the degree of polymerization (i.e. number of glucose monomers (n); e.g., a degree of polymerization equal to 2 means the di- glucose (disaccharide), maltose) these ingredients are simply named “glucosides.” Although these ingredients are most likely the ß-anomers, the names of these alkyl glucosides are not necessarily specific to either anomer. For example, the general decyl glucoside structure shown in Figure 1 is the ten-carbon, alkyl-chain substituted glycopyranoside, wherein n can be 1 (for a mono-glucoside) or more (for di-, tri-, oligo-, and poly-glucosides):

Figure 1. General Decyl Glucoside Structure

Therefore, decyl glucoside may be monomeric or polymeric. “Poly” will be used generically used throughout the rest of this report to refer to di-, tri-, oligo-, poly-glucosides, and mixtures thereof.

CIR Panel Book Page 15 Distributed for Comment Only -- Do Not Cite or Quote

Decyl glucoside, for example, may be comprised of one or more of the following polyglucosides (in this case maltopyranosides) shown in Figure 2:

Figure 2. Examples of Decyl Glucoside Forms

A decyl glucoside with a degree of polymerization of 1.6, for example, would then be a mixture comprised of decyl glucopyranoside and one of the decyl maltopyranosides (with a slightly higher percentage of the maltose derivative) shown in Figure 2. Because many of these fatty alcohols are supplied from natural feed stocks, the designated length may be the average (e.g., median) length (e.g. decyl glucoside may actually be a mixture of C6, C8, C10, C12, C14, and C16 chain lengths, each anomerically attached to a glucopyranose).1 The definitions and structures of the ingredients included in this review are provided in Table 1. Impurities, Constituents, and Physical and Chemical Properties These compounds are typically solids, with solubility in both aqueous and organic solutions. The available impur- ity, constituent, and physical and chemical property information is presented in Table 2. Method of Manufacture The first report of the synthesis of alkyl glucosides, reported by Fischer in 1893, involves reacting glucose with anhydrous ethanol under acidic conditions to produce ethyl glucoside.2 Though not at all new, alcoholysis of glucose and polysaccharides under acidic conditions is still the method of choice. It is considered to be a “green” process that can involve the use of natural and renewable sources (e.g., the alcohols can be obtained from coconut oil or palm oil and the glucose or polysaccharide can be obtained from corn, potato, or wheat starch).3 Of note, the reaction conditions that produce an ether linkage between a fatty alcohol and the anomeric hydroxy group of glucose are known to cause condensation of one molecule of glucose with another molecule of glucose, thereby producing alkyl polyglucosides (APGs) even when an alkyl monoglucosides may be the intended product. USE Cosmetic The alkyl glucosides named in this report function primarily as surfactants.4 A few do not function as surfactants, and are used as a skin conditioning agents, hair conditioning agents, or emulsion stabilizers. Voluntary Cosmetic Registration Program (VCRP) data obtained in 2011 indicate that, of the ingredients reviewed in this safety assessment, decyl glucoside has the highest frequency of uses reported i.e., 492); the majority of these uses i.e., 421), are in rinse-off formulations.5 Cetearyl glucoside, lauryl glucoside, and coco-glucoside have 477, 399, and 350

CIR Panel Book Page 16 Distributed for Comment Only -- Do Not Cite or Quote reported uses, respectively. Cetearyl glucoside is mostly reported to be used in leave-on products. The remaining ingredients that are reported to be used have ≤ 75 uses. Based on data from a survey conducted by the Personal Care Products Council (Council), decyl glucoside has the highest leave-on and rinse-off concentrations of use, at 11 and 33%, respectively.6 Products containing alkyl glucosides are reported to be used on baby skin or applied to the eye area, and mucous membranes may be exposed to these products. Coco-glucoside is reported to be used in a product that could be ingested, and a few of the ingredients are reported to be used in product types that could be inhaled. Since some of the glucosides are reported to be in products that could be inhaled, effects on the lungs that may be induced by aerosolized products containing these ingredients are of concern. The particle size of aerosol hair sprays and in pump hair sprays is around 38 m and >80 m, respectively, and is large compared to respirable particle sizes (≤10 m). Therefore, because of their size, most aerosol particles are deposited in the nasopharyngeal region and are not respirable. Frequency and concentration of use data are provided in Table 3a. In some cases, reports of uses were received in the VCRP, but no concentration of use is available. For example, decyl glucoside is reported to be used in 25 baby products, but no use concentration was available. In other cases, no reported uses were received in the VCRP, but a use concentration was provided in the industry survey. For example, caprylyl glucoside was not reported to be used in non-coloring hair products, but the industry survey indicated that it was used in such products at 4%. It should be presumed that caprylyl glucoside is used in at least one hair care product. The ingredients not listed in the VCRP or by the Council as in use are listed in Table 3b. All of the glucosides named in the report, with the exception of C20-22 alkyl glucoside, are listed in the European Union inventory of cosmetic ingredients.11 Non-Cosmetic Caprylyl glucoside and similar alkyl glucosides are effective solubilizers of lipids and proteins below their critical micelle concentrations (CMC), and are used in various biochemical techniques and membrane research. These ingredients also can be used to reconstitute enzymes of other proteins from crude biological preparations. 12

A C16/18-APG named cetearyl glucoside and cetearyl alcohol has been approved by the FDA as a pharmaceutical excipient.13 The use of decyl glucoside as a stabilizer in nanosuspensions for dermal delivery has been investigated; decyl glucoside was effective as a stabilizer with resveratrol14 and hesperetin ((S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4- methoxy-phenyl)-4H-1-benzopyran-4-one)15 nanosuspensions.

TOXICOKINETICS In an in vitro dermal absorption study using human skin samples, the mean absorbed dose of 10% caprylyl/capryl glucoside was 0.01%. In an oral study in which female mice were dosed by gavage with a 5% aq. solution of caprylyl [U-14C]glucoside, the highest levels of radioactivity at 2 h after dosing were found in the stomach, intestines, liver, and kidneys. The radioactivity in the stomach was primarily unchanged substrate, while only a trace amount found in the liver was unchanged. Labeled glucose was found in all of these organs. In a feeding study in rats in which dietary sucrose was replaced with 10 or 20% ethyl glucoside for 39 days, 60-90% of the ingested ethyl glucoside was recovered in the urine. Absorption, Distribution, Metabolism, and Excretion Dermal Glucoside hydrolases are known to be present in human skin. Therefore, the first step in the metabolism of these ingredients may be breaking down these chemicals to glucose and their respective fatty alcohols.16 In Vitro

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Caprylyl/Capryl Glucoside The dermal penetration of caprylyl/capryl glucoside, diluted to 10% in Hank’s buffered salt solution, pH 6.5, was evaluated in vitro using human skin.17 Two skin samples from each of three donors were used in the study (n=6). After 24 h, the mean recovery was 0.52% of caprylyl/capryl glucoside from two tape strips and 0.30% of caprylyl/capryl glucoside in the further 18 tape strips. The mean absorbed dose of caprylyl/capryl glucoside, as the sum of the amounts found in the viable epidermis, dermis, and receptor medium, was 0.01%. Oral Non-Human Caprylyl Glucoside Three female NMRI mice were given a single oral dose, by gavage, of 37 MBq/mmol caprylyl [U-14C]glucoside in 0.05 ml of a 5% aq. solution of phophatidylcholine.18 The animals were killed 2 h after dosing. The highest levels of radioactivity were found in the stomach, intestines, liver, and kidneys, with most of the radioactivity (81-98%) distributed in the aqueous phase. High levels of radioactivity that were not extractable with chloroform were found in the urine, which, according to the researchers, indicated a high rate of degradation to water-soluble metabolites. In the stomach, 75% of the radioactivity was associated with unchanged substrate. In the kidneys and intestines, 50% of the total radioactivity was unchanged substrate, while only a trace amount found in the liver was associated with unchanged substrate. Labeled glucose was detected in all four of these organs. In the stomach, intestines, and kidneys, 13- 19% of the radioactivity was contained in the chloroform extract, and most of it was derived from caprylyl [U-14C]glucoside. In this extract in the stomach, approximately 2% acylated-labeled substrate was detected. Ethyl Glucoside Groups of 6 male Wistar ST rats were fed, for 39 days, a diet in which sucrose was replaced with 10 or 20% ethyl glucoside, and a control group was fed unaltered (i.e., sucrose-containing) feed.19 A 24-h urine volume was measured weekly. Approximately 60-90% of the ethyl glucoside ingested by treated animals was recovered in the urine. Absorption Enhancement Caprylyl glucoside has been shown to increase the absorption of poorly absorbed drugs (e.g., insulin), both in vitro across human carcinoma monolayers and in vivo through mucosal membranes. In the in vitro study, the enhancement of the permeability of insulin across T84 and Caco-2 cell monolayers by was caprylyl glucoside concentration-dependent; permeability of insulin was not significantly enhanced at concentrations of 0.2 and 0.3%, while it was enhanced with 0.4 and 0.5% caprylyl glucoside.20 In a transmucosal absorption study, the effect of caprylyl glucoside on the nasal, buccal, and rectal absorption of insulin was examined using male Lewis rats.12 A 5% solution of caprylyl glucoside had an enhancing effect on buccal absorption. The effect of other alkyl glycosides, including decyl and lauryl glucoside, on mucosal penetration was also evaluated. A 5% solution of decyl glucoside also enhanced the buccal absorption of insulin, but 5% lauryl glucoside did not have a significant effect. The researchers stated that there was no consistent relationship between alkyl chain length and penetration enhancement.

TOXICOLOGICAL STUDIES In single dose dermal studies with caprylyl/capryl glucoside and C10-16 alkyl glucoside (both 50% a.i., n:1.6) in rabbits, the LD50 was greater than the 2000 mg/kg dose administered. In oral studies with the same test substances, none of the mice dosed with 2000 mg/kg caprylyl glucoside and none of the rats dosed with 5000 mg/kg C10-16 alkyl glucoside died during the study. In 2-wk repeated dose dermal studies in rabbits with 60% active caprylyl/capryl

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glucoside, occlusive applications produced testicular effects, while non-occlusive application did not. In the two occlusive studies, one with 0.09 and 1.8 g a.i./kg and the other with 0.14-1.25 g a.i./kg, an NOEL for testicular effects could not be established. In the non-occlusive study, the NOEL for systemic toxicity was 0.18 g a.i./kg caprylyl/capryl glucoside. Severe dermal irritation was observed in both occlusive studies, while slight to moderate irritation was reported in the non-occlusive study. In oral repeated dose toxicity studies, moderately-dilated renal tubules were observed in 3 of 6 rats fed 20% ethyl glucoside for 39 days, but in none of the rats fed 10% ethyl glucoside. Kidney weights were statistically significantly increased in the test animals. In rats dosed orally with 250-1000 mg/kg C12/16 APG for 13 wks, reversible irritation and ulceration of the stomach mucosa was observed, but there was no systemic toxicity reported for any group.

Single Dose (Acute) Toxicity Dermal Caprylyl/Capryl Glucoside Groups of 5 male and 5 female New Zealand White (NZW) rabbits were given a single dermal dose of 2000 mg/kg bw caprylyl/capryl glucoside, 50% active ingredient (a.i.) (as C8/C10 APG); the degree of polymerization, n, was 1.6.21 Mild to moderate irritant effects, fecal staining, yellowing around the application site, and a few other effects were observed. One animal died of an unrelated infection, and 5 had gross findings at necropsy. C10-16 Alkyl Glucoside Groups of 5 male and 5 female NZW rabbits were given a single dermal dose of 2000 mg/kg bw C10-16 alkyl glucoside, 50% a.i. (as C10-16 APG; n:1.6).21 Slight depression, hunched posture, mild to marked erythema, and marked desquamation were observed. None of the animals died during the study. Oral Caprylyl Glucoside Female NMRI mice were given a single oral dose of 40 mg (2000 mg/kg bw) caprylyl glucoside as a suspension in 0.2 ml of a 5% aq. solution of phosphatidylcholine.18 No toxic effects were observed during a 2-wk post-dose observation period. Growth and behavior were not affected. Caprylyl/Capryl Glucoside Groups of 5 male and 5 female Sprague-Dawley rats were given a single oral dose of 5000 mg/kg bw caprylyl/capryl glucoside (as C8/10 APG; n:1.6, 50% a.i.).21 None of the animals died during the study. C10-16 Alkyl Glucoside Groups of 5 male and 5 female Sprague-Dawley rats were given a single oral dose of 5000 mg/kg bw C10-16 alkyl glucoside (as C10/16 APG; n:1.6, 50% a.i.).21 None of the animals died during the study. Additionally, no mortality was observed upon dosing of 2 male and 2 female Wistar rats with a single oral dose of 2000 mg/kg bw C12/14 APG, n: 1.6 and 60% a.i. Repeated Dose Toxicity Dermal Caprylyl/Capryl Glucoside In a 2-wk study, 10 occluded applications of 0.15 and 0.60 g a.i./kg caprylyl/capryl glucoside in distilled water were made to the skin of rabbits.22 Slight to moderate irritation was reported. No mortality and no signs of toxicity were observed. (Additional details were not provided.) Groups of 6 male and 6 female NZW rabbits were dosed with 4 ml/kg of 0, 0.9, and 1.8 g a.i./kg (0, 22.5, and 45 w/v%, respectively) caprylyl/capryl glucoside (60% active) in distilled water.23 Ten 6-h occlusive applications were made over a 2-wk period. Treatment-related signs of toxicity, such as ataxia, lethargy, and emaciation, were observed in both test groups. One female of the 1.8 g a.i./kg group died after 10 doses, and the death was considered test article-related. Severe 5

CIR Panel Book Page 19 Distributed for Comment Only -- Do Not Cite or Quote dermal irritation was observed in males and females of both test groups by days 5-6 of the study; slight irritation was observed 1 day after the initial dose. Body weights of treated male and female rabbits were significantly less than those of controls, and mean body weight loss was observed for both groups. Significant changes were observed in some hematology and clinical chemistry values; a dose-response relationship was not observed for most of the hematology changes. Compared to controls, absolute testes weights were significantly lower in treated males of both dose groups. No other compound- related changes in organ weights were observed. Microscopic examination of selected male tissues reported very slight to marked testicular degeneration in all rabbits in the 0.9 g a.i./kg group and slight to marked testicular degeneration in four of the rabbits of the 1.8 g a.i./kg group.24 Very slight to moderate atrophy of the prostate and “accessory sex glands” was observed in 3 rabbits of each group. The researchers stated that irritation, inflammation, and stress in these animals were major contributing factors to many, if not all, of the toxicologic effects; however, the researchers also stated that it is possible that caprylyl/capryl glucoside produced some of the effects. 23 A no-observed effect level (NOEL) was not determined. In a similar 2-wk study, but with a non-occlusive application, 2 ml of 0, 0.06, 0.18, or 0.54 g a.i./kg caprylyl/capryl glucoside (60% active) in distilled water (corresponding to concentrations of 0, 3, 9, and 27% a.i., respectively) were applied to the intact skin of the backs of 6 male rabbits/group.25 These doses were selected following a 2-wk pilot study, in which unoccluded exposure to 0.12, 0.23, and 0.45 a.i. g/kg caprylyl/capryl glucoside produced slight to moderate erythema and edema. In the main study, treatment-related signs of toxicity were not observed. Slight dermal irritation was observed in all groups after the initiation of dosing; the irritation became moderate in the high dose group after 3 days of dosing. Body weights of rabbits of the high dose group were slightly, but significantly, decreased compared to controls. Absolute testes weights were slightly, but not significantly, decreased in the high dose group. No treatment-related effects on hematology or clinical chemistry values or organ weights were reported. Microscopically, epithelial hyperplasia, hyperkeratosis, conges- tion, and eschar formation were observed in the skin of rabbits of the high dose group; these changes were not observed in rabbits of the other test groups. No test article-related microscopic changes were observed in the testes or accessory sex glands at any dose. The NOEL for systemic toxicity was 0.18 g a.i./kg caprylyl/capryl glucoside. In another 2-wk study, 10 occlusive applications of 0.14, 0.41, and 1.25 g a.i./kg caprylyl/capryl glucoside (60% active) in distilled water (0, 3.5, 10.4, and 31.1% a.i., respectively) were made to intact skin on the backs of 6 male NZW rabbits/group in order to determine the NOEL for testicular toxicity.22 Two of the high dose animals died during the study, and the 4 surviving animals had signs of treatment-related toxicity. No treatment-related mortality occurred in the low or mid-dose groups. Dermal irritation, which progressed from slight to severe with time, was observed in all test groups., and slight to moderate irritation was observed in the controls. Changes in some hematology and clinical chemistry values were observed, but were attributed to stress of the occlusive procedure, irritation, and body weight loss. A decrease in the mean absolute testicular weights in animals of the mid- and high dose groups was considered treatment-related. A treatment-related loss in body weight was observed in all test groups, and the mean terminal body weights of rabbits of all test groups were decreased compared to controls. Treatment-related microscopic changes, consisting of an increased incidence and se- verity of diffuse bilateral testicular atrophy with necrotic spermatocytes, and atrophy of the prostate and vesicular glands, were observed in the testes, epididymides, prostate, and vesicular glands of the mid and high dose group animals.26 Testicu- lar effects were evident in one rabbit of the low dose group. Changes in the testes and accessory sex glands were attributed to the stresses described above An NOEL was not established.22

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Oral Ethyl Glucoside In a study described earlier under “Toxicokinetics,” in which groups of 6 male Wistar ST rats were fed for 39 days a diet in which sucrose was replaced with 10 or 20% ethyl glucoside, body weight gains, but not final body weights, were statistically significantly decreased in the 20% group when compared to control values.19 All animals survived until study termination. Total water intake was increased with increased ethyl glucoside consumption. In animals fed ethyl glucoside, kidney weights were statistically significantly increased and epididymal and abdominal fatty pad weights were statistically significantly decreased. The renal tubules of 2 and 4 control rats were “not-dilated” and “slightly dilated,” respectively, and the renal tubules of all the rats in 10% group were “slightly dilated.” In the group fed 20% ethyl glucoside, the renal tubules of 3 rats were “slightly dilated,”, while the other 3 had “moderately-dilated” renal tubules. No microscopic damage to renal cells was observed. Alkyl Polyglucosides Groups of 10 male and 10 female Sprague-Dawley rats were dosed orally, by gavage, with 0, 250, 500 and 1000 mg/kg bw C12/16 APG for 13 wks.21 An additional 5 male and 5 female control and high dose rats were used as a recovery group. No treatment-related changes in body weights, organ weights, or biochemistry or hematology parameters were observed. Absolute gonad weights were decreased in all test groups, but the decrease was not considered treatment related by the researchers due to the lack of a dose-response. A dose-dependent, slowly reversible, irritation and ulceration of the forestomach mucosa was observed in animals of the 500 and 1000 mg/kg bw groups. Systemic toxicity was not observed in any group. The no-observed adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg bw. The no-observed effect concentration for “local compatibility” was deduced as 2.5% a.i.

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Dermal application of 60% active caprylyl/capryl glucoside, 0.9-1.8 g a.i./kg, under occlusive conditions may affect the testes and accessory sex glands of rabbits; however, it was not clear if the effects were test-article related or due to stress of the occlusive procedure and resulting irritation and weight loss. Lauryl glucoside, 100-1000 mg/kg by gavage, did not produce adverse reproductive or developmental effects. Lauryl glucoside, 0.1-10,000 nmol, did not have any effects in in vitro estrogenicity assays.

Oral Lauryl Glucoside Groups of 24 female Sprague-Dawley CD rats were dosed orally, by gavage, with 0, 100, 300, or 1000 mg/kg bw/day lauryl glucoside (as C10-14 or C10-16, n: 1.4) on days 6-15 of gestation.27 All animals were killed on day 20 of gestation. No maternal toxicity was observed, and no reproductive or developmental effects were indicated. There were also no differences in external, visceral or skeletal malformations between groups. The NOAELs for maternal toxicity, embryo- toxicity/fetotoxicity, and teratogenicity were all 1000 mg/kg bw/day. Lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) was given orally, by gavage, to groups of 10 male and 10 female Sprague-Dawley rats at doses of 0, 100, 300, and 1000 mg/kg/day, from 2 wks prior to mating to 4 days after delivery.27 No signs of general parental toxicity were observed, and relative and absolute weights of the testes, epididymides, and seminal vesicles were similar for treated and control animals. There were no test article-related effects on reproductive parameters, and no effects on neonates.

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In Vitro Estrogenicity Assays Lauryl Glucoside Lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) was evaluated in the E-Screen assay, in which the induction of cell proliferation in the estrogen-dependent human breast tumor MCF-7 cells is determined, at concentrations of 0.1-10,000 nmol/ml.27 17-β-Estradiol and bisphenol-A were reference substances, and the medium was the negative control. No effects were reported at concentrations up to 105 higher than the concurrent controls. The effects of 0.1-1000 nmol/ml lauryl glucoside (as APG C12-C14 fatty alcohol from renewable sources, n: 1.43) were determined in the MCF-7 reporter gene assay, in which the induction of luciferase activity in stable transfected MCF-7 cells is determined.27 No effects were seen with lauryl glucoside alone, and no anti-estrogenic or other synergistic effects were observed after incubation with 0.01-1000 nmol/ml estradiol:lauryl glucoside (1:1 molar ratio).

GENOTOXICITY Alkyl Polyglucosides The mutagenic potential of APGs (chain length not specified) was determined in two Ames tests at concentrations of 8-500 µg/l and 11-900 µg/plate in distilled water, with and without metabolic activation.21 APGs were not mutagenic. Posi- tive and negative controls gave expected results. The genotoxic potential of C10/16 APG was evaluated in an assay for chromosomal aberrations using Chinese hamster V79 lung fibroblasts, at concentrations of ≤160 µg/ml with and ≤16 µg//ml without metabolic activation.21 C10-16 alkyl glucoside was not clastogenic in this assay. Positive and negative controls gave expected results.

CARCINOGENICITY Published carcinogenicity studies were not found.

IRRITATION AND SENSITIZATION Caprylyl/capryl glucoside, 30% a.i., was slightly irritating to rabbit skin. With APGs of varying chain length (C8/10 to C12/16; 15-70% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from 0.0 to 4.6 in rabbits. (A PDII of 2 was considered a positive responder). In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.0% a.i.) and soap chamber tests (1.0% a.i.), and decyl glucoside was evaluated in an SIOPT (0.5% a.i.). At most, these ingredients were slightly irritating. Glucosides with alkyl chain lengths ranging from C8- C10 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of 1.25-10% for intradermal induction, 5-100% for epidermal induction, and 2.5-50% for challenge, and the local lymph node assay (LLNA) at concentrations of 1.25-50%. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitization at concentrations of 8.4%, 5.9%, and 0.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 20%) and the Magnusson-Kligman protocol (1, 60,and 10% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively). C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. In clinical testing, the sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. aq. coco-glucoside was evaluated in a human repeated insult patch test (HRIPT). These ingredients were not irritating or sensitizing. In alternative system studies for mucosal irritation, decyl, lauryl, C10- 16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating. In a HET-CAM study with APG of varying proportions of alkyl chain length, the ocular irritation potential increased with the increased proportion of shorter-chain APGs. In studies using rabbits, neutralized lauryl glucoside produced slight ocular reactions. Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 10% for 30% a.i. caprylyl/capryl glucoside and 5% for 60% a.i. caprylyl/capryl glucoside.

Dermal irritation and sensitization studies are summarized in Table 4.

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Skin Irritation Non-Human Caprylyl/capryl glucoside, 30% a.i., was slightly irritating to rabbit skin.29-31 With APGs of varying chain length (C8/10 to C12/16; 15-70% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from 0.0 to 4.6 in rabbits.21 (A PDII of 2 was considered a positive responder.) Human In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.0% a.i.) and soap chamber tests (1.0% a.i.), and decyl glucoside was evaluated in a single-insult occlusive patch test (SIOPT) (0.5% a.i.).32,33 At most, these ingredients were slightly irritating. Sensitization Non-Human Glucosides with alkyl chain lengths ranging from C8-C10 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of 1.25-10% for intradermal induction, 5-100% for epidermal induction, and 2.5-50% for challenge, and the local lymph node assay (LLNA) at concentrations of 1.25-50%. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitizations at concentrations of 8.4%, 5.9%, and 0.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 20%) and the Magnusson-Kligman protocol (1, 60,and 10% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively).21 C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. Human The sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. coco-glucoside was evaluated in a human repeat insult patch test (HRIPT).34,35 These ingredients were not irritating or sensitizing. Mucosal Irritation Mucosal irritation studies, in both alternative and non-human systems, are summarized in Table 5. In the alternative systems studies, decyl, lauryl, C10-16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating.21,32 In a comparative study assessing the relationship between ocular irritation potential and alkyl chain length in the hen’s egg test-chorioallantoic membrane (HET-CAM) assay with four decyl glucosides (with varying proportions of alkyl chain length) and one lauryl glucoside, the ocular irritation potential increased 36 with the increased proportion of shorter-chain APGs (C6-C10). The researchers stated that results of a bovine corneal opaci- ty permeability (BCOP) assay suggested that changes in permeability with different alkyl chain lengths could be the main factor for the different eye irritation potentials. In the non-human studies, all of which used rabbits, neutralized lauryl glucoside produced slight reactions.21 Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 10% for 30% a.i. caprylyl/capryl glucoside and 5% for 60% a.i. caprylyl/capryl glucoside.24,29-31,37-43

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Case Studies Decyl Glucoside Case studies with reactions to antiseptic, hair, and sunscreen products that contain decyl glucoside are described in published literature.1,44-48 Subsequent patch testing with decyl glucoside at 0.5-10% had positive results in these cases. Patch testing with other glucosides also produced positive results in these patients.

SUMMARY The 17 alkyl glucosides reviewed in this report are ingredients that consist of anomerically-alkyl-substituted D- glycopyranosides; alkyl substituents range from 2 to 22 carbons in length and the D-glycopyranosides consist of glucose-type mono-, di-, tri-, oligo-, or poly-saccharides. The alkyl glucosides are synthesized by the alcoholysis of glucose and polysaccharides under acidic conditions. Most of these glucosides function in cosmetics as surfactants; a few function as skin conditioning agents, hair conditioning agents, or emulsion stabilizers. In 2011, decyl glucoside was reported to be used in 492 cosmetic formulations, 421 of which are rinse-offs. Decyl glucoside has the highest concentration of use, with 11 and 33% being reported for leave- on and rinse-off formulations. The most frequently use glucoside in leave-on formulations is cetearyl glucoside, with 445 of 477 uses being in leave-on formulations. In an in vitro dermal absorption study using human skin samples, the mean absorbed dose of 10% caprylyl/capryl glucoside was 0.01%. In an oral study in which female mice were dosed by gavage with a 5% aq. solution of caprylyl [U- 14C]glucoside, the highest levels of radioactivity at 2 h after dosing were found in the stomach, intestines, liver, and kidneys. The radioactivity in the stomach was primarily unchanged substrate, while only a trace amount found in the liver was unchanged. Labeled glucose was found in all of these organs. In a feeding study in rats in which dietary sucrose was replaced with 10 or 20% ethyl glucoside for 39 days, 60-90% of the ingested ethyl glucoside was recovered in the urine. In single dose dermal studies with caprylyl/capryl glucoside and C10-16 alkyl glucoside (both 50% a.i., n:1.6) in rabbits, the LD50 was greater than the 2000 mg/kg dose administered. In oral studies with the same test substances, none of the mice dosed with 2000 mg/kg caprylyl glucoside and none of the rats dosed with 5000 mg/kg C10-16 alkyl glucoside died during the study. In 2-wk repeated dose dermal studies in rabbits with 60% active caprylyl/capryl glucoside, occlusive applications produced testicular effects, while non-occlusive application did not. In the two occlusive studies, one with 0.09and 1.8 g a.i./kg and the other with 0.14-1.25 g a.i./kg, an NOEL for testicular effects could not be established. In the non-occlusive study, the NOEL for systemic toxicity was 0.18 g a.i./kg caprylyl/capryl glucoside. Severe dermal irritation was observed in both occlusive studies, while slight to moderate irritation was reported in the non-occlusive study. In oral repeated dose toxicity studies, moderately-dilated renal tubules were observed in 3 of 6 rats fed 20% ethyl glucoside for 39 days, but in none of the rats fed 10% ethyl glucoside. Kidney weights were statistically significantly increased in the test animals. In rats dosed orally with 250-1000 mg/kg C12/16 APG for 13 wks, reversible irritation and ulceration of the stomach mucosa was observed, but there was no systemic toxicity reported for any group. Dermal application of 60% active caprylyl/capryl glucoside, 0.9-1.8 g a.i./kg, under occlusive conditions may affect the testes and accessory sex glands of rabbits; however, it was not clear if the effects were test-article related or due to stress of the occlusive procedure and resulting irritation and weight loss. Lauryl glucoside, 100-1000 mg/kg by gavage, did not produce adverse reproductive or developmental effects. Lauryl glucoside, 0.1-10,000 nmol, did not have any effects in in vitro estrogenicity assays.

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APGs (chain length not specified), tested at 8-500 µg/l and 11-900 µg/plate in distilled water, were not mutagenic in Ames tests with or without metabolic activation. C10-16 APG, tested at concentrations of ≤160 µg/ml with and ≤16 µg/ml without metabolic activation, was not clastogenic. Caprylyl/capryl glucoside, 30% a.i., was slightly irritating to rabbit skin. With APGs of varying chain length (C8/10 to C12/16; 15-70% a.i.), there was a structure-response relationship with irritation potential decreasing with increasing chain length, and, independent of the degree of polymerization, the irritation was concentration-dependent. The primary dermal irritation indices (PDIIs) ranged from 0.0 to 4.6 in rabbits. (A PDII of 2 was considered a positive responder). In clinical studies, the dermal irritation of decyl, lauryl, and coco-glucosides was evaluated in epicutaneous patch (2.0% a.i.) and soap chamber tests (1.0% a.i.), and decyl glucoside was evaluated in an SIOPT (0.5% a.i.). At most, these ingredients were slightly irritating. Glucosides with alkyl chain lengths ranging from C8-C10 to >C18, as well as a C18 branched glucoside, were evaluated for irritation potential in both the guinea pig maximization test (GPMT), at concentrations of 1.25-10% for intradermal induction, 5-100% for epidermal induction, and 2.5-50% for challenge, and the local lymph node assay (LLNA) at concentrations of 1.25-50%. None of the glucosides tested were irritants or sensitizers in the GPMT, but the LLNA indicated that one C12-C18 glucoside, C14 glucoside, and C18 branched glucoside may cause skin sensitization at concentrations of 8.4%, 5.9%, and 0.43%, respectively. The sensitization potential of C12/16 APG was evaluated in studies in guinea pigs using the Buehler method (test concentrations of 20%) and the Magnusson-Kligman protocol (1, 60, and 10% used for intracutaneous induction, epidermal induction, and epidermal challenge respectively). C12/16 APG was not a sensitizer in the Buehler or Magnusson-Kligman studies. In clinical testing, the sensitization potential of 5% a.i. aq. decyl and lauryl glucoside and 1% a.i. aq. coco-glucoside was evaluated in a human repeated insult patch test (HRIPT). These ingredients were not irritating or sensitizing. In alternative system studies for mucosal irritation, decyl, lauryl, C10-16 alkyl, and coco-glucosides were non to slightly irritating, while caprylyl/capryl glucoside was highly irritating. In a HET-CAM study with APG of varying proportions of alkyl chain length, the ocular irritation potential increased with the increased proportion of shorter-chain APGs. In studies using rabbits, neutralized lauryl glucoside produced slight ocular reactions. Caprylyl/capryl glucoside was severely irritating when tested undiluted; the irritation threshold value was 10% for 30% a.i. caprylyl/capryl glucoside and 5% for 60% a.i. caprylyl/capryl glucoside

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TABLES Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Ethyl Ethyl Glucoside is the Skin- Glucoside product obtained from the Conditioning 30285-48-4 condensation of ethyl Agents - alcohol and glucose. Humectant

Butyl Butyl Glucoside is the Surfactants - O O CH3 Glucoside product obtained by the Cleansing Agents H O 5391-18-4 condensation of butyl 41444-57-9 alcohol with glucose. H O OH OH n Caprylyl Caprylyl Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents 29836-26-8 condensation of caprylic alcohol with glucose.

Decyl Decyl Glucoside is the Surfactants - Glucoside product obtained from the Cleansing Agents 58846-77-8 condensation of decyl 68515-73-1 alcohol with glucose. 141464-42-8

Undecyl Undecyl Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents 98283-67-1 condensation of undecyl alcohol with glucose.

Lauryl Lauryl Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents 27836-64-2 condensation of lauryl 110615-47-9 alcohol with glucose.

Myristyl Myristyl Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents 54549-26-7 condensation of myristyl alcohol with glucose.

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Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Arachidyl Arachidyl Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents 144982-05-8 condensation of Arachidyl Alcohol with glucose.

