Safety Assessment of Tissue-Derived Proteins and Peptides as Used in Cosmetics

Transcription

1 Safety Assessment of Tissue-Derived Proteins and Peptides as Used in Cosmetics Status: Draft Report for Panel Review Release Date: May 19, 2017 Panel Meeting Date: June 12-13, 2017 The 2017 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is Lillian J. Gill, DPA. This safety assessment was prepared by Christina L. Burnett, Scientific Analyst/Writer and Bart Heldreth, Ph.D., Chemist CIR. Cosmetic Ingredient Review 1620 L St NW, Suite 1200 Washington, DC ph fax cirinfo@cir-safety.org

2 Commitment & Credibility since 1976 Memorandum To: CIR Expert Panel Members and Liaisons From: Christina L. Burnett, Senior Scientific Writer/Analyst Date: May 19, 2017 Subject: Draft Safety Assessment on Tissue-Derived Proteins and Peptides Enclosed is the Draft Report of the Safety Assessment of Tissue-Derived Proteins and Peptides as Used in Cosmetics. (It is identified as tsupep062017rep in the pdf document). In April 2017, CIR issued the Scientific Literature Review (SLR) for these ingredients, which mainly function as skin and hair conditioning agents in personal care products. The majority of the ingredients in this report were originally included in a larger hydrolyzed source proteins safety assessment from At the time, the Panel determined to divide the ingredients in that larger report into smaller, more manageable safety assessments, with Hydrolyzed Wheat Protein and Hydrolyzed Wheat Gluten being the first ingredients reviewed. The Panel agreed upon the grouping of the 19 ingredients in this current safety assessment in September The Panel has previously reviewed the safety of Hydrolyzed Collagen and concluded that it was safe for use in cosmetics. This safety assessment was published in 1985 and the re-review that reaffirmed the conclusion was published in These publications are included in this report package (identified as tsupep062017oldrep1 and tsupep062017oldrep2). Applicable unpublished data were provided previously by the Personal Care Products Council (Council) for the larger hydrolyzed source proteins safety assessment; the Panel reviewed those data in These data, along with the most current concentration of use survey, have been incorporated into the report and included in this report package as a refresher (identified as tsupep062017data1 through tsupep062017data5). Comments provided by the Council on the SLR have been considered (tsupep062017pcpc). According to 2017 FDA VCRP data, Soluble Collagen is used in 425 formulations; the majority of uses are in leave-on skin care products. Gelatin has the second greatest number of overall uses reported, with a total of 334; the majority of the uses are in rinse-off bath soaps and detergents. The results of the concentration of use survey conducted in 2016 by the Council indicate Collagen has the highest reported maximum concentration of use; it is used at up to 96% in face and neck skin care products. Gelatin is used at up to 66% in bath oils, tablets, and salts. All others are used at much lower concentrations. In 2017, Hydrolyzed Collagen was reported to be used in 543 formulations; the majority of uses are in leave-on skin care products. The number of uses of Hydrolyzed Collagen have declined since the initial safety assessment in 1981 and the rereview in 2002 (923 and 570 uses, respectively). The maximum use concentration of Hydrolyzed Collagen was reported to be 16.5% in hair tonics and dressings in 2017; it was previously reported to be used at concentrations greater than 50%. If no further data are needed, the Panel should formulate a Discussion and issue a Tentative Report. However, if additional data are required, the Panel should be prepared to identify those needs and issue an Insufficient Data Announcement L St NW, Suite 1200, Washington, DC (Main) (Fax) ( ) cirinfo@cir-safety.org (Website)

3 SAFETY ASSESSMENT FLOW CHART INGREDIENT/FAMILY Tissue-Derived Proteins and Peptides MEETING June 2017 Public Comment CIR Expert Panel Report Status Priority List INGREDIENT PRIORITY LIST 60 day public comment period SLR April 13, 2017 Draft Report DRAFT REPORT June 2017 Table Table IDA TR IDA Notice Draft TR IDA DRAFT TENTATIVE REPORT Table Table Tentative Report Issue TR 60 day Public comment period Draft FR DRAFT FINAL REPORT Table Table Different Conclusion PUBLISH Final Report Issue FR

4 Tissue-Derived Proteins and Peptides History April 2017 Scientific Literature Review announced.

5 Tissue-Derived Protein and Peptide Ingredients Data Profile June 2017 Writer, Christina Burnett In-Use Physical/Chemical Properties Molecular Weight Range Method of Manufacturing Composition/Impurities Acute Toxicity Repeated Dose Toxicity Genotoxicity Reproductive and Developmental Toxicity Carcinogenicity Other Relevant Toxicity Studies Irritation/Sensitization - Nonhuman Irritation/Sensitization - Human Ocular/Mucosal Phototoxicity Case Studies Toxicokinetics Ammonium Hydrolyzed Collagen X Atelocollagen X X Calcium Hydrolyzed Collagen Collagen X X X X X X X Elastin X X X X X Fibronectin X X X Gelatin X X X X X X Hydrolyzed Actin X X Hydrolyzed Collagen X X X X X X X X X X X X Hydrolyzed Collagen Extract Hydrolyzed Elastin X X X X X X X Hydrolyzed Fibronectin X Hydrolyzed Gelatin X X Hydrolyzed Reticulin X Hydrolyzed Spongin MEA-Hydrolyzed Collagen X Soluble Collagen X X X X Soluble Elastin X X Zinc Hydrolyzed Collagen X indicates that data were available in the category for that ingredient.

6 Tissue-Derived Proteins and Peptides (prepared by Christina Burnett) Ingredient CAS # InfoB SciFin PubMed ToxNet FDA EU ECHA SIDS ECETOC NICNAS NTIS NTP WHO FAO NIOSH FEMA Web Ammonium Hydrolyzed Collagen (generic to ammonium hydrolyzed proteins) 13 0 No No No No No No Atelocollagen No No No Calcium Hydrolyzed No 29 0 No No No No No No Collagen Collagen , No No No No No No Elastin , No No No No No No Fibronectin , No No No No No No Gelatin , CFR182.70; No No Tier 1 Yes Yes 21CFR349-12; 21CFR Hydrolyzed Actin (generic to No No No No No No animal peptones) Hydrolyzed Collagen (generic to No No No No No No (limited from ) animal peptones); Hydrolyzed Collagen No 27 0 No No No No No No Extract Hydrolyzed Elastin (generic to animal peptones); ; No No No No No No Hydrolyzed Fibronectin (generic to animal peptones); No No No No No No Hydrolyzed Gelatin (specific to CFR No No No No No enzymatic digest product) Hydrolyzed Reticulin (generic to 0 0 No No No No No No animal peptones); Hydrolyzed Spongin No 0 0 No No No No No No MEA-Hydrolyzed Collagen No 0 0 No Annex III/61 No No No No Soluble Collagen No No No No No No No Soluble Elastin No No No No No No No Zinc Hydrolyzed Collagen No 17 0 No Annex III/24 No No No No

7 Search Strategy 3/8/17 all ingredients searched by INCI name and CAS-No, when available. Refinements and those results are listed below ToxLine (excluding PubMed) hits with details Atelocollagen = 8274 hits; refined to cosmetic atelocollagen = 63 hits/2 useful (same as Collagen below) Collagen = 8274 hits; refined to cosmetic collagen = 63 hits/2 useful (same as Atelocollagen above) Elastin = 792 hits; refined to cosmetic elastin = 10 hits/3 useful Fibronectin = 2180 hits; refined to cosmetic fibronectin = 3 hits/1 useful Gelatin = 1730 hits; refined to cosmetic gelatin = 10 hits/0 useful Hydrolyzed Actin = 8 hits/0 useful Hydrolyzed Gelatin = 9 hits/1 useful Soluble collagen = 218; revised to cosmetic soluble collagen = 4 hits/1 useful Soluble elastin = 28 hits/1 useful PubMed hits with details Ammonium hydrolyzed collagen = 13 hits/0 useful Atelocollagen = 492 hits; revised to cosmetic atelocollagen = 4 hits/0 useful Calcium hydrolyzed collagen = 29 hits/1 useful Collagen = 192,707 hits; revised to cosmetic collagen = 1386 hits; revised to cosmetic collagen toxicity = 32 hits OR cosmetic collagen irritation =19 hits/7 useful Elastin = 12,315 hits; revised to cosmetic elastin = 146 hits/1 useful Fibronectin = 38,849 hits; revised to cosmetic fibronectin = 75 hits/0 useful Gelatin = 25,412 hits; revised to cosmetic gelatin = 132 hits/4 useful Hydrolyzed actin = 156 hits/0 useful Hydrolyzed collagen (limited to ) = 190 hits/5 useful Hydrolyzed collagen extract = 27 hits/2 useful Hydrolyzed elastin = 86 hits/1 useful Hydrolyzed fibronectin = 69 hits/0 useful Hydrolyzed gelatin = 249 hits/2 useful Soluble collagen = 5003 hits; revised to cosmetic soluble collagen = 29 hits/4 useful Soluble elastin = 617 hits; revised to cosmetic soluble elastin = 2 hits/0 useful Zinc hydrolyzed collagein = 17 hits/0 useful

8 LINKS online database (self-reminder that this info has been accessed; not a public website) - winci (to cite publicly) - ScfFinder (usually a combined search for all ingredients in report; list # of this/# useful) - PubMed (usually a combined search for all ingredients in report; list # of this/# useful) - ; Also search: PubMed Dietary Supplement Subset and Toxnet databases (usually a combined search for all ingredients in report; list # of this/# useful) (includes Toxline; HSDB; ChemIDPlus; DART; IRIS; CCRIS; CPDB; GENE-TOX) FDA databases (CFR); then, list of all databases: then, (EAFUS); (GRAS); (SCOGS database); (indirect food additives list); (drug approvals and database); (OTC ingredient list); (inactive ingredients approved for drugs) EU (European Union); check CosIng (cosmetic ingredient database) for restrictions and SCCS (Scientific Committee for Consumer Safety) opinions - ECHA (European Chemicals Agency REACH dossiers) IUCLID (International Uniform Chemical Information Database) - OECD SIDS documents (Organisation for Economic Co-operation and Development Screening Info Data Sets)- ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) - HPVIS (EPA High-Production Volume Info Systems) - NICNAS (Australian National Industrial Chemical Notification and Assessment Scheme)- NTIS (National Technical Information Service) - NTP (National Toxicology Program ) - WHO (World Health Organization) technical reports - FAO (Food and Agriculture Organization of the United Nations) - NIOSH (National Institute for Occupational Safety and Health) - FEMA (Flavor & Extract Manufacturers Association) - Web perform general search; may find technical data sheets, published reports, etc Note: ChemPortal can be used to search several of the above databases simultaneously -

9 Safety Assessment of Tissue-Derived Proteins and Peptides as Used in Cosmetics Status: Draft Report for Panel Review Release Date: May 19, 2017 Panel Meeting Date: June 12-13, 2017 The 2017 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is Lillian J. Gill, DPA. This safety assessment was prepared by Christina L. Burnett, Scientific Analyst/Writer and Bart Heldreth, Ph.D., Chemist CIR. Cosmetic Ingredient Review 1620 L St NW, Suite 1200 Washington, DC ph fax cirinfo@cir-safety.org

10 INTRODUCTION The tissue-derived proteins and peptides detailed in this report are described by the International Cosmetic Ingredient Dictionary and Handbook (Dictionary) to function mainly as skin and hair conditioning agents in personal care products. 1 This report assesses the safety of the following 19 tissue-derived ingredients: Ammonium Hydrolyzed Collagen Atelocollagen Calcium Hydrolyzed Collagen Collagen Elastin Fibronectin Gelatin Hydrolyzed Actin Hydrolyzed Collagen Hydrolyzed Collagen Extract Hydrolyzed Elastin Hydrolyzed Fibronectin Hydrolyzed Gelatin Hydrolyzed Reticulin Hydrolyzed Spongin MEA-Hydrolyzed Collagen Soluble Collagen Soluble Elastin Zinc Hydrolyzed Collagen The Cosmetic Ingredient Review (CIR) Expert Panel (Panel) previously reviewed the ingredient Hydrolyzed Collagen and concluded that this ingredient is safe for use in cosmetics: the report was published in 1985and the conclusion was reaffirmed in a re-review that was published in ,3 Summary data from the original safety assessment have been included in this report in italics. Additionally, the safety of several other hydrolyzed proteins as used in cosmetics has been reviewed by the Panel in several previous assessments. The Panel concluded that Hydrolyzed Keratin (finalized in 2016), Hydrolyzed Soy Protein (finalized in 2015), Hydrolyzed Silk (finalized in 2015), Hydrolyzed Rice Protein (published in 2006), and Hydrolyzed Corn Protein (published in 2011) are safe for use in cosmetics. 4-8 The Panel concluded that Hydrolyzed Wheat Gluten and Hydrolyzed Wheat Protein are safe for use in cosmetics when formulated to restrict peptides to a weight-average MW of 3500 Da or less. 9 The CIR is concurrently reviewing the safety of plant-derived and milk-derived proteins in separate reports. Actin, Collagen, Elastin, Fibronectin, Gelatin, and reticulin all occur naturally and are derived from essential components in mammalian tissues. Much of the available published literature evaluated the effects of pharmaceutical or other agents on these proteins in their naturally occurring tissues. These studies were not considered relevant for assessing the safety of the tissue-derived ingredients as used in cosmetics and are not included in this assessment. The sources for these cosmetic ingredients may be from many different land or marine animals. These differing sources could potentially produce or result in tissue-derived proteins with unique properties, which may result in varying compositions and impurities within a single ingredient (e.g., Hydrolyzed Collagen from animals such as cows may have some impurities that are different from Hydrolyzed Collagen obtained from fish). CHEMISTRY Definition The definitions and functions of the tissue-derived proteins and peptides are described in Table 1. General and more specific descriptions of these ingredients are found below and in sub-sections, respectively. Tissue protein derivatives form a broad category of materials that are prepared by extraction from animal tissue and partial hydrolysis to yield cosmetic ingredients. Proteins and protein hydrolysates, including those of animal tissue, are used as conditioning agents in hair and skin products. These proteins are present in many types of tissue, including skin. The most common protein in mammals is Collagen, making up approximately 30% of all proteins by mass. 10,11 The Collagen family is comprised of 28 members (named collagen I to collagen XXVIII) that all have at least one triple helix in their structure at varying degrees (see further description below). 11 The most common are mainly the fibril-forming collagens (types I, II, III, and V) that are found in skin, cartilage, reticulate, and cell surfaces. Most of the other proteins addressed in this report are derivatives of Collagen, are co-located with Collagen in tissues, or are both. Gelatin, for example, is a product obtained by the partial hydrolysis of Collagen derived from the skin, white connective tissue, and bones of animals. 10 Reticulin is a type of fiber in connective tissue composed of type III Collagen secreted by reticular cells. Actin, Elastin, and Fibronectin are discrete in structure from collagens, but are commonly co-located with Collagen in tissue (e.g., Fibronectin commonly provides rigidity on the

11 edges of primarily Collagen-based tissues). Spongin, however, is a Collagen-like protein found only in marine sponges (constituting the small skeletal elements, or spicules, in the animal). The preparation of protein hydrolysates can be afforded via acid, enzyme or other methodologies. These methodologies, and the degree to which they are utilized, may profoundly affect the size and reactivity of such hydrolysates. In most ingredients in this report, even in ingredients without hydrolyzed in the name, the proteins are at least hydrolyzed to some degree as a necessary part of extraction or solubilization. Further steps towards solubilization of these macromolecules are commonly achieved via reaction with an alkaline substance to generate a protein salt (e.g., Calcium Hydrolyzed Collagen). Actin Actin is a major protein of muscle and an important component of all eukaryotic cells. 10 α-actin is found in differentiated muscle cells, while β-actin and γ-actin are in all non-muscle cell types. Collagen Collagen is the main constituent of skin (comprising 70% to 80% dry weight of the dermis) and connective tissue, and is the organic substance of bones and teeth. 10,12 Collagen is primarily responsible for the skin s tensile strength. One Collagen molecule consists of 3 polypeptide chains, each containing approximately 1000 amino acids in a primary sequence that is rich in proline, hydroxyproline, and hydroxylysine. Collagen is not just one discrete, ubiquitous protein sequence, but is a protein superfamily that is diversified across different tissue/function types and source species, including cattle, chicken, and fish. 11,13 The common structural feature of Collagen proteins is the presence of a triple helix. However, the percentage of each protein that this helix makes up can vary across different members of the Collagen superfamily from as little as 10% to nearly 100%. The diversity of the Collagen superfamily is further increased by the presence or absence of several α-chains, the existence of several molecular isoforms and supramolecular structures of specific Collagen types, and the use of different methods of extraction/hydrolysis. Elastin Elastin is the primary component of the elastic, load-bearing fibers of animal connective tissue. 10 It is an insoluble, highly cross-linked hydrophobic protein that is rich in nonpolar amino acid residues, such as valine, leucine, isoleucine, and phenylalanine. There are two types of Elastin: Type 1 is derived from bovine neck ligaments, aorta, skin, and related tissues; Type 2 is derived from cartilage and its derivatives. 14 In skin, Elastin is the intact elastic fiber network that comprises approximately 2% to 4% of the dermis by volume. 12 Fibronectin Fibronectin is a multifunctional glycoprotein found on cell surfaces, in body fluids (especially plasma), in soft connective tissue matrices, and in most basement membranes. 10 Gelatin Gelatin is a heterogeneous mixture of water-soluble proteins of high average molecular weight that are derived from the denaturation and hydrolysis of Collagen. 10 Glycine or alanine accounts for one third to one half of the amino acid residues, while another quarter is composed of proline or hydroxyproline. Reticulin Reticulin is a connective tissue protein that occurs wherever connective tissue forms a boundary 10 Physical and Chemical Properties The molecular weight ranges for some of the tissue-derived proteins and peptides are presented in Table 2. Collagen Collagen for cosmetic use has a ph range of 3.8 to Hydrolyzed Collagen Hydrolyzed Collagen may be a powder or solution. 2 A 10% aqueous solution has a ph of

