Amended Safety Assessment of Biotin as Used in Cosmetics

Status: Re-Review 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, D.P.A. This safety assessment was prepared by Monice M. Fiume, Assistant Director/Senior Scientific Analyst/Writer.

© Cosmetic Ingredient Review 1620 L Street, NW, Suite 1200 ♢ Washington, DC 20036-4702 ♢ ph 202.331.0651 ♢ fax 202.331.0088 ♢

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__________________________________________________________________________________________ 1620 L Street, NW, Suite 1200, Washington, DC 20036

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Memorandum To: CIR Expert Panel Members and Liaisons From: Monice M. Fiume MMF Assistant Director/Senior Scientific Analyst Date: May 19, 2017 Subject: Amended Safety Assessment of Biotin as Used in Cosmetics Enclosed is the Re-Review of the Amended Safety Assessment of Biotin as Used in Cosmetics. (It is identified as biotin062017rep in the pdf.) In 2001, the Panel published a Final Report on the Safety Assessment of Biotin. Based on the available data, the Panel concluded that Biotin is safe as used in cosmetics. No additional ingredients are proposed for inclusion. The reported frequency of use of Biotin in cosmetics has increased since safety was originally reviewed; 71 uses were reported 1998, and 506 uses are reported in 2017. The reported maximum leave-on concentration of use has decreased from 0.6% to 0.1%. Of note, the number of uses near the eye area increased from 2 to 54, and the maximum concentration of use reported for this type of exposure has increased from 0.01% to 0.1%. Updated (2017) VCRP data (biotin062017FDA) and concentration of use data (biotin062017data) are included for your review.

Excerpts from the summary of the 2001 report are disseminated throughout the text of the re-review document, as appropriate, and are identified by italicized text. Additionally, the Discussion from the original report is also included in this document. Some new data were identified in the published literature; these data were similar to data that were included in the original assessment. (Because there is only one ingredient being evaluated, and the data content is not substantial, a data profile was not prepared.)

The original report (identified as biotin062017prev) and the minutes from the original deliberations (biotin062017min) are included.

The Panel should review the document and determine whether the original conclusion should be reaffirmed or reconsidered. If there is a reason to re-open the safety assessment, please identify any data needs and discussion points.

RE-REVIEW FLOW CHART INGREDIENT/FAMILY_____Biotin___________________________________ MEETING _______ June 2017____________________________________________________________

Public Comment CIR Expert Panel Re-Review Rpt Status

announce OR

PRIORITY LIST

DAR

YES NO

Table IDA TAR Yes

No

IDA Notice

Draft TAR

IDA

Admin Book

Table

Tentative Amended Report

60 day Public comment period

Draft FAR

Table Different Conclusion

PUBLISH

Final Amended Report

*If Draft Amended Report (DAR) is available, the Panel may choose to review; if not, CIR staff prepares DAR for Panel Review.

DRAFT AMENDED REPORT

DRAFT TENTATIVE AMENDED REPORT

DRAFT FINAL AMENDED REPORT

Issue TAR

Issue FAR

Table

Table

Table

New Data; or request

Re-review to Panel

June 2017

Are new data cause to reopen?

15 years since last

review

Are new ingredients appropriate for

inclusion/re-open?

RE-REVIEW SUMMARY

No proposed add-ons

IJT 21 (Suppl 4): 1-12; 2001

CIR History: Biotin 2001: the Panel published the Final Report on the Safety Assessment of Biotin with the conclusion: Based on the available data, the Panel concluded that Biotin is safe as used in cosmetics June 2017: the RR document was presented to the Panel; new published data and updated frequency and concentration of use data were included

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BIOTIN RR Ingredient CAS # InfoB SciFin PubMed FDA EU ECHA IUCLID SIDS ECETOC HPVIS NICNAS NTIS NTP WHO FAO NIOSH LLNA db Web biotin 58-85-5 CFR citations 1443 hits/

4 useful VCRP;

CFR; GRAS

no res preR no no no no no no no yes yes no no

Search Strategy PubMed (1995-) (((biotin) OR 58-85-5[EC/RN Number]) AND ("1995"[Date - Publication] : "3000"[Date - Publication])) AND (dermal*) – 51 hits/0 useful (((biotin) OR 58-85-5[EC/RN Number]) AND ("1995"[Date - Publication] : "3000"[Date - Publication])) AND ((all irrita*) OR (all sensiti*)) – 26 hits/0 useful (((biotin) OR 58-85-5[EC/RN Number]) AND ("1995"[Date - Publication] : "3000"[Date - Publication])) AND ((reproductive OR developmental) AND toxicity) – 39 hits (((biotin) OR 58-85-5[EC/RN Number])) AND dermal penetration– 2 hits/0 useful (((biotin) OR 58-85-5[EC/RN Number])) AND carcinogenicity– 6 hits/0 useful (((biotin) OR 58-85-5[EC/RN Number])) AND genotoxicity– 21 hits/0 useful SciFinder (1995-; with qualifiers by document type) dermal toxicity of biotin – 0 hits dermal irritation with biotin – 0 hits dermal sensitization caused by biotin – 3 hits/0 useful irritation caused by biotin – 35 hits/0 useful toxicity of biotin – 6 hits/0 useful reproductive or developmental toxicity caused by biotin – 117 hits/4 useful carcinogenicity caused by biotin – 1240 hits – not diagnostic/imagining/delivery/probe/labeled/expression – 160 hits/0 useful genotoxicity of biotin – 42 hits/0 useful dermal absorption of biotin – 0 hits dermal penetration of biotin – 7 hits/1 useful

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LINKS

InfoBase (self-reminder that this info has been accessed; not a public website) - http://www.personalcarecouncil.org/science-safety/line-infobase wINCI (to cite publicly) - http://webdictionary.personalcarecouncil.org ScfFinder (usually a combined search for all ingredients in report; list # of this/# useful) - https://scifinder.cas.org/scifinder PubMed (usually a combined search for all ingredients in report; list # of this/# useful) - http://www.ncbi.nlm.nih.gov/pubmed ;