Mixtures Caprylyl/ Caprylyl/Capryl Glucoside Surfactants - Capryl is the product obtained by Cleansing Agents Glucoside the condensation of a mix- 68515-73-1 ture of caprylic and decyl alcohols with glucose.

wherein R = an alkyl chain 8 or 10 carbons long C10-16 Alkyl C10-16 Alkyl Glucoside is Surfactants - Glucoside the product obtained by the Emulsifying 110615-47-9 condensation of C10-16 Agents alcohols with glucose.

wherein R = an alkyl chain 10 to 16 carbons long C12-18 Alkyl C12-18 Alkyl Glucoside is Emulsion Glucoside the product obtained by the Stabilizers condensation of C12-18 alcohols with glucose.

wherein R = an alkyl chain 12 to 18 carbons long C12-20 Alkyl C12-20 Alkyl Glucoside is Surfactants - Glucoside the product obtained by the Emulsifying condensation of C12-20 Agents alcohols with glucose.

wherein R = an alkyl chain 12 to 20 carbons long Cetearyl Cetearyl Glucoside is the Surfactants - Glucoside product obtained by the Emulsifying condensation of cetearyl Agents alcohol with glucose.

wherein R = an alkyl chain 16 or 18 carbons long C20-22 Alkyl C20-22 Alkyl Glucoside is Glucoside the product obtained by the condensation of C20-22 alcohols with glucose.

wherein R = an alkyl chain 20 to 22 carbons long Coco- Coco-Glucoside is the Surfactants - Glucoside product obtained by the Cleansing Agents condensation of coconut alcohol with glucose.

wherein R = alkyl chain residue of fatty alcohols derived from Coconut Acid

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Table 1. Definitions, functions, and structures of the alkyl glucosides in this safety assessment. Ingredient CAS No. Definition Function(s) Formula/structure Branched Isostearyl Isostearyl Glucoside is the Surfactants - one example of an “iso” Glucoside product obtained by the Emulsifying 200413-69-0 condensation of isostearyl Agents alcohol with glucose.

Octyldodecyl Octyldodecyl Glucoside is Surfactants - Glucoside the product obtained by the Emulsifying reaction of Octyldodecanol Agents with glucose.

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Table 2. Chemical and physical properties Property Description Reference Decyl Glucoside Appearance cloudy, viscous aq. solution (as Plantacare 2000 UP) (of poly) 49 light yellow aq. solution (as APG 0810) (of poly) 50 molecular weight 340.2 (of mono) 51 390 g/mol (as Plantacare 2000) (of poly) 49 active substance 51-55% (as Plantacare 2000 UP) (of poly) 49 ≥50% (as APG 0810) (of poly) 50 boiling point 467.5°C (of mono) 51 melting point 135.6°C (of mono) 52 critical micelle concentration 2-3 mM (of poly, specifically the maltopyranoside) 12 Viscosity 1000-6000 mPas (20°C) aq. solution (as Plantacare 2000 UP) (of poly) 49 ≤500 mPas (20°C) aq. solution (as APG 0810, a polyglucoside) (of poly) 50 Density 1.14 g/cm3 (at 20°C) (of mono) 51 log P 2.092 (at 25°C) (of mono) 51 may contain (as Plantacare 2000 UP): 53 magnesium oxide max. 500 ppm (of poly) free fatty alcohol max. 1.0% (of poly) sulfate ash max. 3.0% (of poly) (as APG 0810): 50 free fatty acid ash ≤1% (of poly) ≤2% (of poly) Ethyl Glucoside (mono) molecular weight 208.21 51 boiling point 395.1°C 51 melting point 176-179°C 54 Density 1.40 g/cm3 (at 20°C) 51 log P -2.159 (at 25°C) 51 Butyl Glucoside (mono) molecular weight 236.26 51 boiling point 412.0°C 51 melting point 86-87°C 55 Density 1.30 g/cm3 (at 20°C) 51 log P -1.151 (at 25°C) 51 Caprylyl Glucoside Appearance white solid 56 yellowish, slightly cloudy and viscous aq. solution (as Plantacare 810 UP) (of 57 poly) molecular weight 292.37 56 active substance 62-65% (as Plantacare 810 UP) (of poly) boiling point 454.1°C 51 melting point 65-99° (sic) 56 critical micelle concentration 20-25 mM 12 Density 1.18 g/cm3 (at 20°C) 51 log P 0.887 (at 25°C) 51 may contain (as Plantacare 810 UP): 57 fatty alcohol ≤0.7% (of poly)

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Table 2. Chemical and physical properties Property Description Reference Undecyl Glucoside (mono) molecular weight 334.45 51 boiling point 487.8°C 51 Density 1.13 g/cm3 (at 20°C) 51 log P 2.642 (at 25°C) 51 Lauryl Glucoside Appearance viscous pale yellow aq. solution (as APG 1214,) (of poly) 50 molecular weight 343.2 17 348.47 (of mono) 51 420 (as Plantacare 1200 UP) (of poly) 58 active substance 50-53% (as APG 1214 and as Plantacare 1200 UP) (of poly) 50,58 boiling point 499.1°C (of mono) 51 critical micelle concentration 0.13 mM (of poly, specifically the maltopyranoside) 12 Viscosity ≥2000 mPas (20°C) aq. solution (as APG 1214) (of poly) 50 Density 1.12 g/cm3 (at 20°C) (of mono) 51 log P 2.925 (at 25°C) (of mono) 51 may contain (as Plantacare 12900 UP) 58 fatty alcohol ≤0.8% (of poly) ash ≤2%(of poly) (as APG 1214): 50 free fatty acid ≤1% (of poly) ash ≤2%(of poly) Myristyl Glucoside (mono) molecular weight 376.53 51 boiling point 521.5°C 51 Density 1.09 g/cm3 (at 20°C) 51 log P 4.218 (at 25°C) 51 Arachidyl Glucoside (mono) molecular weight 460.69 51 boiling point 586.8°C 51 Density 1.04 g/cm3 (at 20°C) 51 log P 7.406 (at 25°C) 51 Coco-Glucoside (poly) Appearance cloudy, viscous aq. solution (as Plantacare 818 UP) 59 cloudy, viscous pale yellow aq. solution (as APG 0814, a polyglucoside) 50 % active 51-53% (as Plantacare 818 UP) 59 ≥50% (as APG 0814, a polyglucoside) 50 Viscosity 2500-6000 mPas (20°C) aq. solution (as Plantacare 818 UP) 59 ≤2000 aq. solution (as APG 0814, a polyglucoside) 50 may contain (as Plantacare 818 UP) 59 magnesium oxide max. 500 ppm magnesium free fatty alcohol max. 1.0% sulfate ash max. 3.0% may contain (as APG 0814): 50 free fatty acid ≤1% ash ≤2%

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Table 3a. Frequency and concentration of use according to duration and type of exposure

Decyl Glucoside Arachidyl Glucoside C12-20 Alkyl Glucoside # of Uses5 Conc of Use (%)6 # of Uses5 Conc of Use (%)6 # of Uses5 Conc of Use (%)6 Totals* 492 0.002-33 75 0.08-0.6 54 0.1-1 Duration of Use Leave-On 62 0.002-11 73 0.08-0.6 42 0.2-1 Rinse Off 421 0.3-33 2 0.5 12 0.1 Diluted for Use 9 0.5-1 NR NR NR NR Exposure Type Eye Area 12 0.02-1 3 0.08 2 0.8 Possible Ingestion NR NR NR NR NR NR Inhalation NR 0.5-0.6 NR NR NR NR Dermal Contact 379 0.002-33 75 0.08-0.6 48 0.1-1 Deodorant (underarm) NR NR NR NR NR 0.6 Hair - Non-Coloring 94 0.2-7 NR 0.5 6 1 Hair-Coloring 9 2-8 NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane 199 0.3-7 1 NR NR NR Bath Products 9 0.5-1 NR NR NR NR Baby Products 25 NR NR NR NR NR

Caprylyl/Capryl Glucoside Caprylyl Glucoside Cetearyl Glucoside # of Uses5 Conc of Use (%)6 # of Uses5 Conc of Use (%)6 # of Uses5 Conc of Use (%)6 Totals* 58 0.06-3 NR 4 477 0.03-7 Duration of Use Leave-On 27 0.06-0.8 NR 4 445 0.2-7 Rinse Off 31 0.3-3 NR NR 31 0.03-3 Diluted for Use NR NR NR NR 1 NR Exposure Type Eye Area 6 0.2-0.3 NR NR 61 0.6-2 Possible Ingestion NR NR NR NR NR NR Inhalation NR NR NR NR 3 0.6 Dermal Contact 51 0.06-0.9 NR NR 466 0.03-7 Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring 7 0.3-3 NR 4 4 0.3-0.6 Hair-Coloring NR 3 NR NR NR 0.2 Nail NR NR NR NR 2 NR Mucous Membrane 6 NR NR NR 5 0.03 Bath Products NR NR NR NR 1 NR Baby Products NR 0.06 NR NR 3 NR

Coco-Glucoside Ethyl Glucoside Lauryl Glucoside 5 6 5 6 5 6 # of Uses Conc of Use (%) # of Uses Conc of Use (%) # of Uses Conc of Use (%) Totals* 350 0.006-8 24 0.02-0.3 399 0.03-10 Duration of Use Leave-On 42 0.006-2 14 0.02-0.3 22 0.03-8 Rinse Off 294 0.2-8 10 0.02-0.05 347 0.3-10 Diluted for Use 14 NR NR NR 30 0.3-4 Exposure Type Eye Area 6 2-3 2 0.02 NR 5 Possible Ingestion NR 0.5 NR NR NR NR Inhalation 1 0.4 NR NR NR 8 Dermal Contact 275 0.006-8 24 0.02-0.3 310 0.03-10 Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring 59 0.2-8 NR NR 70 0.4-5 Hair-Coloring 16 0.3-5 NR NR 15 0.3-8 Nail NR NR NR NR NR NR Mucous Membrane 163 0.4-8 1 NR 188 0.3-8 Bath Products 14 NR NR NR 30 0.3-4 Baby Products 11 NR NR NR 5 NR 17

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Table 3a. Frequency and concentration of use according to duration and type of exposure

Myristyl Glucoside Hexadecyl D-Glucoside** Octadecyl D-Glucoside** # of Uses5 Conc of Use (%)6 # of Uses5 Conc of Use (%)60 # of Uses5 Conc of Use (%)60 Totals* 5 0.4-0.6 1 3% 1 NR Duration of Use Leave-On 4 0.4-0.6 1 3 1 NR Rinse Off 1 NR NR NR NR NR Diluted for Use NR NR NR NR NR NR Exposure Type Eye Area 2 0.4 NR NR NR NR Possible Ingestion NR NR NR NR NR NR Inhalation NR NR NR NR NR NR Dermal Contact 5 0.4-0.6 1 3 1 NR Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR NR NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR Bath Products NR NR NR NR NR NR Baby Products NR NR NR NR NR NR * Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types my not equal the sum of total uses. ** These ingredients are included in the VCRP, but are not listed in the International Cosmetic Ingredient Dictionary and Handbook NR – none reported

Table 3b. Ingredients Not Reported to be Used Butyl Glucoside C10-16 Alkyl Glucoside C12-18 Alkyl Glucoside C20-22 Alkyl Glucoside Isostearyl Glucoside Octyldodecyl Glucoside Undecyl Glucoside

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Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference IRRITATION STUDIES NON-HUMAN Caprylyl/Capryl Glucoside 30% a.i. rabbits; not provided slightly irritating,; PII 0-2 29-31 no. not specified

C8/10 APG 15% a.i 5 rabbits 4 h application; semi-occlusive patch PDII=0.0 21 C8/10 APG 35% a.i.; n:1.6 3 rabbits 4 h application; semi-occlusive patch PDII = 1.3; no signs of systemic toxicity 21 C8/10 APG 70% a.i.; n:1.6 3 rabbits 4 h application; semi-occlusive patch PDII = 0.8; no signs of systemic toxicity 21

C10/16 APG 20% a.i.; n:1.4 4 rabbits 4 h application; occlusive patch PDII=0.4; no signs of systemic toxicity 21 C10/16 APG 60% a.i.; n:1.4 4 rabbits 4 h application; occlusive patch PDII=4.6; erythema and edema in all 21 animals; 24/48/72 h mean erythema score- Distributed 2.9, mean edema score-2.1 C12/16 APG 50% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.7; no signs of systemic toxicity; 21

erythema and edema in all animals; for CIR

24/48/72 h mean erythema score-2.2, mean Comment edema score-1.6 Panel C12/16 APG 50% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.0; no signs of systemic toxicity; 21 erythema in all animals and edema in 1 Only Book animal; 24/48/72 h mean erythema score- 2.1, mean edema score-0.9 -- Page 21 C12/16 APG 50% a.i.; n:1.4 3 rabbits 4 h application; semi-occlusive patch PDII=3.0; no signs of systemic toxicity; Do

erythema in all animals and edema in 1 Not 33 animal; 24/48/72 h mean erythema score- 1.9, mean edema score-1.1 Cite 21 C8/10 + C12/16 APG C8/10, 21% a.i. 3 rabbits 4 h application; semi-occlusive patch PDII=2.7; erythema and edema in all or

C12/16, 35% a.i. animals; 24/48/72 h mean erythema score- Quote 1.8, mean edema score-0.8 HUMAN Decyl Glucoside 2.0% a.i., pH 6.5 20 subjects epicutaneous patch test; 75 l, 24 h very slightly irritating 32 occlusive application Decyl Glucoside 1.0% a.i., pH 6.5 22 subjects soap chamber test; 100 l applied occlu- slightly irritating 32 sively to the ventral for 24 h on day 1 and 6 h on days 2-5 Decyl Glucoside a.i. not stated; tested at 105 subjects; 14.3% were SIOPT; 40 l was applied for 48 h using AII=0.046; non-irritating 33 0.5% aq. atopic patients Haye’s test chambers

Lauryl Glucoside 2.0% a.i., pH 6.5 20 subjects epicutaneous patch test; 75 l, 24 h slightly irritating 32 occlusive application Lauryl Glucoside 1.0% a.i., pH 6.5 22 subjects soap chamber test; 100 l applied occlu- slightly irritating 32 sively to the ventral for 24 h on day 1 and 6 h on days 2-5

Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference Lauryl Glucoside a.i. not stated; tested at 105 subjects; 14.3% were SIOPT; 40 l was applied for 48 h using AII=0.046; non-irritating 33 0.5% aq. atopic patients Haye’s test chambers Coco-Glucoside 2.0% a.i., pH 6.5 20 subjects epicutaneous patch test; 75 l, 24 h slightly irritating 32 occlusive application Coco-Glucoside 1.0% a.i., pH 6.5 22 subjects soap chamber test; 100 l applied occlu- slightly irritating 32 sively to the ventral for 24 h on day 1 and 6 h on days 2-5 SENSITIZATION NON-HUMAN C8-C10 glucoside a.i. not stated; tested at 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 10% was an irritant (based on ear 61

1.25-25% in acetone/olive thickness); not a sensitizer Distributed oil (4:1) C8-C10 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 5% - intraderm induction 5 controls induction patch application volume of 5% - epiderm induction 0.0625 g/cm2 for CIR 2.5 and 5% - challenge Comment 2 61

Panel C10-C14 glucoside a.i. not stated; tested at 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm not an irritant or sensitizer 1.25-5% in DMF 61 Only Book C10-C14 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 5% - intraderm induction 5 controls induction patch application volume of

2 -- Page 5% - epiderm induction 0.0625 g/cm 2.5 and 5% - challenge Do

62 Not 34 C12/16 APG (may be a.i. not provided 20 guinea pigs Buehler method; 20% tested at induction not a sensitizer; one very weak reaction

similar to C12-18 alkyl and challenge during induction and challenge for one Cite glucoside) guinea pig 62 or

C12/16 APG (may be a.i. not provided 20 guinea pigs Magnusson-Kligman study; 1% used for not a sensitizer; no positive reactions Quote similar to C12-18 alkyl intracutaneous and 60% for epidermal glucoside) induction; 10% for epidermal challenge C12-C18 glucoside a.i. not stated; tested at 2.5- 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 not an irritant or sensitizer 61 (granules) 10% in DMF C12-C18 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 (granules) 10% - intraderm induction 5 controls induction patch application volume of 50% - epiderm induction 0.0625 g/cm2 5 and 10% - challenge C12-C18 glucoside (flakes) a.i. not stated; tested at 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 may cause skin sensitization 61 1.25-10% in DMF C12-C18 glucoside (flakes) a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 10% - intraderm induction 5 controls induction patch application volume of 50% - epiderm induction 0.0625 g/cm2 5 and 10% - challenge C14 glucoside a.i. not stated; tested at 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 all concentrations were irritants (based on 61 1.25-10% in DMF ear thickness); may cause skin sensitization

Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference C14 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 1.25%-intraderm induction 5 controls induction patch application volume of 50% - epiderm induction 0.0625 g/cm2 25 and 50% - challenge C16-C18 glucoside a.i. not stated; tested at 2.5- 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 not an irritant or sensitizer 61 10% in DMF C16-C18 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 10% - intraderm induction 5 controls induction patch application volume of 10% - epiderm induction 0.0625 g/cm2 5 and 10% - challenge >C18 glucoside a.i. not stated; tested at 2.5- 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 not an irritant or sensitizer 61 10% in DMF Distributed >C18 glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 5% - intraderm induction 5 controls induction patch application volume of 10% - epiderm induction 0.0625 g/cm2 for 2.5 and 5% - challenge CIR Comment C18 branched glucoside a.i. not stated; tested at 2.5- 4-5 CBA/j mice LLNA; application volume of 0.025 g/cm2 all concentrations were irritants (based on 61 Panel 50% in DMF ear thickness); may cause skin sensitization C18 branched glucoside a.i. not stated; tested at guinea pigs; 10 treated, GPMT with FCA and SLS; occlusive not an irritant or sensitizer 61 Only Book 2.5% - intraderm induction 5 controls induction patch application volume of 100% - epiderm induction 0.0625 g/cm2 -- Page

6.25 and 12.5% - challenge Do HUMAN Not 35 Decyl Glucoside; tested 5% a.i. 49 subjects HRIPT; 0.2 ml, 24-h semi-occlusive; 3x/wk not an irritant or a sensitizer 34 under 4 tradenames for 3 wks; 10 applications; challenge per- Cite

formed after 2-wk non-treatment period or Lauryl Glucoside; tested 5% a.i. 49 subjects HRIPT, as above not an irritant or a sensitizer 34 Quote under 5 tradenames Coco-Glucoside 52% a.i. 2% aq. Tested 213 subjects HRIPT; 0.2 ml, 24 h occlusive; 3x/wk for 3 not an irritant or a sensitizer 35 wks; 9 applications; challenge performed after 2-wk non-treatment period C8-C10 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction=0.04; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge C10-C14 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge C12-C18 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.10; no 61 (granules) aq. application volume of 0.04 g/cm2 positive reactions at challenge C12-C18 glucoside (flakes) a.i. not stated; tested at 1% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.15; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge C14 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.62; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge

Table 4. Skin irritation and sensitization studies Ingredient/Chain Length % a.i.; n; conc. tested Test Population Method Results Reference C16-C18 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.03; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge >C18 glucoside a.i. not stated; tested at 5% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.21; no 61 aq. application volume of 0.04 g/cm2 positive reactions at challenge C18 branched glucoside a.i. not stated; tested at 6% 50 subjects HRIPT; 20 µl, 9 occlusive applications, irritation index during induction = 0.04; no 61 application volume of 0.04 g/cm2 positive reactions at challenge Abbreviations: a.i. – active ingredient; AII – average index of skin irritation; APG – alkyl polyglucoside; DMF – dimethyl formamide; FCA – Freund’s complete adjuvant; HRIPT – human repeat insult patch test; LLNA – local lymph node assay; n – degree of polymerization; PDII – primary dermal irritation index; SIOPT – single insult occlusive patch test; SLS – sodium lauryl sulfate positive responder: irritation score = 2

Distributed for CIR Comment Panel Only Book -- Page Do Not 36 Cite or Quote

Table 5. Mucosal irritation studies Ingredient/Chain Length % a.i.; pH; conc. tested Animals Method Results Reference ALTERNATIVE STUDIES Decyl Glucoside 1.0% a.i. in PBS; pH 7 RBC not irritating 32 Decyl Glucoside 3.0% a.i.; pH 6.5 , aq. soln HET-CAM assay slightly irritating 32 Decyl Glucoside 0.6% a.i.; pH 7.0 , aq. soln ocular tissue model not irritating 32

Lauryl Glucoside 1.0% a.i. in PBS; pH 7 RBC slightly irritating 32 Lauryl Glucoside 3.0% a.i.; pH 6 , aq. soln HET-CAM assay slightly irritating 32 Lauryl Glucoside 0.6% a.i.; pH 7.0, aq. soln ocular tissue model not irritating 32 Caprylyl/Capryl Glucoside, not specified HET-CAM assay highly irritating 21 as C8/C10 APG Distributed C10-16 Alkyl Glucoside, as pH 7 HET-CAM assay produced slight reactions 21 C10/16 APG C10-16 Alkyl Glucoside, as pH 11.5 HET-Cam assay produced slight reactions 21 C10/16 APG for CIR Comment Coco-Glucoside 1.0% a.i. in PBS; pH 7.4, RBC not irritating 32 Panel aq. Soln Coco-Glucoside 3.0% a.i.; pH 6.5, aq. soln HET-CAM assay slightly irritating 32 Only Book Coco-Glucoside 0.6% a.i.; pH 7.0, aq. soln ocular tissue model not irritating 32 --

Page NON-HUMAN STUDIES Lauryl Glucoside, 12.5% a.i. 6 NZW rabbits Draize test; eyes were not rinsed very slight reactions, in one animal (subsided in 21 Do Not

37 neutralized 48 h); medium to mild conjunctival irritation was

observed in all rabbits; effects were reversible Cite within 7 days in all but one of the rabbits

30 or

Caprylyl/Capryl Glucoside 30% a.i. 9 rabbits 0.1 ml instilled into the conjunctival sac; eyes severely irritating; rinsing reduced the intensity Quote of 3 rabbit rinsed 20-30 sec after dosing and duration Caprylyl/Capryl Glucoside 30% a.i. 9 rabbits 0.1 ml instilled into the conjunctival sac; eyes severely irritating; rinsing reduced the intensity 29 of 3 rabbit rinsed 20-30 sec after dosing and duration Caprylyl/Capryl Glucoside 30% a.i. 9 rabbits 0.1 ml instilled into the conjunctival sac; eyes severely irritating 31 of 3 rabbit rinsed 20-30 sec after dosing Caprylyl/Capryl Glucoside 30% a.i. 9 rabbits 0.1 ml instilled into the conjunctival sac; eyes severely irritating 37 of 3 rabbit rinsed 20-30 sec after dosing Caprylyl/Capryl Glucoside 30% a.i. 9 rabbits 0.1 ml instilled into the conjunctival sac; eyes severely irritating 38 of 3 rabbit rinsed 20-30 sec after dosing Caprylyl/Capryl Glucoside 30% a.i.; 0.1-50% tested 2 rabbits/group; 0.1 ml instilled into the conjunctival sac 0.1, 0.5, 1.0, 5.0%: non or inconsequential irritant 63 4 additional rab- 10%: conjunctival irritation in 6/6 at 4 h; bits dosed w/5 subsided in 4/6 by 72 h; moderate irritant and 10% 20%: moderate irritant 50% substantial to severe irritant Irritation threshold determined to be 10% (v/v); equivalent to 3% solids 23

Table 5. Mucosal irritation studies Ingredient/Chain Length % a.i.; pH; conc. tested Animals Method Results Reference Caprylyl/Capryl Glucoside 60% a.i. rabbits details not provided severely irritating 42 Caprylyl/Capryl Glucoside 60% a.i.; conc. of 0.5, 1.0, 6 rabbits/group 0.1 ml instilled into the conjunctival sac 0.5 and1.0%: no irritation 43 5.0, and 10.0% tested 5%: conjunctival irritation at 4 h, cleared by 72 h 10%: moderate irritation in 6/6, cleared by 72 h 60% a.i.; conc. of 20 and 2 rabbits/group 0.1 ml instilled into the conjunctival sac 20 and 50%: severely irritating 10.0% tested Irritation threshold determined to be 5.0% Caprylyl/Capryl Glucoside 70% a.i.; 40% solution 6 rabbits/study 0.1 ml instilled into the conjunctival sac; two moderately to highly irritating (both studies) 39 studies performed Caprylyl/Capryl Glucoside 70% a.i. not provided not provided; two studies performed moderately to highly irritating (both studies) 40 Caprylyl/Capryl Glucoside 70% a.i.; 40% solution 6 rabbits 0.1 ml instilled into the conjunctival sac; two moderately to highly irritating (both studies) 41

studies performed Distributed C12/16 APG (may be 50% a.i, aq. Solution 4 albino rabbits OECD Guideline 405 24/48/72 h mean scores for the cornea, conjuncti- 21 similar to C12-18 alkyl val erythema, and iris: 0.5/4, 2.08/3, and 0.25/2, glucoside) respectively; moderate to strong reactions in the

conjunctivae did not completely subside within 21 for CIR days in 2 of the animals, persistent corneal effects Comment did not subside in 1 of these rabbits Panel Abbreviations: a.i. – active ingredient; HET-CAM – hen’s egg test-chorioallantoic membrane; PBS – phosphate buffered saline; RBS – red blood cell test Only Book -- Page Do Not 38 Cite or Quote

24 Distributed for Comment Only -- Do Not Cite or Quote

Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference n-Butyl Alcohol ADME can be absorbed through the lungs, gastrointestinal tract, the cornea, and the skin; mainly metabolized by alcohol dehydrogenase and eliminated rapidly from the 64 blood; dogs given i.v. n-butyl alcohol eliminated 15% of the does in CO2(none unchanged) and 2.7% in the urine animal toxicology dermal LD50 (rabbits), 4.2 g/kg; oral LD50 (rats), 0.79-4.36 g/kg short-term oral: 6.9% n-butyl alcohol and 25% sucrose given in drinking water for 3 wks produced some changes in hepatic mitochondria inhalation: results in irritation of the mucous membranes, intoxication, restlessness, ataxia, prostration, and narcosis; high concentrations can be fatal dermal irritation/sensitization no data mucosal irritation 15% n-butyl alcohol produced an ocular irritation score of <5/20 and a 40% solution produced a score of >5/20 in rabbit eyes repro/developmental toxicity fetotoxicity has been demonstrated at maternally toxic levels (1000 mg/kg); no significant behavioral or neurochemical effects were seen in offspring following either maternal or paternal exposure to 3000 or 600 ppm Genotoxicity negative in an Ames test, did not induce sister chromatid exchange 0.1 or (15% aq.) or micronuclei formation, and did not impair chromosome distribution in mitosis Carcinogenicity no data clinical assessment of safety a nail color containing 3% n-butyl alcohol was not a significant irritant or sensitizer in HRIPTs, and this product was not a phototoxin or photoallergen; negative for non-immunological urticaria occupational exposure: n-butyl alcohol (alone or with other solvents) produced complaints of ocular irritation, headache and vertigo, slight irritation of the nose and throat, and dermatitis of the hands and fingers at air concentrations of >50 ppm important Discussion items uses in products other than nail products are at very low concentrations, so there were no toxicity concerns Conclusion safe as used Cetearyl Alcohol animal toxicology no data 65 dermal irritation/sensitization formulation w/3%, mildly irritating (rabbits) mucosal irritation formulation w/3%, not irritating repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety formulation w/3%: not a sensitizer important Discussion items no relevant items identified Conclusion safe as used Cetyl Alcohol ADME in general, long-chain aliphatic alcohols, such as cetyl alcohol, are oxidized to their 65 corresponding fatty acids in mammalian tissues; in rats administered radioactive cetyl alcohol by either stomach tube or thoracic duct fistulas, most of the radioactivity was found in the thoracic duct lymph, indicating good absorption; some of the cetyl alcohol was eliminated unchanged in waste products, but most of the cetyl alcohol was oxidized to palmitic acid and incorporated into triglycerides and phospholipids animal toxicology oral LD50(rats): >8.2 g/kg; formulations w/≤4%, no toxic effects; dermal LD50: >2.6 g/kg; formulation w/5%, 2 g/kg; inhalation: 6-h exposure, 26 ppm (rats, mice, guinea pigs), slight irritation of mucous membranes, but no signs of systemic toxicity or mortality; 6 h exposure, 2220 mg/m3, 100% mortality short-term dermal: 20 day, 11.5%, 5x/day, exfoliative dermatitis, parakeratosis, hyperkeratosis (rabbits); 30 day, 30% in methyl alcohol and propylene glycol, dermal infiltrates of histocytes 3 mos dermal study: formulations w/20%, well-defined erythema, mild edema, no systemic toxicity (rabbits) dermal irritation/sensitization undiluted, minimally to slightly irritating; formulations w/2-4%, no to well-defined erythema and edema mucosal irritation formulations w/≤6.36%, mostly non-irritating mucosal irritation 2%: not irritating to genital mucosa of rabbits repro/developmental toxicity no data Genotoxicity negative, Ames test Carcinogenicity no data clinical assessment of safety 100%: not irritating; formulations w/2-11.5%,:at most, mild irritants formulations w/1-8.4%, not sensitizers 30%: 11.2% of eczema patients (pop. 330) had allergic reactions formulations w/1-4%, not photosensitizers important Discussion items no relevant items identified Conclusion safe as used 25

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Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference Coconut Alcohol animal toxicology no data 66 dermal irritation/sensitization no data mucosal irritation no data repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety no data important Discussion items toxicity and use profiles expected to be similar to coconut oil, coconut acid, hydrogenated coconut oil, hydrogenated coconut acid; addressed use in inhalation products; possible issues with botanicals Conclusion safe as used

65 Isostearyl Alcohol animal toxicology oral LD50: >20 g/kg (rats); formulations w/25-27%, >15 g/kg dermal irritation/sensitization formulation w/5%: mild irritant (rabbits); formulation w/25-27%: barely perceptible erythema 0.2-5%: not a sensitizer mucosal irritation formulations w/5 and 10%, transient irritation; formulations w/25-27%, minimal to mild irritation repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety 100%: not irritating; formulations w/25-28%, not irritating; deodorant formulation w/ 5%, severe irritation in a 21-day cumulative study 25% in 95% isopropyl alcohol: not a sensitizer; formulations w/5%: sensitization reactions occurred important Discussion items no relevant items identified Conclusion safe as used

Myristyl Alcohol animal toxicology oral LD50 (rats): >8 g/kg; formulation w/0.8%, >5 g/kg; dermal LD50: formulation 65 w/0.8%, >2 g/kg inhalation: 3%, 1 h, ataxia and moderate nasal irritation in all animals 10 min after exposure, no mortality dermal irritation/sensitization formulation w/0.8%, non-irritating (rabbits) mucosal irritation formulation w/0.8%: not irritating; formulation w/3%: mildly irritating (rinsed eyes), moderately irritating (unrinsed eyes) repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety formulations w/0.1-0.25%, not irritants; formulations w/0.25-0.8%, not irritating in a 4-wk clinical study formulations w/0.1-0.25%, not sensitizers formulation w/0.1%, not a photosensitizer important Discussion items no relevant items identified Conclusion safe as used

Octyl Dodecanol animal toxicology oral LD50 (rats): >5 g/kg, undiluted; formulation w/10.2%, >25 g/kg; dermal LD50: 67 >3 g/kg dermal irritation/sensitization 100%: irritation score of 0-1.13/4 (rabbits); 30%: irritation score 0/4 (rabbits); formulations w/4 and 10.2%, mild irritation, at most; technical grade: moderate to severe irritation (rabbits, guinea pigs, rats), no irritation (swine, humans) mucosal irritation 100%: irritation score of 1 or 4/110 (24 h) repro/developmental toxicity no data Genotoxicity no data Carcinogenicity no data clinical assessment of safety 100%: mild irritation in 1/40 subjects; undiluted technical grade: no irritation; formulations w/3-10.2%: essentially non-irritating screening patch tests for contact sensitization in large populations: incidence rate of 0.36% (6/1664) formulation w/10.2%: not phototoxic or photoallergenic important Discussion items no Discussion Conclusion safe as used

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Table 6. Summaries of information on fatty alcohols from previous CIR reports Ingredient Parameter Evaluated Outcome Reference Stearyl Alcohol ADME found naturally in various mammalian tissues; readily converted to stearic acid, another common constituent of mammalian tissues; results from several studies 67 indicate that stearyl alcohol is poorly absorbed from the GI tract animal toxicology oral LD50: >8 g/kg; 3 mos dermal study: formulations w/8%,some dermal effects, , no systemic toxicity (rabbits) dermal irritation/sensitization 100%: minimal to mild primary skin irritant (rabbits) formulation w/24%: not a sensitizer mucosal irritation 100%: mildly irritating repro/developmental toxicity no data Genotoxicity negative: Ames test Carcinogenicity did not promote tumor formation in mice when tested with dimethylbenz[a]anthracene clinical assessment of safety 100%: produced mild irritation in 1/80 subjects; formulations w/14-24% were non- to slightly irritating formulations w/14-2%, not sensitizers screening patch tests for contact sensitization in large population: incidence rate of 0.51% (19/3740) important Discussion items Discussion not included in report Conclusion safe as used

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19. Mishima, T., Katayama, Y., Takagi, Y., Ozeki, K., Hayakawa, T., and Tsuge, H. Ethyl alpha-D-glucoside increases urine volume and causes renal morphologic changes in rats. J Nutr Sci Vitaminol.(Tokyo). 2005;51:(1):22- 26.