12 Elastin Purified Elastin is a pale yellow color and is a bluish fluorescence in UV light. 10 It resists acid and alkaline hydrolysis. It is practically insoluble in hydrogen-bond-breaking solvents at temperatures up to 100 ºC. It is nearly impossible to bring into solution except by using hydrolytic reagents capable of rupturing peptide bonds. Unprocessed or native Elastin is reported to be too insoluble for use in cosmetic formulations. 14 Fibronectin Fibronectin can be a solution or a lyophilized powder. 15 Gelatin Gelatin is a vitreous, brittle solid that is colorless to faintly yellow. 10,16 It is practically odorless and tasteless. When Gelatin granules are immersed in cold water, they hydrate into discrete, swollen particles. When warmed, Gelatin disperses into water. Warm-blooded animal sourced Gelatin has a gel point of 30 to 35 ºC while cold-water ocean fish sourced Gelatin has a gel point between 5 and 10 ºC. Gelatin is soluble in aqueous solution of polyhydric alcohols like glycerin and acetic acid and is insoluble in alcohol, chloroform, ether, and most other organic solvents. Soluble Elastin Soluble Elastin is reported to be a cream-colored powder that is soluble in water and ethanol. 14 Method of Manufacturing Methods used to manufacture protein hydrolysates typically yield broad molecular weight distributions of peptides, ranging from 500 to 30,000 daltons (Da) and equating to 4 to 220 amino acids in length. 17,18 Treatment with certain enzymes, such as papain, can routinely yield narrower distributions of 500 to 10,000 Da, equating to 4 to 74 amino acids in length. The available methods of manufacturing for the tissue-derived proteins and peptides are summarized in Table 3. Composition The amino acid composition for Collagen, Soluble Collagen, and Elastin is presented in Table 4. Impurities Several of the ingredients in this safety assessment, including Hydrolyzed Collagen, Hydrolyzed Elastin, and Gelatin, may be bovine sourced. Some bovine materials may be considered risk materials for transmission of infectious agents (i.e. bovine spongiform encephalopathy (BSE)). According to 21 CFR , no cosmetic shall be manufactured from, processed with, or otherwise contain, prohibited cattle materials. Prohibited cattle materials mean specified risk materials, small intestine of all cattle, material from non-ambulatory disabled cattle, material from cattle not inspected and passed, or mechanically separated. Prohibited cattle materials do not include Gelatin or hides and hide-derived products. Cosmetic manufacturers must follow record keeping requirements that demonstrate that the cosmetic is not manufactured from, processed with, or does not otherwise contain prohibited cattle materials. The World Organization for Animal Health (OIE) recommends that when authorizing import or transit of gelatin and collagen prepared exclusively from hides and skins and any products made from these commodities and containing no other tissues from cattle, veterinary authorities should not require any BSE related conditions[i.e. restrictions], regardless of the BSE risk status of the cattle population of the exporting country, zone, or compartment. 19 Collagen An analysis for 3 different Collagen products found the level of arsenic to be less than 1 ppm. 13 Hydrolyzed Collagen The maximum concentrations of iron and heavy metals reported in Hydrolyzed Collagen were 3 ppm and 25 ppm, respectively. 2

13 Elastin and Hydrolyzed Elastin Impurities in commercial Elastin-based preparations include contamination by lipoid substances from the raw materials and products of Collagen degradations. 20 Gelatin According to the Food Chemicals Codex, Gelatin must contain no more than % sulfur dioxide, 10 mg/kg chromium, 1.5 mg/kg lead, and 0.3 mg/kg pentachlorophenol. 16 USE Cosmetic The safety of the cosmetic ingredients included in this assessment is evaluated based on data received from the FDA and the cosmetics industry on the expected use of these ingredients in cosmetics. Use frequencies of individual ingredients in cosmetics are collected from manufacturers and reported by cosmetic product category in the FDA Voluntary Cosmetic Registration Program (VCRP) database. Use concentration data are submitted by Industry in response to surveys, conducted by the Personal Care Products Council (Council), of maximum reported use concentrations by product category. According to 2017 VCRP data, Soluble Collagen is used in 425 formulations; the majority of uses are in leave-on skin care products (Table 5.). 21 Gelatin has the second greatest number of overall uses reported, with a total of 334; the majority of the uses are in rinse-off bath soaps and detergents. The results of the concentration of use survey conducted in 2016 by the Council indicate Collagen has the highest reported maximum concentration of use; it is used at up to 96% in face and neck skin care products. 22 Gelatin is used at up to 66% in bath oils, tablets, and salts. The other in-use ingredients are used at much lower concentrations. Historic and current use data for Hydrolyzed Collagen is reported in Table 5. In 2017, Hydrolyzed Collagen was reported to be used in 543 formulations; the majority of uses are in leave-on skin care products. The number of uses of Hydrolyzed Collagen have declined since the initial safety assessment in 1981 and the re-review in 2002 (923 and 570 uses, respectively 2,3 ). The maximum use concentration of Hydrolyzed Collagen was reported to be 16.5% in hair tonics and dressings in 2016; it was previously reported to be used at concentrations greater than 50%. 2,22 Ingredients with no reported uses in the VCRP or by Council are listed in Table 7. In some cases, reports of uses were received from the VCRP, but no concentration of use data were provided. For example, Elastin is reported to be used in 46 formulations, but no use concentration data were provided. In other cases, no uses were reported to the VCRP, but a maximum use concentration was provided in the industry survey. For example, Ammonium Hydrolyzed Collagen was not reported in the VCRP database to be in use, but the industry survey indicated that it is used in several formulations at concentrations up to 0.12%. Some of these ingredients may be used in products that can come into contact with mucous membranes and the eyes. For example, Gelatin is used in bath oils, tablets and salts at up to 66% and Hydrolyzed Collagen is used in an eyeliner at up to 3.2%. 22 Additionally, some of these ingredients were reported to be used in hair care products, skin care preparations, face powders, and fragrances and could possibly be inhaled. For example, Hydrolyzed Collagen was reported to be used in hair spray at a maximum concentration of 0.28% and Soluble Collagen was reported to be used in face powders at a maximum concentration of %. In practice, 95% to 99% of the droplets/ particles released from cosmetic sprays have aerodynamic equivalent diameters >10 µm, with propellant sprays yielding a greater fraction of droplets/particles below 10 µm compared with pump sprays Therefore, most droplets/particles incidentally inhaled from cosmetic sprays would be deposited in the nasopharyngeal and bronchial regions and would not be respirable (i.e., they would not enter the lungs) to any appreciable amount. 23,24 Conservative estimates of inhalation exposures to respirable particles during the use of loose powder cosmetic products are 400-fold to 1000-fold less than protective regulatory and guidance limits for inert airborne respirable particles in the workplace The tissue-derived protein and peptide ingredients described in this safety assessment are not restricted from use in any way under the rules governing cosmetic products in the European Union; however, monoalkanolamine ingredients must not have a secondary amine content that exceeds 0.5%, and water-soluble zinc salt ingredients must not have more than 1% zinc in ready for use preparations. 30

14 Non-Cosmetic The FDA determined that the use of peptones as direct food substances is generally recognized as safe (GRAS). These GRAS peptones are defined as the variable mixture of polypeptides, oligopeptides, and amino acids that are produced by partial hydrolysis of animal tissue or gelatin (21 CFR ). The FDA requires allergen labeling when one or more of the eight major food allergens, which includes fish, are included in food. 31 Collagen Non-cosmetic uses of Collagen include fibers in sutures, leather substitutes, coatings as a gel in photographic emulsions, and food casings. 10 Gelatin Non-cosmetic uses of Gelatin include uses in food as a stabilizer, thickener, texturizer, firming agent, surface-active agent, or surface-finishing agent. 10,16 Gelatin is also used in the manufacturing of rubber substitute, adhesives, cements, lithographic and printing inks, plastic compounds, artificial silk, photographic plates and films, matches, and light filters for mercury lamps. 10 It is also used as a clarifying agent, in hectographic masters, sizing paper and textiles, and for inhibiting crystallization in culture preparations in bacteriology. In pharmaceuticals, Gelatin is a suspending agent, an encapsulating agent, a tablet binder, and a tablet and coating agent. Gelatin is a category I active ingredient in ophthalmic demulcent over-the-counter (OTC) drug products at up to 0.01% (21CFR ). TOXICOKINETICS Gelatin The bioavailability of Gelatin derived from Nile tilapia scales was determined in an oral pharmacokinetic study in rats. 32 Five groups of six female Sprague-Dawley rats received 4000 mg/kg body weight Gelatin intragastrically (i.g.), 400 mg/kg hydroxyproline i.g., 400 mg/kg hydroxyproline intravenously (i.v.), normal saline i.g., or normal saline i.v. Blood plasma was then drawn from the rats at different times over 24 h to determine the hydroxyproline concentration. The bioavailability of the Gelatin was indirectly measured by the bioavailability of hydroxyproline in Gelatin. The relative and absolute bioavailability of Gelatin was 74.12% and 85.97%, respectively. The amino acid profile of plasma showed 41.91% of the digested Gelatin was absorbed from the intestine in peptide form. The authors of this study concluded that Gelatin had high oral bioavailability. TOXICOLOGICAL STUDIES Acute Animal Dermal Hydrolyzed Collagen Hydrolyzed Collagen at up to 2% in formulation was practically nontoxic when administered dermally in acute toxicity studies in rabbits. 2 Animal - Oral Collagen The safety of a product containing approximately 60% Collagen (type II from chicken sternal cartilage), 20% chondroitin sulfate, and 10% hyaluronic acid was investigated in 5 male and 5 female Sprague-Dawley rats. 33 The rats received a single oral dose of 5000 mg/kg body weight and were observed for clinical signs of toxicity for 14 days. All rats survived the observation period and had normal body weight gains. On the 15 th day of the study, the rats were killed and underwent macroscopic necropsy: no gross pathological lesions were observed in any of the animals. Hydrolyzed Collagen Hydrolyzed Collagen was practically nontoxic when administered orally (up to 100%) in acute toxicity studies of mice and rats. 2

15 Short-Term Toxicity Studies Animal Oral Gelatin In a rat study of the ability of shark skin Gelatin to increase bone mineral density, no adverse effects were reported. 34 The female Wistar rats (n=40) were ovariectomized approximately a week after the start of receiving a low-protein diet and then received shark Gelatin as oral doses of 10, 20, or 40 mg/100 g body weight/day for 2 weeks. Control animals were given ovalbumin at 20 mg/100 g body weight/day. No significant differences between experimental groups and the controls were observed in final body weight, feed intake, femoral bone weight, or femoral bone length. Subchronic Toxicity Studies Animal Dermal Hydrolyzed Collagen Subchronic dermal studies in rabbits and pigs on 2 cosmetic formulations containing 2% Hydrolyzed Collagen were negative for systemic toxicity. 2 Animal - Oral Collagen The safety of a product containing approximately 60% Collagen (type IIfrom chicken sternal cartilage), 20% chondroitin sulfate, and 10% hyaluronic acid was investigated in 40 male and 40 female Sprague-Dawley rats. 33 The rats were divided into groups of 10 animals/sex and received the test material in distilled water at 0, 30, 300, or 1000 mg/kg body weight once daily via gavage for 90 days. Animals were observed twice daily for mortality and detailed observations for clinical signs of toxicity were performed once weekly. Body weight and feed consumption were measured weekly. Hematology samples were collected a week before the end of dosing and the animals were killed at the end of the dosing period. A gross necropsy was performed on all animals and tissues were preserved for histopathological examination. All animals survived until the end of the dosing period and no adverse effects or clinical signs of toxicity were observed during treatment. No significant findings were observed in changes in average body weights, average body weight gain, or hematology parameters. A small but statistically significant decrease in alkaline phosphatase activity in the 1000 mg/kg/day males was observed, but was not considered adverse. Minimal but statistically significant increases in albumin in 300 mg/kg/day males and in globulin in 1000 mg/kg/day females were not considered to be toxicologically significant since these were not dose-related. Statistically significant, but minimal, changes in average brain weight in the low dose females (higher than controls) and spleen to brain weight ratios in the intermediate dose group males (lower than controls) were also not considered to be toxicologically significant. No treatment-related histopathologic changes or gross abnormalities were observed. The researchers concluded that the test material containing Collagen was tolerated well in this rat study. 33 Human - Oral Hydrolyzed Collagen/Gelatin In a 4-month dietary intake study of Hydrolyzed Collagen (interchangeably reported as Gelatin) for the potential role in enhancing bone remodeling in children, no adverse effects were observed. 35 The randomized double-blind study divided the children (ages 6-11) in to 3 groups that received placebo (n=18), Hydrolyzed Collagen (n=20), or Hydrolyzed Collagen+calcium (n=22) daily 250 ml dose. DEVELOPMENTAL AND REPRODUCTIVE TOXICITY (DART) STUDIES No published DART studies on tissue-derived proteins and peptides were discovered and no unpublished data were submitted. GENOTOXICITY No published genotoxicity studies on tissue-derived proteins and peptides were discovered and no unpublished data were submitted.

16 CARCINOGENICITY No published carcinogenicity studies on tissue-derived proteins and peptides were discovered and no unpublished data were submitted. OTHER RELEVANT STUDIES Type 1 Hypersensitivity Skin prick tests and histamine release tests of fish Gelatin and codfish were completed in 30 fish-allergic patients (diagnosed in accordance with European Academy of Allergy and Clinical Immunology Guidelines). 36 Codfish-specific IgE was also measured in the patients and they underwent double-blinded, placebo-controlled food challenges with fish Gelatin. The fish Gelatin used for the study was made through acid extraction of codfish skins and had an average molecular weight of 60,000 Da. All 30 patients had positive skin prick tests, histamine release tests, and specific IgE to codfish. Skin prick tests and histamine release tests with fish Gelatin were positive in 3/30 and 7/30 patients, respectively. Oral challenge resulted in two patients reporting mild subjective reactions. One patient had a mild reaction to the placebo but not the fish Gelatin. The proportion of truly sensitive patients was estimated to be The study authors concluded that the fish Gelatin in the study presented no risk to fish-allergic patients at doses typically used in foods (3.61 g). The potential for tuna skin-derived Gelatin to induce allergic reaction in patients with fish allergy or sensitization was investigated using the serum samples of 100 consecutive allergic patients. 37 Serum IgE antibodies were tested against Gelatin and Hydrolyzed Gelatin extracted from yellowfin tuna skin and compared to extracts of yellowfin tuna flesh and skin and bovine or porcine gelatins. Of the 100 samples tested, only 3 exhibited reactivity to tuna skin-derived Gelatin (1 hydrolyzed, 2 non-hydrolyzed). No cross-reactivity was observed between bovine/porcine Gelatin and fish Gelatin. DERMAL IRRITATION AND SENSITIZATION STUDIES Irritation In Vitro Hydrolyzed Elastin In a MatTek EpiDerm assay, solutions of Hydrolyzed Elastin (concentration not reported; MW = Da) were predicted to be non-irritating. 38 Two forms of the ingredient were tested, with one sourced from fish. The source of the form was not reported. Animal Hydrolyzed Elastin Hydrolyzed Elastin (MW = 3000 Da) was not a primary irritant when tested neat in a Draize primary dermal irritation study in 6 New Zealand white rabbits. 39 The test sites were occluded for 24 h. The primary irritation index score was Hydrolyzed Collagen Primary skin irritation tests in rabbits indicated that Hydrolyzed Collagen was nonirritating or minimally irritating when tested at up to 100%. 2 Human Hydrolyzed Collagen Irritation was not observed in human volunteers with healthy skin at concentrations up to 28%, but moderate irritation was observed in volunteers with dermatitis. 2 Sensitization Animal Hydrolyzed Collagen Hydrolyzed Collagen was nonsensitizing in guinea pig studies at up to 2%. 2

17 Human Hydrolyzed Collagen Formulations containing 0.5% to 28% Hydrolyzed Collagen produced some irritation but no sensitization in human repeated insult patch tests (HRIPTs). 2 Hydrolyzed Elastin In a HRIPT with 52 subjects, Hydrolyzed Elastin (25% w/v in corn oil; MW = 3000 Da) did not produce dermal irritation or dermal sensitization. 40 The test patches were occlusive. Phototoxicity Hydrolyzed Collagen Hydrolyzed Collagen at up to 2% was not phototoxic to guinea pigs and rabbits, nor was it phototoxic or photosensitizing to humans at up to 0.5%. 2 UV-induced erythema was decreased after application of 10% solution of Hydrolyzed Collagen (MW = 1500 Da) onto the skin after irradiation. OCULAR IRRITATION STUDIES In Vitro Hydrolyzed Elastin In a MatTek EpiOcular assay, solutions of Hydrolyzed Elastin (concentration not reported; MW = Da) were predicted to be non-irritating. 38 Two forms of the ingredient were tested, with one sourced from fish. The source of the other form was not reported. Animal Hydrolyzed Collagen Hydrolyzed Collagen was minimally irritating to rabbit eyes when tested full-strength. 2 Hydrolyzed Elastin Hydrolyzed Elastin (MW = 3000 Da) was not a primary irritant when tested neat in a Draize ocular irritation study in 6 New Zealand white rabbits. 39 The treated eyes of the animals were not rinsed. CLINICAL STUDIES Case Reports Elastin A 26-year-old woman with a history of fish allergy experienced urticarial eruptions following use of a cosmetic cream containing codfish-derived Elastin. 41 The patient s serum total IgE level was 442 ku/l, and strong radioallergosorbent test (RAST) scores for specific IgE were observed for tuna, salmon, mackerel, flatfish, codfish, horsemackerel, sardine, and salmon roe. No prick-tests were performed because of the patient s history of severe symptoms. Immunoblot analysis revealed that the patient had IgE antibodies against codfish Elastin, parvalbumin, Collagen, and transferrin. The molecular weight range of the proteins that the patient s serum reacted with was 10,000 to 20,000 Da, which corresponded to the range of codfish Elastin. The company that produced the cosmetic cream reported that the Elastin in the cosmetic cream was derived from the skin and soft tissue of codfish. Atelocollagen and Hydrolyzed Collagen A 30-year-old woman with a history of atopic dermatitis experienced anaphylaxis twice on separate occasions, once after consuming a fortified yogurt containing fish-sourced Hydrolyzed Collagen and once after consuming a gummy candy containing fish-sourced Hydrolyzed Collagen. 42 Fifteen months prior to the anaphylactic episodes, the patient had been applying a moisturizer containing Atelocollagen derived from fish to her impaired facial skin. The Atelocollagen in the product has a molecular weight of 350,000 Da. Skin prick tests on the patient were positive for fish-sourced Hydrolyzed Collagen in the food products, the moisturizer, Atelocollagen, and fish Gelatin. The tests were negative for Gelatin derived from porcine skin or bovine bone. The patient denied anaphylactic reactions following ingestion of raw or cooked fish. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE western blot analyses showed that the patient s serum reacted with an