Also search: PubMed Dietary Supplement Subset https://ods.od.nih.gov/Research/PubMed_Dietary_Supplement_Subset.aspx and https://ods.od.nih.gov/Health_Information/IBIDS.aspx

Toxnet databases (usually a combined search for all ingredients in report; list # of this/# useful) – https://toxnet.nlm.nih.gov/ (includes Toxline; HSDB; ChemIDPlus; DART; IRIS; CCRIS; CPDB; GENE-TOX) FDA databases http://www.ecfr.gov/cgi-bin/ECFR?page=browse (CFR); then, list of all databases: http://www.fda.gov/ForIndustry/FDABasicsforIndustry/ucm234631.htm; then, http://www.accessdata.fda.gov/scripts/fcn/fcnnavigation.cfm?rpt=eafuslisting&displayall=true (EAFUS); http://www.fda.gov/food/ingredientspackaginglabeling/gras/default.htm (GRAS); http://www.fda.gov/food/ingredientspackaginglabeling/gras/scogs/ucm2006852.htm (SCOGS database); http://www.accessdata.fda.gov/scripts/fdcc/?set=IndirectAdditives (indirect food additives list); http://www.fda.gov/Drugs/InformationOnDrugs/default.htm (drug approvals and database); http://www.fda.gov/downloads/AboutFDA/CentersOffices/CDER/UCM135688.pdf (OTC ingredient list); http://www.accessdata.fda.gov/scripts/cder/iig/ (inactive ingredients approved for drugs) LLNA Database (NICEATM Dec. 2013): https://ntp.niehs.nih.gov/iccvam/methods/immunotox/niceatm-llnadatabase-23dec2013.xls EU (European Union); check CosIng (cosmetic ingredient database) for restrictions http://ec.europa.eu/growth/tools-databases/cosing/

and SCCS (Scientific Committee for Consumer Safety) opinions - http://ec.europa.eu/health/scientific_committees/consumer_safety/opinions/index_en.htm ECHA (European Chemicals Agency – REACH dossiers) – http://echa.europa.eu/information-on-chemicals;jsessionid=A978100B4E4CC39C78C93A851EB3E3C7.live1 IUCLID (International Uniform Chemical Information Database) - https://iuclid6.echa.europa.eu/search OECD SIDS documents (Organisation for Economic Co-operation and Development Screening Info Data Sets)- http://webnet.oecd.org/hpv/ui/Search.aspx ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) - http://www.ecetoc.org HPVIS (EPA High-Production Volume Info Systems) - https://ofmext.epa.gov/hpvis/HPVISlogon NICNAS (Australian National Industrial Chemical Notification and Assessment Scheme)- https://www.nicnas.gov.au/ NTIS (National Technical Information Service) - http://www.ntis.gov/ NTP (National Toxicology Program ) - http://ntp.niehs.nih.gov/ WHO (World Health Organization) technical reports - http://www.who.int/biologicals/technical_report_series/en/ FAO (Food and Agriculture Organization of the United Nations) - http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfa-additives/en/ NIOSH (National Institute for Occupational Safety and Health) - http://www.cdc.gov/niosh/ FEMA (Flavor & Extract Manufacturers Association) - http://www.femaflavor.org/search/apachesolr_search/ Web – perform general search; may find technical data sheets, published reports, etc

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BIOTIN – ORIGINAL DELIBERATIONS

March 19-20, 1998 Meeting Dr. Schroeter noted that no responses to the informal data requests issued at the December 8, 1997 Team meetings have been received. Therefore, his Team determined that an Insufficient Data Announcement should be issued at this meeting.

Dr. Belsito recommended deletion of all of the information on Biotin deficiency from the CIR report (pages 12-18, 25-26, and 28). He did not view this information as relevant to the Panel=s safety assessment.

Drs. Andersen and Klaassen suggested that the Panel Ahighlight@ all information in the report that should be deleted, and then make a final decision on deletions during the open session of the May 18-19, 1998 Panel meeting.

The Panel voted unanimously in favor of issuing an Insufficient Data Announcement on Biotin with the following data requests:

1. Current concentration of use data

2. Impurities

3. UV absorption data; if absorption occurs in the UVA or UVB range, photosensitization data are needed

4. Skin irritation and sensitization data at concentration of use

5. Ocular irritation data; if available

6. Genotoxicity testing in a mammalian system; if positive, a 2-year dermal carcinogenicity assay performed using NTP methods may be needed

September 10-11, 1998 Meeting Dr. Belsito recalled that an Insufficient Data Announcement on Biotin was issued at the March 19-20, 1998 Panel meeting, and that only concentration of use data and impurities data were received in response to this announcement. He also recalled that, on the preceding day, Dr. McEwen provided the Panel with data examining the worse case scenario for Biotin absorption and how these data compare with oral ingestion and absorption of Biotin in the normal diet. Dr. Belsito said that based on these absorption data, his Team determined that the available data are sufficient for arriving at a conclusion on the safety of Biotin, which is as follows: Biotin is safe for use in leave-on products at concentrations up to 0.1% and safe as used in rinse-off products.

Dr. Belsito said that the following statements should be included in the report discussion: Assuming maximal absorption through the skin of 10% of the Biotin that was applied and assuming whole body exposure, 0.1% Biotin would still be at a level in the range of what one would be exposed to in the diet. Furthermore, unlike other vitamins, excess Biotin has not been associated with any significant clinical pathology.

Dr. Bergfeld confirmed with the Panel that the data referred to by Dr. Belsito will be incorporated into the Biotin report.

Dr. McEwen requested that the information on absorption received be referred to as a communication and not as either new data or a study. He agreed that the communication could be used in the report discussion to support conclusions on the safety of Biotin in cosmetics.