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28. Yang, J. H., Lu, Y., and Zhang, J. H. [Research on spermicidal action of octyl-beta-D-glucopyranoside (OGP) in vitro] (English abstract.). Shengzhi.Yu Biyun.(Reproduction and Contraception) 2001.Jun.;21(3):173.-5. 2001;(Reproduction and Contraception 2001 Jun;21(3):173-5).

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31. Rohm and Haas Company. Ocular irritation study on Triton CG-110, 30% a.i.. Report No. 80R-105. 7-10-1980. NTIS No. OTS0538042.

32. Mehling A, Kleber M, and Hensen H. Comparative studies on the ocular and dermal irritation potential of surfactants. Food Chem Toxicol. 2007;45:747-758.

33. Corazza M, Lauriola MM, Bianchi A, Zappaterra M, and Virgili A. Irritant and sensitizing potential of eight surfactants commonly used in skin cleansers: an evaluation of 105 patients. Dermatitis. 2010;21:(5):262- 268.

34. Consumer Product Testing Company. Repeat insult patch test on decyl glucoside (5% active) and lauryl glucoside (5% active) under numerous trade names. Experiment Refn. No. C-005-93. 3-2-1993. Unpublished data submitted by the Council on Dec. 14, 2010. (34 pp) Available from CIR.

35. Consumer Product Testing Company. Repeated insult patch test of coco-glucoside. Experiment Ref. No. C05- 0247.01. 2005. Unpublished data submitted by the Council on Dec 14, 2010 (21 pp) Available from CIR.

36. Cho, Sun A., Han, Ju Hee, An, Susun, Lee, Key Hyun, Park, Jae Hak, Kim, Han Kon, and Lee, Tae Ryong. Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants. Cutaneous and Ocular Toxicology. 2010;29:(1):50-56.

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37. Rohm and Haas Company. Ocular irritation study on Triton CG-110, 30% surfactant. Report No. 81R 0046. 5-6- 1981. NTIS No. OTS0538042.

38. Rohm and Haas Company. Ocular irritation study on Triton CG-110, 30% surfactant. Report No. 81R 0047. 5-6- 1981. NTIS No. OTS0538042.

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44. Andersen, K. E. and Goossens, A. Decyl glucoside contact allergy from a sunscreen product. Contact Dermatitis. 2006;54:(6):349-350.

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57. Cognis. Data sheet on Plantacare 810 UP (caprylyl/capryl glucoside). 2010. Submitted by the Council on Dec 14, 2010 (2 pp) Available from CIR.

58. Cognis. Data profile on Plantacare 1200 UP (lauryl glucoside). 2010. Submitted by the Council on Dec 14, 2010. (2 pp) Available from CIR. 30

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59. Cognis.Product data sheet: Plantacare 818 UP. 2011. http://www.cospha.ro/dbimg/Plantacare%20818%20UP.pdf. Accessed 2-14-2011.

60. Personal Care Products Council. Concentration of use by FDA product category: hexadecyl d-glucoside and octadecyl d-glucoside. 3-14-2011. Unpublished data submitted by the Council on March 15, 2011. (2 pp).

61. Garcia C, Ball N, Cagen S, Carrillo J-C, Certa H, Eigler D, Esch H, Graham C, Haux C, Kreiling R, and Mehling A. Comparative testing for the indentification os skin-sensitizing potentials of nonionic sugar lipid surfactants. Regul Toxicol Pharmacol. 2010;58:301-307.

62. Ecology and toxicology of alkyl polyglycosides. 2004. Chapter: 18. Handbook of Detergents, Part B. Environmental Impact. New York, NY: Marcel Dekker.

63. Rohm and Haas Company. Triton CG-110: Rabbit eye irritation tirtration studies. Report No. 81R-131. Protocol No. 80P-439. 4-28-1982. NTIS No. 0538042.

64. Andersen, FA (ed). Final report of the addendum to the safety assessment of n-Butyl Alcohol as used in cosmetics. Int J Toxicol. 2008;27:(Suppl 2):53-69.

65. Elder, RL (ed). Final report o the safety assessment of Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl Alcohol. J Am Coll Toxicol. 1988;7:(3):359-413.

66. Diamante, CD, Andersen, FA, and Cosmetic Ingredient Review Expert Panel. Amended safety assessment of Cocos Nucifera (Coconut) Oil, Coconut Acid, Hydrogenated Coconut Acid, Hydrogenated Coconut Oil, Ammonium Cocomonoglyceride Sulfate, Butylene Glycol Cocoate, Caprylic/Capric/Coco Glycerides, Cocoglycerides, Coconut Alcohol, Coconut Oil Decyl Esters, Decyl Cocoate, Ethylhexyl Cocoate, Hydrogenated Coco-Glycerides, Isodecyl Cocoate, Lauryl Cocoate, Magnesium Cocoate, Methyl Cocoate, Octyldodecyl Cocoate, Pentaerythrityl Cocoate, Potassium Cocoate, Potassium Hydrogenated Cocoate, Sodium Cocoate, Sodium Cocomonoglyceride Sulfate, Sodium Hydrogenated Cocoate, and Tridecyl Cocoate. 2008.

67. Elder, RL (ed). Final report on the safety assessment of Stearyl Alcohol, Oleyl Alcohol, and Octyl Dodecanol. J Am Coll Toxicol. 1985;4:(5):1-29.

CIR Panel Book Page 45

Data

Distributed for Comment Only -- Do Not Cite or Quote

PersonalCare ‘ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

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

FROM: John Bailey, Ph.DZIt j-___Q_-._3(23 1t Industry Liaison to the CIR Expert Panel

DATE: March 23, 2011

SUBJECT: Comments on the Scientific Literature Review on Decyl Glucoside and Other Alkyl Glucosides as Used in Cosmetics

Why does this report have two covers? The first cover indicates that the report was prepared by Bart Heldreth. The second cover indicates that the report was prepared by Monice Fiume and Bart Heidreth. Is there a reason why this report does not include section summaries? p.3 - Please name the ingredient for which no uses were reported to the VCRP, but uses were reported in the Council concentration of use survey. The cosmetic use information is presented in Tables 3a and 3b not 4a and 4b as stated in the text. p.4- - Please indicate what is meant by “and a other toxic effects were observed.” p.7 - Please provide the doses or concentrations tested in the genotoxicity studies of C10-16 Alkyl Glucoside. p.8 - Please also note that irritation was dependent on the concentration of the ingredient tested. It would be helpful to note that all the “non-human” studies were done in rabbits. p.8 - Please include the concentrations that were tested in the alternative eye irritation methods. It would be helpful to be more specific - the “non-animal” mucosal irritation studies were all rabbit eye irritation studies. p.9 - Where is the Summary of this report? p.10-12, Table 1 - Please include the reference(s) with this table. p.17, Table 4-As all of the studies summarized in Table 4 were completed with rabbits, rabbits could be stated once in the title of the table rather than 10 times in the third row. What solvent was used to dilute the test substances? p.18, Table 5 - What solvent was used to dilute the test substances? As all the “non-human” studies were done in rabbits, rabbits should be in the heading of the table. In the results for Lauryl Glucoside, neutralized, is something missing as it currently states “e reversible within 7 days in all but one of the rabbits”? p.19, Table 5 - It would be helpful if the % active ingredient column included the concentrations that were actually tested. What solvent was used to dilute the test substances?

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

CIR Panel Book Page 46 Distributed for Comment Only -- Do Not Cite or Quote

PersonalCare ProductsCouncil Committedto Safety, ua ty nnovation Memorandum

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

FROM: John Bailey, 12—Ic4—10 Industry Liaison to the CIR Expert Panel

DATE: December 14, 2010

SUBJECT: Unpublished Information on Glucoside Ingredients

Cognis, Care Chemicals. 2010. Data profile PLANTACARE 810 UP (Caprylyl/Capryl Glucoside).

Cognis, Care Chemicals. 2010. Data profile PLANTACARE 818 UP (Coco-Glucoside).

Cognis, Cre Chemicals. 2010. Data profile PLANTACARE 1200 UP (Lauryl Glucoside).

Cognis, Care Chemicals. 2010. Data profile PLANTACARE 2000 JP (Decyl Glucoside).

Consumer Product Testing Company. 1993. Repeat insult patch tes. of 5% active Lauryl Gluoside anc Decyl Glucoside. Experiment Reference Number: C-005--93. (Note: In the past, Plantaren was the trade name for product produce tin the United States. Now, the trade namc:worldwide is Plantacare. The former US product has thc same specifications as pmvided for the Pla tacare products.)

Consumei Product Testing Company. 2005. Repeated insult patch Iest of Coco-Glucoside (t% act ie). Experiment Reference Number: C05-0247.01.

11011 7th Street, N.W., Suite 3OO Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personalcarecouncil.org CIR Panel Book Page 47 __

Distributed for Comment Only -- Do Not Cite or Quote l( )O j carechemicals COGMS NATURALM0DFIED

Data Profile PLANTACARE®810 UP

Address

Producer I Supplier Cognis GmbH Care Chemicals D - 40551 Düsseldorf Tel.: +÷49-21 1-7940-0 Fax.: ++49-21 1-798-5457

General characterisation Chemical description C8-10 fatty alcohol glucoside Raw material basis

vegetable (coconut/palm kernel oil, glucose)

Labeling information

INCI name(s) Caprylyl/Capryl Glucoside (EU + CTFA) Composition hints for finished product label

Capryl Glucoside > 60 - 70 % Aqua >30-40%

Ingredient information Ingredient CASR-No. EINECSIELINCS-No. Caprylyl/Capryl Glucoside 6851 5-73-1 no longer polymer

Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at higher temperatu-e.

Rev. 3.0 Cognis, PLANTACARE®810 UP Care Chemicals

CIR Panel1/3 Book Page 48 ZJs Distributed for Comment Only -- Do Not Cite or Quote

Product properties Appearance PLANTACARE®810 UP is a yellowish, slightly cloudy and viscous liquid. Example of use The tirbidity of the product is attributable to a combination of its magnesium oxide content (max. 0,1 % magnesium) and the pH value at which it is supplied. This ttibidity has no negative effects on the products properties and disappears if the pH value is adjusted to below 8.5. PLANTACARE® 810 UP is a nonionic sixfactant with excellent solubility, stability, sirface and interfacial activities. PLANTACARE® 810 UP shows excellent solubilizing properties in highly concentrated sulactant solutions also in the presence of salt and alkalies.

Solubility according to Ph. EUR. Ethylether Solubility: practically insoluble Chloroform Solubility: sparingly soluble Petrolether Solubility: practically insoluble Ethyalcohol Solubility: practically insoluble Paraffinoil Solubility: practically insoluble Water Solubility: practically insoluble

Characteristic values The specifications stated in the paragraphs ‘Quality control data’ and ‘Additional product descriptive data’ finally and conclusively describe the properties of the product. Quality control data (Data which is used for quality release and is certified for each batch.)

Water 35 - 38 % DGF H-Ill 3A Active matter 62- 65 % Difference: 100 % - % Water

pH value (10% in 15% 11,5 - 12,5 DGF H-Ill 1 Isopropanol) Additional product descriptive data (Data vvtiichis proven statistically but not determined regularly.)

Fatty alcohol <= 0,7 % Density (40,0 C) 1,13 - 1,14 g/cm3 Viscosity (dynamic) (40 °C) 500 - 1.500 mPas

Rev. 3.0 Cognis, PLANTACARE 810 UP Care Chemicals

CIR Panel2/3 Book Page 49 ZJs Distributed for Comment Only -- Do Not Cite or Quote

Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present

Storage and transportation

Shelf life 24 months Storage temperature <+ 4000 Store frost-free. Storage conditions In original sealed containers and protected from moistixe. Additional information Upon storage of PLANTACARE® 810 UP separation of the product may occt-, iich has no negative effects on the product quality. The product should be stirred until uniform before use. Containers and storage tanks made of V2A steel (material no. 1.4541) are of only limited use for storing PLANTACARE® 810 UP, because corrosion may occir. Storage containers made of V4A (material no. 1.4571) should therefore be preferred. PLANTACARE® 810 UP has a high pH-value and for this reason the product contains no preservatives (suffix UP = unpreserved).

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

Rev. 3.0 Cognis, PLANTACARE 810 UP Care Chemicals 5 IIIG1: CIR Panel3/3 Book Page 50 ___

Distributed for Comment Only -- Do Not Cite or Quote

carechemicals COGNIS NATURALMODIFIED

Data Profile PLANTACARE®818 UP

Address

Producer I Supplier Cognis GmbH Care Chemicals D - 40551 Düsseldorf Tel.: ++49-21 1-7940-0 Fax.: ++49-21 1-798-5457

General characterisation Chemical description C8-16 fatty alcohol glucoside

Mol weight 390 g/mol Raw material basis

vegetable (coconut/palm kernel oil, glucose)

Labeling information lNCl name(s) Coco-Glucoside (EU + CTFA) Composition hints for finished product label

Coco-Glucoside > 50 - 60 % Aqua >40-50%

Ingredient information Ingredient CASR-No. EINECS!ELINCS-No. Coco-Glucoside 141464-42-8 no longer polymer

Officially listed in I Quality conforms to JCIC: Alkyl(8-16) Glucoside (Ingredient Code: 523007) Bra Miljöval: approved for use in products eco-labelled “Good Environmental Choice” (in Swedish: Bra Miljoval)by the Swedish Society for Nature Conservation

Rev. 12.0 Cognis, PLANTACARE® 818 UP Care Chemicals

CIR Panel1/3 Book Page 51 Distributed for Comment Only -- Do Not Cite or Quote

Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at increased temperature.

Product properties Appearance PLANTACARE®818 UP is a cloudy, viscous, aqueous solution of a C8-C16 fatty alcohol glycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 500 ppm magnesium) and the pH value at which it is supplied. This turbidity has no negative effects on the products properties and disappears if the pH value is adjusted to below 7. PLANTACARE® 818 UP is a nonionic surfactant with a balanced combination regarding the foam volume and the excellent dermatological properties. Therefore it is suitable for use as a base surfactant or a co-surfactant in cosmetic cleansing preparations.

Odor evaluation versus standard corresponds to the standard Visual appearance versus standard corresponds to the standard Water 47-49 % DGF H-Ill 3A

Active matter 51 - 53 % Difference: 100%-%water Viscosity (dynamic) (20 C) 2.500 - 6.000 mPas DIN 53015 pH value (20% in 15% Isopropanol) 11,5 - 12,5 QP1507.1 Additional product descriptive data (Data which is proven statistically but not determined regularly.)

Free fatty alcohol <= 1,0 % Cognis Method 96011701 Sulphate ash <= 3,0 % DAB 10

Rev. 12.0 Cognis, PLANTACARE 818 UP Care Chemicals

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Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present

Storage and transportation

Shelf life 24 months Storage temperature <+ 40°C Storage conditions In original sealed containers and protected from moisture. Additional information The product should not be stored at temperatures below 15° C. If PLANTACARE® 818 UP is stored at temperatures below 15° C crystallization may occur. The product should be heated and stirred until uniform before use. Upon storage also some sedimentation can occur which has no negative effects on the quality. It is recommended to homogenize the product before use by stirring. Containers and storage tanks made of V2A steel (material no. 1.4541) and of V4A (material no. 1.4571) are suitable for storing PLANTACARE® 818 UP. PLANTACARE® 818 UP has a high pH-value and for this reason the product contains no preservatives (suffix UP = unpreserved).

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

Rev. 12.0 Cognis, PLANTACARE®818 UP Care Chemicals

CIR Panel3/3 Book Page 53 Distributed for Comment Only -- Do Not Cite or Quote

carechemicals COGNIS NATURALMODIFIED

Data Profile PLANTACARE®1200 UP

Address

Producer I Supplier Cognis GmbH Care Chemicals D - 40551 Düsseldorf Tel.: ++49-21 1-7940-0 Fax.: ++49-21 1-798-5457

General characterisation Chemical description C 12 - 16 fatty alcohol glucoside

Mol weight 420 g/mol

Raw material basis

Vegetable (coconut / palm kernel oil, glucose)

Labeling information

INCI name(s) Lauryl Glucoside (EU + CTFA) Composition hints for finished product label

Lauryl Glucoside > 50 - 60 % Aqua >40-50%

Ingredient information Ingredient CASR-No. EINECSIELINCSIECIEU NLP-No. Lauryl Glucoside 110615-47-9 600-975-8

Officially listed in I Quality conforms to Bra Miljöval: approved for use in products eco-labelled “Good Environmental Choice” (in Swedish: Bra Miljöval)by the Swedish Society for Nature Conservation

Rev. 12.0 Cognis, PLANTACARE®1200 UP Care Chemicals 4ñis CIR Panel1/3 Book Page 54 Distributed for Comment Only -- Do Not Cite or Quote

Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at increased temperature.

Product properties Appearance PLANTACARE® 1200 UP is a cloudy, viscous, aqueous solution of a C12-C16 fatty alcohol polyglycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 600 ppm magnesium) and the pH value at which it is supplied. This turbidity has no negative effects on the products properties and disappears if the pH value is adjusted to below 7. PLANTACARE® 1200 UP is a nonionic surfactant with good dermatological compatibility and viscosity enhancing effects. Therefore it is suitable for use as an additive or a co-surfactant in cosmetic surfactant cleansing preparations.

Solubility according to Ph. EUR. Ethylether Solubility: practically insoluble Chloroform Solubility: sparingly soluble Petrolether Solubility: practically insoluble Ethyalcohol Solubility: slightly soluble Paraffinoil Solubility: slightly soluble Water Solubility: sparingly soluble

Odor evaluation versus standard corresponds to the standard Visual appearance versus standard corresponds to the standard Water 47,0 - 50,0 % Cognis Method 96002701 Active matter 50,0 - 53,0 % Cognis Method 96002901 pH value (20 Weight% in 11,5 - 12,5 Cognis Method 95004902 isopropanol/water 15 Weight%) Additional product descriptive data (Data which is proven statistically but not determined regularly.)

Fatty alcohol <= 0,8 % Cognis Method 96011701 Ash <= 2,00 % Cognis Method 95014901 Density 40 °C 1,07 - 1,09 g/cm3 DIN 51757V4 Viscosity (dynamic) 40 C 1.000 - 3.000 mPas DIN 53015

Rev. 12.0 Cognis, PLANTACARE® 1200 UP Care Chemicals This CIR Panel2/3 Book Page 55 Distributed for Comment Only -- Do Not Cite or Quote

Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present

Storage and transportation

Shelf life 24 months Storage temperature <+ 45 C Storage conditions In original sealed containers and protected from moisture. Additional information If the product is stored at temperatures below 38 C crystallization may occur. Depending on the storage time sedimentation may occur. Therefore, the product should be heated and stirred until uniform before use. Containers and storage tanks made of V2A steel (material no. 1.4541) and of V4A (material no. 1.4571) are suitable for storing PLANTACARE® 1200 UP. PLANTACARE® 1200 UP has a high pH-value and for this reason the product contains no presevatives (suffix UP = unpreserved). For easier handling the product can be warmed up to max. 50CC for a short duration without influence onto the product specification.

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

Rev. 12.0 Cognis, PLANTACARE®1200 UP Care Chemicals

CIR Panel3/3 Book Page 56 js ___

Distributed for Comment Only -- Do Not Cite or Quote

carechemicals COGNIS NATURALMODIFIED

Data Profile PLANTACARE®2000 UP

Address Producer! Supplier Cognis GmbH Care Chemicals D - 40551 Düsseldorf Tel.: ++49-21 1-7940-0 Fax.: ++49-21 1-798-5457

General characterisation Chemical description C8-16 fatty alcohol glucoside

Mol weight 390 g/mol Raw material basis

vegetable (coconut oil, palm kernel oil, glucose)

Labeling information

INCI name(s) Decyl Glucoside (EU + CTFA) Composition hints for finished product label

Decyl Glucoside > 50 - 60 % Aqua >40-50%

Rev. 12.2 Cognis, PLANTACARE®2000 UP Care Chemicals c©gnis

CIR Panel1/4 Book Page 57 Distributed for Comment Only -- Do Not Cite or Quote

Ingredient information Ingredient CASR-No. EINECS/ELINCS-No. 6851 5-73-1 no longer polymer 110615-47-9 no longer polymer

Officially listed in I Quality conforms to JCIC: Alkyl (8 - 16) Glucoside (Ingredient Code 523007) Bra Miljäval: approved for use in products eco-labelled ‘Good Environmental Choice” (in Swedish: Bra Miljöval) by the Swedish Society for Nature Conservation

Manufacturing procedure Catalytic acetalisation of fatty alcohol with glucose at higher temperature

Product properties Appearance PLANTACARE® 2000 UP is a cloudy, viscous, aqueous solution of a C8-C1 6 fatty alcohol polyglycoside. Example of use The turbidity of the product is attributable to a combination of its magnesium oxide content (max. 500 ppm magnesium) and the pH value at which it is supplied. This turbidity has no negative effects on the product properties and disappears if the pH value is adjusted to below 7. PLANTACARE® 2000 UP is a nonionic surfactant with excellent foaming capacity and good dermatological compatibility. Therefore it is suitable for use as a base surfactant or a co-surfactant in cosmetic surfactant cleansing preparations.

Rev. 12.2 Cognis, PLANTACARE® 2000 UP Care Chemicals Is

CIR Panel2/4 Book Page 58 Distributed for Comment Only -- Do Not Cite or Quote

Odor evaluation versus standard corresponds to the standard Visual appearance versus standard corresponds to the standard Water 45-49 % DGF H-Ill 3A Active matter 51 -55 % Difference: 100% - %water Viscosity (dynamic) (20 °C) 1.000 - 6.000 mPas DIN 53015 Color number < 3 DIN ISO 4630 pH value (20% in 15% isopropanol) 11,5 - 12,5 QP1507.1 Additional product descriptive data (Data which is proven statistically but not determined regularly.)

Fatty alcohol < 1,0 % Sulphate ash < 3,0 %

Stabilising additives I Auxiliaries (type and concentration) Preservatives not added Antioxidants not present Solvents not present Others not present

Rev. 12.2 Cognis, PLANTACARE®2000 UP Care Chemicals *qnis CIR Panel3/4 Book Page 59 Distributed for Comment Only -- Do Not Cite or Quote

Storage and transportation

Shelf life 24 months Storage temperature Temperatures between + 10 °C and + 40 C Storage conditions In original sealed containers and protected from moisture. Additional information Upon storage of PLANTACARE® 2000 UP separation of the product may occur, which has no negative effects on the product quality. The product should be stirred until uniform before use. Containers and storage tanks made of V2A steel (material no. 1.4541) and of V4A (material no. 1.4571) are suitable for storing PLANTACARE®2000 UP. PLANTACARE® 2000 UP has a high pH-value and for this reason the product contains no preservatives (suffix UP = unpreserved).

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

Rev. 12.2 Cognis, PLANTACARE 2000 UP Care Chemicals

CIR Panel4/4 Book Page 60

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Methodology: The upper back between the scapulae served as the treatment site. Approximately 0.2 ml of each test material was applied to the 1” x 1” gauze portion of a webril adhesive dressing.* This was then applied to the appropriate treatment site to form semi-occluded patches.

This procedure was followed three times per week: Monday, Wednesday, and Friday for a total of ten applications. If a participant was unable to report for an assigned test day, one (1) makeup day was permitted. This day was added to the Induction Period. The sites were marked to ensure the continuity of patch application. The participants were instructed to remove these patches after twenty-four hours. The evaluation of each’ site was madejust prior to re-application.

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

At the conclusion of a rest period of approximately fourteen days following the tenth application, a challenge patch was applied to the original site and to a virgin site. Each site was evaluated at twenty- four and forty-eight hours after application. The volar forearm served as the virgin test site.

Evaluation Key: 0 - No visiblereaction 1+ - Mild erythema 2+ - Well-defined erythema, possible presence of barely perceptibleedema 3+ - Erythema and edema 4+ - Erythema and edema with vesiculation and ulceration

Results: The results of each participant are appended. Observations of all treated areas remainednegative throughoutthe testinterval.

Summary: Under the conditions of this study, the following test materials do not indicate a potentialfor dermal irritation or sensitization:

1. VII1-48A 8. VIfl-48H 2. VUI-48B 9. VUI-483 3. Vffl-48C 10. VIll-48K 4. Vffl-48D 11. Vffl-48L 5. ‘VllI-48E 12. VIlI-48M 6. VIII-48F 13. Vlll-48N 7. VIII-48G 14. VIfl-480

*Manufacturedby ProfessionalMedical Products,Inc., Greenwood, SC

CIR Panel Book Page 64 Distributed for Comment Only -- Do Not Cite or Quote crporaor. C-005-93 Page 5

Table I IndividualResults

Vffl-48A

Original Virgin Subject —------InductionExposures —----- Site Site Number 12345678 910 24 48 24 48

1 00000000 0 0 DID NOTCOMPLETE STUDY 2 00000000 00 0 0 0 0 3 00000000 00 0 0 0 0 4 00000000 00 0 0 0 0 5 00000000 00 0 0 0 0 6 00000000 00 0 0 0 0 7 00000000 00 0 0 0 0 8 00000000 00 0 0 0 0 9 00000000 00 0 0 0 0 10 00000000 00 0 0 0 0 11 00000000 00 0 0 0 0 12 00000000 00 0 0 0 0 13 00000000 00 0 0 0 0 14 0 0 0 0 0 0 omo 00 0 0 0 0 15 0 0 0 0 0 0 0 0 00 0 0 0 0 16 00000000 00 0 0 0 0 17 00000000 omo 0 0 0 0 18 00000000 00 0 0 0 0 19 -DiDNOT COMPLETESTUDY 20 00000 0 0 0 0 om 0 0 0 0 21 -DIDNOT COMPLETESTUDY 22 -DIDNOT COMPLETESTUDY — 23 00000 00000 0 0 0 0 24 0 0 0 .0 0 00000 0 0 0 0 25 00000 00000 0 0 0 0 26 00000 0 0 1+ ----PATCHINGDISCONT1NUED* 27 00000 00000 0 0 0 0 28 00000 00000 0 0 0 0 m = Makeup day * =seeText,p.3

CIR Panel Book Page 65 Distributed for Comment Only -- Do Not Cite or Quote p C-OO593 Page 6

Table 1 (continued) IndividualResults

Vffl-48A

, Original Virgin Subject Induction Exposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

29 0 0 0 0 m 0 DID NOT COMPLETESTUDY 30 0 000000000 0 0 0 0 31 0 000000000 0 0 0 0 32 0 0 omo 0 0 0 0 0 o 0 0 •o 0 33 0 DIDNOTCOMPLETESTUDY 34 0 00000 0000 0 0 0 0 35 0 00000 0 0 0 om 0 0 0 0 36 0 0 0 0 omo 0000 0 0 0 0 37 0 0 0 0 omo 0000 0 0 0 0 38 0 00000 oo 0 0 0 0 0 39 0 00000 00 00 0 0 0 0 40 0 00000 00 00 0 0 0 0 41 0 00000 00 00 0 0 0 0 42 0 00000 00 00 0 0 0 0 43 0 00000 00 00 0 0 0 0 44 0 00000 00 00 0 0 0 0 45 0 00000 00 00 0 0 0 0 46 0 0’0 0 00 00 00 0 0 0 0 47 0 0 0 0 00 00 00 0 0 0 0 48 0 0 00 00 00 00 0 0 0 0 A 1. I A A A n A 49 V J V U U U U V V U U V 50 0 0 0 0 00 omo 00 0 0 0 0 51 0.0 0 0 00 00 00 0 0 0 0 52 0 0 0 0 00 00 Oom 0 0 0 0 53 000000 00 00 o 0 0 0 54 0 0 0 0 0 0 00 00 0 0 0 0 55 0 ------DIDNOTCOMPLETESTUDY 56 0 0 0 0 0 0 0 0 0 0 0 0 0 0

m = Makeup day

CIR Panel Book Page 66

m=Makeup

Number

Subject

24 23

=seeText,p.3 27 26

22 21

17 16 11

19 14 13

7 6

3 2 8

00000000

0000000000 00000000

0 0000000000 0000000000 00000000

oooooooooom 00000000 00000000

00000000 00000000 00000000 00000000

00000000 00000000•00 00000000 0 00000000 00000000

00000000 0000000000

000000000 000000000

00000000

1 day

—

—------—-----DID

3 Induction

—

0 4

Distributed

Exposures

0 6

for

DID DID CIR

Comment

00 7

Panel

NOT NOT

NOT

Individual

1+ 8 Only

Book

COMPLETE

COMPLETE COMPLETE

‘IllI-48B

Table

----PATCHING

orno

00 00 00

00 00

00 00 00

00 00

00 00 9 -- Page

Results

0 2 Not

67 DID

Cite

STUDY---- STUDY

STUDY NOT or

Quote

0 0

0 0 0

0 0

0 0 0

0 0

0 0 0

Original

DISCONTINUED*

Site

COMPLETE

0 0

0 0 0

0 0

C-005-93

?age

:-ee:

7 24

—

0 0 0

0 0

0 0 0

0 0

STUDY

Corporation

Virgin

Site

0 0

0 0 0 0

0 0

Number

Subject

40 36 34

45 43 35

48 44 42 38 33

47 32 30

= 50 46 39 37 31 29

56 51 54

An Makeup

—

0 0 0

0 0 0 0 0

0 0 0 U 0 0 0 0

0 0 0 0 0

0 0 0

I’

day

000 2 0

00000000 000 00000000 0 00000000

000 00000000 00000000 00000000 0 00000000

o 00000000 U 00000000 00000000 00000000 0

0000 0 00000000

0 m 0

omo 0 0

0 U 0

0 a

Q111Q

Induction

-U

0 0

0 0 0

0 0 a.

Distributed

00 00 0 U

00 0 0

5 0 0 0

0 m 0

omo

Exposures

0 0 0

00000 n

for --DID CIR

Comment

-DID

7 0 0 0

0000 0000 U 0 0

0000 omo 0 0

Panel

Individual

NOT

U omo

0 0

0 a

8 0

NOT (continued) Only

Book

VIll-48B

Table

0 0

DID

0 a 0

9 0 COMPLETE --

Page COMPLETE

Results

0 0 0 0 0 0 0

0 11 0 0 0 0

0 om 0 0

0 a

NOT 2 Not 68

Cite COMPLETE

or STUDY-

Quote

STUDY

0 o 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0

0 Original

Site

0 0

0 0 0

0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0

(-005-93

Page *ene..

STUDY

0 0 0 0 0

0 0 0 0

0 0 0 0 0

Corporaon

Virgin

Site

0 0 0

0 0 0 0 0

0 0 0 0

0 0

Number

Subject m

21 20

=seeText,p.3 28 26 12

= 27 25 22 11

18 15 13 10

17 14

7 2

5 3 8

Makeup

0000000000

0000000000 0

0000000000

0000000000 0000000000

0000000000 0000000000 0000000000

0000000000 ooo 0 0 0 000mo 0000000000

1 0000000000 0000000000

0000 0 00 m000 0000000000

0000000000

oOOOOOOOO0

0000000000 0000000000

0000000000

day

2345678910

0 0

—

0 Induction

—

0 0

Distributed

Exposures

—--DID ----DID

for

0 DiD CIR

Comment

0 0

Panel

NOT NOT

NOT

Individual

0 1+ Only

Book

VllI-48C

COMPLETE

Table

0 —-PATCHING -- Page

Resulis

0 3 Not

69 DID Cite

STUDY

STUDY NOT

Quote

0 0 Original

0 0 0 0

0 0

0 0 0

0 0

DISCONTINUED*

Site

COMPLETE

0 0

0 0 0 0 0

0 0 0 0 0 0

0 0

Page C-005-93

,‘,‘-‘“-.. 9

—

0 0 0 0

0 0 0 0 0

0 0 0

0 0

STUDY

Virgin

Site

0 0 0 0 0

0 0 0 0

0 0 0

0 0

0 0 0

0 0

Number

Subject

33 30

34 40

41 38

43 42 39

47 46

54 AG

56 = 55

I—

Makeup

0 -—--—

0 0000000000

0 0

0 0000

1 0

0000

0 0 0

flft.