18 approximately 140,000 Da protein of Atelocollagen and a 120,000 Da protein of Gelatin from fish Collagen. Weak reactions were observed with bovine bone Gelatin protein and no reactions were observed to porcine skin Gelatin protein or fish-sourced Hydrolyzed Collagen protein. The researchers of this case study speculated that the Atelocollagen (350,000 Da) was degraded on the skin surface by proteases into smaller peptides and induced sensitization, but did not rule out the possibility that intact Collagen or degradation products with greater than 4500 Da were antigens because of the patient s impaired skin. Hydrolyzed Collagen A 22-year-old female reported contact urticaria following use of a hair conditioner that contained steartrimonium hydrolyzed animal protein. 43 She had a similar, less severe reaction the year before to another hair conditioner that also contained this ingredient. The patient also had a history of hay fever and recurrent hand dermatitis. Prick testing elicited strongly positive wheal and flare response to both hair conditioners, steartrimonium hydrolyzed animal protein, and other hair conditioners that contained protein, including Hydrolyzed Collagen in some products. Negative reactions were observed when the patient was tested with protein-free hair products. Prick tests with the standard series of allergens yielded positive results for grass mix, rye, English plantain, dust mite, cow s milk, soybean, baker s yeast, and wholegrain wheat. Tests with raw meat were negative. The patient s total IgE was 221 ku/l. RASTs were negative to pork, beef, chicken, and mutton. A study of sensitization to protein hydrolysates in hair care products was performed in 3 groups of patients. 44 The first group, which comprised 11 hairdressers with hand dermatitis, submitted to scratch and prick tests with 22 trademarked protein hydrolysates, including Soluble Collagen and Hydrolyzed Collagen, as well as quaternized hydrolyzed proteins. The second test group comprised 1260 consecutive adults with suspected allergic respiratory disease: they were subjected to skin prick tests with 1 to 3 of the protein hydrolysates. The third group of patients comprised 28 adults with atopic dermatitis and was also tested with a protein hydrolysate via a skin prick test. Positive reactions were seen in a total of 12 patients (all female with atopic dermatitis) from 3 of the 22 protein hydrolysates. All 12 had reactions to hydroxypropyl trimonium hydrolyzed collagen. Three of the 12 also had a reaction to one trademarked version of Hydrolyzed Collagen (1% solution), while 1 other had a reaction to hydroxypropyl trimonium hydrolyzed milk protein. 44 SUMMARY This report assesses the safety of 19 tissue-derived ingredients, including Hydrolyzed Collagen, which has been previously reviewed by the Panel. Summary information presented in this safety assessment from the previous report will not be summarized below. Soluble Collagen is used in 425 formulations; the majority of uses are in leave-on skin care products. Gelatin has the second greatest number of overall uses reported, with a total of 334; the majority of the uses are in rinse-off bath soaps and detergents. The results of the concentration of use survey conducted in 2016 by the Council indicate Collagen has the highest reported maximum concentration of use; it is used at up to 96% in face and neck skin care products. Gelatin is used at up to 66% in bath oils, tablets, and salts. A toxicokinetics study of fish-derived Gelatin (4000 mg/kg) in rats found that Gelatin has a high oral bioavailability. A product containing 60% chicken-derived Collagen did not produce acute toxic effects in rats that were given a single oral dose of 5000 mg/kg. No adverse effects were reported in a 2 week oral study of shark skin-derived Gelatin in ovariectomized rats that received the test material at up to 40 mg/100 g daily. In subchronic toxicity studies, rats tolerated daily oral dosing of a test material containing 60% Collagen. No adverse effects were reported in a 4 month study of a dietary supplement containing a 250 ml dose of Hydrolyzed Collagen in human children Gelatin and other tissue-derived proteins may be sourced from fish, which is a major food allergen that can produce Type 1 (immediate) reactions in sensitized individuals. Researchers have reported a low risk of IgEmediated reactions to fish Gelatin in individuals with fish allergies. In vitro dermal irritation studies have predicted solutions of Hydrolyzed Elastin (MW = Da) to be non-irritating. The ingredient (MW = 3000 Da) was not a primary dermal irritant when tested neat in rabbits. In a HRIPT, Hydrolyzed Elastin (25% w/v in corn oil; MW = 3000 Da) did not produce dermal irritation or dermal sensitization. Hydrolyzed Elastin was not predicted to be an ocular irritant in in vitro assays and it was not an ocular irritant in rabbit eyes when tested neat.

19 Case reports of dermal sensitization to cosmetics containing Elastin and Collagen derived from fish have been described in the published literature. Reactions to Hydrolyzed Collagen have been reported as well. No relevant published DART, genotoxicity, or carcinogenicity studies on tissue-derived proteins and peptides were identified in a literature search for these ingredients, and no unpublished data were submitted. To be developed To be determined DISCUSSION CONCLUSION

20 TABLES Table 1. Definitions and functions of the ingredients in this safety assessment. 1 Ingredient CAS No. Definition Function Ammonium Hydrolyzed Collagen Ammonium Hydrolyzed Collagen is the ammonium salt of Hydrolyzed Collagen. hair conditioning agents; skinconditioning agents-misc [generic to ammonium hydrolyzed proteins] Calcium Hydrolyzed Collagen Calcium Hydrolyzed Collagen is the calcium salt of Hydrolyzed Collagen. nail conditioning agents; skinconditioning agents-misc. MEA Hydrolyzed Collagen MEA-Hydrolyzed Collagen is the monoethanolamine salt of Hydrolyzed Collagen. hair conditioning agents; skinconditioning agents-misc. Zinc Hydrolyzed Collagen Zinc Hydrolyzed Collagen is the zinc salt of Hydrolyzed Collagen. hair conditioning agents; skinconditioning agents-misc. Hydrolyzed Collagen [generic to animal peptones] Hydrolyzed Collagen is the hydrolysate of animal or fish collagen derived by acid, enzyme or other method of hydrolysis. It is characterized by a significant level of hydroxyproline residues. hair conditioning agents; nail conditioning agents; skinconditioning agents-misc. Hydrolyzed Collagen Extract Hydrolyzed Collagen Extract is the extract of Hydrolyzed Collagen. skin protectants Soluble Collagen Soluble Collagen is a non-hydrolyzed, native protein derived from the connective tissue of animals. It consists essentially of a mixture of the precursors of mature collagen. It has a triple helical structure and is predominantly not cross-linked. hair conditioning agents; skinconditioning agents-misc. Collagen Atelocollagen Gelatin Hydrolyzed Gelatin [specific to enzymatic digest product] Hydrolyzed Reticulin [generic to animal peptones] Hydrolyzed Actin [generic to animal peptones] Elastin Soluble Elastin Hydrolyzed Elastin [generic to animal peptones] Fibronectin Hydrolyzed Fibronectin [generic to animal peptones] Hydrolyzed Spongin Collagen is the protein found in cartilage and other connective tissues in animals. Atelocollagen is the protein obtained when the telopeptides are enzymatically removed from collagen. Gelatin is a product obtained by the partial hydrolysis of collagen derived from the skin, white connective tissue and bones of animals. Hydrolyzed Gelatin is the hydrolysate of Gelatin derived by acid, enzyme or other method of hydrolysis. Hydrolyzed Reticulin is the hydrolysate of the reticulin portion of animal connective tissue derived by acid, enzyme or other method of hydrolysis. [Reticulin is a type of fiber in connective tissue composed of type III collagen secreted by reticular cells] Hydrolyzed Actin is the hydrolysate of actin derived by acid, enzyme or other method of hydrolysis. Elastin is a fibrous protein found in the connective tissue of animals. Soluble Elastin a water soluble non-hydrolyzed, native protein derived from Elastin. Hydrolyzed Elastin is the hydrolysate of elastin derived by acid, enzyme or other method of hydrolysis. Fibronectin is a glycoprotein found in connective tissues, basement membranes, in plasma and other body fluids. Hydrolyzed Fibronectin is the hydrolysate of Fibronectin derived by acid, enzyme or other method of hydrolysis. Hydrolyzed Spongin is the hydrolysate of spongin derived by acid, enzyme or other method of hydrolysis. [Spongin is a collagen-type protein, common to marine sponges] hair conditioning agents; skinconditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. binders; hair conditioning agents; lytic agents; oral health care drugs; skin-conditioning agents-misc.; viscosity increasing agents-aqueous skin-conditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. skin-conditioning agents-misc. hair conditioning agents; skinconditioning agents-emollient; skin-conditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. hair conditioning agents; skinconditioning agents-misc. skin-conditioning agents-misc.

21 Table 2. Reported molecular weights of tissue-derived proteins 2,10,14,20,45,46 Ingredient Molecular Weight (Da) Range Collagen (native) 130,000 to > 1,000,000 Soluble Collagen 30,000-40,000, but may be up to an average of 300,000 Hydrolyzed Collagen 1000 to 25,000 Hydrolyzed Actin 58.4% < 5000; 41.4% > 5000 and < 30,000 Hydrolyzed Elastin 500 to 150,000 Fibronectin >200,000 Table 3. Method of manufacturing Ingredient Source Procedure Reference Collagen Not reported Prepared by dissolving the mineral part of bones with phosphoric 10 acid. Hydrolyzed Collagen Bovine or fish Prepared by alkaline hydrolysis followed by enzymatic hydrolysis to 2,3 the desired molecular weight Elastin Farm animals such as cattle or goats Prepared from cattle aortas through extraction with sodium hydroxide at 100º C and filtration (which both may be repeated several times), precipitation, neutralization with hydrochloric acid, and washing to remove residual salt. The resultant extract may then be purified by autoclaving or by amylase pretreatment. 14 Elastin Collagen (unspecified) Elastin may be a byproduct of the purification of Collagen Hydrolyzed Elastin Codfish skin or bovine (MW= Da) neck tendons Hydrolyzed Elastin (MW = Da) Hydrolyzed Elastin (MW= Da) Numerous sourced animal ligaments or hides Not reported Prepared by washing and purifying to remove soil and other residual material and then dried. Dried material is then hydrolyzed for several hours until the target molecular weight is reached. The final product is a solution, with the bovine source material being concentrated to a 30% active content. Prepared by enzymatic hydrolysis (by pancreatic elastase, ficin, pepsin or trypsin) or acid hydrolysis at high temperatures (70-100º C, depending on acid) at several 1 hour intervals. Manufactured by enzymatic hydrolysis for a specific duration of time and at an elevated temperature (details not provided). Resultant hydrolyzed protein composed of di- and tri-peptides. Gelatin Collagen (unspecified) Prepared by the acid, alkaline or enzymatic hydrolysis of Collagen. Type A Gelatin is produced by the acid processing of collagenous raw materials and exhibits an isoelectric point between ph 7 and ph 9. Type B Gelatin is produced by the alkaline or lime processing of collagenous raw materials and exhibits an isoelectric point between ph 4.6 and ph 5.2. Soluble Collagen Bovine dermal tissues, bony fish skins, or tropical fish swim bladders Extracted by neutral salt solutions Soluble Elastin Cattle ligaments Obtained by acid treatment at 80º C and a ph less than 4, followed by filtration, grinding, enzymatic treatment at ph 9/13 (alkaline proteases in the presences of urea), and finally neutralizing enzymes at 90º C ,

22 Table 4. Amino acid residue profile of Collagen, soluble Collagen, and Elastin (residues per 1000). 13,14,50 Amino Acid Collagen Soluble Collagen Elastin Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine not determined not determined not determined Valine not determined Methionine not determined not detected Isoleucine Leucine Tyrosine Phenylalanine Histidine Hydroxylysine not determined not detected Lysine Arginine Tryptophan not determined not determined not determined

23 Table 5. Frequency (2017) and concentration of use (2016) according to duration and type of exposure for tissue-derived proteins and peptides. 21,22 # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) Ammonium Hydrolyzed Collagen Collagen Elastin Fibronectin Totals NR NR 2 NR Duration of Use Leave-On NR NR 2 NR Rinse Off NR NR NR NR Diluted for (Bath) Use NR NR NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR NR 55 b ; 94 c b 11 b ; 11 c NR 2 c NR Incidental Inhalation-Powder NR 0.1 a 94 c a 11 c NR 2 c NR Dermal Contact NR NR 2 NR Deodorant (underarm) NR NR NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR Hair-Coloring NR NR 1 NR NR NR NR NR Nail NR NR 4 NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR Baby Products NR NR NR NR NR NR NR NR Gelatin Hydrolyzed Actin Hydrolyzed Elastin Hydrolyzed Fibronectin Totals Duration of Use Leave-On Rinse Off Diluted for (Bath) Use NR NR NR NR NR NR NR Exposure Type Eye Area NR NR Incidental Ingestion 3 NR NR NR NR NR Incidental Inhalation-Spray 19 b ; 6 c 0.03; b ; c 7 b ; 6 c NR 57 b ; 88 c ; b 4 b ; 3 c NR Incidental Inhalation-Powder 1; 6 c c ; a 6 c NR 88 c ; 2 a ; a 3 c NR Dermal Contact NR Deodorant (underarm) NR NR NR NR NR NR NR NR Hair - Non-Coloring NR NR Hair-Coloring NR NR NR NR NR NR NR NR Nail NR NR NR NR NR NR Mucous Membrane NR NR NR NR Baby Products NR NR NR NR 4 NR NR NR

24 Table 5. Frequency (2017) and concentration of use (2016) according to duration and type of exposure for tissue-derived proteins and peptides. 21,22 # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) Hydrolyzed Reticulin MEA-Hydrolyzed Collagen Soluble Collagen* Soluble Collagen Extract Totals NR NR NR Duration of Use Leave-On NR 0.05 NR NR Rinse Off NR NR NR NR Diluted for (Bath) Use NR NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray NR NR NR NR 104 b ; 141 c ; b 1 b ; 1 c NR Incidental Inhalation-Powder NR NR NR 0.1 a 141 c ; a 1 c NR Dermal Contact NR NR NR Deodorant (underarm) NR NR NR NR NR NR NR NR Hair - Non-Coloring NR NR NR NR NR Hair-Coloring NR NR NR NR NR NR Nail NR NR NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR Baby Products NR NR NR NR NR NR NR NR Atelocollagen Totals 118 NS Duration of Use Leave-On 114 NS Rinse Off 4 NS Diluted for (Bath) Use NR NS Exposure Type Eye Area 17 NS Incidental Ingestion 39 NS Incidental Inhalation-Spray 19 b ; 18 c NS Incidental Inhalation-Powder 1; 18 c NS Dermal Contact 66 NS Deodorant (underarm) NR NS Hair - Non-Coloring 1 NS Hair-Coloring NR NS Nail NR NS Mucous Membrane 39 NS Baby Products NR NS NR = Not reported. NS = Survey underway Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses. a. It is possible these products may be powders, but it is not specified whether the reported uses are powders. b. It is possible these products may be sprays, but it is not specified whether the reported uses are sprays. c. Not specified whether a powder or a spray, so this information is captured for both categories of incidental inhalation. * Includes 25 uses listed in the VCRP as soluble animal collagen. Not listed in the INCI Dictionary, possibly the same as Collagen Extract.