Dr. Schroeter said that his Team determined that the available data are insufficient for arriving at a conclusion on the safety of Biotin in cosmetics, and that the following data are needed: impurities, UV absorption, skin irritation and sensitization, ocular irritation, and genotoxicity (dermal carcinogenicity study, if data are positive).

Dr. Schroeter also said that his Team recommends that the data on Biotin deficiency remain in the report, because these data offer biological information on this ingredient.

Dr. Belsito noted that impurities data were received and have been incorporated into the report text. He recalled that the Panel had expressed concern over the skin irritation and sensitization potential of impurities that might be present in Biotin, not Biotin itself. Based on the impurities data submitted, Dr. Belsito said that he has no concerns about sensitization potential.

Regarding the request for UV absorption data, Dr. Belsito said that his Team does not consider Biotin to be a UV absorber based on its chemical structure, but is concerned about impurities that may be present. With respect to potential genotoxicity, Dr. Belsito said that there is no evidence in the medical literature that excess Biotin results in any significant effect.

Dr. Slaga said that if Biotin absorbs UV light, this event may trigger its transformation into a genotoxic chemical.

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Dr. Shank noted that one of the analytical methods for the detection of Biotin depends on fluorometry, suggesting UV absorbance. Therefore, he expressed concern over potential phototoxicity/photosensitization reactions resulting from dermal application of sun tan products or indoor tanning preparations containing Biotin.

Dr. Belsito stated that Biotin is a low UV wavelength absorber, which typically means absorbance in the UVC region. He noted that the structure of Biotin resembles that of a nucleic acid, and that absorbance in the UVC region is typical for chemicals with a nucleic acid-type structure [nucleic acids absorb maximally at 254 nm]. Furthermore, he noted that UVC light is not present in our environment.

Referring to the risk assessment on Biotin provided by industry, Dr. Bronaugh questioned the 5 g/day exposure for body application. He said that if the moisturizer were used on the face only, this amount may be considered reasonable; however, 5 g/day would be an underestimate in terms of whole body application. Dr. Bronaugh said that if 1 or 2 mg of formulation were applied over a body surface area of 17,000 cm2, this would translate into 15 or 30 g/day (30 g is probably too high). He acknowledged that an exposure of 10 or 15 g/day would probably be more accurate in terms of whole body application of the moisturizer.

Dr. Bronaugh added that according to use concentration data in the CIR report, Biotin could be used in cosmetics at concentrations as high as 1%.

Dr. Schroeter noted that skin irritation and sensitization data are not included in the report. However, he acknowledged that he has little concern over the skin irritation potential of Biotin.

Dr. Belsito said that he does not need skin irritation or sensitization data in order to arrive at a conclusion on the safety of Biotin.

Dr. Bailey wanted to know the basis for concluding that Biotin is safe in the absence of skin irritation and sensitization data.

Dr. Belsito said that the basis would be the absence of clinical reports in the literature. Specifically, he said that in the Clinical Assessment of Safety section of the CIR report, there are no reports of Biotin-induced contact hypersensitivity.

Dr. Schroeter suggested that the reason why skin irritation and sensitization data are not being requested should be stated in the report discussion.

Dr. Belsito said that if there were considerable Biotin-induced sensitization in the population, then there should be reports of systemic contact dermatitis. In other words, there should be numerous reports of chronic rashes resulting from Biotin ingestion. Dr. Belsito also said that one does not see reports in the literature of individuals with problems associated with dermal exposure to Biotin.

Dr. Bergfeld said that it is not very scientific to assume that a chemical is safe just because it has not been demonstrated in the medical literature that it is causative of some type of disorder.

Dr. Shank noted that individuals have been handling pure Biotin for some time, manufacturers of the vitamin as well as individuals who take it.

Dr. Schroeter said that it is highly improbable that Biotin-induced sensitization occurs; however, the absence of sensitization data should be addressed in the report discussion.

Dr. Bergfeld said that the more compelling statement that was made earlier is that occupational exposure to 100% Biotin has not caused any problems.

Drs. Slaga and Schroeter said that the Panel really does not know whether this statement is accurate, and that it may be considered undocumented clinical experience.

Dr. Bailey said that his concern relates to the relevance of the available data to products that are on the shelf, under the conditions of formulation and the people who will be using these products (children included). He also said that there should be information in the report indicating why the Panel is or is not concerned about the skin irritation and sensitization potential of Biotin.

Dr. McEwen stated that the Panel must think in terms of how products containing Biotin are going to be used. He also said that if the Panel is going to limit Biotin to a concentration of 0.1% in a formulation, the Panel should consider how strong of a sensitizer it would have to be in order for there to be concern about its sensitization potential. Dr. McEwen agreed that the Panel=s reason(s) for the lack of concern about the sensitization potential of Biotin should be included in the report discussion.

Dr. Bergfeld confirmed that the Panel agrees that the issue of sensitization can be addressed in the report discussion, and that skin irritation and sensitization data should not be requested from industry.

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The Panel voted unanimously in favor of issuing a Tentative Report with an insufficient data conclusion on Biotin. The data needed in order for the Panel to complete its safety assessment of this ingredient will be included in the discussion section of the report as follows:

(1) UV absorption data; if absorption occurs in the UVA or UVB range,

photosensitization data and a genotoxicity study in a mammalian

system with photoactivated Biotin is needed; if the genotoxicity data

are positive, a 2-year dermal carcinogenicity assay performed using

NTP methods may be needed

Dr. Bergfeld noted that the report discussion will include all of the issues under contact allergy and irritancy that were discussed today.

March 3-4, 1999 Dr. Schroeter recalled that a Tentative Report with an insufficient data conclusion was issued at the September 10-11, 1998 Panel meeting because UVA absorption data were needed for completion of the Panel=s safety assessment of Biotin. He also noted that, since that time, a UV spectral analysis indicating no absorption peaks in the UVA range was received. Thus, Dr. Schroeter=s Team proposed that Biotin is safe as used.