0 0 Vt

0 0

day

000000000

0 0 0

0 0

omo

0 0 Sr

0 0

Gmo

0 0 0

0 0

omo

0 (1 0

0 0

0 Induction fl

Distributed

0 0 0

0 0

0 0 0

omo

00 omo

00 00

00 Vt (

000000 00

000000 Exposures--—

for

0 0 Il Vt CIR

Comment

DID

-DID

000 0

000 000

0 000

000

000 000

Panel

000

omo

000 Vt

000

Individual

NOT

0 omo Only

Book

NOT

(continued)

V1I1-48C

Table

DID

9 0

-- 0

Page COMPLETE

COMPLETE

10 Results

Not

0 0

0 0 0

0 0

NOT 0

3 0 Vt

1 0 70

Cite COMPLETE or

Quote

STUDY

Original

STUDY

0 0

0 0 0

0 0

Site

0 0

0 0 0

0 0

0 0 0

C-005-93

Page

STUDY

0 0

0 SI

0 0

Virgin

Site

0 0 0 0

0 0

0 0 0

0 0

Number

Subject

=seeText,p.3

=Makeup

27 26 25

28 21

24 23 22

19 18

15 14 13

6 5

4 2

000

000 000 000 000

000

0 00000000

00000000 00000000 00000000

00000000 00000000

00000000

00000000 00000000 00000000 00000000

00000000

00000000 00000000 00000000

00000000

day

Induction

00 00

00 00 00 00

4 -

Distributed

Exposures

—-DID

00000 00000

00000 00000 0 00000

DID DID for CIR

Comment

NOT NOT NOT

Panel

Individual

COMPLETE

COMPLETE VIfl-48D Only

Book

Table

----PATCHING

00 00

00 00 00 00

00 00

00 00 00 00

00 9 o --

Page 10

Results

o 4

Not DID 71

Cite

STUDY-

SThDY

STUDY NOT

0 0

0 Quote 0 0 0

0 0 0

Original

DISCONTINTJED*

Site

COMPLETE

0 0

0 0 0

0 0

Page

C-005-93

Henkel

0 0 0

0 0

0 0 0

0 0

STUDY

Corporation

Virgin

Site

0 0 0

0 0

0 0 0

0 0 0 0

0 0 0

0 0

Number

Subject

41 34

44 42 37

45 38 36

43 39

56 54

Makeup

0000000000

0000000000 0

0000000000 OOOOOO0000

00 0 m 0 000000 0000000000 00 000 m00000

0000000000 0000000000 m

0000000000 00 0 00m00000

0000000000 0000000000

OOm00000000 0000000000 0000000000

oOom 0 000000

0000000000 0O000G0G0 0000000000

oooo 000 m000 0000000000

0000000000 0000000000 m

0000000000

day

Induction

4 0 —

Distributed

0 m

Exposures

0 6

for DID

CIR 7

Comment

DID Individual

Panel

NOT

(continued)

VIll-48D Table Only

Book

DID

COMPLETE COMPLETE --

Page

10 Results

NOT 4 Not 72

Cite

COMPLETE STUDY

STUDY

0 Original

0 0 0

0 0

0 0 0 0 0

0 0 0 0

0 0

Quote

Site

0 0

0 0 0

0 0

0 0 0

0 0 0 0

C-005-93

Page Henkel

STUDY

0 0

0 0 0 0

0 0 0 0 0

0 0 0

Corporation

Virgin

Site

0 0

0 0 0

0 0 0 0

0 0 0

0 0 0 0

0 0

0 0 0

0 0

Number

Subject m

23 13

LL 12

27 14 11

28 25

24 = 15

=seeText,p.3 19 18 16

7 4 3

6 9

Makeup

-——---Induction

00000000

00000000 00000000

00000000

00000000 00000000 00000000

00000000 00000000

00000000

1 00000000

00000

00000 00000

00000

day

4 0

Distributed

Exposures

00000 0

00000 00000

for

-DID

-DID DID CIR

Comment

omo

0 0

Panel

NOT

Individual

0 2+ Only

Book

VIII-48E

COMPLETE COMPLETE

COMPLETE

Table

0 0

00 0 9

00 00

0 00 00

00 00 00

0 0 m 0 00

00 00 ----PATCHING -- Page

Results

0 0

0 5 Not

73 DID

Cite

STUDY-

STUDY STUDY- NOT or

Quote

0 0 Original

0 0 0

0 0 0 0

0 0

DISCONTINUED

Site

COMPLETE

0 0

0 0 0

0 0 0 0 0

0 0 0

0 0

Page

C:-005-

:-en.e:

0 0 0

0 0 0 0

0 0 0 0 0

0 0

0 0 0 0

STUDY

orporaon

Virgin She

0 0

0 0 0 0 0

0 0 0

0 0 0 0

0 0

Number

Subject m

41 32

40 38 37 34

42 39 36 33

47 44

46 43

48 45 50

54 51

56 55 53 52 Makeup

0000000000

0 000m0 00000 o 0

0000o 000 00m 0000000000

0000000000 000

0000000000 0 0 0 0 000 0moo

0000000000 0000 0 m 00 000

0000000000

000 m 0000000 0000000000

0000000000 0000000000

1 oomo

0000000000 0000000000 0000000000

0 0000000000 0000000m000

000 0000000 m

0000000000

day

0 2 &..n.n_n

—

Induction

oom 000 00 0000000

Distributed

0 m

Exposures

0 6 fi

for

DID 7 CIR

flfl DID

Comment Individual

Panel

NOT

(continued)

VIU-48E Table Only

Book

DID 9

flA

COMPLETE COMPLETE

-- 10

Page

Results

NOT 5 Do Not

74 COMPLETE

Cite

STUDY 24

0 STUDY 0

0 0 0 0 Original

0 0

0 0 0

0 0 0 0 0

0 0 0 fl

0 0 0

Quote

Site

0 0

0 0 0 0

0 0

0 0 0

0 0

Page C

:n.

STUDY

-0O593

0 0 0

0 0

0 0 0 0 0

0 0 0

0 0 A

0 0 0

0 0

oporan

Virgin

Site

0 0 0

0 0 0 0 0

0 0 0

0 0 A 0

0 0 0

0 0

0 0 Distributed for Comment Only -- Do Not Cite or Quote :enke.. (..orpora;on C-OO593 Page 15

Table 6 IndividualResults

VIII-48F

Original Virgin Subject InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

1 00000000 0 0 DID NOT COMPLETESTUDY 2 00000000 00 0 0 0 0 3 00000000 00 0 0 0 0 4 00000000 00 0 0 0 0 5 00000000 00 0 0 0 0 6 00000000 00 0 0 0 0 7 00000000 00 0 0 0 0 8 00000000 00 0 0 0 0 9 00000000 00 0 0 0 0 10 00000000 00 0 0 0 0 11 00000000 00 0 0 0 0 12 00000000 00 0 0 0 0 13 00000000 00 0 0 0 0 14 0 0 0 0 0 0 01110 00 0 0 0 0 15 00000000 00 0 0 0 0 16 00000000 00 0 0 0 0 17 00000000 omo 0 0 0 0 18 00000000 00 0 0 0 0 19 DID NOT COMPLETESTUDY-- 20 00000 0 0 0 oom 0 0 0 0 21 •DIDNOT COMPLETESTUDY• LL -iJiJJINJi LLt1.iLJL)X-- 23 00000 00000 0 0 0 0 24 00000 00000 0 0 0 0 25 00000 00000 0 0 0 0 26 00000 0 0 2+ ----PATCHINGDISCONTINUED* 27 00000 00000 0 0 0 0 28 00000 00000 0 0 0 0 m = Makeup day * =seeText,p3

CIR Panel Book Page 75

Number

Subject

29 45 41 38

47 44 40

34 32 48 46 43 39 36

33 31 42 35

40 37

56 54

55 53 51

Makeup

0 0

0 0 --—

0 0 0 0

0 0 0

0 0

0 0 0

0 0 A

1234567

day

000000000

0 000

000 000 000

000 omo 000 000 000 000

000 000 000 000

000 000 000

000 A

000000000 000

—-Induction

0 rn 0

0 rn 0 A —

Distributed

0 000000 000000

omo 0

000000 Urn 0 000000 000000 000000 000000

000000 000000 000000 000000 0 m 0

000000 000000 000000

000000 Exposures

for

0 0 0 0 CIR

Comment -DID

Panel

0 0

orno

DID

Individual

NOT

omo 0

8910

0 0 Only

Book

NOT

(continued)

VI1I-48F Table

0 DID

0 0

Page COMPLETE

Do COMPLETE

Not

Results

0 rn

0 0

0 m

NOT 6

Cite COMPLETE or

Quote

STUDY-

STUDY

0 0 0

0 0 0 0 Original 0

0 0 0 0 0 0

0 0

Site

0 0 0 0 0 0

0 0 0 0 0

0 0

0 0 0 0 0

c..005-93

Page ;cne..

STUDY

0 0

0 0 0 0 0 0 0 0 0

0 0 0 0

9 0

or2Ora.ofl

Virgin

Site

0 0 0 0 0

0 0 0 0

0 0

Number

Subject rn

26 20

25 22

24 21

=seeText,p.3 =Makeup 28 23

27 13

15 12 10

17 16 14 11

4 2

7 9

5 3

6 8

—------Induction

0000000000 0

0000000000 0000000000

0000000000 0000000000 0000000000

0000000000 0000000000

0000 0000000000

1 0000000000 0000000000 0000000000

0 OOOOOOOO0

------— 0000000000

0000000000 0000000000 m

0 0000000000

0000000000 0000000000

0000000000

clay

0 0

—

3 0

Distributed

0 5

0OO000

—----DID

0 Exposures

for

0 0

CIR

DID DID

Comment

7 0

Panel

NOT

Individual

8 2+ Only

Book

Vffl-48G

COMPLETE

COMPLEFb

COMPLETE

Table

9 0

------PATCHING Page

10 ResulLs

Not 0

7 DID Cite

STUDY

SIUDY STUDY NOT

Quote

0 Original

0 0

0 0 0 0

0 0

0 0 0

0 0

0 0.

DISCONTIN1JED*

Site

COMPLETE

0 0 0 0 0

0 0 0

0 0

C-005-93

Page Hene1

—

0 0 0 0 0

0 0 0 0

0 0

0 0 0

0 0

STUDY

Corporation

Virgin

Site 48

—

0 0

0 0 0 0

0 0

0 0 0 Distributed for Comment Only -- Do Not Cite or Quote :enke.. orporaon (-005.93 Page 18

Table 7 (continued) IndividualResults

Vffl-48G

. Original Virgin Subject ------Induction Exposures —- Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48 29 0000 0m —---DIDNOT COMPLETESTUDY 30 0000 00 0000 0 0 0 0 31 0000 00 0000 0 0 0 0 32 0 0 omo 00 0000 0 0 0 0 33 0 - -DIDNOTCOMPLETESTUDY 34 0000 00 000 0 0 0 0 0 35 0000 00 000 0 0 0 0 36 0000 omo 000 0 0 0 0 0 37 0000 000 0 0 0 0 0 38 0000 00 0 0 0 0 0 39 0000 00 000 0 0 0 0 0 40 0000 00 000 0 0 0 0 0 41 0000 00 000 0 0 0 0 0 42 0000 00 000 0 0 0 0 0 43 0000 00 000 0 0 0 0 0 44 0000 00 000 0 0 0 0 0 45 0000 00 000 0 0 0 0 0 46 0 0m 00 000 0 0 0 0 0 47 0000 00 000 0 0 0 0 0 48 0 0 omo 00 000 0 0 0 0 0 AO A A A A A A A A A A fl A — ‘, ,, ,J I., •S ‘0 t I ‘I V 50 0000 00 omo 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 •0 0 0 0 0 0 53 0 0 0 0 0 0 0. 0 0 0 0 0 0 0 54 00 000000 0 G 0 0 0 0 55 0 — DiD NOT COMPLETESTUDY- 56 0 0 00000000 0 0 0 0 m = Makeup day

CIR Panel Book Page 78 Distributed for Comment Only -- Do Not Cite or Quote

C-005-93 Page 19

Table 8 IndividualResults

VIII-48H

Original Virgin — Subject —InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

1 00000 0000 0 DID NOT COMPLETESTUDY 2 00000 0000 0 0 0 0 0 3 00000 0000 0 0 0 0 0 4 00000 0000 0 0 0 0 0 5 00000 0000 0 0 0 0 0 6 0 0 0 0 0 0000 0 0 0 0 0 7 00000 00000 0 0 0 0 8 00000 00000 0 0 0 0 9 00000 00000 0 0 0 0 10 00000 00000 0 0 0 0 11 00000 00000 0 0 0 0 12 00000 00000 0 0 0 0 13 00000 00000 0 0 0 0 14 00000 0 omo 0 0 0 0 0 0 15 00000 00000 0 0 0 0 16 00000 00000 0 0 0 0 17 00000 0 0 0 omo 0 0 0 0 18 00000 00000 0 0 0 0 19 DID NOT COMPLETESTUDY 20 0 0 0 0 0 0 0 0 0 0m 0 0 0 0 21 DID NOTCOMPLETESTUDY flv S ,fl.i, flt%S S1%T V% St LL --i)LL) 1VIL i I Ui-I I 23 00000 00000 0 0 0 0 24 00000 00000 0 0 0 0 25 00000 00000 0 0 0 0 26 00000 0 0 3+ ----PATCHINGDISCONTINUED* 27 00000 00000 0 0 0 0 28 00000 00000 0 0 0 0 m = Makeupday * =seeText,p.3

CIR Panel Book Page 79 Distributed for Comment Only -- Do Not Cite or Quote

C-OO593 Pace 20

Table 8 (continued) IndividualResults

VllI-48H

Original Virgin Subject InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

29 0 000 omo DID NOT COMPLETESTUDY 30 0 000 00 0000 0 0 0 0 31 0 000 00 0000 0 0 0 0 32 0 0 0m 00 0000 0 0 0 0 33 0 -DIDNOTCOMPLETE STUDY 34 0 00000 000 0 0 0 0 0 35 0 00000 000 On’ o o 0 0 36 0 0 0 0 0m 000 0 o o 0 0 37 0 0 0 0 omo 000 0 0 0 0 0 38 0 00000 0 OfllO 0 0 0 0 0 39 0 00000 000 0 0 0 0 0 40 0 00000 000 0 0 0 0 0 41 0 00000 000 0 0 0 0 0 42 0 00000 000 0 0 0 0 0 43 0 00000 000 0 o o 0 0 44 0 00000 000 0 0 0 0 0 45 0 00000 000 0 o o 0 0 46 0 omo 0 0 0 000 0 0 0 0 0 47 0 00000 000 0 0 0 0 0 48 0 0 omo 0 0 000 0 0 0 0 0 AG 1 00000 ( r A A o 0 0 0 50 0 00000 omo 0 0 0 0 0 0 51 0 00000 000 0 0 0 0 0 52 0 00000 000 om o o 0 0 53 0 00000 000 0 0 0 0 0 54 0 00000 000 0 0 0 0 0 55 0 DIDNOT COMPLETESTUDY 56 0 000000000 0 0 0 0 m = Makeup day

CIR Panel Book Page 80 Distributed for Comment Only -- Do Not Cite or Quote :a;.: C-005-93 Page 21

Table 9 IndividualResults

VIJI-483

Original Virgin Subject InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

1 00000 000 0 0 DIDNOT COMPLETESTUDY 2 00000 000 0 0 0 0 0 0 3 00000 000 0 0 0 0 0 0 4 00000 00000 0 0 0 0 5 0000000000 0 0 0 0 6 0000000000 0 0 0 0 7 0000000000 0 0 0 0 8 0000000000 0 0 0 0 9 0000000000 0 0 0 0 10 0000000000 0 0 0 0 11 0000000000 0 0 0 0 12 0000000000 0 0 0 0 13 0000000000 0 0 0 0 14 0000000m000 0 0 0 0 15 0000000000 0 0 0 0 16 0000000000 0 0 0 0 17 0000000 m00 0 0 0 0 18 0000000000 0 0 0 0 0 19 — DID NOT COMPLETESTUDY 20 0 0 0 0 21 000000000 DID NOT COMPLETESTUDY TT% )TI1’ 1’I.. AThT TT’V C”l’l T17 4. LJ1LJ 4’.J i JIYU A A., .) I JL) 23 0000000000 0 0 0 0 24 m 0 0 0 0 0000000000 25 0000000000 0 0 0 0 26 0 0 0 0 0 0 0 3+ ----PATCHINGDISCONTINUED* 27 0000000000 0 0 0 0 28 0000000000 0 0 0 0 m = Makeup day * =seeText,p.3

CIR Panel Book Page 81 Distributed for Comment Only -- Do Not Cite or Quote zerLe.. c.otpora:.o. ‘C’-005-93 Page 22

Table 9 (continued) IndivduaI Results

VIII-48J

Original Virgin Subject —-----InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

29 0 0 0 0 om0 DID NOT COMPLETESTUDY 30 0 000000000 0 0 0 0 31 0 000000000 0 0 0 0 32 0 0 0m 0 0 0 0 0 0 0 0 0 0 33 0 DIDNOT COMPLETESTUDY 34 0 000 000000 0 0 0 0 35 0 000 0 0 0 0 0 O’’ 0 0 0 0 36 0 000 00 0 0 0 0 0 0 0 0 37 0 000 0m 0 0 0 0 0 0 0 0 38 0 000 0 0 0 omo 0 0 0 0 0 39 0 000 000000 0 0 0 0 40 0 000 000000 0 0 0 0 41 0 000 000000 0 0 0 0 42 0 000 000000 0 0 0 0 43 0 000 000000 0 0 0 0 44 0 000 000000 0 0 0 0 45 0 000 000000 0 0 0 0 46 0 omo 0 000000 0 0 0 0 47 0 000 000000 0 0 0 0 48 0 0 omo 000000 0 0 0 0 A9 A (i A A A A A A A A n A 0 ,. , ., t , I. ‘S ¼? 50 0 000 0 0 omo 0 0 0 0 0 0 51 0 000 000000 0 0 0 0 52 0 00 0 0 0 0 0 0 0m 0 0 0 0 53 0 000 000000 0 0 0 0 54 0 000 000000 0 0 0 0 55 0 — DIDNOT COMPLETESTUDY 56 0 000000000 0 0 0 0 in = Makeupday

CIR Panel Book Page 82

Subject

Number m

24 23 20

26 14 10

28 27 12 =seeText,p.3 hh

= 11

19 13

18 15

7 3

6 9

Makeup

00000000 00000000

00000000

00000000 00000000 0 00000000

00000000 00000000 00000000 00000000 00000000

00000000

00000000 00000000

00 00000000

1 0

00 00

day

—-Induction

00000000

0 0 00 00

Distributed

5 0

Exposures --DID

--Liii)

00000 0

0 00000

00000

for DID CIR

Comment

omo

0 0

Panel NOT

f1UL

NOT

Individual

0 3+ Only

Book

VllI-48K

Table COMPLETE

LUM1’LLIt

COMPLETE

0 0 0 00 00 0

0 00 00 00 0

omo

00 00

0 ----PATCHING -- Page

Results

0 0

0 0 10 0

0 m Not

83 DID

Cite

STUDY

i STUDY NOT or

Quote 24

1)1.)!

0 Original

0 0 0 0

0 0 0

0 0

DISCONTINUED*.

Site

COMPLETE

0 0 0

0 0 0 0

0 0 0

0 0

0 0 0

C-oO5S3

Page

:-ene;,

0 0

0 0 0 0

0 0

0 0 0

0 0

STUDY Corpora.on

—

Virgin

Site

0 0

0 0 0 0 0 0

0 0 0 0 0

0 0

0 0 0 Distributed for Comment Only -- Do Not Cite or Quote :-en;e,, c.oporaon (-005-93 Page 24

Table 10 (continued) IndividualResults

VIU-48K

. Original Virgin Subject — —InductionExposures —---- Site Site Number 1234567 8910 24 48 24 48 29 0000 om 0 DID NOT COMPLETESTUDY 30 0000 000000 0 0 0 0 31 0000 000000 0 0 0 0 32 o o omo 000000 0 0 0 0 33 0 DID NOT COMPLETESTUDY 34 0 00000 0000 0 0 0 0 35 0 00000 0 0 0 om 0 0 0 0 36 0 0 0 0 omo 0000 0 0 0 0 37 0 0 0 0 0O 0000 0 0 0 0 38 0 00000 0 0m 0 0 0 0 0 39 0 00000 0000 0 0 0 0 40 0 00000 0000 0 0 0 0 41 0 00000 0000 0 0 0 0 42 0 00000 0000 0 0 0 0 43 0 00000 0000 0 0 0 0 44 0 00000 0000 0 0 0 0 45 0 00000 0000 0 0 0 0 46 0 0O 0 00 0000 0 0 0 0 47 0 000 00 0000 0 0 0 0 48 0 0 0O 00 0000 0 0 0 0 Afl A A A A A A A A A A A A ‘I V V Id J ., U •I V U V 50 0 000 00 omo 0 0 0 0 0 0 51 0 00 0 00 0000 0 0 0 0 52 0 000 00 0 0 0 om 0 0 0 0 53 0 000 00 0000 0 0 0 0 54 0 000 00 0000 0 0 0 0 55 0 - DID NOTCOMPLETESTUDY 56 0 000000000 0 0 0 0 m = Makeup day

CIR Panel Book Page 84 Distributed for Comment Only -- Do Not Cite or Quote cpo:a.o’. C-005-.93 Page 25

Table 11 IndividualResults

Vffl-48L

Original Virgin Subject Induction Exposures— — Site Site Number 1 2 3 4 5 6 7 8 9 JO 24 48 24 48

1 00000 000 0 0 DID NOTCOMPLETESTUDY 2 00000 000 00 0 0 0 0 3 00000 000 00 0 0 0 0 4 00000 000 00 0 0 0 0 5 00000 000 00 o 0 0 0 6 00000 000 00 0 0 0 0 7 00000 000 00 0 0 0 0 8 00000 000 00 0 0 0 0 9 00000 000 00 0 0 0 0 10 00000 000 00 0 0 0 0 11 00000 000 00 0 0 0 0 12 00000 000 00 o o 0 0 13 00000 000 00 0 0 0 0 14 00000 0 omo 00 o o 0 0 15 00000 000 00 0 0 0 0 16 00000 000 00 0 0 0 0 17 00000 000 omo 0 0 0 0 18 00000 000 00 o 0 0 0 19 ------D1D NOTCOMPLETESTUDY. 20 0 0 0 0 0 0 0 0 0 0m 0 0 0 0 21 - —-DIDNOTCOMPLETESTUDY DID NOT COMPLETESTUDY 23 000 0000000 0 0 0 0 24 000 0000000 0 0 0 0 25 0 0.0 0000000 0 0 0 0 26 000 0 0 0 0 3÷ ----PATCHINGDISCONTINUED* 27 000 0000000 0 0 0 0 28 000 0000000 0 0 0 0 m =Makeup day * =seeText,p.3

CIR Panel Book Page 85

Number

Subject

45 43 41

53 46 44 42 40 38

= 56 47 39 37

54 48

55 51 36 34 32 30

52 50 35 31

33 29

Makeup

0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0

0 0 C)

0 0 0 0 0 0

day

00000

00000 00000

00000 0 000 00000 00000 00 00000 00000 00000 -— 0

00000 000 000 00000 0 00000 0 00000 00000 0

omo 00000

000000000

—--Induction

omo 0

3 0G 0

—

0 0

0 00 Distributed

—

omo

00 0 0 o 0 m 0

0 m 0 5

Exposures

0 0 0 0 for CIR

Comment

DID

0000

0000 0000

0000 0000 0000 0000 0000 0 0000

0 0000 0000 0000 0000 0000 0000 0000

0 0000 0000

0000

0 OmO DID

Panel

Individual

NOT

0 0 m

8 0

NOT (continued) Only

Book

VIU-48L

Table

DID 9

0 0 COMPLETE

-- COMPLETE Page

Results

0 m om 0

11 NOT Not 86

Cite COMPLETE

STUDY- 24

Quote

0 STUDY

0 0

0 0 0

0 0 0 0 0 0 0

0 C) 0 0 0 0 0 0

0 0 0

Original

Site

0 0

0 0 0

0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0

Page

(-005-93

STUDY

0 0 0

0 0 0 0 0 0 0 0 0

0 0

Virgin

Site

0 0

0 0 0 0 A 0

0 0 0 0 0

0 0 0 0

0 0

0 0 0 0

Number

Subject

=Makeup 28 26 24 20

=seeText,p.3 27 25 23 21

16 14 12 10

17 11

19 13

7 3

4 2 9

00000000 00000000 00000 00000000

00000000 00000000 00000 00000000 00000000 0 0 00000000 00000000

00000000 00000000

00000 00000000

00000000 00000 00000 00000 00000000

00000 00000000 00000000

day

2 0

o 0

3 Induction

Distributed

5 0 Exposures

for

--LJUJPiUI

6 00000 00000 0 0

00000 0

00000

-DID DID CIR

Comment

0 omo 0

Panel

NOT NOT

Individual

3+ 0 8 Only

Book

VllI-48M

COMPLETE

LUMkJ11ULJX

COMPLETE

Table

00 00

0 00 00 00 00 00 00

00 00 00 omo 00 00

0 ----PATCHING 00 9 — -- Page

Results

0 m

0 12 Not

87 DID

Cite

STUDY or

STUDY NOT

Quote

0 0

0 0 0 0 0

0 0 0 0 0 0

o 0

Original

D1SCONTINUED*

Site

COMPLETE

0 0

0 0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0

0 0

C-005-93

Page

0 0 0 0

0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0

0 0

STUDY

Virgin

Site

0 0 0

0 0 0 0 0 0

0 0 0 0

0 0 0 0 0

0 0 0 .—

Number

Subject

29 31 30 32 33 34

35 36 37 38 40 39 41 42

43 45 44

47 48

49.

52 54 53 55

56 =

Makeup

0 0000000000 0000000000

1 000m0oooo 0 0000000000 0000000000m 00000m00000 0O00OOOOOO 000

0000000000 0000000000 0000000000

0000000000

0000000000 oom0000 0000000000 0000000000

0000000000 000m000000

0000000m000 0 0000000000

0000O000OO 0000000000

0000000000 0000000000 0

day

—----Induction

00000moo

0 Distributed —

0m 5

Exposures

0 for

DID

DID CIR

0.0 Comment

Individual Panel

NOT

(continued)

VUI-48M

Table

DID 9 Only —----- Book

.00

COMPLETE

Results --

12 Page

NOT Do Not 88

COMPLETE Cite

STUDY or

Original

STUDY 0 0

0 0 0 0

0 0

0 0 0 0

0 0 0

0 Quote

Site

0 0

0 0 C-005- 0

0 0 0 Page

0 0 0

0 0

0 0 0 0

0 0

STUDY

24 93

0 0

0 0 0 0 0

0 0 0 0

0 0 0 0 0

0 Virgin 0

0 0 0

Site

0 0

0 0 0 0

0 0 0

0 0 0 0

0 Distributed for Comment Only -- Do Not Cite or Quote C-005-93 Page 29

Table 13 IndividualResults

VllI-48N

Original Virgin

•Subject InductionExposures Site Site Number 1 2 3 4 5 6 7 8 9 10 24 48 24 48

1 0 00000000 0 DID NOT COMPLETESTUDY 2 0 00000000 0 0 0 0 0 3 0 00000000 0 0 0 0 0 4 0 00000000 0 0 0 0 0 5 0 00000000 0 0 0 0 0 6 0 0. 0 0 0 0 0 0 0 0 0 0 0 0 7 0 00000000 0 0 0 0 0 8 0 00000000 0 0 0 0 0 9 0 00000000 0 0 0 0 0 10 0 00000000 0 0 0 0 0 11 0 00000000 0 0 0 0 0 12 0 00000000 0 0 0 0 0 13 0 0000000 00 0 0 0 0 14 0 0 0 0 0 0 omo 00 0 0 0 0 15 0 0000000 00 0 0 0 0 16 0 0000000 00 0 0 0 0 17 0 0000000 0mb 0 0 0 0 18 0 0000000 00 0 0 0 0 19 — DID NOT COMPLETESTUDY. 20 0 0 0 0 0 0 0 0 0 0m 0 0 0 0 21 DID NOT COMPLETESTUDY 11 rTr Mrvr ((AT L”DTTTh’J 1.dALf a%. A 4.flAVAA £S14_.9t I_PS %_PS S 23 0000 00 0000 0 0 0 0 24 0000 00 0000 0 0 0 0 25 0 0 .0 0 00 0000 0 0 0 0 26 0000 00 0 3+ ----PATCHINGDISCONTINUED* 27 0000 00 0 0 0 0 0 0 0 0 28 0000 00 0000 0 0 0 0 m = Makeup day * =seeTextp.3

CIR Panel Book Page 89

Number

Subject

_._49

43 36 34 31

37 33

44 41 39

35 40 38

48 45

52 50

= 55

Makeup

------Induction

0 0 0 0

0 0 0 0 0

0 0

0 0 0

0 (I

1 0

day

00000000 0m 0 0

000000000 0 000000000 0

000000000

000000000 0

000000000

000000000 000000000

00 000000000 000000000

000000000 0 omo

000 0

000000000

0 000000000

omo 0

0 0

0 omo

00000000

0 0

00 0 Distributed

—

00 5

00 amo 0O

ooo 00 0m

Exposures

for

0fl

0 0 CIR

Comment

DID

-DID

0000

0 omo

Panel

Individual

NOT

omo 0

0 0

NOT (continued) Only

Book

VIll-48N

Table

DID

0 0

0 COMPLETE --

Page COMPLETE —--

Results

13 0

0 NOT Not 90

Cite COMPLETE

or STUDY

Quote

STUDY

0 Originai

0 0

0 0 0 0

0 0 0

0 0

C) 0 0

0 0 0

Site

0 0 0

0 0 0 0 0 0

0 0

0 0 0

0 0

C-005-93

Page

STUDY

0 0

0 0 0

0 0 0 0

0 0

Virgin Site

—

0 0 0 0 0

0 0 0

0 0

0 0 0 0 0

0 0 0

Number

Subject

= 24

=seeText,p.3

28 26

14 12

27 25 10

18 13 11

9 5 3

6 2

Makeup

000000

000000 000000

000000 000000 000000 000000

000000 000000 000000 000000 000000

0000 0 000000

0000 0 000000 000000 000000

0000 0000

0000

day

——Induction

0.0

0 4

Distributed

0 000000

5 000000

000000 0

000000 000000

Exposures

DID DID DID for CIR

Comment

0000 0000 0000

0000 000 000 000 0 0000 0000 omo 0000

0000 000 000 0 0000 0000

0000

NOT

NOT 1ndivdual

Panel

3+ 0

VIll-480

COMPLETE

COMPLETE COMPLETE Table Only

Book

9 ----PATCHING 0 omo --

Page

Results

0 Do

0 0

0 m

14 DID Not 91

Cite

STUDY

STUDY. NOT

0 0

0 0 0 0 0 0 0

0 0 0 0

Original 0

Quote

DISCONTINUED*

Site

COMPLETE

0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0

C-005-93

Page

0 0 0

0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0

STUDY

Virgin

Site

0 0

0 0 0 0 0

0 0 0 0

0 0

0 0 0 0

Number

Subject m

32 29

33 31 30

41 38 35

40 37 34

43 39 36

48 44 42

49 46

= 52

56 53

Makeup

0 0 0

0 0

1 0

0 0 0

0 0

day

000

000 2

00000 o

00000 000 0

00000 0

00000 00000

omo 00000 00000

00000 00000

ff000 00000

a 00000

00000

00000 00000 00000

000000000 00000

omo 0

omo Induction

—------

0 0 0

Distributed

00 omo

00 5

omo

0 O0

Exposures-—

0 0 for CIR

Comment

DID

-DID

0000

000 0000

000 000 0 000

000 000

000 000 000

000

000 000 000

Panel

Individual

NOT

NOT (continued) Only

Book

VIll-480

Table

DII)

0 COMPLETE

-- COMPLETE Page

Results

0 14

om 0

0 0

0 0 0

0 0

A 0

0 0

om NOT Not 92

Cite COMPLETE

STUDY---. 24

Quote

STUDY

o 0 0 0

o o

0 0 o Original

0 0 0 o

0 o

o o

o 0

Site

0 0

0 0 0 0 o 0 0

0 0

o 0

0 o

0 0 0

o 0

C-005-93

Page

STUDY

0 0 0 0

0 0 0

0 0 0 0

0 0

0 0 0

Virgin

Site

0 0

0 0 0 0

0 0

0 0 0

0 0

0 0 0 0 Distributed for Comment Only -- Do Not Cite or Quote opoaon C-005-93 Page 33

Table 15

SubjectData

Subject Number Initials Age Sex

1 WC 41 M 2 A.H 61 F 3 HB 47 F 4 IH 61 F 5 DD 63 F 6 IM 41 F 7 SF 65 F 8 LR 59 F 9 JC 64 F 10 PL 63 F 11 NV 32 F 12 PA 65 M 13 MA 64 F 14 AM 40 F 15 PS 46 F 16 LH 56 F 17 LG 30 F 18 DR 26 F 19 LO 24 M 20 CA 50 F 21 CP 22 F 22 .BW 33 M 23 iN 37 F TY JI 25 WH 62 M 26 DO 29 F 27 MP 47 F 28 LG 40 F

CIR Panel Book Page 93

Number

Subject

31 29

44 42 40 36 34

45 43 41 35

49 38

48 39 37 33

50 46

55 53

56 54 52

Initials

BH LH GB

JR MP

CE TK LD

BR MP LI

RR MM LH RD RC GB

DM MD KI MW IC

MS CP SC

RM SV Distributed for CIR Comment

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(continued) Table Only

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41 45 41

58 48 32 54 31

33 28 24 48 25 44

53 53 46 31 38

61 43 30 36

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Cognis Deutschland GmbH & Co. KG. C05-0247.01 Page 3

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

Participants: Two hundred twenty-seven (227) qualified subjects, male and female, ranging in age from 16 to 79 years, were selected for this evaluation. Two hundred thirteen (213) subjects completed this study. The remaining subjects discontinued their participation for various reasons, none of which were related to the application of the test material.