25 Table 6. Historic and current frequency and concentration of use according to duration and type of exposure for Hydrolyzed Collagen. 21,22 # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) 1981 uses/concentrations 2002 uses/2004 concentrations 2017 uses/2016 concentrations Totals 923 <0.1 - > d Duration of Use Leave-On 284 <0.1 - < Rinse Off 633 <0.1 - > Diluted for (Bath) Use 6 > NR 1 NR Exposure Type Eye Area 40 <0.1 - < Incidental Ingestion 15 < Incidental Inhalation-Spray 7; 96 a ; 46 b <1; >0.1 - >50 a ; <10 b 3; 108 a ; 38 b a ; b 116 a ; 139 b ; a Incidental Inhalation-Powder 5; 46 b <1; <10 b 4; 38 b 0.5; b 3; 139 b ; 2 c c Dermal Contact 207 <0.1 - < Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring 609 >0.1 - > Hair-Coloring 46 <0.1 - <5 4 NR Nail 18 <0.1 - <50 9 NR Mucous Membrane 24 > Baby Products 1 <0.1 NR NR 4 NR NR = Not reported. Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses. a.. It is possible these products may be sprays, but it is not specified whether the reported uses are sprays. b. Not specified whether a powder or a spray, so this information is captured for both categories of incidental inhalation. c. It is possible these products may be powders, but it is not specified whether the reported uses are powders d. Majority of the uses were categorized as Hydrolyzed Animal Protein in the VCRP database. Table 7. Ingredients not reported in use. Calcium Hydrolyzed Collagen Zinc Hydrolyzed Collagen Hydrolyzed Collagen Extract Hydrolyzed Gelatin Soluble Elastin Hydrolyzed Spongin

26 References 1. Nikitakis J and Lange B. International Cosmetic Ingredient Dictionary and Handbook. 16 ed. Washington, DC: Personal Care Products Council, Elder RL. Final Report on the Safety Assessment of Hydrolyzed Collagen. JACT. 1985;4(5): Andersen FA (ed.). Annual Review of Cosmetic Ingredient Safety Assessments /2005. Int J Toxicol. 2006;25(Suppl. 2): Burnett CL, Heldreth B, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Liebler DC, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Gill LJ. Safety Assessment of Keratin and Keratin-Derived Ingredients as Used in Cosmetics L St NW, Suite 1200, Washington, DC 20036, Cosmetic Ingredient Review Burnett CL, Heldreth B, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Liebler DC, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Gill LJ. Safety Assessment of Soy Proteins and Peptides as Used in Cosmetics L St NW, Suite 1200, Washington, DC 20036, Cosmetic Ingredient Review Johnson WJ, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Liebler DC, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Gill LJ. Safety Assessment of Silk Protein Ingredients as Used in Cosmetics L St NW, Suite 1200, Washington, DC 20036, Cosmetic Ingredient Review Andersen FA (ed.). Amended Final Report on the Safety Assessment of Oryza Sativa (Rice) Bran Oil, Oryza Sativa (Rice) Germ Oil, Rice Bran Acid, Oryza Sativa (Rice) Bran Wax, Hydrogenated Rice Bran Wax, Oryza Sativa (Rice) Bran Extract, Oryza Sativa (Rice) Extract, Oryza Sativa (Rice) Germ Powder, Oryza Sativa (Rice) Starch, Oryza Sativa (Rice) Bran, Hydrolyzed Rice Bran Extract, Hydrolyzed Rice Bran Protein, Hydrolyzed Rice Extract, and Hydrolyzed Rice Protein. Int J Toxicol. 2006;25(Suppl 2): Andersen FA, Bergfeld WF, Belsito DV, Klaassen CD, Marks JG, Shank RC, Slaga TJ, and Snyder PW. Final Report of the Safety Assessment of Cosmetic Ingredients Derived from Zea Mays (Corn). Int J Toxicol. 2011;30(Suppl. 1):17S-39S. 9. Burnett CL, Heldreth B, Boyer IJ, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Liebler DC, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Gill LJ. Safety Assessment of Hydrolyzed Wheat Protein and Hydrolyzed Wheat Gluten as Used in Cosmetics L St NW, Suite 1200, Washington, DC 20036, Cosmetic Ingredient Review O'Neil MJ. The Merck Index. 15thth ed. Royal Society of Chemistry; Ricard-Blum S. The Collagen Family. Cold Spring Harb Perspect Biol. 2011;3(1):a004978

27 12. Waller JM and Maibach HI. A quantitative approach to age and skin structure and function: Protein, glycosaminoglycan, water, and lipid content and structure. Chapter: 23. Barel O, Paye M, and Maibach HI. In: Handbook of Cosmetic Science and Technology. Third ed. New York: Informa Healthcare USA, Inc.; 2009: Peng Y, Glattauer V, Werkmeister JA, and Ramshaw JAM. Evaluation for collagen products for cosmetic application. J Cosmet Sci. 2004;55: Lower ES. Elastin in cosmetics. Drug Cosmet Ind. 1987;141: Wein E. Biofactors for skin care. Cosmet Toiletries. 1986;101: Council of Experts, United States Pharmacopeial Convention. Food Chemicals Codex. 8th ed. Rockville, MD: United States Pharmacopeia (USP), Stern ES and Johnsen VL. Studies on the molecular weight distribution of cosmetic protein hydrolysates. J Soc Cosmet Chem. 1977;28: Geetha G and Priya M. Ultrasonic studies on halide doped amino acids. Arch Phy Res. 2011;2(4): World Organization for Animal Health (OIE). Terrestrial Animal Health Code; Chapter 11.4 Bovine Spongiform Encephalopathy; Article Last Updated Date Accessed Langmaier F, Mládek M, Kolomazník K, and Sukop S. Isolation of elastin and collagen polypeptides from long cattle tendons as raw material for the cosmetic industry. Int J Cosmet Sci. 2002;24: Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA. 22. Personal Care Products Council Concentration of Use by FDA Product Category: Collagen, Hydrolyzed Collagen and Related Proteins. Unpublished data submitted by Personal Care Products Council. 23. Bremmer HJ, Prud'homme de Lodder LCH, and Engelen JGM. Cosmetics Fact Sheet: To assess the risks for the consumer; Updated version for ConsExpo Report No. RIVM /2006. pp Rothe H, Fautz R, Gerber E, Neumann L, Rettinger K, Schuh W, and Gronewold C. Special aspects of cosmetic spray safety evaluations: Principles on inhalation risk assessment. Toxicol Lett. 2011;205(2): Rothe H. Special Aspects of Cosmetic Spray Evalulation Unpublished data presented at the 26 September CIR Expert Panel meeting. Washington, D.C. 26. Johnsen MA. The Influence of Particle Size. Spray Technology and Marketing. 2004;14(11): CIR Science and Support Committee of the Personal Care Products Council (CIR SSC) Cosmetic Powder Exposure. Unpublished data submitted by the Personal Care Products Council. 28. Aylott RI, Byrne GA, Middleton J, and Roberts ME. Normal use levels of respirable cosmetic talc: Preliminary study. Int J Cosmet Sci. 1976;1(3):

28 29. Russell RS, Merz RD, Sherman WT, and Siverston JN. The determination of respirable particles in talcum powder. Food Cosmet Toxicol. 1979;17(2): European Union. Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on Cosmetic Products Internet site accessed September 13, Food and Drug Administration (FDA). Guidance for Industry: A Food Labeling Guide (6. Ingredient Lists). ngnutrition/ucm htm. Last Updated Date Accessed Wang L, Wang Q, Liang Q, He Y, Wang Z, He S, Xu J, and Ma H. Determination of bioavailability and identification of collagen peptide in blood after oral ingestion of gelatin. J Sci Food Agric. 2015;95: Schauss AG, Merkel DJ, Glaza SM, and Sorenson SR. Acute and subchronic oral toxicity studies in rats of a hydrolyzed chicken sternal cartilage preparation. Food Chem Toxicol. 2007;45(2): Nomura Y, Oohashi K, Watanabe M, and Kasugai S. Increase in bone mineral density through oral administration of shark gelatin to ovariectomized rats. Nutrition. 2005;21(11-12): Martin_Baurtista E, Martin-Matillas M, Martin-Lagos JA, Miranda-Leon MT, Muñoz-Torres M, Ruiz- Requena E, Rivero M, Quer J, Puigdueta I, and Campy C. A nutritional intervention study with hydrolyzed collagen in pre-pubertal Spanish children: Influence on bone modeling biomarkers. J Pediatr Endocr Met. 2011;24(3-4): Hansen TK, Poulsen LK, Stahl Skov P, Hefle SL, Hlywka JJ, Taylor SL, Bindslev-Jensen U, and Bindslev- Jensen C. A randomized, double-blinded, placebo-controlled oral challenge study to evaluate the allergenicity of commercial, food-grade fish gelatin. Food Chem Toxicol. 2004;42: André F, Cavagna S, and André C. Gelatin prepared from tuna skin: A risk ractor for fish allergy or sensitization? Int Arch Allergy Immunol. 2003;130(1): Anonymous Summaries of Dermal and Ocular Irritation Tests of Hydrolyzed Protein Ingredients (including proteins hydrolyzed to amino acids). Unpublished data submitted by Personal Care Products Council. 4 pages. 39. Consumer Product Testing Co Primary dermal irritation in rabbits; primary ocular irritation in rabbits; acute oral toxicity in rats: Hydrolyzed Elastin (MW ~ 3,000 Da) Experiment Reference No: Unpublished data submitted by Personal Care Products Council. 40. CPTC Inc Repeated insult patch test: Hydrolyzed Elastin (MW ~ 3,000 Da) Experiment Reference No.: C Unpublished data submitted by Personal Care Products Council. 41. Nishida K, Tateishi C, Tsuruta D, Shimauchi T, Ito T, Hirakawa S, and Tokura Y. Contact urticaria caused by a fish-derived elastin-containing cosmetic cream. Contact Dermititis. 2012;67(3): Fujimoto W, Fukuda M, Yokooji T, Yamamoto T, Tanaka A, and Matsuo H. Anaphylaxis provoked by ingestion of hydrolyzed fish collagen probably induced by epicutaneous sensitization. Letter to the Editor. Allergol Int. 2016;65(4): Freeman S and Lee M-S. Contact urticaria to hair conditioner. Contact Dermititis. 1996;35: Niinimaki A, Niinimaki M, Makinen-Kiljunen S, and Hannuksela M. Contact urticaria from protein hydrolysates in hair conditioners. Allergy. 1998;53:

29 45. Idson B. Natural moisturizers for cosmetics. Drug Cosmet Ind. 1985;136: Personal Care Products Council Information on Hydrolyzed Actin. Unpublished data submitted by Personal Care Products Council. 1 pages. 47. Arch Personal Care Products LP Solu-Lastin 30 (Hydrolyzed Elastin) Manufacturing process. Unpublished data submitted by Personal Care Products Council. 8 pages. 48. Arch Personal Care Products LP Solu-Mar Elastin (Hydrolyzed Elastin) Manufacturing process. Unpublished data submitted by Personal Care Products Council. 8 pages. 49. Brooks GJ. Collagen. Chapter: 16. Schlossman ML. In: The Chemistry and Manufacture of Cosmetics. Vol. 3 - Ingredients. 3rd ed. Carol Stream, IL: Allured Publishing Corp.; 2002: Todd R. Soluble collagen. Soap Perfum Cosmet. 1974;47:

30 journa1 OF THE AMERICAN COLLEGE OF TOXICOLOGY Volume 4, Number 5, 1985 Mary Ann Liebert, Inc., Publishers 7 Final Report on the Safety Assessment of Hydrolyzed Collagen Hydrolyzed Collagen is a hydrolysate derived from animal byproducts. It is used in cosmetic products as a conditioner or moisturizer at concentrations less than 5 percent. Hydrolyzed Collagen was practically nontoxic when administered orally or dermally in acute animal toxicity studies. This ingredient was minimally irritating to rabbit eyes when tested full-strength. Primary skin irritation tests in rabbits indicated that Hydrolyzed Collagen was nonirritating or minimally irritating when tested full-strength. Subchronic dermal studies on 2 cosmetic formulations containing 2 percent Hydrolyzed Collagen were negative for systemic toxicity. Hydrolyzed Collagen was nonsensitizing in guinea pigs. In clinical studies, Hydrolyzed Collagen produced no skin irritation, sensitization, or indication of phototoxicity. On the basis of the available animal and clinical data, it is concluded that Hydrolyzed Collagen is safe as a cosmetic ingredient in the present practices of use and concentration. T INTRODUCTION his report presents the available information published between 1940 and the present and the unpublished cosmetic industry data on Hydrolyzed Collagen (formerly Hydrolyzed Animal Protein). CHEMICAL AND PHYSICAL PROPERTIES Hydrolyzed Collagen is defined as a collagen hydrolysate derived by acid, enzyme, or other method of hydrolysis. (I) It is a polypeptide of molecular weight 1000 to 10,000 with the following generalized structure:(2) 199

31 200 COSMETIC INGREDIENT REVIEW HO H I H I H H I L I Jn The typical amino acid composition is given in Table 1 along with the corresponding structures of R, R, and R. Hydrolyzed Collagen is an off-white to white hygroscopic powder. It is also available as a viscous, amber aqueous solution. t2) The physicochemical properties of Hydrolyzed Collagen and its solution are given in Table 2. Hydrolyzed Collagen can be prepared by any 1 of 3 methods: alkaline hydrolysis of bovine skin products followed by enzymatic hydrolysis to the desired molecular weight, enzymatic hydrolysis of fresh animal byproducts or bovine-derived leather, or acid or alkaline hydrolysis of chrome leather fold splinters with inorganic acids or lyes to a defined molecular weight. The hydrolysate produced by the latter method is purified in an aqueous solution and then by precipitation and filtration to effectively remove the heavy ions.( ) Acid hydrolysis tends to split the polypeptide bond between proline (or hydroxyproline) and other amino acids, with very little specificity for which amino acid donates its amino group to the peptide bond.(3) The spectrum of amino acids resulting from the hydrolysis of collagen differs substantially from that of other proteins by its high content of glycine and proline and low content of histidine, tryptophan, and cystine (Table 1). Collagen also contains 2 amino acids, hydroxyproline and hydroxylysine, not found in other proteins. (4) Hydrolyzed Collagen is analyzed primarily by column chromatography. The literature cites numerous chromatographic methods. (5-g1 Its solution can be positively identified by comparison to a standard infrared spectrum.( O) The 2 amino acids found only in collagen, hydroxyproline and hydroxylysine, allow for differentiation between collagen hydrolysates and other protein hydrolysates.(4) Hydrolyzed Collagen is stable under the conditions of normal cosmetic use. However, the addition of perfumes high in aldehyde content may cause color reactions and odor changes due to the reaction of active carbonyl groups with the amino group of the hydrolysate. *, Burnett (11) has found that cosmetics containing proteins are better preserved at successively lower ph values, whereas Frech et ai. found that sodium acetate is an effective preservative in protein hydrolysate solutions. Hydrolyzed Collagen has shown a 15 to 45 percent reduction in viscosity after storage for 1 month at room temperature.(13

32 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 201 TABLE 1. Typical Amino Acid Composition of Hydrolyzed Collagen(* Amino Acid R, R, or R Typical Composition * I%) Clycine Alanine Serine Threonine Proline Hydroxyproline Valine lsoleucine Leucine Phenylalanine Tyrosine Cystine/cysteine Methionine Aspartic acid Glutamic acid Arginine Histidine Lysine Hydroxylysine -H CH, 8.0-l 1.O -CH,OH CHOHCH, 1 B-2.6 CYCLIC CYCLIC CHCH,CH, CHCH,CH,CH, 1.3-l.8 -CH,CHCH,CH, CHKsH CH&H,OH 0.2-l.o -+/-CH,-SH CH,CH,SCH, CHZCOZH CH~CHICOZH CH,CHKH,CfNH),NH, NHCHNCHtCH,- 0.7-l.o -CH,CH#ZH,CHINH~ (CHIKHOHCHZNHZ 0.7-l.2 *Prepared by alkaline hydrolysis of bovine skin products to form a food-grade gelatin, followed by enzyme hydrolysis to the desired molecular weight. +These do not conform to the generalized formula. Proline: CH z- CH, Hydroxyproline: HOCH,- CH, I I I I 1 CHCO,H P / NH Cystine: S-CH,CHCO,H I NH, S-CHKHCOIH I USE Cosmetic Use Hydrolyzed Collagen is used in cosmetics, primarily hair and skin care products, because of its conditioning and moisturizing properties. It is generally used

33 202 COSMETIC INGREDIENT REVIEW TABLE 2. Physicochemical Properties of Hydrolyzed Collagen 2 Value Property Powder So/u tion Molecular weight 1000 to 10,000 Solids content (105W16 hours) - Moisture content (vacuum oven 9O C/6 hours) 0.0% max. PH - Nitrogen 12.0% min. Ash content 12.0% max. Iron - Heavy metals % min (10% aqueous solution) 8.0% min. 5.0% max. 3 ppm max. 25 ppm max. at concentrations <5 percent in the following product categories: baby shampoos, bath, eye makeup, hair, hair coloring, makeup, manicuring, personal cleanliness, shaving, skin care, and tanning preparations ) Table 3 presents the FDA product formulation data for Hydrolyzed Collagen. (Is) The cosmetic product formulation computer printout that is made available by the Food and Drug Administration (FDA) is compiled through voluntary filing of such data in accordance with Title 21 part of the Code of Federal Regulations. Ingredients are listed in prescribed concentration ranges under specific product type categories. Since certain cosmetic ingredients are supplied by the manufacturer at < 100 percent concentration, the value reported by the cosmetic formulator may not necessarily reflect the actual concentration found in the finished product; the actual concentration in such a case would be a fraction of that reported to the FDA. The fact that data are only submitted within the framework of preset concentration ranges also provides the opportunity for overestimation of the actual concentration of an ingredient in a particular product. An entry at the lowest end of a concentration range is considered the same as one entered at the highest end of that range, thus introducing the possibility of a 2- to lo-fold error in the assumed ingredient concentration. In 1981, approximately 95 percent of the 923 formulations containing Hydrolyzed Collagen incorporated the hydrolysate at concentrations of <5 percent: 23 percent of these at concentrations 10.1 percent, 50 percent at concentrations >O.l to 1 percent, and 22 percent at concentrations > 1 to 5 percent. Hair preparations accounted for 66 percent of the total product listings of Hydrolyzed Collagen, with the second highest listing (16 percent) found in skin care products. (15) The formulation data presented in Table 3 indicate that cosmetic products containing Hydrolyzed Collagen may contact all external body surfaces and hair, as well as the eyes. These products may be used daily or occasionally over a period of up to several years. The frequency and length of application could result in continuous exposure,