Dr. Belsito recalled that the Panel may have originally limited the concentration of Biotin to 0.1% in cosmetics.

Dr. Bergfeld said that current data received from CTFA indicate that Biotin is used at concentrations up to 0.6%.

Dr. Schroeter said that the fact that the maximum use concentration of Biotin is now 0.6% and not 0.1% should be mentioned in the report discussion. He also recalled that his Team recommended that a statement on the poor absorption of Biotin should be included in the discussion as well. Considering that the available data indicate slight absorption of Biotin, Dr. Schroeter=s Team determined that the degree of absorption does not generate any safety concerns.

Dr. Klaassen said that 0.1% Biotin amounts to twice the daily dose and that 0.6% Biotin amounts to 12x the daily dose. Given this level of exposure and knowing the overall toxicity of Biotin, Dr. Klaassen noted that Dr. Belsito=s Team had no concerns about Biotin toxicity.

Dr. Belsito recalled that, originally, the proposed 0.1% limitation was based on the complete absorption of Biotin and equaling the daily dose. Based on Dr. Klaassen=s preceding comments, he agreed that a concentration limit is not necessary.

The Panel voted unanimously in favor of issuing a Final Report on Biotin with a safe as used conclusion.

In response to Ms. Fise’s concern about the meaning of safe as used, Dr. Bergfeld proposed that the use concentration range that corresponds with this conclusion should be included in the discussion section of CIR reports.

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Amended Safety Assessment of Biotin as Used in Cosmetics

Status: Re-Review 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, D.P.A. This safety assessment was prepared by Monice M. Fiume, Assistant Director/Senior Scientific Analyst/Writer.

© Cosmetic Ingredient Review 1620 L Street, NW, Suite 1200 ♢ Washington, DC 20036-4702 ♢ ph 202.331.0651 ♢ fax 202.331.0088 ♢

cirinfo@cir-safety.org

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INTRODUCTION

The Cosmetic Ingredient Review (CIR) Expert Panel (Panel) published the Final Report on the Safety Assessment of Biotin in 2001.1 Based on the available data, the Panel concluded that Biotin is safe as used in cosmetics. In accordance with its Procedures, the CIR evaluates the conclusions of previously-issued reports every 15 years; therefore this re-review document has been prepared.

According to the web-based International Cosmetic Ingredient Dictionary and Handbook (wINCI Dictionary), Biotin (which is also known as vitamin B7, vitamin H, and coenzyme R) is reported to function in cosmetics as a hair conditioning agent and a skin conditioning agent-miscellaneous;2 these reported functions are the same as those indicated in the 2001 assessment.

Excerpts from the summary of the 2001 report are disseminated throughout the text of this re-review document, as appropriate, and are identified by italicized text. Additionally, the Discussion from the original report is also included in this document. The complete report is available on the CIR website. (http://www.cir-safety.org/ingredients)

CHEMISTRY Definition and Structure

Biotin, a naturally occurring carboxylaze coenzyme, is a bicyclic thieno-imidazol-one that conforms to the formula:

Figure 1. Biotin

Physical and Chemical Properties Biotin had low nonspecific absorption in the ultraviolet A (UVA) and UVB range and into the visible spectrum.1 It is gradually destroyed by UV radiation. Biotin occurs as a practically white, crystalline powder, and it has a molecular weight of 244.31.3-5 It can also occur as colorless crystals. Biotin is very slightly soluble in water and in alcohol, dissolves in dilute solutions of alkali hydroxides, is practically insoluble in acetone, and is insoluble in other common organic solvents. It occurs naturally as the D(+)-isomer and has a specific rotation between +89º and +93º.

Methods of Manufacture dl-Biotin has been synthesized from 4-benzamido-3-ketotetrahydrothiophene and methyl y-formylbutyrate and from 3,4-diamino-2-carbomethoxythiophene.1

Natural Occurrence Biotin occurs naturally as the D-isomer and is present in minute amounts in every living cell.1 Biotin occurs in animal and plant tissues primarily in combined forms that are liberated by enzymatic hydrolysis during digestion. Food sources of Biotin include organ meats, egg yolk, milk, fish, and nuts.

Impurities One company reported that it requires that Biotin contain <0.25% diamino derivative, biotinylbiotin, and monobenzyl biotin.1 According to the Food Chemical Codex3 and the United States Pharmacopeia4, the acceptance criteria for Biotin is not less than (NLT) 97.5% and not more than (NMT) 100.5% C10H16N2O3S, and the acceptance criteria for the presence of lead is NMT 2 mg/kg. According to the British Pharmacopoeia, Biotin contains NLT 98.5% and NMT the equivalent of 101.0% of 5-[(3aS-4S,6aR)-2-oxohexahydrothieno [3,4-d]imidazol-4-yl]pentanoic acid, calculated with reference to the dried sub-stance.5 Possible impurities include di[3-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]propyl acetic acid, 4-(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d] imidazol-4-yl]butane-1,1-dicarboxylic acid, 5-(3,4-diamino-2-thienyl)pentanoic acid, 2-methyl-5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid, and 5-[(3aS,4S,6aR)-3-benzyl-2-oxohexahydrothieno[3,4-d]imidazol-4-yl] pentanoic acid and 5-[(3aS,4S,6aR)-1-benzyl-2-oxohexahydrothieno[3,4-d]imidaz-ol-4-yl]pentanoic acid; the amounts that may be present were not specified.

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USE Cosmetic

The safety of the cosmetic ingredient addressed in this assessment is evaluated based on data received from the U.S. Food and Drug Administration (FDA) and the cosmetics industry on the expected use of this ingredient in cosmetics. Use frequencies of individual ingredients in cosmetics are collected from manufacturers and reported by cosmetic product category in FDA’s Voluntary Cosmetic Registration Program (VCRP) database. Use concentration data are submitted by the cosmetic industry in response to a survey, conducted by the Personal Care Products Council (Council), of maximum reported use concentrations by product category.