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

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

Test Material: C-SAT 050015

Study Schedule: Panel# Initiation Date Completion Date

20050143 March 23, 2005 May 5, 2005 20050147 March 28, 2005 May 5, 2005 20050155 March 28, 2005 May 6, 2005 20050160 March 30, 2005 May 5, 2005

aWith parental or guardian consent

CIR Panel Book Page 97 Distributed for Comment Only -- Do Not Cite or Quote

Cognis Deutschland GmbH & Co. KG. C05-0247.O1 Page 4

Methodology: Prior to the initiation of this study, the test material was prepared as follows:

The pH was adjusted to 6.5 (± 0.5) with HCL. A 2% dilution was then prepared. (i!)e.-.l vIc lp)

The upper back between the scapulae served as the treatment area. Approximately 0.2 ml of the test material, or an amount sufficient to cover the contact surface, was applied to the 3/4” x 3/4” absorbent pad portion of an adhesive dressing*. This was then applied to the appropriate treatment site to form an occlusive patch. The test material was refrigerated, when not in use.

Induction Phase:

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

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

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

ChaI1erne Phase:

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

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

CIR Panel Book Page 98 Distributed for Comment Only -- Do Not Cite or Quote

Cognis Deutschland GmbH & Co. KG. C05-0247.01 Page 5

Evaluation Key: 0 No visible skin reaction = Barely perceptible or spotty erythema 1 = Mild erythema covering most of the test site 2 = Moderate erythema, possible presence of mild edema 3 = Marked erytherna,possible edema 4 = Severe erythema, possible edema, vesiculation, bullae andlor ulceration

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

Observations remained within normal limits throughout the test interval.

Summary: Under the conditions of this study, test material, C-SAT 050015, did not indicate a potential for dermal irritation or allergic contact sensitization.

CIR Panel Book Page 99 24* 28 27 24 Number 26 23 25 20 22 21 Subject 17 19 18 13 16 15 14 11 10 12 9 7 6 4 3 5 2 8 1 = Supervised 24*hr 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 removal 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 1 of 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 Induction Distributed 0 0 0 0 0 0 0 0 0 0 — 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 and DII) for 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 4 Induction CIR Challenge Individual Comment Panel C-SAT NOT Panel DID Table 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 5 #20050143 COMPLETE Book Only Phase Patch NOT 050015 Results 1 Page -- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 Do COMPLETE Not 100 Cite STUDY 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 Page C05-0247.01 Cognis or Quote STUDY 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 Deutschland 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 1 9 GmbH Virgin 24*hr 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 & Site Challenge Co. 72 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 KG. hr — Distributed for Comment Only -- Do Not Cite or Quote

Cognis Deutsehiand GmbH & Co. KG. C05-0247.01 Page 7

Table 1 (continued) Panel #20050143

Individual Results

C-SAT 050015

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

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

24* = Supervisedremovalof 1st Inductionand ChallengePatch m = Additionalmakeupday grantedat the discretionof the clinicsupervisor DNC = Did not completestudy

CIR Panel Book Page 101 Distributed for Comment Only -- Do Not Cite or Quote

Cognis Deutschland GmbH & Co. KG.

C05-0247.0 1 Page 8

Table 1 (continued) Panel #20050147

Individual Results

C-SAT 050015

Virgin Challenge Subject Induction Phase Site Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72br

1 0 0 0 0 0 0 0 0 0 0 0 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 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 0 0 0 0 18 0 0 0 0 0 0 0 0 0 0 0 0 19 DID NOT COMPLETE STUDY 20 0 0 0 0 0 0 0 0 0 0 0 0 21 0 0 0 0 0 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 0 0 26 0 0 0 0 0 0 0 0 0 0 0 0 27 0 0 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof c1 Inductionand ChallengePatch DNC = Did not completestudy

CIR Panel Book Page 102 Distributed for Comment Only -- Do Not Cite or Quote

Cois Deutschland GmbH & Co. KG. C05-0247.01 Page 9

Table 1 (continued) Panel #20050 147

Individual Results

C-SAT 050015

Virgin Challenge Subject Induction Phase Site Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72hr

30 0 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 38 0 0 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 0 41 0 0 0 0 0 0 0 0 0 0 0 0 42 0 0 0 0 0 0 0 0 0 0 0 0 43 0 0 0 0 0 0 0 0 0 0 0 0 44 0 0 0 0 0 0 0 0 0 0 0 0 45 0 0 0 0 0 0 0 0 0 0 0 0 46 0 0 0 0 0 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 0 0 0 0 0 53 0 0 0 0 0 0 0 0 0 0 0 0 54 0 0 0 0 0 0 0 0 0 0 0 0 55 0 0 0 0 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 0 0 0 0 57 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof Inductionand ChallengePatch

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Table 1 (continued) Panel #20050155

Individual Results

C-SAT 050015

Virgin Challenge Subject Induction Phase — Site Number 24*1w 1 2 3 4 5 6 7 8 9 24*hr 72hr

1 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 0 0 17 - 0 0 0 0 0 0 DID NOT COMPLETE STUDY--- 18 0 0 0 0 0 0 0 0 0 0 0 0 19 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 21 0 0 0 0 0 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 0 0 23 0 0 0 0 0 0 DID NOT COMPLETE STUDY 24 0 0 0 0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 0 0 26 0 0 0 0 0 0 0 0 0 0 0 0 27 0 0 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof 1st InductionandChallengePatch - = Subjectnot presentfor supervisedremoval

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Table I (continued) Panel #20050155

Individual Results

C-SAT 050015

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

30 0 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 38 0 0 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 0 41 0 0 0 0 0 0 0 0 0 0 0 0 42 0 0 0 0 0 0 0 0 0 0 0 0 43 0 0 0 0 0 0 0 0 0 0 0 0 44 0 0 0 0 0 0 0 0 0 0 0 0 45 0 0 0 0 0 0 0 0 0 0 0 0 46 0 0 0 0 0 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 0 0 0 0 0 53 0 0 0 0 0 0 0 0 0 0 0 0 54 0 0 0 0 0 0 0 0 0 0 0 0 55 0 0 0 0 0 0 0 0 0 0 0 0 56 — DID NOT COMPLETE STUDY 57 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof 1 Inductionand ChallengePatch - = Subjectnot presentfor supervisedremoval

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Table 1 (continued) Panel #20050160

Individual Results

C-SAT 050015

Virgin Challenge Subject Induction Phase Site Number 24*hr 1 2 3 4 5 6 7 8 9 24*hr 72 hr

1 0 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 DID NOT COMPLETE STUDY 6 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 DID NOT COMPLETE STUDY 15 0 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 0 0 0 0 18 0 0 0 0 0 0 0 0 0 0 0 0 19 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 21 0 0 0 0 0 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 0 0 0 0 0 23 0 DID NOT COMPLETE STUDY 24 0 0 0 0 0 0 0 0 0 0 0 0 25 0 0 0 0 0 0 0 0 0 0 0 0 26 0 0 0 0 0 0 0 0 0 0 0 0 27 0 0 0 0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervisedremovalof l InductionandChallengePatch

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Table 1 (continued) Panel #20050160

Individual Results

C-SAT 050015

Virgin Challenge Subject Induction Phase Site 24*1w Number 1 2 3 4 5 6 7_8 9 24*1w 721w

30 0 0 0 0 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 38 0 0 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 0 0 0 0 0 41 0 0 0 0 0 0 0 0 0 0 0 0 42 0 0 0 0 0 0 0 0 0 0 0 0 43 0 0 0 0 0 0 0 0 0 0 0 0 44 DID NOT COMPLETE STUDY 45 0 0 0 0 0 0 0 0 0 0 0 0 46 0 0 0 0 0 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 51 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 0 0 0 0 0 53 0 0 0 0 0 0 0 0 0 0 0 0 54 0 0 0 0 0 0 0 0 0 0 0 0 55 0 0 0 0 0 0 0 0 0 0 0 0 56 0 DID NOT COMPLETE STUDY 57 0 0 0 0 0 0 0 0 0 0 0 0

24* = Supervised removal of 1 Induction and ChallengePatch

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Table 2 Panel #20050 143

Subject Data

Subject Number Initials Age Sex

1 GV 32 F 2 SH 46 F 3 JR 42 F 4 KS 61 F 5 PS 61 M 6 VR 33 F 7 MK 66 F 8 NE 57 M 9 JS 36 F 10 WE 73 M 11 JC 55 F 12 DZ 41 F 13 MT 21 F 14 CS 43 F 15 SB 50 F 16 MP 17 F 17 CW 74 F 18 SN 43 M 19 IP 38 F 20 ES 58 F 21 CR 46 F 22 FR 55 M 23 DG 30 F 24 JH 50 M 25 NH 50 F 26 RG 25 M 27 MC 49 F 28 DQ 52 M

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Table 2 (continued) Panel #20050 143

Subject Data

Subject Number Initials Age Sex

29 KI 44 M 30 JT 24 F 31 KS 55 F 32 PM 43 F 33 RH 49 M 34 PW 38 F 35 AW 62 F 36 CH 65 F 37 ES 49 F 38 BF 23 F 39 MH 66 F 40 DJ 74 M 41 MS 60 F 42 CV 49 F 43 EC 45 M 44 RD 48 F 45 KP 46 F 46 RC 30 F 47 DC 49 F 48 EH 58 F 49 FS 63 M 50 NR 46 F 51 AC 50 F 52 MA 27 F 53 EM 69 F 54 BR 73 F 55 HT 72 M 56 KL 45 F

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C05-0247.0 1 Page 16

Table 2 (continued) Panel #20050147

Subject Data

Subject Number Initials Age Sex

1 NM 42 M 2 BM 40 F 3 LR 71 F 4 PD 65 F 5 PL 75 F 6 JF 70 F 7 HF 74 M 8 HP 75 F 9 LS 49 M 10 LS 48 F 11 LL 76 F 12 AL 78 M 13 BS 49 F 14 JO 64 M 15 DS 74 M 16 PR 28 F 17 JD 58 F 18 GM 55 M 19 SK 18 M 20 JS 67 F 21 EC 42 F 22 AS 79 M 23 ES 76 F 24 EN 47 F 25 DW 51 F 26 GP 71 F 27 RN 56 F 28 SH 43 F 29 RM 61 F

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Table 2 (continued) Panel #20050147

Subject Data

Subject Number Initials Age Sex

30 KP 45 F 31 MG 58 M 32 HF 42 M 33 KC 35 F 34 RS 65 F 35 MR 44 F 36 OB 56 F 37 KU 23 F 38 MC 40 F 39 GM 42 F 40 DR 38 F 41 AD 70 F 42 AD 75 M 43 MA 57 F 44 CF 40 F 45 KF 21 M 46 LL 45 F 47 EM 63 F 48 MV 19 F 49 WL 66 F 50 RF 68 F 51 JO 69 M 52 MP 59 F 53 VL 46 F 54 RG 32 F 55 MM 42 F 56 SC 41 F 57 RL 57 F

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Table 2 (continued) Panel #20050155

Subject Data

Subject Number Initials Age Sex

1 LN 35 M 2 YG 29 F 3 BL 35 F 4 RG 54 M 5 RR 62 F 6 LA 51 F 7 PA 53 M 8 DM 39 F 9 CC 68 F 10 PR 47 M 11 AV 58 F 12 JA 54 F 13 PS 46 F 14 DH 64 M 15 EB 60 F 16 NT 43 F 17 KR 19 F 18 JT 42 F 19 CO 61 F 20 MG 37 F 21 TR 17 F 22 DC 47 F 23 CS 23 F 24 PC 63 F 25 EL 66 F 26 FS 16 M 27 AJ 31 F 28 RC 29 F 29 RV 21 M

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Table 2 (continued) Panel #200501 55

bject Data

Subject Number Initials Age Sex

30 SG 70 M 31 PV 60 M 32 AA 47 M 33 AA 53 F 34 MM 71 F 35 SH 38 F 36 DB 56 F 37 EB 58 M 38 JP 62 M 39 CL 46 F 40 JA 42 M 41 GC 40 F 42 AC 39 M 43 HH 64 F 44 RL 47 M 45 LS 45 F 46 DR 59 F 47 ES 53 F 48 NG 50 F 49 EB 45 F 50 ZP 41 F 51 CP 47 F 52 DP 46 M 53 CB 26 F 54 RW 38 F 55 MV 47 F 56 AV 47 M 57 JG 31 M

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Table 2 (continued) Panel #20050160

Subject Data

Subject Number Initials Age Sex

1 MK 41 M 2 LO 61 F 3 SV 55 F 4 TB 55 M 5 MS 56 F 6 MO 21 F 7 MF 56 M 8 JB 55 F 9 SS 40 F 10 KL 41 F 11 CO 39 F 12 RM 66 M 13 MP 59 F 14 HF 23 F 15 JA 45 F 16 LC 52 F 17 LA 50 M 18 LC 59 F 19 CM 58 F 20 VS 31 F 21 JS 68 M 22 DB 38 F 23 MS 21 F 24 MC 69 F 25 ER 41 F 26 DR 38 F 27 AD 40 M 28 DM 62 F 29 LH 71 F

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Table 2 (continued) Panel #20050160

Subject Data

Subject Number Initials Age Sex

30 IM 65 F 31 FR 66 M 32 MD 47 F 33 DW 65 F 34 RW 68 M 35 AS 72 F 36 JS 72 M 37 ES 41 M 38 AB 48 F 39 IA 55 M 40 CD 73 F 41 MD 76 M 42 DP 33 F 43 NM 60 F 44 LC 38 F 45 ES 71 F 46 FN 71 F 47 RM 70 F 48 YH 26 F 49 SS 40 F 50 MK 51 F 51 FR 76 F 52 JM 66 F 53 NM 57 F 54 CS 59 F 55 TD 35 F 56 CB 23 F 57 SB 50 M

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PersonalCare ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

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

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

DATE: January 7, 2011

SUBJECT: Concentration of Use by FDA Product Category: Glucoside Ingredients

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

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Concentration of Use by FDA Product Category Decyl Glucoside, Arachidyl Glucoside, Butyl Glucoside, C1O-16 Alkyl Glucoside, C12-18 Alkyl Glucoside, C12-20 Alkyl Glucoside, C20-22 Alkyl Glucoside, CaprylyL/Capryl Glucoside, Caprylyl Glucoside, Cetearyl Glucoside, Coco-Glucoside, Ethyl Glucoside, Isostearyl Glucoside, Lauryl Glucoside, Lauroyl Ethyl Glucoside, Myristoyl Ethyl Glucoside, Myristyl Glucoside, Octyldodecyl Glucoside, Oleoyl Ethyl Glucoside, Palm Kernel/Coco Glucoside, Phytosteryl Glucoside, Rapeseed Glucoside, Safflower Glucoside, Tallowoyl Ethyl Glucoside, Tocopheryl Glucoside and Undecyl Glucoside*

Ingredient Product Category Concentration of Use

Decyl Glucoside Bubble baths 0.5-0.8%

Decyl Glucoside Other bath preparations 1%

Decyl Glucoside Eyeliner 0.2%

Decyl Glucoside Eye lotion 0.02-1%

Decyl Glucoside Eye makeup remover 0.3-6%

Decyl Glucoside Mascara 0.05%

Decyl Glucoside Hair conditioners 0.4-6%

Decyl Glucoside Hair sprays (aerosol fixatives) 0.6%

Decyl Glucoside Shampoos (noncoloring) 1-7%

Decyl Glucoside Tonics, dressings and other hair grooming aids 0.2-2%

Decyl Glucoside Other hair preparations (noncoloring) 1%

Decyl Glucoside Hair dyes and colors (all types requiring caution 2-8% statement and patch tests)

Decyl Glucoside Foundations 2%

Decyl Glucoside Other makeup preparations 0.2%

Decyl Glucoside Bath soaps and detergents 0.3—7%

Decyl Glucoside Other personal cleanliness products 2-3%

Decyl Glucoside Shaving cream (aerosol, brushless and lather) 2%

Decyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.8-33% liquids and pads)

Decyl Glucoside Face and neck creams, lotions and powders 0.002-0.5%

Decyl Glucoside Body and hand creams, lotions and powders 0.5-11%

Decyl Glucoside Body and hand sprays 0.5%

Decyl Glucoside Moisturizing creams, lotions and powders 0.07%

Decyl Glucoside Night creams, lotions and powders 0.09%

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Decyl Glucoside Paste masks (mud packs) 2%

Decyl Glucoside Skin fresheners 0.002%

Decyl Glucoside Other skin care preparations 0.6-2%

Decyl Glucoside Suntan gels, creams and liquds 0.2%

Decyl Glucoside Indoor tanning preparations 0.8%

Arachidyl Glucoside Eye shadow 0.08%

Arachidyl Glucoside Tonics, dressings and other hair grooming aids 0.5%

Arachidyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.5% liquids and pads)

Arachidyl Glucoside Face and neck creams, lotions and powders 0.2-0.6%

Arachidyl Glucoside Body and hand creams, lotions and powders 0.2-0.5%

Arachidyl Glucoside Moisturizing creams, lotions and powders 0.5%

Arachidyl Glucoside Night creams, lotions and powders 0.5%

Arachidyl Glucoside Indoor tanning preparations 0.5%

C12-20 Alkyl Glucoside Eye shadow 0.8%

C12-20 Alkyl Glucoside Other hair preparations (non-coloring) 1%

C12-20 Alkyl Glucoside Deodorants (underarm) 0.6%

C12-20 Alkyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.1% liquids and pads)

C12-20 Alkyl Glucoside Face and neck creams, lotions and powders 0.6%

Cl 2-20 Alkyl Glucoside Other skin care preparations 1%

CaprylyllCapryl Glucoside Other baby products’ 0.06%

CaprylyllCapryl Glucoside Eye lotion 0.2%

Caprylyl/Capryl Glucoside Eye makeup remover 0.3%

Caprylyl/Capryl Glucoside Hair conditioners 0.3%

Caprylyl/Capryl Glucoside Shampoos (noncoloring) 3%

Caprylyl/Capryl Glucoside Tonics, dressings and other hair grooming aids 0.5%

Caprylyl/Capryl Glucoside Hair dyes and colors (all types requiring caution 3% statement and patch test)

Caprylyl/Capryl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.9% liquids and pads)

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Caprylyl/Capryl Glucoside Face and neck creams, lotions and powders 0.8%

Caprylyl/Capryl Glucoside Body and hand creams, lotions and powders 0.3%

Caprylyl/Capryl Glucoside Night creams, lotions and powders 0.8%

CaprylyllCapryl Glucoside Suntan gels, creams and liquids 0.3%

Caprylyl Glucoside Other hair preparations (noncoloring) 4%

Cetearyl Glucoside Eye shadow 1-2%

Cetearyl Glucoside Eye lotion 0.6-2%

Cetearyl Glucoside Other fragrance preparations 0.6%

Cetearyl Glucoside Hair conditioners 0.3-0.6%

Cetearyl Glucoside Hair dyes and colors (all types requiring caution 0.2% statement and patch testing)

Cetearyl Glucoside Bath soaps and detergents 0.03%

Cetearyl Glucoside Aftershave lotions 0.2-0.6%

Cetearyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.03-0.9% liquids and pads)

Cetearyl Glucoside Depilatories 3%

Cetearyl Glucoside Face and neck creams, lotions and powders 0.6-2%

Cetearyl Glucoside Body and hand creams, lotions and powders 0.2-7%

Cetearyl Glucoside Moisturizing creams, lotions and powders 0.6%

Cetearyl Glucoside Night creams, lotions and powders 0.2-0.8%

Cetearyl Glucoside Paste masks (mud packs) 0.6-0.8%

Cetearyl Glucoside Other skin care preparations 0.3-0.7%

Cetearyl Glucoside Suntan gels, creams and liquids 0.2%

Cetearyl Glucoside Indoor tanning preparations 0.2-0.6%

Cetearyl Glucoside Other suntan preparations 0.3%

Coco-Glucoside Eyeliner 2%

Coco-Glucoside Eye makeup remover 3%

Coco-Glucoside Hair conditioners 0.2%

Coco-Glucoside Shampoos (noncoloring) 0.4-8%

Coco-Glucoside Hair dyes and colors (all types requiring caution 5% statement and patch testing)

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Coco-Glucoside Other hair coloring preparations 0.3%

Coco-Glucoside Dentifrices (aerosol, liquid, pastes and powders) 0.5%

Coco-Glucoside Bath soaps and detergents 0.4-3%

Coco-Glucoside Other personal cleanliness products 0.7-8%

Coco-Glucoside Shaving cream (aerosol, brushless and lather) 0.6%

Coco-Glucoside Skin cleansing (cold creams, cleansing lotions, 0.4-8% liquids and pads)

Coco-Glucoside Face and neck creams, lotions and powders 0.02-2%

Coco-Glucoside Body and hand creams, lotions and powders 0.006-2%

Coco-Glucoside Other skin care preparations 2%

Ethyl Glucoside Eye makeup remover 0.02%

Ethyl Glucoside Rouges 0.02%

Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.05% liquids and pads)

Ethyl Glucoside Body and hand creams, lotions and powders 0.04%

Ethyl Glucoside Moisturizing creams, lotions and powders 0.3%

Ethyl Glucoside Paste masks (mud packs) 0.05%

Lauryl Glucoside Bath oils, tablets and salts 0.5%

Lauryl Glucoside Bubble baths 0.3-4%

Lauryl Glucoside Eye shadow 5%

Lauryl Glucoside Hair conditioners 0.4%

Lauryl Glucoside Shampoos (noncoloring) 1-5%

Lauryl Glucoside Hair dyes and colors (all types requiring caution 0.3-2% statement and patch test)

Lauryl Glucoside Hair color sprays (aerosol) 8%

Lauryl Glucoside Hair shampoos (coloring) 2%

Lauryl Glucoside Other makeup preparations 0.03%

Lauryl Glucoside Bath soaps and detergents 0.3-8%

Lauryl Glucoside Other personal cleanliness products 0.6-2%

Lauryl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.6-10% liquids and pads)

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Lauryl Glucoside Face and neck creams, lotions and powders 2%

Lauryl Glucoside Body and hand creams, lotions and powders 0.3%

Lauryl Glucoside Moisturizing creams, lotions and powders 2%

Lauryl Glucoside Other skin care preparations 0.2-2%

Lauroyl Ethyl Glucoside Bath soaps and detergents 1%

Lauroyl Ethyl Glucoside Other personal cleanliness products 2%

Lauroyl Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions 2% liquids and pads)

Myristyl Glucoside Eye lotion 0.4%

Myristyl Glucoside Face and neck creams, lotions and powders 0.5-0.6%

Myristyl Glucoside Night creams, lotions and powders 0.6%

Safflower Glucoside Hair conditioners 0.1%

Safflower Glucoside Hair sprays (aerosol fixatives) 0.005%

Safflower Glucoside Other hair preparations (noncoloring) 0.1% *Ingredients included in the title of the table but not found in the body of the table were incluoed in the concentration of use survey, but no uses were reported. ‘0.06% in a leave-on baby product Information collected in 2010 Table prepared January 6, 2011

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PersonalCare ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

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

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

DATE: January 28, 2011

SUBJECT: Updated Concentration of Use by FDA Product Category: Glucoside Ingredients

1101 17th Street, N.W., Suite 300 Washington, D.C. 20036-4702 202.331.1770 202.331.1969 (fax) www.personalcarecouncil.org

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Concentration of Use by FDA Product Category Decyl Glucoside, Arachidyl Glucoside, Butyl Glucoside, C1O-16 Alkyl Glucoside, C12-18 Alkyl Glucoside, C12-20 Alkyl Glucoside, C20-22 Alkyl Glucoside, CaprylyllCapryl Glucoside, Caprylyl Glucoside, Cetearyl Glucoside, Coco-Glucoside, Ethyl Glucoside, Isostearyl Glucoside, Lauryl Glucoside, Lauroyl Ethyl Glucoside, Myristoyl Ethyl Glucosidc, Myristyl Glucoside, Octyldodecyl Glucoside, Oleoyl Ethyl Glucoside, Palm Kernel/Coco Glucoside, Phytosteryl Glucoside, Rapeseed Glucoside, Safflower Glucoside, Tallowoyl Ethyl Glucoside, Tocopheryl Glucoside and Undecyl Glucoside’

Ingredient Product Category Concentration of Use

Decyl Glucoside Bubble baths 0.5-0.8%

Decyl Glucoside Other bath preparations 1%

Decyl Glucoside Eyeliner 0.2%

Decyl Glucoside Eye lotion 0.02-1%

Decyl Glucoside Eye makeup remover 0.3-6%

Decyl Glucoside Mascara 0.05%

Decyl Glucoside Hair conditioners 0.4-6%

Decyl Glucoside Hair sprays (aerosol fixatives) 0.6%

Decyl Glucoside Shampoos (noncoloring) 1-7%

Decyl Glucoside Tonics, dressings and other hair grooming aids 0.2-2%

Decyl Glucoside Other hair preparations (noncoloring) 1%

Decyl Glucoside Hair dyes and colors (all types requiring caution 2-8% statement and patch tests)

Decyl Glucoside Foundations 2%

Decyl Glucoside Other makeup preparations 0.2%

Decyl Glucoside Bath soaps and detergents 0.3—7%

Decyl Glucoside Other personal cleanliness products 2-3%

Decyl Glucoside Shaving cream (aerosol, brushless and lather) 2%

Decyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.8-33% liquids and pads)

Decyl Glucoside Face and neck creams, lotions and powders 0.002-0.5%

Decyl Glucoside Body and hand creams, lotions and powders 0.5-11%

Decyl Glucoside Body and hand sprays 0.5%

Decyl Glucoside Moisturizing creams, lotions and powders 0.07%

Decyl Glucoside Night creams, lotions and powders 0.09%

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Decyl Glucoside Paste masks (mud packs) 2%

Decyl Glucoside Skin fresheners 0.002%

Decyl Glucoside Other skin care preparations 0.6-2%

Decyl Glucoside Suntan gels, creams and liquds 0.2%

Decyl Glucoside Indoor tanning preparations 0.8-1%

Arachidyl Glucoside Eye shadow 0.08%

Arachidyl Glucoside Tonics, dressings and other hair grooming aids 0.5%

Arachidyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.5% liquids and pads)

Arachidyl Glucoside Face and neck creams, lotions and powders 0.2-0.6%

Arachidyl Glucoside Body and hand creams, lotions and powders 0.2-0.5%

Arachidyl Glucoside Moisturizing creams, lotions and powders 0.5%

Arachidyl Glucoside Night creams, lotions and powders 0.5%

Arachidyl Glucoside Indoor tanning preparations 0.2-0.5%

C12-20 Alkyl Glucoside Eye shadow 0.8%

C12-20 Alkyl Glucoside Other hair preparations (non-coloring) 1%

C12-20 Alkyl Glucoside Deodorants (underarm) 0.6%

C12-20 Alkyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.1% liquids and pads)

C12-20 Alkyl Glucoside Face and neck creams, lotions and powders 0.6%

C12-20 Alkyl Glucoside Other skin care preparations 1%

C12-20 Alkyl Glucoside Suntan gels, creams and liquids 0.2%

C12-20 Alkyl Glucoside Indoor tanning preparations 0.3%

C12-20 Alkyl Glucoside Other suntan preparations 0.5%

Caprylyl/Capryl Glucoside Other baby products’ 0.06%

Caprylyl/Capryl Glucoside Eye lotion 0.2%

Caprylyl/Capryl Glucoside Eye makeup remover 0.3%

Caprylyl/Capryl Glucoside Hair conditioners 0.3%

Caprylyl/Capryl Glucoside Shampoos (noncoloring) 3%

Caprylyl/Capryl Glucoside Tonics, dressings and other hair grooming aids 0.5%

CaprylyllCapryl Glucoside Hair dyes and colors (all types requiring caution 3%

Page 2 of 5

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statement and patch test)

Caprylyl/Capryl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.9% liquids and pads)

Caprylyl/Capryl Glucoside Face and neck creams, lotions and powders 0.8%

CaprylyllCapryl Glucoside Body and hand creams, lotions and powders 0.3%

Caprylyl/Capryl Glucoside Night creams, lotions and powders 0.8%

CaprylyllCapryl Glucoside Suntan gels, creams and liquids 0.3%

Caprylyl Glucoside Other hair preparations (noncoloring) 4%

Cetearyl Glucoside Eye shadow 1-2%

Cetearyl Glucoside Eye lotion 0.6-2%

Cetearyl Glucoside Other fragrance preparations 0.6%

Cetearyl Glucoside Hair conditioners 0.3-0.6%

Cetearyl Glucoside Hair dyes and colors (all types requiring caution 0.2%

statement and patch testing) -

Cetearyl Glucoside Bath soaps and detergents 0.03%

Cetearyl Glucoside Aftershave lotions 0.2-0.6%

Cetearyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.03-0.9% liquids and pads)

Cetearyl Glucoside Depilatories 3%

Cetearyl Glucoside Face and neck creams, lotions and powders 0.6-2%

Cetearyl Glucoside Body and hand creams, lotions and powders 0.2-7%

Cetearyl Glucoside Moisturizing creams, lotions and powders 0.6%

Cetearyl Glucoside Night creams, lotions and powders 0.2-0.8%

Cetearyl Glucoside Paste masks (mud packs) 0.6-0.8%

Cetearyl Glucoside Other skin care preparations 0.3-0.7%

Cetearyl Glucoside Suntan gels, creams and liquids 0.2%

Cetearyl Glucoside Indoor tanning preparations 0.2-0.6%

Cetearyl Glucoside Other suntan preparations 0.3%

Coco-Glucoside Eyeliner 2%

Coco-Glucoside Eye makeup remover 3%

Coco-Glucoside Hair conditioners 0.2%

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Coco-Glucoside Shampoos (noncoloring) 0.4-8%

Coco-Glucoside Hair dyes and colors (all types requiring caution 5% statement and patch testing)

Coco-Glucoside Other hair coloring preparations 0.3%

Coco-Glucoside Dentifrices (aerosol, liquid, pastes and powders) 0.5%

Coco-Glucoside Bath soaps and detergents 0.4-3%

Coco-Glucoside Other personal cleanliness products 0.7-8%

Coco-Glucoside Shaving cream (aerosol, brushless and lather) 0.6%

Coco-Glucoside Skin cleansing (cold creams, cleansing lotions, 0.4-8% liquids and pads)

Coco-Glucoside Face and neck creams, lotions and powders 0.02-2%

Coco-Glucoside Body and hand creams, lotions and powders 0.006-2%

Coco-Glucoside Body and hand sprays 0.4%

Coco-Glucoside Other skin care preparations 2%

Coco-Glucoside Suntan, gels, creams and liquids 0.7%

Coco-Glucoside Indoor tanning preparations 0.4%

Coco-Glucoside Other suntan preparations 1%

Ethyl Glucoside Eye makeup remover 0.02%

Ethyl Glucoside Rouges 0.02%

Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.05% liquids and pads)

Ethyl Glucoside Body and hand creams, lotions and powders 0.04%

Ethyl Glucoside Moisturizing creams, lotions and powders 0.3%

Ethyl Glucoside Paste masks (mud packs) 0.05%

Lauryl Glucoside Bath oils, tablets and salts 0.5%

Lauryl Glucoside Bubble baths 0.3-4%

Lauryl Glucoside Eye shadow 5%

Lauryl Glucoside Hair conditioners 0.4%

Lauryl Glucoside Shampoos (noncoloring) 1-5%

Lauryl Glucoside Hair dyes and colors (all types requiring caution 0.3-2% statement and patch test)

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Lauryl Glucoside Hair color sprays (aerosol) 8%

Lauryl Glucoside Hair shampoos (coloring) 2%

Lauryl Glucoside Other makeup preparations 0.03%

Lauryl Glucoside Bath soaps and detergents 0.3-8%

Lauryl Glucoside Other personal cleanliness products 0.6-2%

Lauryl Glucoside Skin cleansing (cold creams, cleansing lotions, 0.6-10% liquids and pads)

Lauryl Glucoside Face and neck creams, lotions and powders 2%

Lauryl Glucoside Body and hand creams, lotions and powders 0.3%

Lauryl Glucoside Moisturizing creams, lotions and powders 2%

Lauryl Glucoside Other skin care preparations 0.2-2%

Lauroyl Ethyl Glucoside Bath soaps and detergents 1%

Lauroyl Ethyl Glucoside Other personal cleanliness products 2%

Lauroyl Ethyl Glucoside Skin cleansing (cold creams, cleansing lotions 2% liquids and pads)

Myristyl Glucoside Eye lotion 0.4%

Myristyl Glucoside Face and neck creams, lotions and powders 0.5-0.6%

Myristyl Glucoside Night creams, lotions and powders 0.6%

Safflower Glucoside Hair conditioners 0.1%

Safflower Glucoside Hair sprays (aerosol fixatives) 0.005%

Safflower Glucoside Other hair preparations (noncoloring) 0.1% *Ingredients included in the title of the table but not found in the body of the table were incluued in the concentration of use survey, but no uses were reported. 10.06% in a leave-on baby product Information collected in 2010 Table prepared January 6, 2011 Table updated January 28, 2011 (Decyl Glucoside: added high concentration to Indoor tanning preparations; Arachidyl Glucoside: added low concentration to indoor tanning preparations; C 12-20 Alkyl Glucoside: added 3 tanning product categories; Coco-Glucoside: added Body and hand sprays and 3 tanning product categories)

Page 5 of 5

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Persona Care ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

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

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

DATE: March 15, 2011

SUBJECT: Concentration of Use by FDA Product Category: Hexyldexyl d-Glucoside and Octadecyl d-Glucoside

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

CIR Panel Book Page 128 Distributed for Comment Only -- Do Not Cite or Quote

Concentration of Use by FDA Product Category Hexadecyl d-Glucoside and Octadecyl dG1ucoside*

Ingredient Product Category Concentration of Use

Hexadecyl d-Glucoside Moisturizing creams, lotions and powders 3% I *Ingredients included in the title of the table but not found in the table were included in the concentration of use survey, but no uses were reported. Information collected in 2011 Table prepared March 14, 2011

CIR Panel Book Page 129 Distributed for Comment Only -- Do Not Cite or Quote

PersonalCare ProductsCouncil Committedto Safety, Quality& Innovation Memorandum

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

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

DATE: April 8, 2011

SUBJECT: In Vitro Dermal Penetration Study: Caprylyl/Capryl Glucoside

Across Barriers GmbH. 2009. In vitro permeation and penetration of Capryl glucoside (INCI: Caprylyl/Capryl Glucoside) from the Product Plantacare 810 UP in human skin under GLP conditions. GLP Study Report STP 044/00. Report number: C-10335-244-1 107.