34 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 203 Noncosmetic Use Hydrolyzed Collagen is used in soaps to impart a soft texture to skin. ) It is also used in a treatment for duodenal and gastric ulcers and gastritis. A protective film-forming composition containing Hydrolyzed Collagen is also applied to cow teats. (l*) GENERAL BIOLOGY Su bstantivity The literature contains many studies documenting the substantivity of Hydrolyzed Collagen to human hair and skin. Hydrolyzed Collagen generally is adsorbed on hair; the amount of protein adsorbed is measured by hydroxyproline analysis. *3,19) Kim et al. found that adsorbed Hydrolyzed Collagen increased the tensile strength and elongation of hair. Increasing concentrations of Hydrolyzed Collagen increased adsorption. (19,*0) Hydrolyzed Collagens of average molecular weight 1000 and 2000 have greater substantivity to hair and skin, respectively, than those of average molecular weight 10,000. (13,*1) Brooks ** stated that Hydrolyzed Collagen substantivity is due to the distribution of terminal amino groups along (primarily at the lysine and arginine residues) and at the ends of the polypeptide. Cooperman and Johnsen (23) studied the penetration of Hydrolyzed Collagen into both unbleached and bleached hair strands. In the hair strands treated with the lower molecular weight Hydrolyzed Collagens, the cuticle contained the highest percentage of protein. However, the cortex, by virtue of its greater bulk, contained the greater amount of protein. Hair strands treated with the higher molecular weight Hydrolyzed Collagens had equal quantities of protein in the cuticle and cortex. Protein penetration increased with increasing damage to hair. As a hygroscopic compound, Hydrolyzed Collagen helps bind water to the hair and skin surfaces. (11.13,24.25) Brooks(22) found that Hydrolyzed Collagen binds water better at higher relative humidities and at ph 5 rather than ph 7 or 9. Hydrolyzed Collagen potentiates epidermal metabolism by providing a suitable, moist environment on the surface of the epidermis for healthy skin and hair. The amphoteric nature of Hydrolyzed Collagen makes it an acceptable buffering agent for alkali in permanent waving products. It is incorporated in waving formulations to avoid or minimize damage to hair. A protective application of Hydrolyzed Collagen is sometimes made prior to waving. Hydrolyzed Collagen is also used in hair dyes to insure uniformity in dyeing. General Effects Various enzymes will hydrolyze collagen: trypsin, (26) intracellular proteolytic enzymes of Oidiodendron kalari, (* ) Streptomyces griseus protease, (28,29) collagenases of genera Bacteroides, Clostridium, and Peptostreptococcus, 30.3 ) rat hepatic lysosomal extracts, 32 collagenases from rabbit synovial fibroblasts, 33 and cathepsin B and collagenolytic cathepsin from human placenta.(34)

35 TABLE 3. Product Formulation Data r5) Product Category Total No. of Total No. No. of Product Formulations Within Each Concentration Range (~6) Formulations Containing in Category Ingredient >50 >25-50 >lo-25 >5-10 >l-5 >O. J-I 10.1 Hydrolyzed Collagen Baby shampoos 35 1 Bath oils, tablets, and salts Bubble baths Other bath preparations Eyeliner Eye shadow Mascara Other eye makeup preparations Hair conditioners Hair sprays (aerosol fixatives) Hair straighteners 64 7 Permanent waves Hair rinses (noncoloring) Hair shampoos (noncoloring) Tonics, dressings, and other hair grooming aids Wave sets Other hair preparations (noncoloring) Hair tints Hair rinses (coloring) a

36 Hair bleaches Other hair coloring preparations Blushers (all types) Face powders Makeup foundations Lipstick Makeup bases Cuticle softeners Nail creams and lotions Nail polish and enamel Nail polish and enamel remover Other manicuring preparations Bath soaps and detergents Aftershave lotions Skin cleansing preparations (cold creams, lotions, liquids, and pads) Face, body, and hand skin care preparations (excluding shaving preparations) Moisturizing skin care preparations Night skin care preparations Paste masks (mudpacks) Skin fresheners Wrinkle smoothers (removers) Other skin care preparations z 3 a g 2 1 i y: - * 2 w 2 a - z z 4 E E s? TOTALS

37 206 COSMETIC INGREDIENT REVIEW In other studies, a proteinase from Pseudomonas aeruginosa catalyzed the hydrolysis of collagen,(35 whereas anthocyanosides isolated from Vaccinium myrtillis decreased collagen hydrolysis. (36) Hydrolyzed Collagen induced the activity of an extracellular collagenase produced by a marine Vibrio,(37) and a secondary vitamin K deficiency in rats increased the hydrolysis of collagen.(3s) The epimerization of trans-4-hydroxy-l-proline to cis-4-hydroxy-d-proline during acid hydrolysis of collagen has also been documented. 3g Collagen hydrolysates have been studied for their stimulatory effect on the healing of open wounds. (40) The ears of 1 rabbit were incised: 1 was kept as a control and 1 was treated with Hydrolyzed Collagen. The same rate of healing was observed for the first few days; thereafter, the treated ear healed more rapidly. 16 Animal Toxicology Acute Toxicity Oral Hydrolyzed Collagen (100 percent) was analyzed for acute oral toxicity in rats in 2 tests. LDSOs were > 10 g/kg and > 15 g/kg, respectively. In each study, investigators concluded that Hydrolyzed Collagen was nontoxic by ingestion 41,42 (Table 4). Two shampoo formulations, each containing 2 percent Hydrolyzed Collagen, were tested for acute oral toxicity in mice and rats. LD50s were > 15 ml/kg in both mice and rats. The investigators concluded that each formulation was practically nontoxic(43*44) (Table 4). A hair conditioner containing 0.5 percent Hydrolyzed Collagen had an oral LDso of >40.0 ml/kg in rats, estimated by interpolation from the probit response curve. The investigators observations included decreased activity, rales, diarrhea, salivation, and an increase in body weight(46) (Table 4). Ocular Irritation Four lots of Hydrolyzed Collagen (100 percent) were evaluated for ocular irritation by a modified Draize eye test. A 0.1 ml sample of Hydrolyzed Collagen TABLE 4. Acute Oral Toxicity of Hydrolyzed Collagen Compound Species LDso Results/Comments Reference Hydrolyzed Collagen, 100% Rat >lo g/kg Nontoxic 41 Hydrolyzed Collagen, 100% Rat > 15 g/kg Nontoxic 42 Hydrolyzed Collagen, 2% in a shampoo Mouse > 15 g/kg Practically nontoxic 43 Hydrolyzed Collagen, 2% in a shampoo Rat >15 g/kg Practically nontoxic 44 Hydrolyzed Collagen 0.5% in a hair conditioner Rat >4O.O ml/kg - 45

38 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 207 was instilled into the conjunctival sac of 1 eye of each of 6 albino rabbits. Eyes were scored by Draize classification (max, 110) at 1, 2, 3, 4, and 7 days or until negative. The 4 lots had average irritation scores of 5, 4, 3, 0; 6, 3, 1, 0; 5, 3, 3, 0; and 6, 3, 3,0 on Days 1, 2, 3, and 4, respectively. As judged by the Draize classification of eye irritation, Hydrolyzed Collagen was mildly irritatingf4 (Table 5). Hydrolyzed Collagen was evaluated as a 50 percent aqueous solution in a modified Draize eye irritation test. A 0.1 ml sample of the solution was instilled into the conjunctival sac of 1 eye of each of 6 albino rabbits; the other eye served as a control. The eyes were examined for 7 days or until negative. Average irritation scores were 1 and 0 (max, 110) on Days 1 and 2, respectively. As judged by the Draize classification of eye irritation, the test solution was minimally irritating 48 (Table 5). A shampoo formulation containing 2 percent Hydrolyzed Collagen was tested for ocular irritation in 9 albino rabbits. The shampoo was diluted to a concentration of 20 percent (w/v) with distilled water, and a 0.1 ml sample was instilled into the conjunctival sac of the left eye. Each right eye served as a control. TABLE 5. Ocular Irritation of Hydrolyzed Collagen Compound Number of Rabbits/Test Groups Results/Comments Reference Hydrolyzed Collagen, 100% (4 lots) Hydrolyzed Collagen, 50% aqueous solution 6 Average irritation scores (max, 110) on Days 1, 2, 3, and 4, respectively were: Lot 1-5, 4, 3, 0 Lot 2-6, 3, 1, 0 Lot 3-5, 3, 3, 0 Lot 4-6, 3, 3, 0; Hydrolyzed Collagen was concluded to be mildly irritating 6 Average irritation scores (max, 110) were 1 and 0 on Days 1 and 2, respectively; test solution concluded to be minimally irritating Hydrolyzed Collagen, 0.2% in a shampoo, tested as a 20% (w/v) aqueous dilution 91 3 rinsed 6 unrinsed No irritation observed in rinsed eyes; unrinsed eyes had mean total scores (max, 130) of 3.33, 0.67, 0.0, and 0.0 at 24, 48, and 72 hours and 7 days, respectively; concluded that shampoo was a nonirritant but could cause minimal irritation under test conditions 49 Hydrolyzed Collagen, 0.5% in a hair conditioner 91 3 unrinsed 3 rinsed 3 received 1:9 dilution with water Conjunctival scores of 2 or less (max, 110) seen in all unrinsed eyes, 1 rinsed, and 1 dilution eye; effects cleared by 72, 72, and 48 hours, respectively; concluded that hair conditioner was a mild, transient irritant under all test conditions 50

39 208 COSMETIC INGREDIENT REVIEW The eyes of 3 rabbits were rinsed with water 2 seconds after application; the other 6 were not rinsed. Eyes were scored at 24,48, and 72 hours and 7 days. The unrinsed treated eyes had conjunctival irritation at 24 hours consisting of slight erythema (6 rabbits), very slight edema (3 rabbits), and slight discharge (1 rabbit). Irritation decreased progressively, and all unrinsed eyes were normal at 72 hours. No irritation was observed in the rinsed eyes throughout the 7-day period. Mean total scores for the unrinsed eyes were 3.33, 0.67, 0.0, and 0.0 (max, 130) at 24, 48, and 72 hours and 7 days, respectively. The shampoo was found to be a nonirritant. However, under test conditions, it could cause minimal eye irritation 49 (Table 5). A hair conditioner containing 0.5 percent Hydrolyzed Collagen was tested for ocular irritation in 9 albino rabbits. A 0.1 ml sample was applied to the right eye of each rabbit. The rabbits were divided into groups of 3: Group I received the product full-strength, Group II received a full-strength application followed by rinsing 4 seconds later, and Group III received a 1:9 dilution of the conditioner with water. Ocular reactions were recorded 24, 48, and 72 hours and 7 days after instillation of the test material. Conjunctival effects (score of 2 or less, max, 110) were seen in all 3 rabbits of Group I and in 1 rabbit of Groups II and III. These effects had disappeared by 72 hours in Groups I and II and by 48 hours in Group III. The investigators concluded that the hair conditioner was a mild, transient irritant when applied full-strength with or without rinsing, or diluted 1:9 with water( ) (Table 5). DermalPrimary Skin irritation Hydrolyzed Collagen (100 percent) was tested for primary skin irritation using 6 rabbits. The backs of the rabbits were clipped and divided into 2 sections, of which 1 was abraded. Hydrolyzed Collagen was applied to both sections under gauze pads for 24 hours. Sites were scored upon patch removal and 48 hours later. No reactions were noted. The investigators concluded that Hydrolyzed Collagen was nonirritatingt41) (Table 6). Hydrolyzed Collagen was applied full-strength to the shaved backs of 9 rabbits in a modified Draize primary skin irritation test. A 0.1 ml sample was applied to each rabbit by occlusive filter disc for 24 hours. Four lots of Hydrolyzed Collagen were evaluated. Sites were graded upon disc removal and 48 hours later. Average irritation socres (max, 4) for the 4 lots were 0.25, 0.20, 0.16, and The investigators concluded the Hydrolyzed Collagen was minimally irritatingt5 (Table 6). Hydrolyzed Collagen was evaluated as a 50 percent aqueous solution for primary skin irritation using 9 albino rabbits. Samples of 0.1 ml were applied by occlusive filter disc to the shaved skin of the rabbits for 24 hours. Applications were made for 3 consecutive days. Sites were scored for irritation 24 hours after each application. The maximum average irritation response was 1.33 (max, 4); the test solution was considered mildly irritating(521 (Table 6). Various concentrated solutions of a soap containing 26 to 28 percent Hydrolyzed Collagen were brushed onto the skin of guinea pigs (number unspecified). Skin changes were monitored clinically and microscopically. Body weights were recorded and necropsies performed on a number of the animals. No internal in-

40 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 209 juries were noted. The soap was considered significantly less irritating than a lauryl sulfate solution of the equivalent concentration(53 (Table 6). A shampoo formulation containing 2 percent Hydrolyzed Collagen was evaluated for skin irritation and acute dermal toxicity. Three groups of 4 rabbits each received doses of 1.5, 3, and 6 ml/kg of the test shampoo, and a fourth group received 6 ml/kg of a regular shampoo as a comparative control. Backs of all rabbits were clipped; 2 in each group also were abraded. The shampoos were applied full-strength under an occlusive wrap for 24 hours. Sites were evaluated according to Draize at 24, 48, and 72 hours and 7 and 14 days. At 24 and 48 hours, moderate erythema (score of 2 on 0 to 4 scale) was seen in all control rabbits and 1 high-dose nonabraded rabbit. Slight erythema (score of 1) was noted in all others, and no edema was noted. The skin appeared darkened at 48 hours in 1 control rabbit and in the high-dose rabbit with moderate erythema. At 72 hours, no erythema or edema was noted. However, the same 2 rabbits still had darkened skin, whereas peeling and scaling of the skin was observed in the others. At 7 and 14 days, treated skin sites appeared normal except for areas of pustular dermatitis due to a secondary infection; 1 high-dose rabbit had darkened, thickened skin (Day 7), and 1 had dry, cracked, and peeling skin (Day 14). The investigators reported that application of the shampoo caused erythema and burning of the skin of the rabbit. Its application, under exaggerated conditions, was responsible for the degeneration and necrosis of the skin of some rabbits and was followed by secondary bacterial infection in others as manifested by a pustular dermatitis. There were no significant differences in hematological and urinalysis values. A dose-related decrease in feed consumption and body weight gain was observed. However, there were no other signs of systemic toxicity. Necropsy of the 3 rabbits that died during the study (2 control, 1 high dose) indicated that acute pneumonia was the prime contributing factor(54 (Table 6). Two other shampoo formulations, each containing 2 percent Hydrolyzed Collagen, were evaluated for skin irritation using identical tests. The first shampoo was administered full-strength, whereas the second was diluted to 20 percent (w/v) in distilled water. SampJes of 0.5 ml of the shampoo or dilution were administered to the intact and abraded skin of 6 rabbits and occluded for 24 hours. Sites were scored according to Draize at 24, 48 (shampoo only), and 72 hours. Moderate to severe irritation was observed in the rabbits treated with the full-strength shampoo; very slight to mild irritation was seen in the rabbits receiving the dilution treatments (Table 6). The shampoo and dilution had primary irritation scores (PIS) of 4.67 and 1.46, respectively, where a score of 5 (max, 8) is considered to be a primary skin irritant. The investigators concluded that the shampoo was a dermal irritant to the intact and abraded skin of rabbits, whereas the dilution was classified as a nonirritant. However, it was noted that mild irritation can occur under these test conditions.(55.56) A hair conditioner containing 0.5 percent Hydrolyzed Collagen was evaluated for dermal irritation in a combined irritation and phototoxicity test. Occlusive patches containing 0.20 ml of the conditioner were applied to the backs of 6 clipped rabbits for 48 hours. Sites were scored for erythema and edema at 49, 72, and 96 hours; all rabbits had a score of 0 (max, 8). It was concluded that the hair conditioner was not a primary dermal irritantc5 ) (Table 6).