The frequency of use of Biotin has increased since safety was originally reviewed, from 71 reported uses in 19981 to 506 reported uses in 20176 (Table 1). The reported maximum concentration of use has decreased; the maximum leave-on concentration of use reported was 0.6% (in face and neck preparations) in 1999,1 and the results of the survey conducted by the Council in 2015 now indicate that the maximum leave-on use concentration is 0.1% (in several formulation types).7 Notably, the number of uses near the eye area increased from 2 to 54, and the maximum concentration of use reported for this type of exposure has increased from 0.01% to 0.1%.

Biotin is used in products that are used near the eye at maximum concentrations up to 0.1% (in eyeliners; maximum use concentration reported in 1999 was 0.01%) and in those that can come in contact with mucous membranes at maximum concentrations up to 0.001% (in bath soaps and detergents).7 Additionally, Biotin is used in aerosol and pump hair sprays formulations at concentrations up to 0.009 and 0.1%, respectively, and in face powders at a maximum concentrations of 0.1%; these product-types could possibly be inhaled. 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 <10 µm compared with pump sprays.8,9 Therefore, most droplets/particles incidentally inhaled from cosmetic sprays would be deposited in the nasopharyngeal and thoracic regions of the respiratory tract and would not be respirable (i.e., they would not enter the lungs) to any appreciable amount.10,11 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.12-14

Biotin is not restricted from use in any way under the rules governing cosmetic products in the European Union (EU).15

Non-Cosmetic According to the U.S. FDA, Biotin is generally recognized as safe (GRAS) as a nutrient for human consumption when used in accordance with good manufacturing practices (GMP).[21CFR182.8159] The recommended daily intake (RDI) for infants 12 mos old and younger is 6 µg, children ages 1-3 is 8 µg, adults and children 4 yrs old or older is 30 µg, and pregnant or lactating women is 35 µg. According to the Food and Agricultural Organization of the United Nations and the World Health Organization (FAO/WHO), the Reference Nutrient Intake (RNI) for adult males is 30 µg.16

Biotin is also GRAS in animal nutrients or dietary supplements when used in accordance with good manufacturing or feeding practice.[21CFR582.5159]

According to 21CFR310.527, Biotin has been marketed as an ingredient in over-the-counter (OTC) drug products for external use as hair growers or for hair loss prevention. There is a lack of adequate data to establish general recognition of the safety and effectiveness of Biotin as intended for OTC external use as a hair grower or for hair loss prevention. Based on evidence currently available, all labeling claims for OTC hair grower and hair loss prevention drug products for external use are either false, misleading, or unsupported by scientific data. Therefore, any OTC drug product for external use containing an ingredient offered for use as a hair grower or for hair loss prevention cannot be considered generally recognized as safe and effective (GRASE) for its intended use.

Biotin has also been present in OTC drug products for weight control; it has not been established as GRASE for this use.[21CFR310.545]

TOXICOKINETIC STUDIES Biotin functions in carbon dioxide fixation reactions in intermediate metabolism, transferring the carboxyl group to acceptor molecules; it acts similarly in decarboxylation reactions.1 Biotin is also essential in human metabolism for its part in catalyzing deamination of amino acids and in oleic acid synthesis. Biotin is a cofactor for the enzymatic carboxylation of pyruvate, acetyl coenzyme A (CoA), propionyl CoA, and 8-methylcrotonyl CoA, and, therefore, plays an important role in carbohydrate and fat metabolism.

Biotin functions as a co-enzyme within several carboxylases.17 In humans and other mammals, three of the four Biotin-dependent carboxylases are mitochondrial, and one is found in both the mitochondria and the cytosol.

Dermal Penetration Dermal penetration studies data on Biotin were not found in the published literature, and unpublished data were not

submitted.

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Absorption, Distribution, Metabolism, and Excretion (ADME) Biotin was administered orally to humans for 90 days; a significant increase in the Biotin concentration in the blood and urine was observed after 6 and 13 wks of dosing, and these values returned to baseline values when measured 13.5 weeks after dose termination.1 It was reported that in both humans and rats, total excretion of Biotin was greater than dietary intake, and it was postulated that Biotin synthesis occurs by microorganisms in the colon.

Animal In dietary studies in rats, urinary and fecal excretion of Biotin increased with increasing dietary intake.18,19 Serum free Biotin content also increased with increased intake. The following parameters increased in a dose-dependent manner: free content in the serum; total and free content in the liver (but the liver content of the protein-bound content did not change); and content in the skeletal muscle, brain, heart kidney, lung, spleen, and testes.

TOXICOLOGICAL STUDIES The oral LD50 of Biotin for mice and rats was > 10 g/kg and > 1.45 mmol/kg, respectively.1 The intravenous LD50 for mice and the intraperitoneal LD50 for rats and cats was >4.1, >0.12, and >0.001 mmol/kg, respectively. Biotin was not toxic in oral short-term or subchronic toxicity studies.1

Short-Term Toxicity Studies Groups of four male Wistar rats were fed a control diet (20% casein diet containing 0.00002% Biotin) or control diet supple-mented with 0.04, 0.08, 0.10, 0.20, 0.50, 0.80, or 1.0% Biotin for 28 days.18 Feed consumption and body weight gains were statistically significantly decreased in all groups, except the 0.04% group, compared to controls. Diarrhea was reported for animals fed >0.50% Biotin. One animal of the 0.80% and two of the 1.0% group died during the study. Because of the mortality rate in the high dose group, results from that group were not considered when examining the effect on organ weights. Liver, heart, kidney, lung, spleen, and testis weights decreased in a dose-dependent manner in all test groups, except the 0.04% group, when compared to controls. The no-observed-adverse-effect level (NOAEL) was at 0.04% in the diet (calculated as 38.4 mg/kg bw/day), and the lowest-observed-adverse-effect level (LOAEL) was at 0.08% Biotin in the diet (calculated as 79.2 mg/kg bw/day).

DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES In a number of reproductive studies using rats, the number of resorptions in animals dosed with Biotin by subcutaneous (s.c.) injection was increased as compared to controls, and fetal, uterine, and placental weights were decreased.1 Dosing with estrogen generally prevented the resorptions. In a reproductive study in which mice were dosed with Biotin orally and by s.c. injection, significant differences were not observed between treated and control groups.

Groups of male Wistar rats were fed a control diet (containing 0.00004% Biotin; n=5) or a diet supplemented with 0.01% (n=6), 0.1% (n=6), or 1.0% Biotin (n=12), and a pair-fed group (n=12) was given control diet equal in calories to that ingested by the 1.0% Biotin group.19 Half of the animals in the 1.0% Biotin and the pair-fed group were fed for 8 wks, and the other animals were given their respective diets for 6 wks. All animals were killed at the termination of dosing (i.e., at 56 or 42 days, respectively). There were no differences in feed consumption or body weights in the low or mid-dose group when compared to controls, but growth in the high-dose group was less than in controls. Hair loss and mucosal erosion were observed in the high dose animals after 4 wks of dosing, and one high-dose animal died within the 6 wk feeding period. Liver and spleen weights were statistically significantly decreased in the 0.01% (but not the 0.1%) group when compared to controls, and kidney, brain, and testis weights were statistically significantly decreased in the 1.0% group as compared to the pair-fed group. Most hematology parameters did not change, but total cholesterol, total protein, and albumin were statistical-ly significantly decreased in the 1.0% group compared to the pair-fed group.

Administration of 1% Biotin in the diet resulted in a decreased sperm count; the sperm count in the caudal epididymis of rats of the pair-fed group was 34 ± 25 x 106/ml, and it was <1 sperm/ml in rats of the 1% Biotin group. There was a statistically significant increase in the number of sperm with an abnormal (mostly round) head in the 1% group, and only a few spermato-gonia and no spermatocytes were present in this group. The diameters of the seminiferous tubules were statistically signifi-cantly lower in the 1.0% Biotin group, as compared to the pair-fed group (230.0 ± 24.5 µm vs. 342.5 ± 26.3 µm). Apoptosis was not observed, and there were no significant differences in testosterone levels in the testes among the groups. (No results for these parameters were presented for the low and mid-dose groups.)

The effect of Biotin-supplementation on the mouse female reproductive system was evaluated by feeding BALB/cAnN Hsd mice a diet supplemented with 410 µmol/kg diet for 9 wks.20 A statistically significant increase in serum estradiol concentra-tions (73 ± 6.0 vs. 35 ± 2.2 pmol/l in controls) and in CYP1A2 activity (40 ± 7.1 vs. 32 ± 4.6 pmol/mg/protein/min in con-trols) was observed. Primary and Graafian follicles were decreased approximately 50% compared to controls. There were no significant changes in in the estrous cycle, progesterone concentrations, or in the number of corpora lutea in the ovary.

GENOTOXICITY STUDIES Biotin was not mutagenic in an Ames test or an RK bacterial test, but was mutagenic in a Tradescantia-Micronucleus test.1

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CARCINOGENICITY STUDIES Carcinogenicity data on Biotin were not found in the published literature, and unpublished data were not submitted.

DERMAL IRRITATION AND SENSITIZATION STUDIES

Intradermal injection of 0.1 cc Biotin into guinea pigs did not produce skin irritation.1

OCULAR IRRITATION STUDIES Biotin, 0.1% and pH 7.3, produced slight and transient ocular irritation in rabbits.1

CLINICAL STUDIES Case Reports

In a case study, Biotin produced an urticarial reaction, but other clinical studies produced no adverse reactions.1

SUMMARY OF NEW DATA The Panel has previously reviewed the safety of Biotin, and in 2001 published a safety assessment with the conclusion that Biotin is safe as used in cosmetics. This re-review document was prepared in accordance with CIR Procedures to re-evaluate previous conclusions every 15 years. Biotin (which is also known as vitamin B7, vitamin H, and coenzyme R) is reported to function in cosmetics as a hair conditioning agent and a skin conditioning agent-miscellaneous.

The reported frequency of use of Biotin in cosmetics has increased since safety was originally reviewed, from 71 reported uses in 1998 to 506 reported uses in 2017. The reported maximum leave-on concentration of use has decreased from 0.6% to 0.1%.

Biotin is generally recognized as safe (GRAS) in the U.S. as a nutrient for human consumption when used in accordance with GMP, and it is also GRAS in animal nutrients or dietary supplements when used in accordance with good manufacturing or feeding practice. Biotin has been marketed as an ingredient in OTC drug products for external use as hair growers or for hair loss prevention, but there is a lack of adequate data to establish general recognition of the safety and effectiveness of Biotin as intended for these OTC uses, and any OTC drug offered for these uses containing Biotin cannot be considered GRASE. Additionally, Biotin has also been present in OTC drug products for weight control, and it has not been established as GRASE for this use.

In dietary studies in rats, urinary excretion of Biotin increased with increasing dietary intake of Biotin. In a 28-day dietary study in rats with 0.04-1.0% Biotin, the NOAEL was at 0.04% in the diet (calculated as 38.4 mg/kg bw/day) and the LOAEL was at 0.08% Biotin in the diet (calculated as 79.2 mg/kg bw/day). One animal of the 0.80% and two of the 1.0% group died during the study.

Dietary administration of 1% Biotin to male rats for 6 to 8 wks resulted in a decreased sperm count, an increase in the number of sperm with an abnormal (mostly rounded) head, and the presence of only a few spermatogonia and no spermato-cytes. Apoptosis was not observed, and there were no significant differences in testosterone levels in the testes among the groups.