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

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In vitro permeation and penetration of Capryl glucoside from c yiiI/L... (/icscIe) the Product Plantacare 810 UP in human skin1 under GLP conditions / GLP Study Report STP 044100

Sponsor: Cognis GmbH Henkelstral3e 40589 Düsseldorf GERMANY

Study Monitor: Annette Mehling Phone: ±49 211-7940-9209 Fax: ±49 211-2006-19209 E-mail: [email protected]

Test Facility: Across Barriers GmbH Science Park 1 661 23 Saarbruecken GERMANY

Report number: c-i0335-244-1 107

Study Director: Dr. Monika Kaca Phone: ±49681/95918821 Fax: ±49681/95918802 E-mail: [email protected]

Responsible Scientist: Dr. Iris Zengerly

Author: Dr. Iris Zengerly

Date of report: 30.04.2009

CIR Panel Book Page 131 9O429FinalreportClO335244llO7STPO44.doc Function: Further The Mnaging Tabular The Dr.’ Saarbruecken, Authentication Final

Draft Good cordance I Eleónore final undersigned report: final Laboratory staff Director timeline report to report: Haitner-Ukomadu GLP involved Q includes Scientist Scientist Scientist Laboratory Laboratory Laboratory Practice’ study as of defined Oj study in director the the 30.04.2009 08.01.2009 £Y( Distributed with study: report assistant assistant assistant in correct the the

herewith ( Tawuik Saarbruecken, Head German STP methods for and CIR Comment of 044/00 Jalal confirms complete Quality Panel “Chemikaliengesetz” described. Book Assurance Only Name: that results Page -- Do the Not 132 OOsaarbruecken, of present Cite the Jessica Alesandro Phillip Nazende Natalie Nicole or study. and Quote study Study Dr. the Koch Kiet3ling Lenz Gimmler Gunday Monika GLP was Marangon “OECD Director Study performed Kaca

Principles )RIGINAL SW 044-00 in of ac (jg Distributed for Comment Only -- Do Not Cite or Quote

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Contents

1. Summary 6 1.1. Objectives 6 1 .2. Summarized results 8 1.2.1. HPLCmethod 8

1 .2.2. In vitro permeation study 1 1 1 .2.3. In vitro penetration study 1 2 1 .3. Conclusion 1 3

2. Materials and methods 14 2.1. Materials 14 2.2. Equipment and software 16 2.3. Buffers and media 17 2.4. Methods 18 2.4.1. HPLCmethod development and validation 18 2.4.2. Sample preparation for HPLCmethod 22 2.4.3. Liquid Scintillation counting 25 2.4.4. Preparation of test solution 10% CG (application solution) 25 2.4.5. Determination of CG content in the test solution 25 2.4.6. In vitro experiments 26 2.4.7. Permeation studies with the test product 28 2.4.8. Penetration experiments with the test product 31 2.4.9. Establishment of the extraction method 31 2.5. MEA:Qualification of skin membranes 33 2.6. Calculations 34

3. Results 36 3.1. HPLCmethod 36 3.1 .1. System suitability test 36 3.1 .2. Selectivity 38 3.1 .3. Linearity in KRB-/- pH 7.4 41 3.1 .4. Accuracy and Precision 42 3.1 .5. Stability of the test product in donor and acceptor media 43 3.2. Determination of CG content in the test solution 47 3.3. Permeation study and penetration studies 47 3.3.1. Establishment of extraction method for penetration study 48 3.3.2. Results from permeation and penetration studies 50 3.4. Quality control of the utilized skin samples (MEA) 54

4. Discussion 56 4.1. HPLCmethod development and validation 56 4.2. Determination of CG content in the test solution 56 4.3. Establishment of the extraction method for penetration study 56 4.4 Permeation study 56 4.5. Penetration study 57 4.6. Quality control of the utilized skin (MEA) 58

5. References 59 6. Related guidelines 59

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7. Data storage and archiving 60

8. QAU Statement 62

9. Appendix 63 9.1. HPLCmethod 63 9.2. Establishment of the extraction method 67 9.2.1. Establishment of the extraction method with MeOH!water (50:50, v/v %) 67 9.2.2. Establishment of the extraction method with water 68 9.3. Franz cell volumes and areas 69 9.4 Thickness of the human skins 69 9.4. Quality control of the human skin (MEA) 70 9.4.1. Transport of Caffeine (1%) through skin 70 9.5. Transport studies with the test formulation (permeation results) 71 9.5.1. Transport of CG from test solution through 3 different skin donors into the receptor medium 71

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Abbreviations

A area of exposed skin ACB-ID Across Barriers identification number ACC accuracy

TC Test compound

BMI Body mass index c concentration

D dermis CG Capryl glucoside DS deeper skin

E Epiderms eq. equation

FZ Franz diffusion cell HBSS Hank s buffered salt solution HBSS- HBSS without glucose HPLC high pressure liquid chromatography

ID inner diameter KLP Calibration sample KRB Krebs Ringer bicarbonate buffer KRB-/- KRBwithout glucose and HEPES

KS Cryo-cuts LSC Liquid scintillation counting

initial mass in donor compartment

MEA Multiple endpoint analysis mean arithmetic mean MeOH methanol MW molecular weight n number of repeats within an experiment OECD Organization for Economic Co-operation and Development

Papp apparent permeability coefficient PDA Photo diode array

Q transported mass rpm rotations per minute

RT room temperature

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RSD relative standard deviation SC Stratum corneum SCCP Scientific Committee on Cosmetic Products SD standard deviation SOP Standard operating procedure SST System suitability STB stability

T temperature t time

TC Tenside concentration TS Tape strip

UV Ultra violet absorption 0V donor volume

VA acceptor volume

/xQI/xt permeability rate (transported mass of the substrate against the time)

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1. Summary

1.1. Objectives

The aim of the present study was to investigate the in vitro permeation and penetration of capryl glucoside (GC)from the product Plantacare 810 UP at human skin of 3 different donors in vitro according to SCCP requirements and to OECDGuideline 428. The product provided by the cus tomer was diluted with HBSS- buffer to the concentration of 10 % CG and the pH was adjusted to 6.5. This formulation was used as the test solution in all experiments. The first step of the study was to establish an analytical method for quantification of GC. The described analytical method was developed to quantify the compound in the used biological acceptor and extraction media. The method was validated under GLPconditions for this purpose. At the same time the stability of CG from the test solution was tested over 48 hours at 32 ±

2 °C, 25 ± 5 °C and 4 ± 2 °C in biological acceptor medium (KRB buffer without HEPES and glucose) and in the donor medium (HBSS without glucose) used for the permeation study. Fur thermore the stability of the test compound was also investigated in the extraction medium em ployed in the penetration studies (MeOH/water, 50:50 v/v, %).

At the beginning of the study, the CG content in the test solution without skin contact in tripli cate and after skin contact (one-fold) was determined. The permeability of CG from the test solution on human skin was investigated on fresh human skin from 3 donors in two fold (n=2) for each donor (totalizing n=6) conformable the SOP M 005 at Across Barriers. The skin specimen was internally encoded according to the corresponding SOP A 005 at Across Barriers. Dermatomized (to approximately 500 pm) skin was used. The skin thickness was measured immediately before performing the studies. 8 samples were taken from the acceptor medium during a period of 24 hours to obtain information about permeability of CG. At the end of the permeation experiment the remaining CG content in the test solution was determined. For that goal the test formulation left on the skin surface was collected with cotton swabs and transferred to the falcon tube with the extraction medium, this is the so-called wash procedure. After removing residual formulation, the concentration of CG in the skin, in the Stratum corneum and deeper skin layers, was quantified. The upper corneous layer of the skin was stripped off and the residual skin was cryo-sectioned. After the in vitro study a mass recovery was carried out to determine the mass balance and local distribution of CG in the different skin compartments. For that goal a quotient of total mass of CG at the end of the study on the skin surface in test solution, in Stratum corneum, Epidermis/Dermis and acceptor compartment versus the applied amount of CG in the formulation at the start of the study was calculated. The permeability of

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Caffeine (MEA:multiple endpoint analysis) was carried out at a concentration of 10 1mgL in Krebs-Ringer-buffer (KRB)at pH 7.4 (n=2 for 3 skin donors) for the study C-i 0335-227-1107. Since the same skin donors were employed in the present study, the permeability of caffeine was not performed. Caffeine is a recommended marker molecule of the OECDGuideline for the quality control of human skin. The results of the Caffeine permeation were compared with the permeability coefficients of previous studies on historical human skin membranes of different origin at Across Barriers.

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1.2. Summarized results

1.2.1. HPLC method

The results of the analytical method development and validation are summarized in the following three tables.

Tab. 1: Analytical parameters.

System Agilent 1100 [C MSD

Waters Atlantis 18 3 pm Column C 2.1 x 50 mm (Length. ID);ACBID: S387

A: Water ± 0.1% formic acid Mobile Phase B: Methanol ± 0.1% formic acid Flow 0.4 1mLmin Mode Gradient

Temperature 30 C (Column), 30 C (Autosampler)

Injection volume 20 pL

ESI positive MS detection SIR m/z 343.20

Tab. 2: Gradient profile for CG analytical method.

Time [mini A [%] B [%] Flow ImL•min] 0.00 100 0 0.4

1.00 100 0 0.4

2.50 0 100 0.4 7.00 0 100 0.4

7.01 100 0 0.4

9.00 100 0 0.4

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Tab. 3: Summarized results of the HPLC method validation.

Parameter Detail limits Result Compliance

System suitability 6 injections of standard RSD = 10 % 2.18 % yes test solution

Visual comparison of No interferences No interferences chromatograms of the test with eluent mixture, with eluent mix- Selectivity compound in eluent mix- extraction medium ture, extraction yes ture, extraction medium and acceptor me- medium and ac and acceptor medium dium ceptor medium 2 Order regression model y — b + m x + o x2 , = 0.99 0.99302 yes weighting: 21/x mm. 5 calibration levels Theoretical val ues: 0.0986- Linearity in KRB* 10.2678 buffer pH 7.4 Linear range - pg 1mL yes measured values: 0.091-11.488 pg mL’

LLOQ:7.07 % Deviation conc. Meas- LLOQ:± 20% Others: ured/ theor. conc.: Others: ± 15% yes -11.88 until 14.55%

3 concentration levels Recovery Recovery: CON1-CON3 Accuracy in accep- 100 ± 20 °‘ CON1: 85.14 % tor medium KRB* n=3 (LLOQ) yes pH7.4 CON2:88.95% deviation mean value/true 100 ± 15 % CON3: 84.94 % value (others)

3 concentration levels RSD: RSD (n=3) Precision in accep- CON1-CON3 CON1: 0.58% ± KRB* 15% (others) yes tor medium n==3 CON2: 2.12 % ± 20 % (LLOQ) RSD% CON3:12.86% Measured value: S/N: = 5:1 0.088 pg 1mL concentration near LLOQ (CON3) accuracy: S/N: = 5:1 Limit of 100 ±20% accuracy: I,..,3 yes quantification precision: 86.59% S/N RSD=20% precision: accuracy/precision RSD = 8.58 %

* without HEPES and glucose

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Tab. 3: Summarized results of the HPLC method validation (continuation),

Parameter Detail Limits Result Compliance

Recovery Recovery Acceptor medium

. Recovery > 85 °‘ . 4°C124h: 95.5 % in acceptor medium: RT, < yes 4°C and 32°C over 24h Precision 15% RT/24h: 96.5 %

n=3 32°C/24h: 91.5 % Robustness (sta bility of the test compound in KRB* 7.4) Recovery Recovery

in acceptor medium: Recovery > 85 % Acceptor medium

. . 4 °C/48h: 89.3 % RT, 4 C and 32 C over Precision < 15 % yes 48h RTI48h: 89.2%

n=3 32°C/48h: 91.8 %

Recovery Recovery Recovery > 85 % Donor medium in donor medium: RT 4 °i’ °C124h: 89.3 yes 32CC and 4° C over 24h Precision < 15 /o RTI24h: 95.0 % n=3 32C/24h: 91 .0 % Robustness (St a bility of the test compound in HBSS** 6.5) Recovery Recovery Donor medium Recovery > 85 % °C/48h 87.8 % in donor medium: RT Precision < yes 32CC and 4° C over 48h 15 °‘ RT/48h: 86.8 %

n=3 32°C/48h: 91.8 %

* without HEPES and glucose ** without glucose

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1.2.2. In vitro permeation study

Tab. 4: Summarized results from the permeation study

Theoretical content CG transport into the Skin Test product Test substance of CG in the donor acceptor after 24 h number 1mg] [%]

336-01-0808 CG 3.09 0.00* Test solution 10% 337-01 -0908 CG 3.09 0.00* CG 340-01 -0908 CG 3.09 0,00* * Values below analytical LLOQ(0.088 iig 1).mL

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1.2.3. In vitro penetration study

The results of the in vitro transport studies are summarized in Tab. 5 and Tab. 6. Tab. 5: Mean amount [pg ]2cm- of CG in the samples from the three in vitro experiments. Cumulative amount [pg . J2cm of CG in the samples Skin 336-01 -0808 Skin 337-01-0908 Skin 340-01-0908 All skins used Sample Mean SD Mean SD Mean SD Mean SD dose ap plied 1011.12 10.87 1098.37 4.10 1084.09 16.10 1064.53 42.79 receptor 0.00* 0.00 0.00* 0.00 0.00* 0.00 0.00* 0.00 skin wash 1283.58 67.12 1283.38 122.21 1437.39 160.40 1334.78 123.90 2 Tape strips 5.94 1.03 7.03 2.18 3.76 1.25 5.58 1.92 18 Tape strips 2.18 0.08 8.02 4.19 3.28 3.02 4.49 3.61 cryocuts 0.00* 0.00 0.43 0.47 0.00* 0.00 0.14 0.31 * Values below the LLOQoi the analytical method.

Tab. 6: Mean recovery rate and dose absorbed [%]of CG in the samples from the three in vitro experi ments.

Recovery and dose absorbed [%1of CG in the samples

Sample Skin 336-01 -0808 Skin 337-01 -0908 Skin 340-01-0908 All skins used Mean SD Mean SD Mean SD Mean SD receptor 0,00* 0.00 0.00* 0.00 0.00* 0.00 0.00* 0.00* skinsurface 126.92 5.27 116.82 10.69 132.71 16.77 125.49 11.68 2 Tape strips 0.59 0.11 0.64 0.20 0.35 0.11 0.52 0.18 18 Tape strips 0.22 0.01 0.73 0.38 0.30 0.27 0.30 0.32 cryocuts 0.00* 0.00 0.04 0.04 0.00* 0.00 0.01 0.03 Dose absorbed 0.00* 0.00* 0.04 0.04 0.00* 0.00* 0.01 0.03 Total recovery 127.72 5.17 118.23 10.06 133.36 16.38 126.44 11.23

* Values below the LLOQof the analytical method.

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1.3. Conclusion

The HPLCmethod was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRBbuffer without HEPES and glucose). Furthermore, the test compound has demonstrated good stability over the period of 48 hours at RTand 4 °C in the extraction medium employed for the penetration experiments

(MeOH/water (50:50, v/v %))and good stability in the acceptor (KRB-/-) and donor media

(HBSS-) at 32 °C, 4°C and RTover the period of 48 hours, which covers the experiment and measurements duration.

The content of CG in the test solution without skin contact was determined in triplicate and the mean value was 96.63 ± 1 .56 %.The test solution was also applied to skin and the recovery reached 90.10 %. This complies to the acceptance criteria of 100 ± 10 %.

The results from the extraction method has shown that water/MeOH (50:50 v/v, %)was most suitable medium for performing the penetration experiments presenting mean recovery values which has ranged between 88.65 % and 112.28 %, which complies to the acceptance limit of 100±20%. The permeated amounts of CG through three different human skins (no. 336-01 -0808, 337-

01 -0908 and 340-09 -0908) have presented values in the receptor below the LLOQof the vali dated analytical method. This low absorption was confirmed through the mass recovery calcula tion, where 125.49 % (mean value for 6 Franz cells) of surfactant were determined in the test solution which remained at the skin surface after 24 hours. The mass recovery calculations present amounts and percentages of compound which perme ated, penetrated and remained in the donor compartment. The mean amount of CG removed from the skin surface (skin wash) ranged from 109.26 % to 144.57 % of the dose applied. The mean recovery (mean value for 6 Franz cells) in the two first tape strips was 0.52 % during all performed experiments. In the further 18 tape strips a mean recovery of 0.30 % was docu mented.

The mean absorbed dose of CG, sum of the amounts found in the viable epidermis, dermis and receptor medium, were considered as 0.01 %.

The mean Papp values measured for Caffeine forthe human skins no. 340-01 -0808, 337-01 -

0908 and 340-01 -0908 are in good agreement with app values determined for a variety of skin specimens from different donors under comparable conditions. The result are reported in detail in the report C-i 0335-227-9 107.

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2. Materials and methods

2.1. Materials

Tab. 7: Skin sample parameters used for the CG in vitro studies and quality control experiments.

Species Gender Age Region BMI Storage Skin number

Human Female 48 Abdomen 28.1 - 20 C 336-01-0808

Human Female 43 Abdomen 22.7 - 20 C 337-01 -0908

Human Female 30 Abdomen 25.7 - 20 C 340-01 -0908 Human Female 38 Abdomen 29.1 -20 C 306-01-1107

The skin samples were excised during surgical operations. The skin was not removed to provide samples for these in vitro investigations. The hospital had the prior consent of the patients that the tissue could be used for scientific research.

Tab. 8: Test product.

Name Plantacare 81 OUP Compound Capryl glucoside

ACB-ID K8828 Supplier Cognis GmbH Batch CE73510004

Tenside content 62.8 %

Tab. 9: Test compound from test product. Name Capryl glucoside Molecular weight 343.2 g!mol

Molecular structure n = 1,6; R C8-1 0

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Tab. 10: Further chemicals and reagents used.

Compound I reagent Batch ACB-ID

Ethanol K38999227 L9781

KRB pH 74* - - Methanol 440807 L9454

Formic acid K38550564 L8986

HBSS pH 55** - - Caffeine 066K0085 [8070

[3H] Caffeine 080109 L8689

*without HEPES and gIucose **without glucose

Tab. 11: Other consumables.

Article Application Specifications glass diffusion chambers for the acceptor volume: approx. 20 mL Franz cells . transport assays diffusion. area: approx. 3 cm 2 donor volume: variable glass diffusion chambers for the acceptor volume: approx. 12 mL Franz cells transport assays (MEA) diffusion area: approx. 1.77 2cm donor volume: variable Syringes sampling single use fine dosage

Needles sampling disposable

Plates Scintillation counting 4 x 6 wells

Disposable scalpels cutting of skin Bayha sterile surgical blade

. stripping of stratum cor- . Tape film 19 mm wide, Beiersdorf, product number 57330 neum

HPLC vials sampling -

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2.2. Equipment and software lab. 12: HPLC equipment and software.

Device I accessories Specification Supplier

HPLC Agilent 1100 [C Agilent

Detectors MSD Agilent

HP G2710 [C/MS ChemStation Software Agilent Software lab, 13: Utilized equipment.

Device Type I Specification Manufacturer

. SBC22/BCBC100 Analytical Balance Scaltec d*=0,01/0.1 mg)

Centrifuge 4K1 5C Sigma

Cryo microtome MEV SLEE

Dermatom GA 630 Aesculap

Fridge-freezer Santo (4 C/-20 C) AEG

Heating cabinet Equ.Nr. 20001740 Heraeus

Magnetic stirrer Vario-Mag Mono grau H±P [abortechnik

Magnetic stirrer Stirring Drive HP 15 H÷P [abortechnik

Mini Shaker MS2 IKA[abortechnik

pH-meter 4-7 model 330 Orion 10-100 p[ 398077 50-200 p[ 399199 Piston-stroke pipettes Eppendorf 100-1000 p[ 383566 500-5000 p[ 459937

Saarbruecken model developed by Prof. H. [oth -

Shaker KS-i 6 Edmund BUhIer

Shaker KS-i 5 Edmund Buhler

Skin thickness gage ND 221 B Heidenhain

Steaming cabinet 20001740 / 32 C Heraeus

SG Wasseraufbereitung + Ultra-pure water system Ultra Clear . Regenerierstation Ultrasonic bath Sonorex Super RK1O6 Bandelin

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2.3. Buffers and media

Krebs Ringer buffer without HEPES and glucose, pH 7.4

The Krebs Ringer buffer without HEPES and glucose (KRB-!-)was prepared referring to SOP M

003 and used as modified form for acceptor medium in permeation experiments.

Tab. 14: Preparation of the Krebs Ringer buffer pH 7.4 without HEPESand glucose (KRB-/-)

Substance Weighted sample [g] per 1 1 of water 6HMgCI 0.224 20CaCI 0.274 2 206H NaCI 6.663

KCI 0.375 Na2HHPO 0.294 2HPO240NaH 0.047 240 3NaHCO 1.680

The pH value was adjusted with formic acid to 7.4.

HBSS buffer without glucose, pH 6.5

The content of the HBSS without glucose (HBSS-) buffer is summarized below.

Tab. 15: HBSS- bufferatpH 6.5

Substance Weighed sample [g] per 1 1 of water 4MgSO 0,098 2CaCI 6 20H 0.209 NaCI 7.988

KCI 0.400 24NaHPO 20H 0.069 K2HHPO 0.101 240 MES 1.952

The pH-value was adjusted with formic acid to the value of 6.5.

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Mobile phase A

Water ± 0.1% formic acid

Mobile phase B

Methanol ± 0.1% formic acid

2.4. Methods

2.4.1. HPLC method development and validation

The first step of the study was to establish an analytical HPLC method for quantification of CG in the used acceptor media (KRB-!-buffer). The HPLCmethod development was not carried out under GLPconditions. The validation of the method was investigated in accordance with the FDA

Guideline ,,Bioanalytical Method Validation” (2001) and it was performed under GLPconditions.

System suitability test

A system suitability test is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system. This test was performed for one reference substance CG in six fold determination. Relative standard deviation below 10% was accepted.

Selectivity

The specificity of the method was investigated by means of eluent mixture, acceptor medium and extraction media. The relevant chromatograms with and without CG were compared and scanned for possible interferences with extraneous components by visual inspection.

Linearity

The linearity was checked for KRB-!-buffer with 8 concentration levels of the test compound. The absorption values of the standard solutions were plotted against their corresponding theo retical concentrations. The purity of the analytical standard was considered for the calculation of its concentration. The data were subjected to regression analysis to provide information on the slope, the intercept, the determination coefficient 2),(r and the back-calculated calibration stan dard concentrations.

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Regression model:

,weighting:y=b+nl.x+ox 21/x Eq. 1 m: Slope first order 2 0: Slope second order b: Intercept y: Absorption values I Areas x: Concentration levels / Amount jiglmL

Accuracy and precision

In order to assess the accuracy of the method the mean value of 3 replicate samples was de termined for 3 different concentration levels. The difference between the determined mean value and the true (theoretical) value was calculated and expressed as percent of the true value:

%Acc=100%+ •100% Eq. 2 CONtheory

% Acc accuracy as percent value

CONtheorytheoretical concentration value (true value) CON, single determined concentration value of a particular concentration level

The precision corresponds with the standard deviation of the 3 replicates of each concentration level. The following concentration levels were prepared:

CON1: high concentration: 70 %-90 % of the highest calibration standard

CON2: mean concentration: 3-5-fold value of the LLOQ

CON3: low concentration: close to LLOQ

The demanded values at the LLOQare = 20 % for the precision (%RSD) and 100 ± 20 % for the accuracy. Regarding concentration values above the LLOQvalues of 15 % are demanded for the precision, whereas the accuracy has to be within a range of 100 ± 15 %.

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Quantification limit

For the determination of the quantification limit 3 samples in the range of the LLOQwere ana

lyzed in series. Regarding a signal noise ratio (SIN) 5 : 1, precision values (% RSD) = 20 %

and accuracy values of 100 ± 20 % were accepted. A SIN ratio of 3 : 1 is required for the de tection limit.

Stability in donor medium (HBSS without glucose)

Aliquots of the test compound were dissolved in the donor medium used (HBSS-) stored for

48 h at 4 °C ± 2 °C, 25 °C ± 5 °C and 32°C ± 2 °C. Subsequently they were analyzed by HPLC. The stability was determined as the recovery difference between three replicates treated sam ples and three untreated samples.

Stability in the permeation matrix (KRB without Glucose and HEPES)

As biological acceptor medium KRB-I-buffer at a pH of 7.4 was used. For the stability determi nation aliquots of the test compound were dissolved in this matrix, stored for 48 h at 4 °C

± 2 °C, 25 °C ± 5 °C and 32 °C ± 2 °C. Subsequently they were analyzed by HPLC.The stability was determined as the recovery difference between three replicate treated samples and three untreated samples.

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HPLC validation plan

Tab. 16: Summarized target parameters for HPLC analysis of in vitro sample.

Parameter Description Limits

SST 6 injections of standard solution RSD = 10 %

Comparison of chromatograms. no interferences with eluents and Selectivity Test compound in matrix vs. matrix, extraction medium, slight interference eluents and extraction medium with the acceptor medium

deviation conc. measured / theor. At least 5 concentration levels Conc.: (external standards) Linearity LLOQ:± 20 % 25t order regression model others: ± 15 % 21/x r = 0.99

3 concentration levels deviation: (CON1 CON3) Accuracy - n=3 LLOQ:100 ± 20 %

deviation mean value! true value others: 100 ± 15 %

RSD (n=3): 3 concentration levels Precision LLOQ:± 20 % (CON1 — CON3) n=3, RSD others: ± 15%

Concentration near LLOQ S/N: = 5:1 Limit of quantification (CON3) n=3 accuracy: 100 ± 20%

accuracy/precision precision: ± 20 %

Test conditions: 4 °C, 25 C and Stability of the test Recovery > 85 % 32 C for 48 h compound Precision < 15 %

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2.4.2. Sample preparation for HPLC method

Allsamples for the validation of the HPLC method were diluted to acceptable concentrations (within the calibration range).

Sample preparation for the system suitability test

The test compound was firstly dissolved in KRB-!- buffer pH 7.4 (stock solutions) and subse quently diluted to appropriate yield concentrations. The purity of the compound was considered in the calculations.

Tab. 17: Preparation of the stock solutions for the reference samples.

I I Sample Purity Weighted I Final volume c I Compound I ACB-IDI Solvent

I name I [%i Isample [mgi I I Emil [ig.mL

I 1 KRB-/- SST1 CG K8828 62.8 70.28 I 50 i 882.72 buffer

Tab. 18: Preparation of the reference samples.

Samples to assess the linearity

Tab. 19: Preparation of the stock solutions for the linearity assessment.

The standard solution from the test compound was prepared in the range of 0.0986 — 102.6780 .1pgmL The solution preparation is presented in the table Tab. 20.

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Tab. 20: Preparation of the analytical solutions used to assess the linearity

Stock solution Volume SI - Final volume c Sample name Diluted with 1 (SL) [ml] [mIl [pg-mL I KLP2 1A 2.5 KRB-/- pH 7.4 25 102.678

KLP3 1A 2 KRB-/- pH 7.4 25 82.1424

KLP4 1A 1.5 KRB-/- pH 7.4 25 61 .6068

KLP5 1A 1.5 KRB-/- pH 7.4 50 30.8034

KLP6 2A 2 KRB-/- pH 7.4 20 10.2678

KLP7 2A 2 KRB-/- pH 7.4 25 8.2142

KLP8 7A 3 KRB-/- pH 7.4 10 2.4643

KLP9 7A 1 KRB-/- pH 7.4 5 1.6248

KLP1O 7A 2.5 KRB-/- pH 7.4 20 1.0268

KLP11 7A 1 KRB-/- pH 7.4 10 0.8214

KLP12 8A 1 KRB-/- pH 7.4 5 0.4929

KLP13 8A 1 KRB-/- pH 7.4 10 0.2464

KLP14 8A 1 KRB-/- pH 7.4 20 0.1232

KLP1S 8A 1 KRB-/- pH 7.4 25 0.0986

* Only the analytical solutions KLP6, KLP7, KLP8, KLP9, KLP11, KLP12, KLP14, and KLP15 were em ployed for linearity determination.

Samples to assess accuracy and precision in acceptor medium

For the determination of accuracy and precision in the acceptor medium (KRB-/-) 3 sarrples were prepared and diluted for 3 different concentrations. The preparation of the stock solutions follow the table below:

Tab. 21: Summary of samples preparation to assess accuracy in acceptor medium,

Stock Weighted Final volume c Compound* ACB-ID Solvent solution sample 1mg] [ml] 1[ig-mL KRB-/- buffer ] CONC1 CG K8828 68.04 25 1222,00 pH 7.4

KRB-/- buffer CONC2 CG K8828 64.55 25 1159.32 pH 7.4

KRB-I-buffer CONC3 CG K8828 64.88 25 1165.24 pH 7.4

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Each stock solution was diluted to yield 3 different concentrations according to presented on the table below.

Tab. 22: Summary of standard solutions prepared from stock solutions for assessment of accuracy and precision.

C Sample name Ipg.ml’

ACCCON 1 8.8272

ACCCON2 2.2068

ACCCON 3 0.0883

Sample for assess stability in KRB buffer (without glucose and HEPES) pH 7.4 and HBSS buffer (without glucose) pH 6.5

The samples for assessment of stability were prepared and stored at the temperature of 4°C, RT and 32 °C during 48 hours.

Tab. 23: Preparation of the stock solutions for the reference samples.

Sample Weighted Final volume c Compound* ACB-lD Solvent name sample [mgi [mu 1IpgmL Methanol/wate KRBSTB 1 CG K8828 75.29 50 l 945.64 (50:50)

HBSS STB 1 CG K8828 797.18 HBSS** 5 100125.81 [ **Without glucose

Tab. 24: Preparation of the reference samples.

Stock solution Volume SI Final vol- c Sample name Diluted with (SI) [ml] ume [ml] 1[pg.mL KRB*buffer ] KRBSTB2 KRBSTB1 1 100 9.4564 pH 7.40 KRB*buffer KRBSTB 3 KRBSTB 2 5 20 2,3641 pH 7.40

HBSSSTB2 HBSSSTB1 1 HBSS** 100 1001.2581

HBSS STB3 HBSS STB 2 1 HBSS** 50 20.0252

HBSS S184 HBSSSTB3 1 HBSS** 10 2.0025 Without HEPESand glucose. *Without glucose

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2.4.3. Liquid Scintillation counting

Caffeine was quantified by liquid scintillation counting. The samples (300 were transferred into 24 well plates and mixed with 500 liquid scintillation cocktail. After an equilibration pe riod of at least 1 -hour radiation was measured for 2 minutes per well in a scintillation counter. The recorded counts in a blank sample were subtracted from the value of each test sample.

Tab. 25: Scintillation counting equipment.