41 TABLE 6. Primary Skin IrritationlDermal Toxicity of Hydrolyzed Collagen Compound Test Method Number and Species of Animals Results/Comments Reference Hydrolyzed 100% Collagen, Applied under gauze pads for 24 hours. I, A* 6 rabbits No reactions Pllt of 0; nonirritating 41 Hydrolyzed Collagen, 100% (4 lots) Occlusive disc for 24 hours-l 9 rabbits Average irritation scores fmax, 4) for 4 lots were 0.25, 0.20, 0.16, and 0.26; concluded to be minimally irritating 51 Hydrolyzed Collagen, 50% aqueous solution Occlusive disc for 24 hours, 3 consecutive applications- I 9 rabbits Maximum average irritation response was 1.33 (max, 4); test solution considered mildly irritating 52 Hydrolyzed Collagen, 26-28% in a soap Various concentra- tions brushed on- Guinea pigs, unspecified number Considered significantly less irritating than a lauryl sulfate solution of the equivalent concentration; no internal injuries noted 53 Hydrolyzed Collagen, 2% in a shampoo Occlusive patch for 24 hours-l, A- doses of 1.5, 3, and 6 ml/kg 12 rabbits Moderate erythema observed in all control rabbits and 1 high-dose rabbit (score of 2, max, 4), slight erythema (score of 1) in all others, clearing by 72 hours; no edema noted; several rabbits had darkened or cracked and peeling skin, pustular dermatitis due to secondary infection in others; dose-related decrease in feed con- 54

42 sumption and body weight gain; no other signs of systemic toxicity; conclusion: under exaggerated conditions, caused erythema and burning of rabbit skin and was responsible for the degeneration and necrosis of skin, allowing secondary infection Hydrolyzed Collagen, Occlusive patch for 2% in a shampoo 24 hours-l, A 6 rabbits Moderate to severe and well-defined erythema was observed in 5 and 1 rabbits, respectively, at 24 hours, with similar results at 72 hours; very slight, slight, and moderate edema was observed in 1, 3, and 2 rabbits, 55 respectively, at 24 hours, decreasing minimally by 72 hours; PISS of 4.67 (max, 8); concluded to be dermal irritant on I and A skin Hydrolyzed Collagen, 2% in a shampoodiluted to 20% (w/v) in distilled water Occlusive patch for 24 hours-l, A 6 rabbits Well-defined and very slight erythema was observed in 3 and 3 rabbits, respectively, at 24 hours, diminishing to only very slight scores in 5 rabbits at 72 hours; slight and very slight edema was observed in 1 and 3 rabbits, respectively, at 24 hours, clearing totally by 72 hours; PIS of 1.46 (max, 8); concluded to be a nonirritant on I and A skin but can cause mild irritation under study conditions 56 Hydrolyzed Collagen, 0.5% in a hair conditioner Occlusive patch for 48 hours-l 6 rabbits No erythema or edema; all rabbits had individual score of 0 (max, 8); concluded to be not a primary dermal irritant 57 *I, intact; A, abraded. tpll, primary irritation index. *PIS, primary irritation scores

43 212 CbSMETlC INGREDIENT REVIEW Subchronic Toxicity Dermal A hair preparation containing 2 percent Hydrolyzed Collagen was tested for subchronic dermal toxicity. Three groups of 2 male and 2 female rabbits received 100, 1000, or 3200 mg/kg of the test shampoo. Control groups received 2000 mg/kg of a marketed antidandruff shampoo, 3220 mg/kg of the test shampoo without the active drug ingredient, and 1 ml water/kg. All test and control applications were made daily for 30 days, were left on the skin for 15 minutes, and were then removed with water. The skin of 1 male and 1 female in each group was abraded weekly. The only treatment-related finding was local skin irritation. No deaths, abnormal behavior, gross or microscopic lesions were associated with treatment.(58) Another shampoo containing 2 percent Hydrolyzed Collagen was tested for dermal toxicity using Yorkshire pigs (white). Three groups of 2 male and 2 female pigs (1 of each sex abraded twice weekly) received applications of 0.5, 1.O, and 2.0 ml/kg. Control groups received 2.0 ml/kg saline and 2.0 ml/kg of another marketed shampoo. Applications were made to the clipped back of each pig twice daily for 4 weeks. Treated sites were rinsed with warm water 1 hour after each application. All pigs were given a general physical examination before and at 4 weeks observation. Body weights were recorded weekly, and blood samples were obtained for routine hematological and serum chemistry evaluations. All animals were necropsied. The abraded skin of 1 high-dose female was slightly irritated; the skin of all others appeared unremarkable. No dose-related effects were determined by physical examination, hematological and serum chemistry evaluations, necropsy examination, and histopathological evaluation. A statistically significant dose-regression relationship for male gonad weights was not considered indicative of systemic toxicity due to the immaturity and variation in size of the testes of these young pigs. Minimal focal inflammatory cellular infiltration was noted in the treated dermis of 1 middle-dose and 1 high-dose pig. However, this same condition was observed in the untreated skin of 1 saline control and 1 high-dose pig. 5g) Sensitization Hydrolyzed Collagen was tested for sensitization using 2 male white guinea pigs. A 0.1 percent solution of Hydrolyzed Collagen in physiological saline was injected intracutaneously into the clipped back or flank of each guinea pig every other day or 3 times weekly for a total of 10 injections. The first injection consisted of 0.05 ml of the test solution; each succeeding injection consisted of 0.1 ml. Sites were scored for diameter, height, and color 24 hours after each injection. After a 2-week rest period, a challenge injection of 0.05 ml was administered into a different site. Induction injections gave average diameter scores of 7.6 and 8.9 mm, average heights of 0.3 and 0.2 mm, and an average color of pink for the 2 guinea pigs. On challenge, 1 animal had no reaction, whereas the second had a reaction of diameter 5 mm, no height, and a pink color. Both animals had a sensitization score of 0 (9 or above is severely sensitizing), classifying Hydrolyzed Collagen as a nonsensitizer.(41) A shampoo formulation containing 2 percent Hydrolyzed Collagen was

44 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 213 tested for sensitization using a modification of the Buehler and Griffithf60) method. A total of 3 inductive applications, 1 per week, were made to the clipped back of 10 guinea pigs. The first inductive application consisted of 0.5 ml of a 1, 5, and 10 percent dilution (distilled water v/v) occlusively patched on 3 separate sites on the right side of the animal for 24 hours. The second and third inductions consisted of 0.5 ml of a 10 percent aqueous dilution occlusively patched on the left side of the animal for 6 hours. A 24hour challenge patch was applied 2 weeks later to an untreated site on the animal s right side. Sites were scored for erythema 24 hours after each application. Dinitrochlorobenzene (DNCB) was tested as a positive control. The first and third inductive patches produced no erythema, and the second produced very slight erythema in 2 guinea pigs. No erythema was observed at challenge. The investigators concluded that the shampoo formulation did not cause sensitization.(45) Phototoxicity A shampoo formulation containing 2 percent Hydrolyzed Collagen was tested for phototoxicity using 2 guinea pigs. The shampoo was tested as a 20 percent (w/v) mixture in distilled water, and 8-methoxypsoralen was tested undiluted as a positive control. The back of each animal was clipped and divided into 4 site:; 2 received 0.1 ml of the shampoo and 2 received 0.05 ml of 8-methoxypsoralen. Fifteen to twenty minutes later, the right side of each animal was shielded with cardboard while the animals were irradiated for 1 hour with UVA light (320 to 400 nm) using a No. F40 BL 40W Westinghouse Blacklight. Sites were graded for erythema (max, 4) 24 hours after exposure. All exposed and unexposed sites treated with the shampoo dilution had scores of 0. 8-Methoxypsoralen gave a mean score of 3.5 for the irradiated sites and a score of 0 for the nonirradiated sites. The investigators concluded that the shampoo formulation was not phototoxic in guinea pigs. A hair conditioner containing 0.5 percent Hydrolyzed Collagen was also analyzed for phototoxicity using 6 rabbits. One rabbit received 8-methoxypsoralen as a positive control. A 0.20 ml sample of the conditioner was applied to a gauze patch, evaporated for 5 minutes, and then placed on the clipped back of the rabbit and occluded. Two patches were applied to each animal. Two hours later, 1 patch was removed and the other protected with aluminum foil while the animals were irradiated for 15 minutes with Sylvania lights No. F-40-BLB. The patches were then replaced until 48 hours posttreatment, at which time all patches were removed. Sites were scored for erythema and edema (max, 8) 1 hour later and at 72 and 96 hours. Each rabbit had an individual score of 0 for both irradiated and nonirradiated sites. The hair conditioner was neither a primary dermal irritant nor a phototoxic irritant to rabbit skin.ts7) CLINICAL ASSESSMENT OF SAFETY Irritation and Sensitization Hydrolyzed Collagen (100 percent) was tested for skin irritation on 20 humans. A single patch containing 0.1 ml of Hydrolyzed Collagen was applied to the volar forearm or the inner aspect of the arm of each subject. A standard con-

45 214 COSMETIC INGREDIENT REVIEW trol was also tested. Reactions were recorded 2 and 24 hours after patch removal. The irritation score for Hydrolyzed Collagen and the controls in all 20 subjects was 0 (max, 4). No significant difference in irritancy potential existed between Hydrolyzed Collagen and the control 62 (Table 7). Patch tests were performed on 33 subjects (18 men and 15 women) using Hydrolyzed Collagen at concentrations of 2 and 20 percent. Occlusive patches containing Hydrolyzed Collagen at each concentration were applied to the breast or arm for 24 hours. Sites were scored at 24, 48, and 72 hours; no reactions were observed 3 (Table 7). A 21 -day cumulative irritation test was conducted on a hair conditioner containing 0.5 percent Hydrolyzed Collagen. Semiocclusive patches with 0.5 ml of the conditioner were applied to the upper part of the back of 20 subjects for 24 hours. Patches were then removed, evaluated 30 minutes later, and a new patch was applied. These procedures were repeated for 15 applications, allowing for 21-day continuous exposure. Mineral oil was used as the standard control. Of the 17 subjects who completed the study, only 1 had any reaction, giving a cumulative irritation score of 0.5 (max, 84). The mean cumulative irritation score of 0.03 was exactly comparable to that of mineral oil (0.03). The investigators concluded that the product as used by label directions would not present any medical hazard to the consumer 64) (Table 7). Various compositions of a soap containing 26 to 28 percent Hydrolyzed Collagen were applied daily to the skin as a 5 percent solution for 10 to 48 days. A large number of healthy subjects and people with dermatitis were used. A low degree of irritation was seen even at high concentrations of the least irritating composition. No sensitization was observed. The treated skin area was examined microscopically; those with acute dermatitis had moderate irritation 53 (Table 7). Five cosmetic formulations were evaluated for irritation and sensitization in repeated insult patch tests (RIPT). Three of these, a morning cream, a suntan lotion, and a night cream, containing 3.0, 2.2, and 3.0 percent Hydrolyzed Collagen, respectively, were tested in the same manner. A series of hour occlusive patches containing the undiluted formulation was applied to the back of each subject. Sites were graded after each removal and 24 hours after removal of the tenth patch (morning cream and suntan lotion only). After apprcximately an 1 l-day rest period, challenge patches were applied, occluded for 48 hours, and scored upon removal and 24 hours later. Scattered irritant responses after the third application were seen in the 103 subjects who completed the induction phase for the morning cream and suntan lotion. The maximum number of responses seen at any 1 reading for the suntan lotion was 11 with erythema and 8 with very mild erythema. Maximum response to the morning cream was 5 with erythema and 10 with very mild erythema. Of the 96 subjects who completed the challenge phase, 1 had erythema and 1 had very mild erythema to the suntan lotion at 24 hours. However, the panelist with erythema had a negati-e reaction on rechallenge. No reactions were observed on challenge with the morning cream. Two of the 113 panelists completing the induction phase testing of the night cream had irritant responses: 1 had very mild erythema and 1 had erythema. One of the 103 subjects completing the challenge phase had erythema. The investigators concluded that the morning cream, suntan lotion, and night cream were mildly irritating, definitely irritating, and nonirritating, respectively, whereas none of the formulations gave significant evidence of sensitization(65,66) (Table 7).

46 TABLE 7. Clinical Irritation and Sensitization ingredient Type of Test Number of Humans ResultKomments Reference Hydrolyzed 100% Collagen, Single patchtype and duration not specified 20 Average irritation score of 0 for all 20 subjects (max, 4); no significant difference between test material and control 62 Hydrolyzed Collagen, 2 and 20% Single, 24.hour occlusive patch 33 No reactions were observed 63 Hydrolyzed Collagen, 0.5% in a hair con- 21-day cumulative irrita- 17 One subject had a reaction, giving a cumulative irritation score of 0.5 (max, 84); mean cumulative irritation score 64 ditioner tion test of 0.03 was exactly comparable to control; product should not present any medical hazard; nonirritant Hydrolyzed Collagen, 26-28% in a soaptested as 5% solution of the soap Applied daily to skin for 1 O-48 days Large number (unspeci fied), both healthy subjects and some with dermatitis Low degree of irritation, no sensitization was observed; those with acute dermatitis showed moderate irritation 53 Hydrolyzed Collagen, 3.0% in a morning cream RIPT* 103-l+ 96-C Scattered irritant responses, maximum response at any one reading was 10 very mild erythemas and 5 erythemds; no reactions on challenge; mildly irritating dnd nonsensitiring 65 Hydrolyzed Collagen, 2.2% in a suntan lotion RIPT 103-l 96-C Scattered irritdnt responses, maximum response at dny one redding was 8 very mild erythemas and 11 erythemas; 2 reactions to the challenge, 1 very mild erythema and 1 erythema; negative reaction on rechallenge; definitely irritating and nonsrnsitizing 65 Hydrolyzed Collagen, 3.0% in a night cream RIPT 113-l 103-c Two irritant responses: 1 very mrld erythcma and I erythema; 1 erythema reaction on challenge; nonirritating and nonsensitizing bh Hydrolyzed Collagen, 0.5% in a mascara RIPT 205 One subject exhibited faint erythemd during the induction phase; no reactions at chdtlenge; nonirritating and nonsensitizing 67

47 TABLE 7. (Continued) Ingredient Type of Test Number of Humans Results/Comments Reference Hydrolyzed Collagen, 0.5% in a hair conditioner tested as a 0.1 % dilution RIPT 207-I 201 -c Mean cumulative irritation scores (max, 50) as follows: 167 subjects had score of 0 22 subjects had score of 1 12 subjects had score of subject had score of 3 1 subject had score of 4 3 subjects had score of 5 1 subject had score of 6; one subject exhibited erythema at challenge; however, this panelist reacted to 10 of the other 13 products and had no reaction on rechallenge Hydrolyzed Collagen, 0.5% in a mascara Controlled use 27 test, 4 weeks of daily use No reactions were observed 69 *Repeated insult patch test. +I, induction; C, challenge.

48 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 217 The fourth formulation tested by RIPT was a mascara containing 0.5 percent Hydrolyzed Collagen. Occlusive patches containing the undiluted mascara were applied to the upper backs of 205 subjects on Monday, Wednesday, and Friday for 3 consecutive weeks. Patches were removed and sites graded just prior to the next scheduled patch application. Following a 2-week rest, 2 consecutive 48-hour challenge patches were applied to adjacent sites on the back. Sites were graded at 48 and 96 hours. One subject had faint erythema during the induction phase; no reactions were observed at challenge. The mascara was found to be neither an irritant nor a sensitizer(67) (Table 7). The fifth formulation evaluated by RIPT was a hair conditioner containing0.5 percent Hydrolyzed Collagen. A 0.1 percent dilution of the conditioner was applied using semioccluded patches to the upper backs of the subjects for 48 hours. Patches were then removed, sites evaluated, and new patches applied for a total of 10 applications. Following a 2-week rest period, challenge patches were applied to the subjects thighs. Mean cumulative irritation scores (max, 50) were as follows: 167 subjects had a score of 0, 22 subjects had a score of 1, 12 subjects had a score of 2, 3 subjects had scores of 3,4, and 6 (each), and 3 subjects had a score of 5. Of the 201 subjects completing the challenge phase of the study, 1 had erythema. However, this panelist also reacted to 10 of the other 13 substances being tested. A rechallenge on the other thigh produced no reaction(68) (Table 7). A mascara formulation containing 0.5 percent Hydrolyzed Collagen was evaluated by a 4-week controlled use test. Twenty-seven women used the product daily as per normal instructions. No reactions were observed(69) (Table 7). A prospective study of cosmetic-induced dermatitis by 11 dermatologists of the North American Contact Dermatitis Group (NACDG) identified 1 case of dermatitis associated with use of Hydrolyzed Collagen from among a total of 487 cases. ( O) Phototoxicity/Photosnsitization A mascara formulation containing 0.5 percent Hydrolyzed Collagen was tested for phototoxicitylphotosensitization on a panel of 23 humans. Occlusive patches containing 0.1 g/cm* of the mascara were applied to the backs of the subjects for 24 hours. Patches were then removed, evaluated, and irradiated with 3 times the individual s MED using a xenon arc solar simulator (150 W) filtered to produce a continuous UVA-UVB emission spectrum (290 to 400 nm). Sites were evaluated 48 hours later, and the procedures of application, patching, and irradiation were repeated for a total of 7 applications. No reactions were observed. The investigators concluded that the mascara was neither phototoxic nor a photosensitizer. ( l) Domsch et al.( ) have found that UV-induced erythema was decreased by rubbing Hydrolyzed Collagen (mean molecular weight of 1500) into the skin. A 10 percent solution of Hydrolyzed Collagen applied immediately and 24 hours after irradiation decreased erythema by 20 percent at 24 hours and 25 percent at 48 hours.

49 218 COSMETIC INGREDIENT REVIEW SUMMARY Hydrolyzed Collagen is a collagen hydrolysate derived by acid, enzyme, or other method of hydrolysis. It is a white to off-white hygroscopic powder of molecular weight 1000 to 10,000 and is also available as a viscous, amber aqueous solution. Hydrolyzed Collagen can be prepared by any 1 of 3 methods: alkaline hydrolysis of bovine skin products followed by enzymatic hydrolysis to the desired molecular weight, enzymatic hydrolysis of fresh animal byproducts or bovine-derived leather, or acid or alkaline hydrolysis of chrome leather fold splinters with inorganic acids or lyes to a defined molecular weight. The hydrolysis of collagen yields a high content of glycine and proline compared to other proteins, as well as 2 unique amino acids, hydroxyproline and hydroxylysine. Hydrolyzed Collagen is usually analyzed by column chromatography. Hydrolyzed Collagen is used in cosmetics, primarily hair and skin care products, because of its conditioning and moisturizing properties. It is usually incorporated at concentrations ~5 percent and was in 923 formulations reported in Cosmetic products containing Hydrolyzed Collagen may contact all external body surfaces and hair, as well as the eyes. Frequency and length of application could result in continuous exposure. Many studies have documented the substantivity of Hydrolyzed Collagen to human hair and skin. Hydrolyzed Collagen generally is adsorbed on hair, although it has been shown to penetrate the cuticle and cortex. Increasing concentrations of Hydrolyzed Collagen increased adsorption, as did increasing damage to hair. Hydrolyzed Collagen also binds water to the hair and skin surfaces and is used as a buffering agent for alkali in permanent waving preparations. Acute toxicity studies found Hydrolyzed Collagen and formulations containing Hydrolyzed Collagen practically nontoxic when administered orally to mice and rats. Dermal studies gave no indication of systemic toxicity when formulations containing Hydrolyzed Collagen were applied to rabbits and guinea pigs. However, a shampoo formulation (2 percent Hydrolyzed Collagen) administered to rabbits under exaggerated conditions did cause erythema and burning, leading to degeneration and necrosis of the skin. Hydrolyzed Collagen was minimally irritating to rabbit eyes when tested fullstrength and in formulation. Primary skin irritation tests in rabbits indicated that Hydrolyzed Collagen was nonirritating or minimally irritating when tested fullstrength, whereas a 50 percent aqueous solution of Hydrolyzed Collagen was mildly irritating. Shampoo formulations containing Hydrolyzed Collagen (2 percent) were generally nonirritating when tested as dilutions. However, these were irritating under the exaggerated conditions of a full-strength application. A soap (26 to 28 percent Hydrolyzed Collagen) and hair conditioner (0.5 percent Hydrolyzed Collagen) produced no dermal irritation in guinea pigs and rabbits, respectively. Subchronic dermal studies on 2 cosmetic formulations containing 2 percent Hydrolyzed Collagen were negative for systemic toxicity in rabbits and Yorkshire pigs. Hydrolyzed Collagen was nonsensitizing in guinea pigs. Cosmetic formulations containing Hydrolyzed Collagen (2, 2, and 0.5 percent) were also nonsensitizing and nonphototoxic in guinea pigs and rabbits.