Biotin-supplementation in female mice had several effects on the reproductive system. A statistically significant increase in serum estradiol concentration and in CYP1A2 activity, and a decrease in the number of primary and Graafian follicles, were observed. There were no significant changes in in the estrous cycle, progesterone concentrations, or in the number of corpora lutea in the ovary.

DISCUSSION FROM THE FINAL SAFETY ASSESSMENT OF BIOTIN (2001)1 The Expert Panel recognized that data on the irritation and sensitization potential of Biotin were absent from the report, but the Panel noted that a large number of people are exposed to Biotin daily. The Expert Panel was of the opinion that if Biotin had a strong potential for irritation or sensitization, case reports would be available in the published literature. The lack of such case reports was an indicator to the Expert Panel that Biotin did not have a strong potential for skin irritation or sensitization. UV absorption data demonstrated that phototoxicity would not be a concern.

Several of the available studies that assessed reproductive and developmental toxicity suggested effects of Biotin and one genotoxicity study was positive, but other studies suggested no effects. Assuming that a product containing 0.6% Biotin was applied at a rate of 15 g/day, and approximately 10% of the Biotin was absorbed, it was postulated that the daily exposure to Biotin would be approximately twelve-fold the total potential dietary intake. Based on the low toxic potential of Biotin, however, and on Biotin's rapid metabolism and excretion, such that accumulation would not be a problem, the Expert Panel was of the opinion that no toxic effects would be expected.

Distributed for comment only -- do not cite or quote

DISCUSSION To be developed if the report is re-opened.

CONCLUSION To be determined if the report is re-opened.

Distributed for comment only -- do not cite or quote

Table 1. Current and historical frequency and concentration of use of butyl polyoxyalkylene ethers according to duration and exposure # of Uses Max Conc of Use (%) 20176 19981 2015-20167 19991 # Totals* 506 71 0.0000002-0.1 0.001-0.6 Leave-On 365 34 0.0000002-0.1 0.0001-0.6 Rinse-Off 140 36 0.000001-0.1 0.0001-0.01 Diluted for (Bath) Use 1 1 NR NR Eye Area 54 2 0.0000002-0.1 0.001-0.01 Incidental Ingestion NR NR NR NR Incidental Inhalation-Spray 141a; 106b 20a; 2b 0.001-0.1; 0.001-0.1a 0.001-0.005a; 0.002-0.6b Incidental Inhalation-Powder 106b 2b 0.1; 0.000004-0.1c 0.002-0.6b Dermal Contact 322 31 0.0000002-0.1 0.0001-0.6 Deodorant (underarm) NR NR NR NR Hair - Non-Coloring 166 40 0.0000002-0.1 0.0001-0.01 Hair-Coloring 5 NR 0.0003 NR Nail 6 NR 0.0001-0.1 NR Mucous Membrane 1 3 0.000006-0.001 NR Baby Products NR NR NR NR

*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. #at the time of the original safety assessment, concentration of use data were not reported by the FDA; however, some concentration of use data were received from industry a It is possible these products are sprays, but it is not specified whether the reported uses are sprays.. b Not specified whether a spray or a powder, but it is possible the use can be as a spray or a powder, therefore the information is captured in both categories c It is possible these products are powders, but it is not specified whether the reported uses are powders NR – no reported use

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REFERENCES 1. Andersen FA (ed). Final Report on the Safety Assessment of Biotin. Int J Toxicol. 2001;20(Suppl 4):1-12.

2. Nikitakis J and Lange B (eds). Web-Based Ingredient Dictionary (wINCI): Biotin. http://webdictionary.personalcarecouncil.org/jsp/IngredientDetail.jsp?monoid=300. Washington, D.C. Last Updated 2017. Date Accessed 4-19-2017.

3. Council of Experts. Food Chemicals Codex. 10th ed. Rockville, MD: United Stated Pharmacopeial Convention, 2016.

4. Council of Experts. The United States Pharmacopeia (USP 32). Rockville, MD: The United States Pharmacopeial Convention, 2009.

5. Council of Europe. British Pharmacopoeia. London: The Stationery Office, 2007.

6. Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database. 2017.

7. Personal Care Products Council. 2-17-2016. Concentration of Use by FDA Product Category: Biotin. Unpublished data submitted by Personal Care Products Council.

8. Johnsen MA. The influence of particle size. Spray Technol Marketing. 2004;14(11):24-27.

9. Rothe H. Special Aspects of Cosmetic Spray Evalulation. 9-26-2011. Unpublished data presented at the 26 September CIR Expert Panel meeting. Washington, D.C.

10. Bremmer HJ, Prud'homme de Lodder LCH, and Engelen JGM. Cosmetics Fact Sheet: To assess the risks for the consumer; Updated version for ConsExpo 4. 2006. Report No. RIVM 320104001/2006. pp. 1-77.

11. 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):97-104.

12. CIR Science and Support Committee of the Personal Care Products Council (CIR SSC). 11-3-2015. Cosmetic Powder Exposure. Unpublished data submitted by the Personal Care Products Council.

13. Aylott RI, Byrne GA, Middleton, J, and Roberts ME. Normal use levels of respirable cosmetic talc: preliminary study. Int J Cosmet Sci. 1979;1(3):177-186. PM:19467066.

14. Russell RS, Merz RD, Sherman WT, and Sivertson JN. The determination of respirable particles in talcum powder. Food Cosmet Toxicol. 1979;17(2):117-122. PM:478394.

15. European Commission. CosIng database; following Cosmetic Regulation No. 1223/2009. http://ec.europa.eu/growth/tools-databases/cosing/. Last Updated 2016. Date Accessed 3-15-2017.