Device I accessories Specification Supplier

Liquid scintillation counter Microbeta WallacTM Perkin Elmer Scintillation Cocktail TMOptiphase Supermix Perkin Elmer

2.4.4. Preparation of test solution 10 % CG (test solution)

The test product provided by the customer was diluted with HBSS buffer (without glucose) to concentration of 10 % CG and pH value was adjusted to 65. The HBSS- buffer was employed instead the KRB-/- buffer because HBSS- buffer shows better buffer capacity at the pH 6.5. The term “test solution” used in this report applies to the 10% CG solution in HBSS (without glu cose).

2.4.5. Determination of CG content in the test solution

At the beginning of the study the drug content in the test solution was determined by HPLCin triplicate with an appropriate extraction method. The mean recovery of 100 ± 10% was ac cepted.

For the determination of CG content, the following solutions were prepared:

Compound I Weighted Final Sample name Stock solu- ACB-ID sample I Solvent volume IpgmL tion (SI) Volume SL Emil j

SOL1 CG K8828 796.24mg HBSS- pH 6.5 5 10000774

SOL2 SOL1 - 2 mL MeOH/water (50:50) 200 1000.0774

SOL3 SOL2 - 2 mL MeOH/water (50:50) 200 10.0008

SOL4 SOL3 - 2 mL MeOH/water (50:50) 20 1 .0001

The S0L1 was diluted until to obtain a theoretical concentration of 1 .0001 ig/mL. This last solu tion was employed for the AS content determination (For sample name see table Tab. 26 CG1,

CG 2 and CG 3).

Additionally 30 jL of the application solution (10% CG) were applied to human dermatomized skin (sample named as CG 4), that was clamped into a Franz cell. The tenside was immediately

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removed with cotton balls and washed twice with 1 mL of water/methanol (50:50, v/v %).The cotton balls and the wash solution were pooled in a 50 mL volumetric flask and completed with water/methanol (50:50, v/v %).850 pL of this solution were diluted with the extraction medium to 50 mL. The table below summarizes the respective amount in mg and the theoretical surfac tant content.

Tab. 26: Summary of corresponding weight of samples and theoretical tenside content in the test solu tion.

Theoretical Amount Test formula- weighed* tenside Compound Skin contact Sample name concentration [mg] . mL1 CG1 2033 1.0001

no CG2 2033 1.0001 Test solution CG 10%CG CG3 2033 1.0001

yes CG4 30.50 1.0201 *Corresponds to 30 p1 of the test solution CG4 ano 2m1of the test solution CG1-3

2.4.6. In vitro experiments

As an introduction a definition of the dermal absorption according to SCCP guideline and its adaption and more detailed use to/in the present report is presented. The percutaneous/dermal absorption process is a global term, which describes the passage of compounds across the skin. This process can be divided into three steps:

- penetration, which is the entry of a substance into a particular layer or structure such as the entrance of a compound into the stratum corneum (in the present report entrance of a test compound into the stratum corneum, entrance of a compound into the viable epidermis and into the dermis);

- permeation, which is the penetration through one layer into another, which is both functionally and structurally different from the first layer (in the present report penetration of the test com pound through skin layers into the receptor medium);

- resorption, which is the uptake of a substance into the vascular system (lymph and/or blood vessel), which acts as the central compartment (not investigated in the present study).

Introduction anatomy of human skin

Human skin comprises the epidermis, the dermis and the underlying subcutaneous tissue, with sebaceous and sweat glands running throughout. The outermost epidermal layer, i.e. the Stra

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Across Barriers GmbH Seite 27 von 71 Intelligent Drug Profiling turn corneum (SC) or corneous layer is the main barrier to skin permeation for dermally applied drug formulations. The structure of the Stratum corneum itself can be explained in terms of the so-called brick and mortar model in which corneous keratinocytes (corneocytes) represent the bricks while the intercellular lipids and water-retaining natural moisturizing factors act as the mortar.

— 8 — 4. 5OOpm r

Fig. 1: Cross-section through human skin (stratum corneum and dermis)

The main gender-specific difference in abdominal skin is the higher number of hair follicles in male samples. Because it is important not to cut the hair follicles when preparing the subcutis, female hairless abdominal skin is preferable for in vitro studies.

Skin membranes

During the study human skin biopsies of 3 donors were used.

Preparation of the skin

Transport and preparation of full-thickness skin

The skin was made available in accordance with the relevant statutory and contractual provi sions. The excised skin from the surgery was cooled to 4 °C and its surface was dried before being transported. Care was taken to ensure that the subcutaneous fatty tissue does not get into contact with the surface of the skin. Once in the laboratory, the skin was separated from the subcutaneous fatty layer and then issued by Across Barriers with an encrypted identification number.

Encryption was performed in accordance with the Across Barriers SOP A 005. The encrypted identification numbers are always used to refer to the skin specimens in laboratory records and

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Across Barriers GmbH Seite 28 von 71 IntelligentDrugProfiling reports. Across Barriers assured to the hospital that information on the patients and the surgery can only be accessed via a tile held by Across Barriers. The suitability of the skin biopsies was assessed according to the following criteria: Inclusion criteria:

• No pathological findings

• Hospital must have patient’s consent that tissue may be used for scientific purposes Exclusion criteria: • Skin damage, strongly marked scarring or stretch marks

Working under controlled storage conditions (4°C), the subcutaneous fatty layer was removed within two hours after reception of the abdominal skin. Once separated from the subcutaneous fatty tissue, the residual full-thickness skin was stored at -20°C. According to the OECDGuide line, storage of skin at -20 °C for a period of up to six months does not alter its permeability. The skin was used immediately after thawing. Repeated freeze-thaw cycles were avoided.

Dermatomization of the skin

The human skin was cut into 4 cm broad stripes. Subsequently skin sections with a thickness of approximately 500 pm were prepared from the full-thickness skin samples using an Aesculap GA 630 dermatome. The skin surface with intact Stratum corneum is directed towards the dernm tome. The use of the dermatome is described in SOP G 004. Prior to use, the thickness of the dermatomized skin was checked with a Heidenhain thickness gage (SOP G 026) at five different places of each skin sample.

The measured thickness was not exactly the 500 pm scheduled in the study plan, as it strongly depends on the skin’s elasticity and cannot be estimated prior to dermatomization. The slightly varying thickness, however, has no influence on the permeability of the marker compound Caf feine [Bock et al. 20021. Because the stratum corneum represents the principle absorption bar rier a significant alteration of the test compounds’ transport kinetics by remaining deeper skin layers is not expectable.

2.4.7. Permeation studies with the test product

The cylindrical glass Franz cell is a diffusion chamber comprising an upper and a lower part be tween which the skin specimen will be clamped. The two halves of the cell are held together by means of a ball-and-socket clamp. The lower (receptor) chamber has a volume of approximately

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20 mL, while the volume of the upper (donor) chamber is variable. The skin specimens was punched out (area of approximately 4 )2cm immediately prior to insertion in the Franz cells.

Fig.2: Franzcell viewedfrom above (left)and from the side (right).

The skin is always inserted with the dermal layer oriented downwards so that the skin’s horny layer is uppermost.

Franz diffusion cells Franz diffusion cells with a diffusion area of approximately 3 2cm and an acceptor volume of approximately 20 mL were used. The stirring speed and the temperature was set at 400 rotation per minute and 32 °C ± 2 C, respectively. At each sampling point the stirring speed and tem perature were documented. For more details regarding the used Franz cell diffusion area and volume see appendix topic 9.3.

Dose

30 jiL of the test solution 10 % CG were applied at the diffusion skin area and homogenously spread over the skin surface. In order to avoid any changes in the formulation, Franz diffusion cells were kept covered with Parafllm®over the period of 24 hours. The attribution of the particu lar test formulations and the applied amount to the different Franz cells is depicted in Tab. 27.

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Tab. 27: Assignment of test formulation, applied amounts and skin thickness to the Franz cells.

Thickness of skin Theoretical Applied vol Donor ume per Skin num- GC content Formulation FZ mass* diffusion ber Mean [mg] in do- RSD [%] [mg. cm- area 1pm] ]2 nor ]2[p11cm 525.40 5.11 12 0.97 3,05 10.64 336-01 - 0808 538.60 3.45 5 0.97 3.05 10.42

503.40 6,79 129 1.03 3.05 9.74 Test solution 337-01- 10%CG 0908 563.60 5.93 133 1.04 3.05 9.90

572.60 2.11 125 1.06 3.05 10.68 340-01- 0908 572.40 6.31 14 0.96 3.05 10.64

* Value in mg which corresponds to 30 ‘ of test solution 10 % CG

Acceptor medium

As acceptor medium for the studies with the test preparation, KRB buffer at pH 7.4 without glu cose and HEPES was used. The content of this medium is presented in the Tab. ‘14.

Sampling times

The permeation study was performed over a period of 24 hours, after 0.5, 2, 4, 6, 20, 22, 23 and 24 hours. The samples with a volume of 200 pL were taken from the acceptor compart ments and analyzed for the drug content by HPLC. The taken amount was replaced with a fresh medium preheated to the temperature of 32 C. In total 8 samples were analysed for each time point (3 donors, n=2).

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Tab. 28: Sampling times for the in vitro study.

Sample ID Run Time [h]

1 0.5

2 2

3 4

4 6

5 21

6 22

7 23 8 24

2.4.8. Penetration experiments with the test product

After removing the formulation from the skin surface, the skin was partitioned into horizontal segments to determine the distribution profile of CG in the skin.

2.4.9. Establishment of the extraction method

Prior to the penetration studies an appropriate extraction method was established under non GLP conditions. In order to detect a possible adsorption of the test compound to the Tape film or to the skin in the extraction matrix, test solution samples were incubated with Tape strips, Tape strips ± Stratum corneum and deeper skin layers, respectively. For penetration experiments a scotch tape purchased from Beiersdorf (Germany, 19 mm wide, product number 57330) was used. The skin No. 306-01 -1 107 was used for the establishment of the extraction method.

For this purpose a skin sample was stripped by means of the Saarbruecken model (Fig. 3). Mo reover, Tape strips without skin contact and cryosections of deeper skin layers (epidermis and dermis) were prepared. Two different concentrations of CG (about 3.6 pglmL and 0.9 ig/mL) were added to the samples, which subsequently were agitated for 1 hour with extraction matrix.

Two solutions were tested as extraction media: water and Methanol/water 50:50 (v/v, %).Finally the recovery of the test compound was determined by comparing the extracted and the initially applied amounts. An appropriate extraction medium for the scheduled in vitro penetration stud ies was defined in this experiment.

Penetration studies

The residual formulation on the skin surface (skin wash) was removed and the concentration of the test substance was quantified. Two segmenting techniques, Tape stripping and Cryosection ing, were applied to separate different skin layers parallel to the upper surface of the sample and

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Across Barriers GmbH Seite 32 von 71 Intelligent Drug Profiling thus provide information on the extent to which the test compound has penetrated into the dif ferent skin areas.

Fig. 3: Saarbruecken model (SB) used for penetration studies. left: stripping of the Stratum corneum of the skin right: cryo sectioning of deeper skin layer

After removing the residual formulation, the skin biopsies were transferred into a stripping appa ratus. Additionally to the SOP M 01 9 the skin sample may be placed onto the Styrofoam for the stripping procedure. The upper corneous layers (Stratum corneum) of the skin were stripped off using Tape film. The stripped skin area was 1 .767 2cm ± 5 %. In total 20 tape strips were per formed per each skin biopsy (see Tab. 29). The first two strips are always considered separtately due to potential contaminations by residual drug on the surface of the skin. The samples for 2 tape-strips and 1 8 tape-strips were extracted in 3 mL and 5 ml of the extraction medium, re spectively and quantified by HPLC. Methanol/water (50:50, v/v %) was used as an extraction medium.

After stripping, the skin biopsies were punched out and frozen at —80 C. The residual skin was cryo-sectioned to determine the amount of test compound, which had penetrated into the deeper skin layers. The skin biopsy was cut into surface parallel sections with a thickness of 25 pm. The surface area of a single cut was approximately 1 .327 .2cm All cuts were collected into one beaker. All samples were extracted in 3 mL of the extraction medium and quantified by

HPLC. Methanol/water (50:50 v/v, %)was used as extraction medium.

Tab. 29: Samples from the penetration study.

Number of tape strips Sample no. Tesa Strip no. Cryosections (total of 20)

1-2 1 2 -

3-20 2 18 -

Rest - - Allcryocuts

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Fig. 4 and Fig. 5 give a schematic view of the stripping procedure and cryo-sectioning, respec tively.

sample vials I skin

50 *0 40 20

stripping apparatus stopwatch

Fig. 4: Schematic view of tape stripping (thesis Heike Wagner).

Fig. 5: Schematic view of cryo-sectioning (thesis Heike Wagner).

2.5. MEA:Qualification of skin membranes

The skin donors employed in this experiment was the same employed for the last study C-

10335-227-1 107, where the quality control for the skin samples were carried out. For this rea son it was not necessary to perform again the skin quality control experiments. The apparent permeability coefficients determined in the present study were compared to the

Caffeine Papp measured using dermatomized skins from different donors. The Franz cells em-

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Across Barriers GmbH Seite 34 von 71 IntelligentDrugProfiling ployed for MEAwere different in diffusion area and volume from the Franz cells from the per meations studies. These information are detailed in the report C-i 0335-227-1 107.

2.6. Calculations

In the calculations the individual values of Franz cells used in the experiments were considered.

Allcalculations were performed using the program EXCEL2000, SP-2 from Microsoft Corpora tion, USA.

The calculations were performed using the full number of digits handled by the EXCEL2000 pro gram. The results were rounded only for printing and for the presentation in the report.

Apparent permeability coefficient (Papp)

The apparent permeability coefficient (Papp)was calculated from the linear part of the slope of the cumulative transport of the substance. The lag phase was estimated out of the transport curves.

The Pappwas calculated according to Eq. 3.

P i” = —-----”VD [cms’] Eq.3 At 10m A AQIz\t permeability rate (steady state transport rate) obtained from the profile of the trans ported amount of substrate versus time [s]. Calculated by the linear regression of time and concentration A area of the exposed skin ]2[cm m00 initial mass of test compound in the donor compartment [pg] or [dpm] 0V donor volume ]3[cm

Mass recovery

Results of experiments with test item are presented as a mass balance including the amount and percentage of the active compound permeated, penetrated and remaining in the donor compartment after the experiments. Based on the results of the three compartments a mass balance was determined according to Eq. 4 and 5. 7RC = 4m + rnosc + f0lfl + lflpQ + 1PDS {j.ig] Eq. 4 m mass of test compound in the three compartments after the experiment [pg] m mean value of the amount of test compound permeated through the tissue into the acceptor medium [pg]

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m05 mean value of the amount of remaining test compound on the skin in the test formula tion after the expermient [pg] mosc mean value of the amount of test compound content in two first tape strips [jig]

mpsc mean value of the amount of test compound penetrated into the Stratum corneum (18 tape strips) [jig] 5m mean value of the amount of test compound penetrated into the deeper skin layers [jig]

RC=--1OO [%] Eq.5 D0111 RC total recovery of the active compound after the experiment [%] m mass of test compound in the three compartments after the experiment [jig] m00 initial mass of test compound in the donor compartment [jig]

The overall mass recovery in the range of 100 ± 15 % should be reached according to SCCP.

Dose absorbed

Dose absorbed was calculated according to the Eq. 6.

DA A PDS [%] Eq.6

DA dose absorbed of test compound after the experiment [%] r recovery of test compound permeated through the tissue into the acceptor medium in

relation to the applied formulation amount [%] r0 recovery of test compound penetrated into the deeper skin layers (cryosections) in rela tion to the applied formulation amount [%]

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3. Results

3.1. HPLC method

3.1.1. System suitability test

Tab. 30 summarizes the results of the system suitability test. The test was performed by 6 injec tions from the same vial containing standard solution of CG in KRB-/- buffer.

Tab. 30: Summarized results of system suitability test (SST).

Sample Name lnj. No. Compound Retention time Area

SST 1 1 CG 5.800 2035000 2.2068 pg/mL

SST 2 2 CG 5.793 2064000 2.2068 pg/mL

SST 3 3 CG 5.800 2151000 2.2068 pg/mL

SST 4 4 CG 5.794 2093000 2.2068 pg/mL

SST 5 5 CG 5,793 2112000 2.2068 pg/mL SST 6 6 CG 5.800 2147000 2.2068 pg/mL

Mean - - - 2100333.33

SD - - - 45876.65

RSD% - . - 2.18

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I S

Fig. 6: First SST chromatogram of CO 2.2068 Jg/mL standard solution in matrix.

4 0

Fig. 7: Second SST chromatogram of CG 2.2068 pg/mL standard solution in matrix.

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3.1.2. Selectivity

Figures 7-1 2 compare the MS-chromatograms of the test compound in acceptor medium (KRB

without Glucose and HEPES), in MeOH/Water (50:50 v/v, %)in the donor medium and the blank chromatograms (eluents). With exception of the acceptor medium (slight interferences) there were no interferences with the compound.

- i-.- --- .

Fig. 7: Chromatogram of GCstandard solution in KRBbuffer (without Glucose and HEPES).The injec tion volume amounted to 20 jL.

4eooo

t4QO-

0 4

Fig. 8: Blank-chromatogram of KRB7.4 without Glucose and HEPES.

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ll : —I, 1IL L V I 1V

Fig. 9: Blank-chromatogram of methanol / water 50:50.

I =_ 4 :::

Fig. 10: Blank-chromatogram of eluent B (Methanol + 0.1% formic acid).

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I’ L\f \A \i

Fig. 11: Blank-chromatogram of eluent A (Water dest. ± 0.1% formic acid).

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3.1.3. Linearity in KRB-I- pH 7.4

The linearity data for the established HPLC method in KRB-/- pH 7.4 are depicted in the follow ing table and figure.

Tab. 31: Statistics of the calibration curve for CG KRB-/- buffer pH 7.4.

Response accord- Theoretical c ing to internal c found Deviation* Sample name standard 1[pg.mL 1[ig.mL 1%] ] [Area] KLP15 0.0986 593700 0.091 7.707

KLP14 01232 622000 0.12 2.597 KLP12 0.4929 1045000 0.548 -11.178 KLP11 0.8214 1343000 0,861 -4.821

KLP9 1.6428 2031000 1.627 0.961 KLP8 2.4643 2766000 2.528 -2.584

KLP7 8.2142 5242000 7.019 14.550

KLP6 10.2678 6160000 11.488 • -11.883

Slope second order 0 -43125.131 Slope first order m 500768.77

Intercept b 1 .02034e6

Correlation coefficient r 0.99651 Determination coefficient r2 0.99302

Weighting 121x * calculated by means of linear 2 regression.

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Sn, ! r.nw1D 2 -

y.IJII,h

0 4 0 0 I

Fig. 10: Calibration curve CG in KRB-/- pH 7.4.

The calibration curve is quadratic in the range 0.0986 — 10.2678 pg .1mL The deviation of the concentration found from the nominal concentration is within the limit (20 % for LLOQand 1 5 % for other concentrations).

3.1.4. Accuracy and Precision

The results from determination of accuracy and precision of CG in KRB-/- buffer are presented in the tables below:

Tab. 32: Accuracy and precision of the analytical method: CG in KRBwithout HEPES and glucose pH 7.4.

Concentration level CON1 (high) CON2 (mean) CON3 (low) ni 1IpgmL 7.545 1.930 0.068 n2 1[pg.mL 7.534 1.950 0.086 n3 ]1[pg.mL 7.465 2,01 0.071 Mean] IpgmLl 7.515 1.963 0.075 SD 1[pq.mL 0.043 0.042 0.010 RSD] [%J 0.577 2.121 12.858 Theoretical concentration 1[ig.mL 8.827 2.2068 0.0883 Accuracy [%] ] 85.14 88.95 84.94

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The values for precision and accuracy have complied the limits established in the FDA Guideline (RSD ± 15%forCONl -C0N3andaccuracyloo ± 15%).

Quantification limit

The limit of quantification for CG with a signal to noise ratio (SIN)= 5:1 amounts to 0.088 pg. 1mL in KRB pH 7.4 without HEPES and glucose for the validated HPLC method. The accuracy at the LLOQconcentration complies the limit of 100 ± 20 % (86.59 %)and precision RSD ± 20% (8.58 %).

3.1.5. Stability of the test product in donor and acceptor media

The stability of CG was tested over a period of 48 hours and measured at 3 time points (after 0,

24 and 48 hours) as presented in the tables below: Tab. 33: Stability of CGin KRBbuffer (2.3641 pg )1mL without glucose and HEPESat 0 hour. Medium KRB-/-buffer

Storage time Lhl 0

Storage temperature 4 C RT 32° C ni IpgmL1 2.145 2.298 2.302 n2 IpgmL1 2.331 2.322 2.275 n3 1g.mL- 2,305 2.356 2.259 Mean J1[pgmL 2.260 2.325 2.279 SD 1[pgmL 0.101 0.029 0.022 J 4.456 RSD I%1 1.253 0.954 Theoretical c J1[igmL 2.3641 2.3641 2.3641 Recovery (related to theoreti- 98.4 96.4 cal c)_[/o]

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Tab, 34: Stability of CC in KRB buffer without HEPES and glucose over 24 hours.

Medium KRB-/

Storage time [h] 24

Storage temperature 4 C RI 32° C ni [pg•mL’l 2.073 2.269 2.084 n2 1[ig.mL 2.248 2.155 2.063 J 2.155 n3[pg.mL 2.307 2.112 MeanJ1 1[pg.mL 2.159 2.244 2.086 SD 1[pgmL] 0.088 0.079 0.025 J 4.056 RSDI%1 3.526 1.178 Initial c 1IpgmL 2.260 2.325 2.279 Recovery (related to initial c) 95 5 96 5 91 5 L%1

Tab. 35: Stability of CC in KRB buffer without HEPES and glucose over 48 hours.

Medium KRB-/ Storage time [hj 48

Storage temperature 4 C RI 32° C ni 1[ig.mL 2.026 2.029 2.104 n2 1][pg•mL 2.061 2.013 2,135 n3 1[pg.mL 1.970 2.178 2.037 J 2.019 Mean 1[pgmL 2.073 2,092 J 0.046 SD 1[pg.mL 0.091 0,050 RSD[%]] 2.274 4.389 2.394 Initial c 1IpgmL 2.260 2.325 2.279 Recovery (relatedJ to initial c) 89 3 89 2 91 8 I%1

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Tab, 36: Stability of CG in HBSS buffer without glucose at 0 hour.

Medium HBSS- buffer

Storage time [hi 0

Storage temperature 4 C RT 32° C ni 1[ig.mL 2.144 2.101 2116 n2 1i[pg.mL 2.101 2.104 2.104 J 2.100 n3 1[pg.mL 2.072 2.091 Mean] 1[ig.mL 2.115 2.092 2.104 SD 1[pgmLi 0.025 0.018 0.013 RSDi [%] 1.188 0.845 0.594 Theoretical c 1[pgmL 2.0025 2.0025 2.0025 Recovery (related ]to theoreti- 105.6 104.5 105.1 cal c) [%]

Tab. 37: Stability of CO in HBSS- buffer without glucose over 24 hours.

Medium HBSS- buffer

Storage time [hi 24

Storage temperature 4 C RT 32° C ni 1[lJg.mL 1 .886 1,963 1.923 J 1 .898 1 1 n2 1IigmL .996 .926 n3 1[pg.mL 1 .851 2.003 1 .923 Mean] IpgmL’i 1 .878 1.987 1 .924 SD 1[pg.mL 0.024 0.021 0.002 RSDi 1%i 1.300 1.075 0.090 Initial c 1IpgmL 2.115 2.092 2.104 Recovery (related to initial c) i 89 3 95 0 91 0 [%j . .

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Tab. 38: Stability of CG in HBSS buffer without glucose over 48 hours.

Medium HBSS

Storage time IhI 48

Storage temperature 4 C RT 32° C ni J1[pgmL 1.884 1.814 1.935 n2 1[pgmL 1.855 1.792 1.956 1.829 n3 1[pg•mL.j 1.842 1 .905 Mean] 1[pg.mL 1.856 1.816 1 .932 SD 1[pg•mL 0.028 0.025 0,026 RSDI%1l 1.482 1.380 1.327 Initial c 1IpgmL 2.115 2.092 2.104 Recovery (relatedI to initial c) 87.8 86.8 91.8 [%J

The results demonstrate, that the test compound was stable over a period of 48 h at 32°C, room temperature and at 4 C in all tested media.

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3.2. Determination of CG content in the test solution

The results of the determination of the CG content in the test solution are summarized in the following table.

Tab. 39: Summary of CGcontent in the test solution, amount of 2000 p1 test solution 10 % CG (n=3),

Sample Weighted Theoretical Theoretical Measured Recovery [%J amount 1mg] amount lmgImL] amount after amount [ig!mL] dilution [pg/mi] n=1 2033 1.0001 1.0001 0.952 95.2 n=2 2033 1.0001 1.0001 0.964 96.4 n=3 2033 1.0001 1.0001 0,983 98.3

Mean - - - 0.97 96.63

SD - - - 0.016 1.563

The mean recovery was within the limits of 100 ± 10 %. Therefore the extraction procedure of CG from the test solution (10% CG) was successfully established.

Tab. 40: OlDcontent in the test solution after skin contact, amount of 30 l’ test solution 10 % CG

Sample Weighted amount Theoretical amount Measured amount Recovery 1%] [mgi Ipglmi] EigImi1 n=1 30.50 1.0201 0.901 90.1

The recovery of CG after application of 30 pL test solution 10 % CG was lower than in samples without any skin contact, but also within the limit of 100 ± 10 %.

3.3. Permeation study and penetration studies

Samples from in vitro experiments with the test item

In the Tab. 41 a short explanation of the samples further discussed in the report is depicted.

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Tab. 41: Samples from the in vitro experiments with the test item.

Sample name Description

dose applied amount of the test compound applied at the skin surface

receptor cumulative test compound content in the receptor medium after the permeation experiment

. test compound content remaining on the skin surface at the end of the skin wash permea tion experiment (formulation)

2 Tape strips test compound content in the two first tape strips (Stratum corneum) strips* 18 Tape test compound content in the 18 further tape strips (Stratum corneum)

Cryocuts* test compound content in the deeper skin (viable epidermis ± dermis)

dose absorbed sum of the test compound content in the receptor medium and in the deeper skin

total recovery mass recovery of the test compound content * Tape strip no. 3 — 20: the first third of stratum corneum; Cryocuts: residual skin (rest stratum corneum, epidermis, parts of dermis).

3.3.1. Establishment of extraction method for penetration study

To develop the extraction method for the in vitro penetration experiments, two different extra c tion media were tested: water and MeOH!water (50:50 vlv,%).The experiments were performed under non-GLP conditions. The table below presents an explanation from the denomination of the samples investigated.

Tab. 42: Denominationof samples employed in the extraction method experiments.

Sample name Description

2 2 tape-strips without skin

18 18 tape-strips without skin

2/SC 2 tape-strips withstratum corneum

18/SC 18 tape-strips with skin stratum corneum

Rest deeper skin (deeper epidermis ± dermis)

Recovery of CG with water as extraction medium

The Fig. 11 presents the recovery values of CG at two different concentrations: 3.62 .tg/mL and 0.89 ig/mL after the simulated extraction procedure with water as extraction medium.

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160

140

120

100

l3,622 [pg/mL] 80 D 0.897 [pg/mL]

0 2 18 2/Sc 18/Sc Rest

Number of strips I skin fraction

Fig. 11: Recoveryof CGafter the simulated extraction procedure with water as extraction medium. The values shown are the arithmetic mean values ± SD (n=2).

With water as extraction medium the mean recovery values of CG have shown a variation from

53.44 % to 86.25 % at the higher concentration (3.62 ig!mL) whereas the samples at lower concentration (0.89 .tglmL) the mean recovery valued have varied from 58.42 %to 1 20.9 2 %.

The lowest recovery values were found in the samples with 1 8 tape-strips without skin either at higher or lower concentration.

Recovery of CG with MeOHlwater (50:50 v/v.%)as extraction medium

The figure below presents the recovery values of CG at concentration 0.928 ig/mL and

3.643 ig/mL after the simulated extraction procedure with MeOH/water (50:50 v/v, %).

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140

120

100

80 I.. • 3.643 [pg/mL] 0 D0.928 [pg/mL] C) 60

0 2 18 2/sc 18/Se Rest

Number of strips I skin fraction

Fig. 12: Recovery of CG after the simulated extraction procedure with MeOH/water (50:50 v/v, %) as

extraction medium. The values shown are the arithmetic mean values ± SD (n=2).

With MeGH/water (50:50 vlv,%)as extraction medium the mean recovery values of CG have shown a variation from 88.65 % to 93.70 % for the samples higher concentrated (3.643 ig!mL), whereas the samples at 0.928 tg!mL have demonstrated recovery mean values from 94.40 % to 112.28 %. These results have complied to the specified in the SCCP Guide line (100 ±20 %),therefore MeOH/water (50:50 v/v %)was the selected extraction medium for performing the penetration experiments.

3.3.2. Results from permeation and penetration studies

Cumulative transport of CG from test product through skin no. 336-01 -0808, 337-01- 0908, 340-01 -0908

The CG amounts in the receptor over an incubation period of 24 hours were below of the LLOQ

(0.088 tg/mL) of the analytical method for the 3 skin donors (The area of he peaks found were similar to the negative control (pure acceptor medium)). This low absorption is confirmed by the recovery values found for the test solution, which remained on the skin surface (skin wash sam

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pies), which was 1 2549 % (mean of all 6 Franz-cells). This demonstrates that the CG has mostly remained on the skin surface for all the samples.

The results of the penetration study of CG from the test solution 10 % CG through skin no. 336-

01 -0808 are presented in the lab. 43. Tab. 43: Amount [pg ]2cm of CGin the samples from the in vitro experiment using skin no. 336-01 -0808. Cumulative amount [pg ]2cm of CG in the samples Sample FZ12 FZ5 Mean SD Diffusion area ]2[cm 3.03 3.08 3.06 0.03 dose applied 1018.81 1003.43 1011.12 10.87 receptor 0.00 0.00* 0.00* 0.00 skin wash 1331.04 1236.12 1283.58 67.12 2 Tape strips 5.21 6.67 5.94 1.03 18 Tape strips 2.24 2.12 2.18 0.08 cryocuts 0.00* 0,00* 0.00* 0.00 * Values under the LLOQof the analytical method.

The mass recovery of the test compound in the analysed samples is shown in Tab. 44.

Tab. 44: Recoveryand dose absorbed [%] of CGin the samples from the in vitro experiment using skin no. 336-01-0808,

Recovery and dose absorbed 1%]of CG in the samples from the in vitro experiment using skin no. 336-01 -0808

Sample FZ12 FZ5 Mean SD receptor 0.00* 0.00* 0.00* 000 skin wash 130.65 123.19 126.92 5.27

2 Tape strips 0.51 0.66 0.59 0.11 18 Tape strips 0.22 0.21 0.22 0.01 cryocuts 0.00* 0.00* 0,00* 0.00 Dose absorbed 0.00* 0.00* 0.00* 0.00* Total recovery 131.38 124.07 127.72 5.17 * Values under the LLOQof the analytical method

The mean total mass recovery of CG after the in vitro experiment using skin no. 336-01 -0808 sum up to 127.72 %. After the 24-hours incubation no quantifiable amount of CG has been found in the receptor medium and cryocuts.

The results of the penetration study of CG from the test solution 10 % CG through skin no. 337-

01-0908 are presented in the Tab. 45.

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Tab. 45: Amount [pg ]2cm of CG in the samples from the in vitro experiment using skin no. 337-01 - 0908, Cumulative amount Ipg ]2cm of CG in the samples Sample FZ 129 FZ 133 Mean SD Diffusion area ]2[cm 2.81 2.82 2.81 0.01 dose applied 1101.28 1095.47 1098,37 4.10 receptor 0.00* Q,QQ* 0.00* 0.00

skin wash 1369.80 11 96.96 1283.38 122.21

2 Tape strips 5.48 8.57 7.03 2.18 18 Tape strips 5.06 10.98 8.02 4,19 cryocuts 0.10 0.76 0.43 0.47 * Values below the LLOQof the analytical method.

The mass recovery of the test compound in the analyzed samples is shown in the table below.

Tab. 46: Recovery and dose absorbed [%]of CG in the samples from the in vitro experiment using skin no. 337-01 -0908,

Recovery and dose absorbed [%1of CG in the samples from the in vitro experiment using skin no. 337-01-0908

Sample FZ 129 FZ 133 Mean SD receptor 0.00* 0.00* 0.00* 0.00 skin surface 124.38 109,26 116.82 10.69 2 Tape strips 0.50 0.78 0.64 0.20

18 Tape strips 0.46 1.00 0.73 0.38 cryocuts 0.01 0.07 0.04 0.04 Dose absorbed 0.01 0.07 0.04 0.04 Total recovery 125.35 111.12 118.23 10.06 * Values below the LLOQof the analytical method.

The mean mass recovery of CG after the in vitro experiment using skin no. 337-01 -0908 sum up to 1 18.23%. After the 24-hours incubation 0.04 % of CG have been absorbed.