50 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN 219 In clinical studies, Hydrolyzed Collagen produced no skin irritation. Formulations containing Hydrolyzed Collagen at concentrations ranging from 0.5 to 28 percent produced some irritation. However, no significant evidence of sensitization was observed in any study. No phototoxicity or photosensitization was evident in a study of a mascara containing 0.5 percent Hydrolyzed Collagen. It has been reported that UV-induced erythema was decreased by rubbing Hydrolyzed Collagen into the skin after irradiation. CONCLUSION On the basis of the available animal and clinical data presented in this report, the Panel concludes that Hydrolyzed Collagen is safe as a cosmetic ingredient in the present practices of use and concentration. ACKNOWLEDGMENT Elizabeth Meerman Santos, Scientific Analyst and writer, prepared the Iiterature review and technical analysis used by the Expert Panel in developing this report. REFERENCES 1. ESTRIN, N.F., CROSLEY, P.A., and HAYNES, C.R. (eds.). (1982). The CTFA Cosmetic Ingredient Dictionary, 3rd ed. Washington, DC: The Cosmetic, Toiletry and Fragrance Association. 2. COSMETIC, TOILETRY AND FRAGRANCE ASSN. (CTFA). (September 16, 1982). Submission of unpublished data by CTFA: CTFA cosmetic ingredient chemical description ( ).* 3. LAST, J.A., BAER, J., and MILLSON, C. (1976). Site of hydrolysis of collagen by hot trichloroacetic acid. Connect. Tissue Res. 4(3), ROGERS, R. (August 1, 1978). Monograph on Collagen. NTIS No. PB Prepared by Informatics, Inc., Rockville, MD, for the Food and Drug Administration. Contract No. FDA BORE, M., and ARNAUD, M. (1975). Control of protein hydrolysatesand amino acids in cosmetic preparations. Labo-Pharma-Probl. Tech. 23(240), BOREL, J.P., DESANTI, M., CUILLAUME, C., and BARASCUT, J.L. (1968). A method for the detection of peptides separated by column chromatography: Application to the analysis of collagen hydrolysates. Bull. Sot. Chim. Biol. (Paris) 50(11), ZARKADAS, C.G. (January, 1975). A simple chromatographic method for the determination of the methylated basic amino acids in proteins. Can. J. Biochem. 53(l), MILLER, E.J., and PIEZ, K.A. (August 1966). An accelerated single-column procedure for the automatic analysis of amino acids in collagen and elastin hydrolyzates. Anal. Biochem. 16(2). 9. CUNNINGHAM, L.W., FORD, J.D., and SEGREST, J.P. (May 25, 1967). The isolation of identical hydroxylysyl glycosides from hydrolysates of soluble collagen and from human urine. J. Biol. Chem. 242(10) l 10. ESTRIN, N.F., HAYNES, C.R., and WHELAN, J.M. (eds.). (1982). Cosmetic Ingredient Descrrptrons: Compendium of Cosmetic ingredient Composition. Washington, DC: Cosmetic, Toiletry and Fragrance Association. *Available upon request: Administrator, Cosmetic Ingredient Review, Suite 810, 1110 Vermont Avenue, N.W., Washington, DC

51 220 COSMETIC INGREDIENT REVIEW 11. BURNETT, R.S. (October 1963). Proteins in cosmetics. Am. Perfumer Cosmetics 78, FRECH, C., ALLEN, L.V., STILES, M., and LEVINSON, R.S. (December 1979). Sodium acetate as a preserva- tive in protein hydrolysate solutions. Am. 1. Hosp. Pharm. 36, SCAFIDI, A.A., and NEGHME, B.M. (April 1980). Protein hydrolysates and condensates in creams and lo- tions. Cosmet. Toilet. 95, COOPERMAN, E.S. (1972). Protein hydrolysates as skin moisturizers. Am. Cosmetics Perfume 87(3), FOOD AND DRUG ADMINISTRATION (FDA). (December 1981). Product formulation data. Computer printout. 16. RISO, R. (April 1974). Protein derivatives in cosmetics. Cosmetics Perfume 89, CONSTANTINESCU, M., DOBRESCU, D., VOINESCU, M.V., and CONSTANTINESCU, M. (July 23, 1980). Product from Collagen for Treating Gastritis and Gastric and Duodenal Ulcers. Rom. Patent No (In- stitutul de Cercetari Pielarie si Incaltaminte). 18. EASTON, I.A., GLEN, J.M., and KIERAN, A.C. (March 25, 1981). Film Forming Composition and Its Uses. Eur. Pat. Appl. Patent No (Devro, Inc.). 19. KIM, C.D., YANG, S.J., and LEE, O.S. (October 1979). Effect of ginseng saponin on hair. Cosmet. Toilet. 94, 49-50, 52, 56, 60, 62, KARJALA, S.A., WILLIAMSON, J.E., and KARLER, A. (1966). Studies on the substantivity of collagen-derived polypeptides to human hair. J. Sot. Cosmet. Chem. 21. STERN, ES., and JOHNSEN, V.L. (August 1977). Studies on the molecular weight distribution of cosmetic protein hydrolysates. J. Sot. Cosmet. Chem. 28, BROOKS, G.J. (July 1979). Use of protein in bath and shower products. Cosmet. Toilet. 94, COOPERMAN, E.S., and JOHNSEN, V.L. (July 1973). Penetration of protein hydrolysates into human hair strands. Cosmetics Perfume 88, KARJALA, S.A.. BOUTHILET, R.J., and WILLIAMSON, J.E. (May 1966). Some factors affecting the substantivity of proteins to hair. The Toilet Goods Association, Proceedings of the Scientific Section. 25. SCHRADER, K. (1981). Studies on water-retaining cosmetics on the skin. Pa&em. Kosmet. 62(g), GUMPRECHT, J.A. (June 8, 1977). Polypeptides for Cosmetic Use. Ger. Offen. Patent No (Red- ken Laboratories, Inc.). 27. CINO, P.M., and TEWARI, R.P. (September 1975). Proteolytic activity of Oidiodendron kalari. Can. J. Mi- crobiol. 21(g), KARPENKO, G.F., and KASTRIKINA, T.F. (May-June 1977). Study of bone tissue insoluble collagen hydrol- ysis by Streptomyces griseus protease using the method of N-terminal analysis. Ukr. Biokhim. Zh. 49(3), MYSHUNIN, I.F., TSYPEROVYCH, O.S., and KUZNETSOVA, I.M. (March-April 1975). Hydrolysis of insol- uble collagen of bull bones by Streptomycesgriseus crystalline protease. Ukr. Biokhim. Zh. 47(2), STEFFEN, E.K., and HENTGES, D.J. (August 1981). Hydrolytic enzymes of anaerobic bacteria isolated from human infections. J. Clin. Microbial. 14(2), OPPENHEIM, F., and FRANZBLAU, C. (1978). A modified procedure for the purification of clostridial col- Jagenase. Prep. Biochem. 8(5), COFFEY, J.W., FIEDLER-NAGY, C., GEORGIADIS, A.G., and SALVADOR, R.A. (September 10, 1976). Di- gestion of native collagen, denaturated collagen, and collagen fragments by extracts of rat liver lysosomes. J. Biol. Chem. 251(17), WERB. Z., and REYNOLDS, J.J. (December 1975). Purification and properties of a specific collagenase from rabbit synovial fibroblasts. Biochem. J. 151(3), EVANS, P., and ETHERINGTON, D.J. (February 1, 1978). Characterization of cathepsin 8 and collagenoly- tic cathepsin from human placenta. Eur. J. Biochem. 83(l), CARRICK, K., JR., and BERK, R.S. (June 24, 1975). Purification and partial characterization of a col- Jagenolytic enzyme from Pseudomonas aeruginosa. Biochim. Biophys. Acta 391(2), ROBERT, A.M., GODEAU, G., MOATI, F., and MISKULIN, M. (1977). Action of anthocyanosides of Vaccinium myrti//is on the permeability of the blood-brain barrier, J. Med. 8(5), DRElSBACH, J..H., and MERKEL, J.R. (August 1978). Induction of collagenase production in Vibrio J. Bacterial. 135(2), SHARAEV, P.N., BOGDANOV, N.G., and IAMALDINOV, R.N. (June 1976). Collagen metabolism in the skin with different vitamin K regimens. Biull. Eksp. Biol. Med. (Bulletin of Experimental Biology and Medi- cine) DZIEWIATKOWSKI, D.D., HASCALL, V.C., and RIOLO, R.L. (October 1972). Epimerization of trans-4.hydroxy-l-proline to cis-4-hydroxy-d-proline during acid hydrolysis of collagen, Anal. Biochem. 49(2),

52 FINAL REPORT: SAFETY ASSESSMENT OF HYDROLYZED COLLAGEN NOSOVA, I.M., PISARZHEVSKII, S.A., ABOIANTS, R.K., and KARELIN, A.A. (August 1981). Mechanism of action of collagen on processes of regeneration. Bull. Exp. Biol. Med. (Russian) 92(8), CTFA. (August 19, 1975). Submission of unpublished data by CTFA. Primary skin irritation, sensitization, and acute oral toxicity tests (2-19-3).* 42. CTFA. (April 29, 1974). Submission of unpublished data by CTFA. Acute oral toxicity test ( ).* 43. CTFA. (December 6, 1977). Submission of unpublished data by CTFA. Acute oral toxicity test ( l).* 44. CTFA. (December 6, 1977). Submission of unpublished data by CTFA. Acute oral toxicity test ( ).* 45. CTFA. (March 3, 1978). Submission of unpublished data by CTFA. Guinea pig sensitization test ( ) 46. FOOD AND DRUG RESEARCH LABORATORIES. (FDRL). (May 1, 1978). Acute oral toxicity test (2-19-8).* 47. CTFA. (September 4, 1973). Submission of unpublished data by CTFA. Acute eye irritation test ( ).* 48. CTFA. (April 27, 1972). Submission of unpublished data by CTFA. Acute eye irritation test ( ).* 49. CTFA. (December 13, 1978). Submission of unpublished data by CTFA. Acute eye irritation test ( ).* 50. FDRL. (April 24, 1978). Acute eye irritation test (2-19.6).* 51. CTFA. (September 4, 1973). Submission of unpublished data by CTFA. Primary skin irritation test ( ).* 52. CTFA. (April 28, 1972). Submission of unpublished data by CTFA. Primary skin irritation test ( ).* 53. NILZEN, A. (1965). Two Types of Soap of Medical interest. Allergies Laboratories, Karolinska sjukhuset, Stockholm.* 54. TOXICOLOGY AND PATHOLOGY SERVICES (TPS). (February 9, 1978). Acute dermal toxicity test ( ). 55. CTFA. (December 3, 1977). Submission of unpublished data by CTFA. Primary skin irritation test ( ).* 56. CTFA. (March 5, 1978). Submission of unpublished data by CTFA. Primary skin irritation test (2-l g-17).* 57. FDRL. (May 21, 1979). Primary dermal phototoxic irritation study (2-19-7).* 58. CTFA. (January 25, 1979). Submission of unpublished data by CTFA. Subchronic dermal toxicity test ( ).* 59. TPS. (February 15, 1978). Subchronic dermal toxicity test (2-19-lo).* 60. BUEHLER, E.V., and GRIFFITH, F. (1975). In: Maibach (ed.). Animal Models in Dermatology. Edinburgh: Churchill Livingston, pp CTFA. (January 6, 1978). Submission of unpublished data by CTFA. Guinea pig phototoxicity test ( ).f 62. CTFA. (April 3, 1972). Submission of unpublished data by CTFA. Clinical skin irritation test ( ).* 63. CTFA. (July 9, 1971). Submission of data in support of safety of potassium-coca-hydrolyzed animal protein and related compounds. Human patch test. Tokyo Medical and Dental University Dermatology Dept., Toshikatsu Irimajiri.* 64. CONCORDE LABORATORIES, LTD. (January 22, 1982). Twenty-one-day primary dermal cumulative irritation (2-19-S).* 65. TESTKIT LABORATORIES, INC. (March 31, 1981). Clinical irritation and sensitization test (2-19-l).* 66. TESTKIT LABORATORIES, INC. (July 31, 1980). Clinical irritation and sensitization test (2-19-2).* 67. CTFA. (September 17, 1979). Submission of unpublished data by CTFA. Clinical repeat insult patch test (RIPT) ( ).* 68. CONCORDE LABORATORIES, LTD. (January 22, 1982). Clinical irritation and sensitization (2-1 g-4).* 69. CTFA. (September 17, 1979). Submission of unpublished data by CTFA. Clinical controlled use test ( ).* 70. NORTH AMERICAN CONTACT DERMATITIS GROUP (NACDG). (1982). Prospective study of cosmetic reactions: Am. Acad. Dermatol. 6, CTFA. (September 17, 1979). Submission of unpublished data by CTFA. Clinical phototoxicity and photosensitization tests ( ).* 72. DOMSCH, A., POSPISCHIL, H., SCHUSTER, G., and TRONNIER, H. (1980). Cosmetic dermatological effects of protein hydrolyzates. Pa&em. Kosmet. 61(g),

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143 2017 FDA VCRP RAW DATA 03D - Eye Lotion COLLAGEN 9 03E - Eye Makeup Remover COLLAGEN 1 03F - Mascara COLLAGEN 10 03G - Other Eye Makeup Preparations COLLAGEN 11 05A - Hair Conditioner COLLAGEN 13 05C - Hair Straighteners COLLAGEN 1 05E - Rinses (non-coloring) COLLAGEN 1 05F - Shampoos (non-coloring) COLLAGEN 10 05G - Tonics, Dressings, and Other Hair Grooming COLLAGEN 8 Aids 05I - Other Hair Preparations COLLAGEN 6 06D - Hair Shampoos (coloring) COLLAGEN 1 07C - Foundations COLLAGEN 3 07E - Lipstick COLLAGEN 5 07I - Other Makeup Preparations COLLAGEN 4 08C - Nail Creams and Lotions COLLAGEN 1 08E - Nail Polish and Enamel COLLAGEN 1 08G - Other Manicuring Preparations COLLAGEN 2 10A - Bath Soaps and Detergents COLLAGEN 1 10E - Other Personal Cleanliness Products COLLAGEN 2 11A - Aftershave Lotion COLLAGEN 1 11E - Shaving Cream COLLAGEN 1 12A - Cleansing COLLAGEN 7 12C - Face and Neck (exc shave) COLLAGEN 79 12D - Body and Hand (exc shave) COLLAGEN 15 12F - Moisturizing COLLAGEN 26 12G - Night COLLAGEN 15 12H - Paste Masks (mud packs) COLLAGEN 20 12I - Skin Fresheners COLLAGEN 4 12J - Other Skin Care Preps COLLAGEN 39 13A - Suntan Gels, Creams, and Liquids COLLAGEN 2 01B - Baby Lotions, Oils, Powders, and Creams HYDROLYZED COLLAGEN 2 01C - Other Baby Products HYDROLYZED COLLAGEN 2 02A - Bath Oils, Tablets, and Salts HYDROLYZED COLLAGEN 1 03D - Eye Lotion HYDROLYZED COLLAGEN 4 03F - Mascara HYDROLYZED COLLAGEN 6 03G - Other Eye Makeup Preparations HYDROLYZED COLLAGEN 13 04C - Powders (dusting and talcum, excluding HYDROLYZED COLLAGEN 3 aftershave talc) 05A - Hair Conditioner HYDROLYZED COLLAGEN 48 05C - Hair Straighteners HYDROLYZED COLLAGEN 2 05D - Permanent Waves HYDROLYZED COLLAGEN 2 05E - Rinses (non-coloring) HYDROLYZED COLLAGEN 1 05F - Shampoos (non-coloring) HYDROLYZED COLLAGEN 42 05G - Tonics, Dressings, and Other Hair Grooming HYDROLYZED COLLAGEN 9