The results from the in vitro penetration experiment across skin 340-01 -0908 are depicted in the table below.

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Tab. 47: Amount [pg ]2cm of CG in the samples from the in vitro experiment using skin no. 340-01- 0908.

Cumulative amount [pg . 21cm of CG in the samples Sample FZ 125 FZ 14 Mean SD Diffusion area ]2[cm 2.82 2.88 2.85 0.04 Dose applied 1095.47 1072.71 1084,09 16.10 receptor 0.00* 0,00* 0.00* 0.00 skin wash 1323.97 1550.81 1437,39 160.40 2lapestrips 4.64 2.88 3.76 1.25 18 Tape strips 5.42 1.15 3,28 3.02 cryocuts 0.00* 0.00* 0,00* 0.00 * Values below the LLOQof the analytical method.

The mass recovery of the test compound in the analyzed samples is shown in table below.

Tab. 48: Recovery and dose absorbed [%]of CG in the samples from the in vitro experiment using skin no. 340-01 -0908.

Recovery and dose absorbed 1%]of CG in the samples from the in vitro experiment using skin no. 340-01 -0908

Sample FZ 125 FZ 14 Mean SD receptor 0,00* 0,00* 0.00* 0.00 skin surface 120.86 144.57 132.71 16.77 2 Tape strips 0.42 0.27 0.35 0.11 18 Tape strips 0.49 0.11 0.30 0.27 cryocuts 0.00* 0.00* 0.00* 0.00 Dose absorbed 0.00* 0.00* 0.00* 0.00 Total recovery 121.78 144.95 133.36 16.38 * Values below the LLOQof the analytical method.

The mean mass recovery of CG after the in vitro experiment using skin no. 340-01 -0908 sum up to 133.36 %. After the 24-hours incubation no quantifiable amount of CG has been found in the receptor medium and in the deeper skin layers.

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3.4. Quality control of the utilized skin samples (MEA)

The mean apparent permeability coefficients (Papp)measured for the Caffeine reference marker Q.8 was for skin no. 336-01 -0808 in mean 2.39 ± 0.86 1 1cm•s (n=2), for skin no. 337-01 - 0908 in mean 5.67 ± 0.11 8110cms (n=2) and for skin no. 340-01 -0908 in mean 6.38 ± 0.53 10 cms (n=2). The Pappwas calculated under steady state conditions from 0.5 to 24 hours.

The results of the Caffeine transport studies through all three dermatomized human skins are shown figure below.

10,00

9.00

8,00

7,00 -

6,00 - E

b 5,00 -

0.a 0 4,00 -

3,00

2,00

1,00

O,00i - Skin 336-01 -0808 Skin 337-01 -0908 Skin 340-01 -0908 Skin number

Fig. 13: Mean apparent permeability coefficients for the transport of Caffeine through human skin no. 336-01-0808, 337-01-0908 and 340-01-0908. The values shown are the arithmetic mean values ± SD (n’=2).

In an earlier study, the permeability of Caffeine from a 1 % aqueous solution across full- thickness skin, dermatomized skin, heat-separated epidermis and isolated SC had been studied at Across Barriers to establish quality assurance benchmarks for skin integrity [Bock et al., 2002]. Subsequent quality control studies have been performed on a range of human skin sam

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Across Barriers GmbH Seite 55 von 71 Intelligent Drug Profiling pies. The table below provides an overview of Caffeine permeabilities through dermatomized skin specimens measured at Across Barriers and includes the values determined in the present study.

Tab. 49: Comparison of apparent permeability coefficients for Caffeine through different dermatomized skin specimens with intact SC. Skin no. 336-01 -0808, 337-01-0908 and 340-01-0908 were used in the present study.

Skin number Mean Papplcms (n=3) RSD 1%] 061-01-0701 ]1 9.38E-08 29 153-01 -01 04 8.41 E-08 28

059-01 -0601 8.24E-08 39

060-01 -0601 5.63E-08 6

062-01-0701 5.34E-08 18

155-01-0204 5.03E-D8 27

209-01-0605 3,82E-08 7 157-01-0304 3,23E-08 27

057-01-0601 3.21E-08 16 150-01-1203 2.54E-08 56

058-01-0601 2.OOE-08 7

336-01-0808 2.39E08* 36

337-01-0908 5.67EO8* 3

340-01-0908 6.38EO8* 8 *n2

The mean Pappvalues measured for Caffeine in the present study are in good agreement with values determined for a variety of skin specimens from different donors under comparable conditions. This demonstrates the integrity of the skin biopsies used and their suitability for use in transport studies with the test formulation.

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4. Discussion

4.1. HPLC method development and validation

The HPLCmethod was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRB-/- buffer). There were minor but acceptable interferences with the acceptor medium (KRB-!-). In addition, CG has presented good stability in the acceptor and donor media at 3 different temperatures (4 °C, RTand 32 °C) over the period of 48 hours.

4.2. Determination of CG content in the test solution

The content of CG was determined in triplicate in the test solution without skin contact employed in the experiments. The mean concentration value was 0.97 ig/mL which corresponds to the recovery mean value of 96.63 ± 1 .563. The content of the test solution after the skin contact was 0.901 jiglmL,corresponding to 90.10 %. This complies with the acceptance value 100 ± 10%.

4.3. Establishment of the extraction method for penetration study

Two extraction media were investigated for the penetration studies: MeOH/water (50:50 v/v, %) and water. With water as extraction medium the mean recovery values of CG has shown a varia tion from 53.44 % to 120.12 % (considering all the samples) whereas with MeOH/water (50:50 v/v, %)the mean recovery values of CG have ranged from 88.65 % to 112.28 % (considering all the samples). The results demonstrated that MeOH/water (50:50 v/v, %)was the most suitable extraction medium for the penetration experiments, since the recovery values have complied to the limit of 100 ± 20 % specified in the SCCP Guideline.

4.4. Permeation study

The results from the in vitro permeation of CG from the test product through skin no. 336-01 - 0808, 337-01 -0908 and 340-01 -0908 have demonstrated that the test compound permea tion after 24 hours was below the LLOQof the analytical method (0.088 j.iglmL)(The area of he peaks found were similar to the negative control (pure acceptor medium)). Another fact which support this low permeation are the recovery values found for the test product which remained on the skin surface. These values have varied in the 6 Franz-cells from 109.20 % to 144.57 %, which demonstrated that the CG has mostly remained on the skin surface for all the samples.

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4.5. Penetration study

The mean amounts of CG found in the samples from the three in vitro transport experiments are summarized in the table below. Tab. 50: Mean amount [pg ]2cm- of CG in the samples from the three in vitro experiments. Cumulative amount [pg ]2cm of CG in the samples Skin 336-01-0808 Skin 337-01-0908 Skin 340-01-0908 All skins used Sample Mean SD Mean SD Mean SD Mean SD dose ap plied 1011.12 1087 1098.37 4,10 1084.09 16.10 1064.53 42.79 receptor 0.00* 0.00 0.00* 0.00 0.00* 0.00 0.00* 0.00 skin wash 1283.58 67.12 1283,38 122.21 1437.39 160.40 1334.78 123.90 2 Tape strips 5.94 1.03 7.03 2.18 3.76 1.25 5.58 1.92 18 Tape strips 2.18 0.08 8.02 4.19 3.28 3.02 4.49 3.61 cryocuts 0.00* 0.00* 0.43 0.47 0.00* 0.00* 0.14 0.31 * Values below the LLOQof the analytical method.

Tab. 51: Mean recovery rate and dose absorbed [%]of CG in the samples from the three in vitro experi ments.

Recovery and dose absorbed [%]of CG in the samples

Sample Skin 336-01-0808 Skin 337-01-0908 Skin 340-01-0908 All skins used Mean SD Mean SD Mean SD Mean SD receptor 0.00* 0.00 0.00* 0.00 Q•QQ* 0.00 0.00* 0.00 skin surface 126.92 5.27 116.82 10.69 132.71 16.77 125.48 10.66 2 Tape strips 0.59 0.11 0.64 0.20 0.35 0.11 0.52 0.18 18 Tape strips 0.22 0.01 0.73 0.38 0.30 0.27 0.30 0.32 cryocuts 0.00* 0.00 0.04 0.04 0,00* 0.00 0.01 0.03 Dose absorbed 0.00* 0.00 0.04 0.04 0.00* 0.00 0.01 0.03 Total recovery 127.72 5.17 118.23 10.06 133.36 16.38 126.44 11.23

* Values below the LLOQof the analytical method.

The mean amount of CGremoved from the skin surface (skin wash) ranged from 1 16.82 % to 132.71 % of the dose applied in the mean values of the 3 skin donors. This demonstrates that the CG has mostly remained on the skin surface. The amounts in the receptor could not be

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quantified for the skin donors 336-01 -0808 and 340-01 -0908 since it was below the analytical

LLOQ.The recovery value of the dose absorbed for the skin 337-01 -0908 was accounted to 0.04 %. Together with skin no. 340-01 -0908 (comparable P values) skin sample no. 337-01 - 0908 also exhibited the highest permeation rates for caffeine.

The mean recovery in the two first tape strips was 0.52 % during all performed experiments. In the further 18 tape strips a mean recovery of 0.30 % was documented. The recovery values for the cryocuts have accounted 0.04 % only for the skin 337-01 -0908. For the other skin donors

CG was not detectable in the samples with cryocuts (below the analytical LLOQ).

The mean absorbed dose of CG, sum of the amounts found in the viable epidermis, dermis and receptor medium was 0.] %, which has accounted only the dose absorbed in the skin 337-01 - 0908.

The calculated total recovery rate of CG for the three different skin donors used was 126.44 %. The mean recovery values have varied from 11 8.23 % until 133.36 %, which does not comply to the aspired acceptance criteria of 100 ± 15 %. The reason for this could be an interference of skin components that liberated during the permeation experiment and disturb the measure ment or the adding up of the deviations of the single analytical validation steps and of the in vitro experiment itself.

4.6. Quality control of the utilized skin (MEA)

The transport rates of caffeine demonstrate that the utilized human skins no. 336-01 -0908, 337-01 -0908 and 340-01 -0408 represent intact tight barrier properties, which are congruent to data obtained on other human skin biopsies under comparable study design.

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5. References

Bock, U., Schmitz, S. and Haltner, E.: In vitro systems to characterize dermal permeation and penetration, in: The essential Stratum corneum edited by Ronald Marks, Jean-Luc [eve

que and Rainer Voegeli Martin Dunitz 2002, page 157-1 60.

Kuhn, A. and Neubert, R.: Characterization of mixtures of alkyl polyglycosides (Plantacare) by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrome try, in: Pharmaceutical research, Vol. 21, No. 12, 2004, page 2347-2353.

Wagner, Heike: Charakterisierung des Arzneistofftransportes in Humanhaut unter in-vitro und in vivo Bedingungen unter Berucksichtigung des Einflusses zweier in-vitro Testsysteme, Uni versität des Saarlandes 2001

6. Related guidelines

Organization for Economic Co-operation and Development (OECD), OECD Guideline for the test

ing of chemicals 428 (2004): Skin Absorption: in vitro Method.

Organization for Economic Co-operation and Development (OECD), Guidance Document for the conduct of skin absorption studies No. 28, 2004.

European Commission, The SCCPs (Scientific Committee on Cosmetic Products) Notes of Guid ance for the testing of cosmetic ingredients and their safety evaluation. Adopted at 10th

plenary meeting of 19 December 2006.

European Commission, The Scientific Committee on Cosmetic Products (SCCP), Opinion on Ba sis Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients- up dated on March 2006.

U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER)and Center for Veterinary Medicine (CVM):Guidance for Industry: Bioanalytical Method Validation (May 2001)

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Data storage and archiving

Alldocuments and raw data willbe archived in the project file C-i 0335-244-1 107 in print ver sion and in the GLP archive of Across Barriers for 15 years.

Archived documents at Across Barriers: • German study plan and English Translation (md. Amendments)

• Relevant correspondence between sponsor and Across Barriers

• Cover sheets of the records of all inspections performed by the QAduring the study

• Exact copy of relevant lab journal pages

• Method parameters for HPLC

• Raw data

• Data calculation and evaluation

• GLP study report

The test product will be archived until expiration date at Across Barriers.

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7. Time Line

Tab. 52: Time line of the study.

Date planned Topic Responsibility

03.11.2008 HPLC method transfer ACB - 14.11.2008 (non GLP)

18.11.2008 Establisment of the extraction ACB - 21.11 .2008 method (non GLP)

18.11.2008 Validation of analytical method ACB - 28.11.2008

21 .11 .2008 Assay, drug content in ACB 24.11.2008 test solution

In vitro experiments:

permeability, CG in 28.1 1 .2008 content the CO in ACB - 03.12.2008 formulation, content Stra- turn corneurn, CO content in the deeper skin

Multiple endpoint analysis 16.10. 2008 -21.10.2008 (MEA) (see study C-10335-227- ACB 1107)

18.11.08 Studyaudit ACB

08.01 .09 Draft Report ACB

30.04.09 Final Report ACBI Sponsor

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c) C)

9O429_FinaIreport_cjo335_24411O7STP_O44.doc TawfikJalal

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9. Appendix

9.1. HPLC method

Fig. 14: Chromatogram of CG standard solution in KRB buffer (without glucose and HEPES). The injection volume amounted to 20 pL.

Fig. 15: Chomatogram of the acceptor matrix (KRB-/-) during permeation study.

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Fig. 16: Representative permeation chromatogram of the acceptor sample, transport of CG after 0.5 hours (skin no.336-01 -0808).

I z______)

Fig. 17: Representative chromatogram of CG sample (skin wash) after the penetration study.

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—

______j__j__-_

Fig. 18: Representative chromatogram of CG sample extracted with methanol / water (50:50) from the Stratum Corneum 2 TS/ penetration study.

2 2 4 0 7 S

Fig. 19: Representative chromatogram of CG sample extracted with methanol / water (50:50) from the Stratum Corneum 18 TS/ penetration study.

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\\\j _11

- 7

Fig. 20: Representative chromatogram of CG sample extracted with methanol / water (50:50) from the deeper skin layers (cryo-cuts/ penetration study).

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9.2. Establishment of the extraction method

The following table shows which samples were pooled and extracted together for the establish ment of the extraction medium.

Sample schedule

Sample name Description

p_ Transport solution (Reference solution)

2 2 Tape strips without Stratum corneum

18 18 Tape strips without Stratum corneum

2ISC 2 Tape strips with Stratum corneum

18!SC 18 Tape strips with Stratum corneum

Rest Deeper Skin (Cryo-cuts)

9.2.1. Establishment of the extraction method with MeOHlwater (50:50, vlv%)

Recovery of CO (3.643 pqlmL) with MeOHlwater (50:50, viv %) n=1 n=2 MW(n=2) Sample ro 1 SD L%1 pglmL % pglmL % 1’°i 2 3.21 87.98 3.29 90.37 89.17 1.19 18 3.32 91.02 3.25 89,10 90.06 0.96 215C 3.56 97.78 3.27 89.62 93.70 4.08 18ISC 3.35 91.82 3.43 94.02 92.92 1.10 Rest 3.14 86.11 3.32 91,19 88.65 2.54

Recovery of CG (0.928 .igImL) with MeOHIwater (50:50, vlv %) n=1 n=2 MW(n=2) Sample SD 1%] igImL % pglmL [“°J 2 0.84 90.63 0.95 101.83 96.23 5.60 18 0.85 91.16 0.91 97.63 94.40 3.23 2!SC 1,03 110.99 1.03 110.99 110.99 0.00 18!SC 0.91 97.95 1.18 126,62 112.28 14.33 Rest 0.74 79.20 1.14 122.63 100.92 21.71

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9.2.2. Establishment of the extraction method with water

Recovery of CG (3.622 pglml) with water n=1 n=2 MW(n=2) Sample SD [%] pglmL % pglmL 2 3.01 83,13 2.97 81.89 82.51 0.62 18 1.97 54.47 1.90 52.40 53.44 1.04 2ISC 3.13 86.44 3.12 86.06 86.25 0.19 1815C 2.90 80.15 2.76 76.15 78.15 2.00 Rest 3.06 84.43 2.79 77.11 80.77 3.66

Recovery of CG (0.897 pglmL) with water n=1 n=2 MW(n=2) Sample SD [%1 pglmL % pg!mL 2 0.82 91.75 0.76 84.39 88.07 3.68 18 0.53 59.53 0.51 57.30 58.42 1.11 2ISC 0.83 91.97 0.83 92.98 92.47 0.50 1815C 0.88 97.66 1.28 142.59 120.12 22.46 Rest 0.71 78.60 0.70 78.48 78.54 0.06

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9.3. Franz cell volumes and areas Franz cell no. Acceptor volume [ml] Exposed area ]2[cm 5 20.46 3.08 12 20.31 3.03

129 19.61 2.81 133 19.81 2.82 125 19.79 2.82 14 18.09 2.88

9.4 Thickness of the human skins

Measured thickness [pm] of skin no. 336-01-0808 FZ 1 2 3 4 5 Mean SD RSD [%]

12 530.00 542.00 487.00 512.00 556.00 525.40 26.87 5.11

5 526.00 532.00 522.00 545.00 568.00 538.60 18.60 3.45

Measured thickness [pm] of skin no. 337-01-0908 FZ 1 2 3 4 5 Mean SD RSD [%J 129 523.00 51 6.00 493.00 449.00 536.00 503.40 34.18 6.79

133 578.00 585.00 596.00 51 5.00 544.00 563.60 33,40 5.93

Measured thickness [pm] of skin no. 340-01 -0908 FZ 1 2 3 4 5 Mean SD RSD [%] 125 574.00 587.00 580.00 556.00 566.00 572.60 12.07 2.11

14 579.00 618.00 548.00 526.00 591.00 572.40 36.12 6.31

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9.4. Quality control of the human skin (MEA)

9.4.1. Transport of Caffeine (1%) through skin

Concentration Ipg•mL of Caffeine in the acceptor during transport through 3 1 skin donors Time [hi l 336-01 -0808 337-01-0908 340-01 -0908 All skins Mean SD Mean SD Mean SD Mean SD 0.50 0,00 0.00 0.13 0.10 0.14 0.03 0.09 0.04 2.00 0.00 0.00 0.22 0.20 0.29 0.16 0.17 0.12 4.00 0.33 0.07 0.85 0.05 0,98 0.30 0.72 0.14 6.00 0.59 0.41 1.15 0.31 1.58 0.44 1.11 0.39 21.00 2.60 0.80 5.77 0.30 6.37 0.44 4.91 0.51 22.00 2.34 0.95 6.06 0.12 6.95 0.11 5.12 0.39 23.00 2.98 1.35 6.65 0.49 7.78 0.24 5.80 0.69 24.00 2.89 1.39 7.09 0.20 8.51 0.73 6.16 0.77

Cumulative transport 2[pg.cm of Caffeine through through 3 skin donors Time [hi 336-01-0808 337-01-0908] 340-01-0908 All skins Mean SD Mean SD Mean SD Mean SD 0.50 0.00 0.00 0.85 0.71 0.93 0.16 0.59 0.29 2.00 2.04 0.00 1.48 1.37 1.87 1.02 1.80 0.80 4.00 2.20 0.36 5.76 0.33 6.45 1.87 4.80 0.85 6.00 3.95 2.62 7.81 2.13 10.43 3.13 7.40 2.62 21.00 17.56 4.72 39.04 2.20 42.02 3.71 32.87 3.54 22.00 15.79 5.80 40.99 0.65 45.82 1.60 34.20 2.68 23.00 20.06 8.37 45.02 3.49 51.25 2.59 38.78 4.82 24.00 19.43 8.64 47.98 1.57 56.10 5.91 41.17 5.37

-8 1 2.39E- 8.58E- 5.67E- 1.09E- 6.38E- 5.30E- 4.81E- 4.99E- Papp[10 cms 08 09 08 09 08 09 08 09

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9.5. Transport studies with the test formulation (permeation results)

9.5.1. Transport of CG from test solution through 3 different skin donors into the re ceptor medium

Concentration [jig.mL’] of CG after transport from the test solution through 3 skin donors

Time [hi FZ 5 FZ 12 FZ 129 FZ 133 FZ 125 FZ 14 050 000* 000* 000* 000* 000* 000*

2 00 0 QQ* 0 00* 0 00* 0 00* 0 00* 0 O0

4 00 0 QQ* 0 00* 0 QQ* 0 00* 0 00* 0 00*

6 00 0 00* 0 00* 0 00* 0 00* 0 00* 0 00*

21 00 0 00* 0 00* 0 00* 0 QQ* 0 00* 0 00* 2200 000* 000* 000* 000* 000* 000* 2300 000* 000* 000* O00 000* 000* 24.00 0.00* 0.00* 0.00* 0.00* 0,00* 0.00* * Values below LLOQ

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DECYL GLUCOSIDE 6 01A - Baby Shampoos DECYL GLUCOSIDE 1 01B - Baby Lotions, Oils, Powders, and Creams DECYL GLUCOSIDE 18 01C - Other Baby Products DECYL GLUCOSIDE 6 02B - Bubble Baths DECYL GLUCOSIDE 3 02D - Other Bath Preparations DECYL GLUCOSIDE 1 03B - Eyeliner DECYL GLUCOSIDE 1 03D - Eye Lotion DECYL GLUCOSIDE 4 03E - Eye Makeup Remover DECYL GLUCOSIDE 4 03F - Mascara DECYL GLUCOSIDE 2 03G - Other Eye Makeup Preparations DECYL GLUCOSIDE 86 05F - Shampoos (non-coloring) DECYL GLUCOSIDE 4 05G - Tonics, Dressings, and Other Hair Grooming Aids DECYL GLUCOSIDE 4 05I - Other Hair Preparations DECYL GLUCOSIDE 9 06D - Hair Shampoos (coloring) DECYL GLUCOSIDE 2 07C - Foundations DECYL GLUCOSIDE 1 07H - Makeup Fixatives DECYL GLUCOSIDE 1 07I - Other Makeup Preparations DECYL GLUCOSIDE 141 10A - Bath Soaps and Detergents DECYL GLUCOSIDE 58 10E - Other Personal Cleanliness Products DECYL GLUCOSIDE 3 11E - Shaving Cream DECYL GLUCOSIDE 1 11G - Other Shaving Preparation Products DECYL GLUCOSIDE 106 12A - Cleansing DECYL GLUCOSIDE 15 12C - Face and Neck (exc shave) DECYL GLUCOSIDE 2 12D - Body and Hand (exc shave) DECYL GLUCOSIDE 1 12F - Moisturizing DECYL GLUCOSIDE 7 12H - Paste Masks (mud packs) DECYL GLUCOSIDE 4 12J - Other Skin Care Preps DECYL GLUCOSIDE 1 13C - Other Suntan Preparations

ARACHIDYL GLUCOSIDE 2 03D - Eye Lotion ARACHIDYL GLUCOSIDE 1 03G - Other Eye Makeup Preparations ARACHIDYL GLUCOSIDE 1 11A - Aftershave Lotion ARACHIDYL GLUCOSIDE 2 12A - Cleansing ARACHIDYL GLUCOSIDE 18 12C - Face and Neck (exc shave) ARACHIDYL GLUCOSIDE 10 12D - Body and Hand (exc shave) ARACHIDYL GLUCOSIDE 1 12E - Foot Powders and Sprays ARACHIDYL GLUCOSIDE 15 12F - Moisturizing ARACHIDYL GLUCOSIDE 8 12G - Night ARACHIDYL GLUCOSIDE 4 12J - Other Skin Care Preps ARACHIDYL GLUCOSIDE 6 13A - Suntan Gels, Creams, and Liquids ARACHIDYL GLUCOSIDE 6 13B - Indoor Tanning Preparations ARACHIDYL GLUCOSIDE 1 13C - Other Suntan Preparations

C12-20 ALKYL GLUCOSIDE 1 03D - Eye Lotion C12-20 ALKYL GLUCOSIDE 1 03G - Other Eye Makeup Preparations C12-20 ALKYL GLUCOSIDE 3 05A - Hair Conditioner C12-20 ALKYL GLUCOSIDE 1 05F - Shampoos (non-coloring) C12-20 ALKYL GLUCOSIDE 2 05G - Tonics, Dressings, and Other Hair Grooming Aids C12-20 ALKYL GLUCOSIDE 7 12A - Cleansing C12-20 ALKYL GLUCOSIDE 20 12C - Face and Neck (exc shave) C12-20 ALKYL GLUCOSIDE 6 12D - Body and Hand (exc shave) C12-20 ALKYL GLUCOSIDE 5 12F - Moisturizing C12-20 ALKYL GLUCOSIDE 1 12H - Paste Masks (mud packs) C12-20 ALKYL GLUCOSIDE 1 12I - Skin Fresheners C12-20 ALKYL GLUCOSIDE 4 12J - Other Skin Care Preps C12-20 ALKYL GLUCOSIDE 2 13B - Indoor Tanning Preparations

CAPRYLYL/CAPRYL GLUCOSIDE 1 03D - Eye Lotion CAPRYLYL/CAPRYL GLUCOSIDE 3 03E - Eye Makeup Remover CAPRYLYL/CAPRYL GLUCOSIDE 2 03G - Other Eye Makeup Preparations CAPRYLYL/CAPRYL GLUCOSIDE 2 05A - Hair Conditioner CAPRYLYL/CAPRYL GLUCOSIDE 5 05F - Shampoos (non-coloring)

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CAPRYLYL/CAPRYL GLUCOSIDE 1 07I - Other Makeup Preparations CAPRYLYL/CAPRYL GLUCOSIDE 3 10A - Bath Soaps and Detergents CAPRYLYL/CAPRYL GLUCOSIDE 3 10E - Other Personal Cleanliness Products CAPRYLYL/CAPRYL GLUCOSIDE 15 12A - Cleansing CAPRYLYL/CAPRYL GLUCOSIDE 8 12C - Face and Neck (exc shave) CAPRYLYL/CAPRYL GLUCOSIDE 2 12D - Body and Hand (exc shave) CAPRYLYL/CAPRYL GLUCOSIDE 3 12F - Moisturizing CAPRYLYL/CAPRYL GLUCOSIDE 2 12G - Night CAPRYLYL/CAPRYL GLUCOSIDE 7 12J - Other Skin Care Preps CAPRYLYL/CAPRYL GLUCOSIDE 1 13A - Suntan Gels, Creams, and Liquids

CETEARYL GLUCOSIDE 2 01B - Baby Lotions, Oils, Powders, and Creams CETEARYL GLUCOSIDE 1 01C - Other Baby Products CETEARYL GLUCOSIDE 1 02D - Other Bath Preparations CETEARYL GLUCOSIDE 1 03C - Eye Shadow CETEARYL GLUCOSIDE 33 03D - Eye Lotion CETEARYL GLUCOSIDE 5 03F - Mascara CETEARYL GLUCOSIDE 22 03G - Other Eye Makeup Preparations CETEARYL GLUCOSIDE 3 04E - Other Fragrance Preparation CETEARYL GLUCOSIDE 1 05F - Shampoos (non-coloring) CETEARYL GLUCOSIDE 2 05G - Tonics, Dressings, and Other Hair Grooming Aids CETEARYL GLUCOSIDE 1 05I - Other Hair Preparations CETEARYL GLUCOSIDE 2 07C - Foundations CETEARYL GLUCOSIDE 1 07F - Makeup Bases CETEARYL GLUCOSIDE 1 07I - Other Makeup Preparations CETEARYL GLUCOSIDE 1 08B - Cuticle Softeners CETEARYL GLUCOSIDE 1 08C - Nail Creams and Lotions CETEARYL GLUCOSIDE 3 10A - Bath Soaps and Detergents CETEARYL GLUCOSIDE 2 10E - Other Personal Cleanliness Products CETEARYL GLUCOSIDE 1 11A - Aftershave Lotion CETEARYL GLUCOSIDE 11 12A - Cleansing CETEARYL GLUCOSIDE 64 12C - Face and Neck (exc shave) CETEARYL GLUCOSIDE 60 12D - Body and Hand (exc shave) CETEARYL GLUCOSIDE 164 12F - Moisturizing CETEARYL GLUCOSIDE 37 12G - Night CETEARYL GLUCOSIDE 14 12H - Paste Masks (mud packs) CETEARYL GLUCOSIDE 17 12J - Other Skin Care Preps CETEARYL GLUCOSIDE 3 13A - Suntan Gels, Creams, and Liquids CETEARYL GLUCOSIDE 22 13B - Indoor Tanning Preparations CETEARYL GLUCOSIDE 1 13C - Other Suntan Preparations

COCO-GLUCOSIDE 3 01A - Baby Shampoos COCO-GLUCOSIDE 8 01C - Other Baby Products COCO-GLUCOSIDE 2 02A - Bath Oils, Tablets, and Salts COCO-GLUCOSIDE 10 02B - Bubble Baths COCO-GLUCOSIDE 2 02D - Other Bath Preparations COCO-GLUCOSIDE 1 03D - Eye Lotion COCO-GLUCOSIDE 3 03E - Eye Makeup Remover COCO-GLUCOSIDE 2 03G - Other Eye Makeup Preparations COCO-GLUCOSIDE 4 05A - Hair Conditioner COCO-GLUCOSIDE 52 05F - Shampoos (non-coloring) COCO-GLUCOSIDE 3 05I - Other Hair Preparations COCO-GLUCOSIDE 15 06A - Hair Dyes and Colors (all types requiring caution statements and patch tests) COCO-GLUCOSIDE 1 06D - Hair Shampoos (coloring) COCO-GLUCOSIDE 113 10A - Bath Soaps and Detergents COCO-GLUCOSIDE 3 10C - Douches COCO-GLUCOSIDE 47 10E - Other Personal Cleanliness Products COCO-GLUCOSIDE 1 11D - Preshave Lotions (all types) COCO-GLUCOSIDE 3 11E - Shaving Cream COCO-GLUCOSIDE 1 11G - Other Shaving Preparation Products COCO-GLUCOSIDE 48 12A - Cleansing COCO-GLUCOSIDE 7 12C - Face and Neck (exc shave) COCO-GLUCOSIDE 12 12D - Body and Hand (exc shave)

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COCO-GLUCOSIDE 1 12E - Foot Powders and Sprays COCO-GLUCOSIDE 5 12F - Moisturizing COCO-GLUCOSIDE 3 12J - Other Skin Care Preps

ETHYL GLUCOSIDE 2 03D - Eye Lotion ETHYL GLUCOSIDE 1 10A - Bath Soaps and Detergents ETHYL GLUCOSIDE 8 12A - Cleansing ETHYL GLUCOSIDE 4 12C - Face and Neck (exc shave) ETHYL GLUCOSIDE 3 12F - Moisturizing ETHYL GLUCOSIDE 1 12G - Night ETHYL GLUCOSIDE 1 12H - Paste Masks (mud packs) ETHYL GLUCOSIDE 1 12I - Skin Fresheners ETHYL GLUCOSIDE 3 12J - Other Skin Care Preps

HEXADECYL D-GLUCOSIDE 1 12F - Moisturizing

LAURYL GLUCOSIDE 4 01A - Baby Shampoos LAURYL GLUCOSIDE 1 01C - Other Baby Products LAURYL GLUCOSIDE 1 02A - Bath Oils, Tablets, and Salts LAURYL GLUCOSIDE 15 02B - Bubble Baths LAURYL GLUCOSIDE 14 02D - Other Bath Preparations LAURYL GLUCOSIDE 1 05A - Hair Conditioner LAURYL GLUCOSIDE 1 05E - Rinses (non-coloring) LAURYL GLUCOSIDE 65 05F - Shampoos (non-coloring) LAURYL GLUCOSIDE 1 05G - Tonics, Dressings, and Other Hair Grooming Aids LAURYL GLUCOSIDE 2 05I - Other Hair Preparations LAURYL GLUCOSIDE 13 06A - Hair Dyes and Colors (all types requiring caution statements and patch tests) LAURYL GLUCOSIDE 1 06B - Hair Tints LAURYL GLUCOSIDE 1 06D - Hair Shampoos (coloring) LAURYL GLUCOSIDE 131 10A - Bath Soaps and Detergents LAURYL GLUCOSIDE 2 10C - Douches LAURYL GLUCOSIDE 55 10E - Other Personal Cleanliness Products LAURYL GLUCOSIDE 2 11A - Aftershave Lotion LAURYL GLUCOSIDE 72 12A - Cleansing LAURYL GLUCOSIDE 3 12C - Face and Neck (exc shave) LAURYL GLUCOSIDE 3 12D - Body and Hand (exc shave) LAURYL GLUCOSIDE 4 12F - Moisturizing LAURYL GLUCOSIDE 1 12G - Night LAURYL GLUCOSIDE 1 12H - Paste Masks (mud packs) LAURYL GLUCOSIDE 5 12J - Other Skin Care Preps

MYRISTYL GLUCOSIDE 2 03D - Eye Lotion MYRISTYL GLUCOSIDE 1 12A - Cleansing MYRISTYL GLUCOSIDE 1 12D - Body and Hand (exc shave) MYRISTYL GLUCOSIDE 1 12F - Moisturizing

OCTADECYL D-GLUCOSIDE 1 12F - Moisturizing

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