144 Aids 05H - Wave Sets HYDROLYZED COLLAGEN 3 05I - Other Hair Preparations HYDROLYZED COLLAGEN 14 06A - Hair Dyes and Colors (all types requiring HYDROLYZED COLLAGEN 1 caution statements and patch tests) 06G - Hair Bleaches HYDROLYZED COLLAGEN 1 06H - Other Hair Coloring Preparation HYDROLYZED COLLAGEN 1 07C - Foundations HYDROLYZED COLLAGEN 5 07D - Leg and Body Paints HYDROLYZED COLLAGEN 1 07E - Lipstick HYDROLYZED COLLAGEN 3 07F - Makeup Bases HYDROLYZED COLLAGEN 1 07I - Other Makeup Preparations HYDROLYZED COLLAGEN 5 08A - Basecoats and Undercoats HYDROLYZED COLLAGEN 1 08E - Nail Polish and Enamel HYDROLYZED COLLAGEN 4 08G - Other Manicuring Preparations HYDROLYZED COLLAGEN 2 09B - Mouthwashes and Breath Fresheners HYDROLYZED COLLAGEN 2 10A - Bath Soaps and Detergents HYDROLYZED COLLAGEN 5 10E - Other Personal Cleanliness Products HYDROLYZED COLLAGEN 11 12A - Cleansing HYDROLYZED COLLAGEN 10 12C - Face and Neck (exc shave) HYDROLYZED COLLAGEN D - Body and Hand (exc shave) HYDROLYZED COLLAGEN 11 12F - Moisturizing HYDROLYZED COLLAGEN 88 12G - Night HYDROLYZED COLLAGEN 10 12H - Paste Masks (mud packs) HYDROLYZED COLLAGEN 48 12I - Skin Fresheners HYDROLYZED COLLAGEN 3 12J - Other Skin Care Preps HYDROLYZED COLLAGEN 46 13A - Suntan Gels, Creams, and Liquids HYDROLYZED COLLAGEN 1 13B - Indoor Tanning Preparations HYDROLYZED COLLAGEN 2 13C - Other Suntan Preparations HYDROLYZED COLLAGEN 1 05F - Shampoos (non-coloring) SOLUBLE ANIMAL COLLAGEN 1 07F - Makeup Bases SOLUBLE ANIMAL COLLAGEN 22 12D - Body and Hand (exc shave) SOLUBLE ANIMAL COLLAGEN 1 12J - Other Skin Care Preps SOLUBLE ANIMAL COLLAGEN 1 03D - Eye Lotion SOLUBLE COLLAGEN 18 03F - Mascara SOLUBLE COLLAGEN 24 03G - Other Eye Makeup Preparations SOLUBLE COLLAGEN 9 05A - Hair Conditioner SOLUBLE COLLAGEN 4 05F - Shampoos (non-coloring) SOLUBLE COLLAGEN 4 05G - Tonics, Dressings, and Other Hair Grooming SOLUBLE COLLAGEN 5 Aids 05H - Wave Sets SOLUBLE COLLAGEN 1 06G - Hair Bleaches SOLUBLE COLLAGEN 1 07C - Foundations SOLUBLE COLLAGEN 11 07E - Lipstick SOLUBLE COLLAGEN 6 07F - Makeup Bases SOLUBLE COLLAGEN 1 07G - Rouges SOLUBLE COLLAGEN 1 07I - Other Makeup Preparations SOLUBLE COLLAGEN 7

145 10A - Bath Soaps and Detergents SOLUBLE COLLAGEN 3 11A - Aftershave Lotion SOLUBLE COLLAGEN 2 12A - Cleansing SOLUBLE COLLAGEN 25 12C - Face and Neck (exc shave) SOLUBLE COLLAGEN D - Body and Hand (exc shave) SOLUBLE COLLAGEN 30 12F - Moisturizing SOLUBLE COLLAGEN 75 12G - Night SOLUBLE COLLAGEN 21 12H - Paste Masks (mud packs) SOLUBLE COLLAGEN 18 12I - Skin Fresheners SOLUBLE COLLAGEN 3 12J - Other Skin Care Preps SOLUBLE COLLAGEN 21 12D - Body and Hand (exc shave) SOLUBLE COLLAGEN EXTRACT 1 12F - Moisturizing SOLUBLE COLLAGEN EXTRACT 1 03D - Eye Lotion ELASTIN 1 03G - Other Eye Makeup Preparations ELASTIN 1 05A - Hair Conditioner ELASTIN 1 05F - Shampoos (non-coloring) ELASTIN 1 05I - Other Hair Preparations ELASTIN 2 07I - Other Makeup Preparations ELASTIN 1 11A - Aftershave Lotion ELASTIN 1 12A - Cleansing ELASTIN 3 12C - Face and Neck (exc shave) ELASTIN 9 12D - Body and Hand (exc shave) ELASTIN 2 12F - Moisturizing ELASTIN 7 12G - Night ELASTIN 2 12H - Paste Masks (mud packs) ELASTIN 1 12I - Skin Fresheners ELASTIN 1 12J - Other Skin Care Preps ELASTIN 12 13A - Suntan Gels, Creams, and Liquids ELASTIN 1 01B - Baby Lotions, Oils, Powders, and Creams HYDROLYZED ELASTIN 2 01C - Other Baby Products HYDROLYZED ELASTIN 2 03D - Eye Lotion HYDROLYZED ELASTIN 2 03F - Mascara HYDROLYZED ELASTIN 1 03G - Other Eye Makeup Preparations HYDROLYZED ELASTIN 7 05A - Hair Conditioner HYDROLYZED ELASTIN 21 05F - Shampoos (non-coloring) HYDROLYZED ELASTIN 16 05G - Tonics, Dressings, and Other Hair Grooming HYDROLYZED ELASTIN 11 Aids 07C - Foundations HYDROLYZED ELASTIN 4 07E - Lipstick HYDROLYZED ELASTIN 2 07F - Makeup Bases HYDROLYZED ELASTIN 22 07I - Other Makeup Preparations HYDROLYZED ELASTIN 2 10E - Other Personal Cleanliness Products HYDROLYZED ELASTIN 3 11A - Aftershave Lotion HYDROLYZED ELASTIN 1 12A - Cleansing HYDROLYZED ELASTIN 20 12C - Face and Neck (exc shave) HYDROLYZED ELASTIN 59 12D - Body and Hand (exc shave) HYDROLYZED ELASTIN 29

146 12F - Moisturizing HYDROLYZED ELASTIN 34 12G - Night HYDROLYZED ELASTIN 10 12H - Paste Masks (mud packs) HYDROLYZED ELASTIN 7 12J - Other Skin Care Preps HYDROLYZED ELASTIN 21 13B - Indoor Tanning Preparations HYDROLYZED ELASTIN 2 12C - Face and Neck (exc shave) FIBRONECTIN 2 03G - Other Eye Makeup Preparations HYDROLYZED FIBRONECTIN 1 12C - Face and Neck (exc shave) HYDROLYZED FIBRONECTIN 2 12D - Body and Hand (exc shave) HYDROLYZED FIBRONECTIN 1 12F - Moisturizing HYDROLYZED FIBRONECTIN 2 12G - Night HYDROLYZED FIBRONECTIN 2 12H - Paste Masks (mud packs) HYDROLYZED FIBRONECTIN 1 12J - Other Skin Care Preps HYDROLYZED FIBRONECTIN 1 05A - Hair Conditioner GELATIN 3 05E - Rinses (non-coloring) GELATIN 1 05F - Shampoos (non-coloring) GELATIN 1 05G - Tonics, Dressings, and Other Hair Grooming GELATIN 4 Aids 05H - Wave Sets GELATIN 1 05I - Other Hair Preparations GELATIN 2 07B - Face Powders GELATIN 1 07C - Foundations GELATIN 2 08E - Nail Polish and Enamel GELATIN 6 08F - Nail Polish and Enamel Removers GELATIN 27 09B - Mouthwashes and Breath Fresheners GELATIN 3 10A - Bath Soaps and Detergents GELATIN E - Other Personal Cleanliness Products GELATIN 6 12A - Cleansing GELATIN 10 12C - Face and Neck (exc shave) GELATIN 4 12D - Body and Hand (exc shave) GELATIN 2 12F - Moisturizing GELATIN 12 12H - Paste Masks (mud packs) GELATIN 1 03D - Eye Lotion HYDROLYZED ACTIN 1 03F - Mascara HYDROLYZED ACTIN 3 03G - Other Eye Makeup Preparations HYDROLYZED ACTIN 1 05A - Hair Conditioner HYDROLYZED ACTIN 1 05F - Shampoos (non-coloring) HYDROLYZED ACTIN 1 05G - Tonics, Dressings, and Other Hair Grooming HYDROLYZED ACTIN 5 Aids 12C - Face and Neck (exc shave) HYDROLYZED ACTIN 6 12F - Moisturizing HYDROLYZED ACTIN 1 12G - Night HYDROLYZED ACTIN 1 03D - Eye Lotion ATELOCOLLAGEN 2 03F - Mascara ATELOCOLLAGEN 12

147 03G - Other Eye Makeup Preparations ATELOCOLLAGEN 3 05F - Shampoos (non-coloring) ATELOCOLLAGEN 1 07A - Blushers (all types) ATELOCOLLAGEN 1 07B - Face Powders ATELOCOLLAGEN 1 07C - Foundations ATELOCOLLAGEN 12 07E - Lipstick ATELOCOLLAGEN 39 07I - Other Makeup Preparations ATELOCOLLAGEN 4 12A - Cleansing ATELOCOLLAGEN 3 12C - Face and Neck (exc shave) ATELOCOLLAGEN 17 12D - Body and Hand (exc shave) ATELOCOLLAGEN 1 12F - Moisturizing ATELOCOLLAGEN 14 12G - Night ATELOCOLLAGEN 3 12J - Other Skin Care Preps ATELOCOLLAGEN 3 13B - Indoor Tanning Preparations ATELOCOLLAGEN 2

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180 Memorandum TO: FROM: Lillian Gill, D.P.A. Director - COSMETIC INGREDIENT REVIEW (CIR) Beth A. Lange, Ph.D. Industry Liaison to the CIR Expert Panel DATE: February 11, 2016 SUBJECT: Concentration of Use by FDA Product Category: Collagen, Hydrolyzed Collagen and Related Proteins

181 Concentration of Use by FDA Product Category Collagen, Hydrolyzed Collagen and Related Proteins* Ammonium Hydrolyzed Collagen Calcium Hydrolyzed Collagen MEA-Hydrolyzed Collagen Zinc Hydrolyzed Collagen Hydrolyzed Collagen Hydrolyzed Collagen Extract Collagen Soluble Collagen Gelatin Hydrolyzed Gelatin Hydrolyzed Reticulin Hydrolyzed Actin Elastin Soluble Elastin Hydrolyzed Elastin Fibronectin Hydrolyzed Fibronectin Hydrolyzed Spongin Ingredient Product Category Maximum Concentration of Use Ammonium Hydrolyzed Hair conditioners 0.05% Collagen Ammonium Hydrolyzed Collagen Face and neck products Not spray 0.1% Ammonium Hydrolyzed Collagen Moisturizing products Not spray 0.12% Ammonium Hydrolyzed Collagen Night products Not spray 0.12% Ammonium Hydrolyzed Paste masks and mud packs 0.03% Collagen MEA-Hydrolyzed Collagen Hair conditioners 0.06% MEA-Hydrolyzed Collagen Face and neck products Not spray 0.1% MEA-Hydrolyzed Collagen Moisturizing products Not spray 0.12% MEA-Hydrolyzed Collagen Night products Not spray 0.12% MEA-Hydrolyzed Collagen Paste masks and mud packs 0.03% Hydrolyzed Collagen Eyebrow pencils 0.001% Hydrolyzed Collagen Eyeliners % Hydrolyzed Collagen Eye lotions % Hydrolyzed Collagen Mascara % Hydrolyzed Collagen Other eye makeup preparations 0.02% Hydrolyzed Collagen Hair conditioners % Hydrolyzed Collagen Hair sprays Aerosol 0.28% % Pump spray Hydrolyzed Collagen Hair straighteners 0.2% Hydrolyzed Collagen Permanent waves % Hydrolyzed Collagen Rinses (noncoloring) % Hydrolyzed Collagen Shampoos (noncoloring) % Hydrolyzed Collagen Tonics, dressings and other hair grooming aids Not spray Aerosol % % % Hydrolyzed Collagen Wave sets 0.039%

182 Hydrolyzed Collagen Other hair preparations (noncoloring) 0.015% Hydrolyzed Collagen Hair dyes and colors 1.2% Hydrolyzed Collagen Hair tints 0.15% Hydrolyzed Collagen Hair lighteners with color 0.25% Hydrolyzed Collagen Hair bleaches 0.25% Hydrolyzed Collagen Foundations % Hydrolyzed Collagen Lipstick % Hydrolyzed Collagen Nail polish and enamel % Hydrolyzed Collagen Nail polish and enamel removers % Hydrolyzed Collagen Bath soaps and detergents % Hydrolyzed Collagen Other personal cleanliness products Hand wash 0.06% Hydrolyzed Collagen Skin cleansing (cold creams, cleansing lotions, 0.029% liquids and pads) Hydrolyzed Collagen Face and neck products Not spray % Hydrolyzed Collagen Body and hand products Not spray Spray % 0.029% Hydrolyzed Collagen Moisturizing products Not spray % Hydrolyzed Collagen Night products Not spray % Hydrolyzed Collagen Paste masks and mud packs % Hydrolyzed Collagen Other skin care preparations 0.5% Hydrolyzed Collagen Indoor tanning preparations % Collagen Eyebrow pencils 0.2% Collagen Eyeliners 0.2% Collagen Eye shadows % Collagen Eye lotions % Collagen Mascara 0.02% Collagen Hair conditioners 1.3% Collagen Shampoos (noncoloring) 1.3% Collagen Tonics, dressings and other hair grooming aids Not spray % Collagen Blushers 0.2% Collagen Foundations % Collagen Lipstick % Collagen Bath soaps and detergents 0.67% Collagen Aftershave lotions % Collagen Collagen Collagen Skin cleansing (cold creams, cleansing lotions, % liquids and pads) Face and neck products Not spray % Moisturizing products Not spray 0.001%

183 Collagen Night products Not spray % Soluble Collagen Bath oils, tablets and salts % Soluble Collagen Bubble baths 0.005% Soluble Collagen Eyeliners % Soluble Collagen Eye shadows % Soluble Collagen Eye lotions % Soluble Collagen Mascara % Soluble Collagen Other eye makeup preparations 0.002% Soluble Collagen Other fragrance preparations % Soluble Collagen Hair conditioners % Soluble Collagen Hair sprays Aerosol % Pump spray % Soluble Collagen Rinses (noncoloring) % Soluble Collagen Shampoos (noncoloring) % Soluble Collagen Tonics, dressings and other hair grooming aids Not spray % % Soluble Collagen Hair dyes and colors % Soluble Collagen Blushers % Soluble Collagen Face powders % Soluble Collagen Foundations % Soluble Collagen Lipstick % Soluble Collagen Makeup bases % Soluble Collagen Rouges % Soluble Collagen Other makeup preparations % Soluble Collagen Nail creams and lotions % Soluble Collagen Soluble Collagen Soluble Collagen Soluble Collagen Soluble Collagen Skin cleansing (cold creams, cleansing lotions, liquids and pads) Face and neck products Not spray Spray Body and hand products Not spray Spray % % % % % Moisturizing products Not spray % Night products Not spray % Soluble Collagen Paste masks and mud packs % Soluble Collagen Skin fresheners % Soluble Collagen Other skin care preparations % Soluble Collagen Suntan products Not spray % Gelatin Bath oils, tablets and salts % Gelatin Other bath preparations 0.21%

184 Gelatin Eyeliners 0.5% Gelatin Eye lotions 1% Gelatin Other fragrance preparations 0.03% Gelatin Hair conditioners % Gelatin Rinses (noncoloring) 0.5% Gelatin Shampoos (noncoloring) 0.5% Gelatin Tonics, dressings and other hair grooming aids % Gelatin Blushers % Gelatin Makeup bases 0.01% Gelatin Basecoats and undercoats (manicuring 0.001% preparations) Gelatin Nail creams and lotions 0.02% Gelatin Nail polish and enamel 0.001% Gelatin Bath soaps and detergents % Gelatin Other personal cleanliness products % Gelatin Skin cleansing (cold creams, cleansing lotions, % liquids and pads) Gelatin Face and neck products Not spray 1-2% Gelatin Body and hand products Not spray % Gelatin Foot products 0.016% Gelatin Moisturizing products Not spray % Gelatin Paste masks and mud packs 9.5% Hydrolyzed Reticulin Moisturizing products Not spray 0.05% Hydrolyzed Reticulin Paste masks and mud packs 0.025% Hydrolyzed Actin Mascara 1.6% Hydrolyzed Actin Hair conditioners % Hydrolyzed Actin Shampoos (noncoloring) % Hydrolyzed Elastin Eyebrow pencils 0.2% Hydrolyzed Elastin Eyeliners 0.2% Hydrolyzed Elastin Eye shadows 0.2% Hydrolyzed Elastin Eye lotions % Hydrolyzed Elastin Eye makeup removers 0.01% Hydrolyzed Elastin Other eye makeup preparations 0.5% Hydrolyzed Elastin Hair conditioners % Hydrolyzed Elastin Hair sprays Pump spray % Hydrolyzed Elastin Permanent waves 0.001% Hydrolyzed Elastin Rinses (noncoloring) % Hydrolyzed Elastin Shampoos (noncoloring) % Hydrolyzed Elastin Tonics, dressings and other hair grooming aids Aerosol % 0.005% Hydrolyzed Elastin Blushers 0.2%

185 Hydrolyzed Elastin Face powders % Hydrolyzed Elastin Foundations % Hydrolyzed Elastin Lipstick % Hydrolyzed Elastin Makeup bases % Hydrolyzed Elastin Skin cleansing (cold creams, cleansing lotions, % liquids and pads) Hydrolyzed Elastin Face and neck products Not spray % Hydrolyzed Elastin Body and hand products Not spray % Hydrolyzed Elastin Moisturizing products Hydrolyzed Elastin Not spray % Night products Not spray 0.06% Hydrolyzed Elastin Paste masks and mud packs 0.06% Hydrolyzed Elastin Skin fresheners 0.06% Hydrolyzed Elastin Other skin care preparations 0.5% Hydrolyzed Fibronectin Moisturizing products Not spray 0.05% Hydrolyzed Fibronectin Paste masks and mud packs 0.025% *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 Table prepared February 11, 2016

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