16. Allen L, de Benoist B, Dary O, and Hurrell R (eds). Guidelines on food fortification with micronutrients. http://www.who.int/nutrition/publications/guide_food_fortification_micronutrients.pdf?ua=1. Geneva, Switzerland. Last Updated 2006. Date Accessed 4-19-2017.

17. World Health Organization (WHO) Food and Agriculture Organization (FAO) of the United Nations. Human Vitamin and Mineral Requirements. http://www.fao.org/docrep/004/y2809e/y2809e09.htm#bm9. Rome. Last Updated 2001. Date Accessed 4-19-2017.

18. Sawamura H, Fukuwatari T, and Shibata K. Effects of excess biotin administration on growth and urinary exccretion of water-soluble vitamins in young rats. Biosci Biotechnol Biochem. 2007;71(12):2977-2984.

19. Sawamura H, Ikeda C, Shimada R, Yoshii Y, and Watanabe T. Dietary intake of high-dose biotin inhibits spermatogenesis in young rats. Congenital Anomalies. 2015;55(1):31-36.

20. Baez-Saldana A, Camacho-Arroyo I, Espinosa-Aguirre JJ, Neri-Gomez T, Rojas-Ochoa A, Guerra-Araiza C, Larrieta E, Vital P, Diaz G, Chavira R, and Fernandez-Mejia C. Biotin deficiency and biotin excess: Effects on the female reproductive system. Steroids. 2009;74(10-11):863-869.

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Final Report on the Safety Assessment of Biotin1

Biotin is a water-soluble vitamin used as a hair-conditioning agent and a skin-conditioning agent in many cosmetic products at concentrations ranging from 0.0001% to 0.6%. Although Biotin does absorb some ultraviolet (UV) radiation, the absorption shows no peaks in the UVA or UVB region. Biotin is rapidly metabolized and excreted in urine. Little acute oral toxicity is seen in animal tests. Short-term and subchronic toxicity studies likewise found no evidence of toxicity. Although intradermal injection of a small quantity of Biotin (0.1 ml) into guinea pig skin did not produce skin irritation, Biotin (0.1% at pH 7.3) did produce slight, tran­sient ocular irritation in rabbit eyes. Biotin was not mutagenic in bacterial tests, but positive results were found in a Tradescantia mi­cronucleus test. There was evidence of an increase in the number of resorptions in rats receiving Biotin by subcutaneous injection, with concomitant decreases in fetal, uterine, and placental weights. An­other study of mice receiving Biotin orally or by subcutaneous in­jection found no differences between control and treatment groups. Although there is one case study reporting an urticarial reaction in the literature, there are a very large number of individuals exposed to Biotin on a daily basis, and there is not a parallel appearance of irritation, sensitization, or other adverse reactions. Based on these available data, it was concluded that Biotin is safe as used in cos­metic formulations.

INTRODUCTION Biotin, vitamin B7 (Lewis 1993) or vitamin H (Wenninger,

Canterbery, and McEwen 2000), is a water-soluble vitamin (Mock 1991) that functions as a hair-conditioning agent and a skin-conditioning agent-miscellaneous in cosmetic products (Wenninger, Canterbery, and McEwen 2000).

CHEMISTRY

Definition and Structure Biotin (CAS No. 58-85-5) is the organic compound that con­

forms generally to the formula (Wenninger, Canterbery, and McEwen 2000) shown in Figure 1.

Biotin is also known as:

Received 19 September 2001; accepted 11 October 2001. 1 Reviewed by the Cosmetic Ingredient Review Expert Panel. Mon­

ice Zondlo Fiume, former Scientific Analyst/Report Management Co­ordinator, prepared this report. Address correspondence to Dr. F. Alan Andersen, Director, Cosmetic Ingredient Review 1101 17th Street, NW, Suite 310, Washington, DC 20036, USA.

International Journal of Toxicology, 20(Suppl. 4): 1-12, 2001 Copyright© 2001 Cosmetic Ingredient Review 1091-5818/01 $12.00 + .00

• (+)-Biotin; d-Biotin; d-( +)-Biotin (Lewis 1993) • [3aS-(3aa,4b,6aa )]-Hexahydro-2-0xo-IH-Thieno[3,

4-d]Imidazole-4-Pentanoic Acid (Wenninger, Canter­bery, and McEwen 2000; Budavari 1989)

• IH-Thieno[3,4-d]Imidazole-4-Pentanoic Acid, Hexa­hydro-2-0xo-, [3aS-(3aa,4b,6aa)]-; (Wenninger, Can­terbery, and McEwen 2000)

• 1H-Thieno[3,4-d]Imidazole-4-Pentanoic Acid, Hexa­hydro-2-0xo-, [3aS-(3aa,4,B,6aa)]-; (3aS,4S,6aR)­Hexahydro-2-0xo-lH-Thieno[3,4-d]Imidazole-4-Val­eric Acid (U.S. Pharmacopeia! Convention, Inc. 1995)

• Hexahydro-2-0xo-IH-Thieno-[3,4-d]Imidazole-4-Pentanoic Acid (Bonjour 1991; Sax 1979)

• cis-Hexahydro-2-0xo-1H-Thieno[3,4]Imidazole-4-Valeric Acid; cis-Tetrahydro-2-0xothieno[3,4-d]-Imi­dazoline-4-Valerie Acid (Budavari 1989; Informatics, Inc. 1974)

• cis-Hexahydro-2-0xothieno[3,4-d]Imidazole-4-Val­eric Acid (Gennaro 1990)

• (+)-Cis-Hexahydro-2-Keto-IH-Thieno-(3 ,4 )-Imida­zole-4-Valerie Acid (Life Science Research Office [LSRO] 1978)

• 2'-Keto-3,4-Imidazolido-2-Tetrahydrothiophene-8-n­Valeric Acid (Taylor 1988)

• 2-Keto-3,4-Imidazolido-2-Tetrahydrothiophene-n­Valeric Acid (Grant 1972)

• Vitamin B7 (Lewis 1993) • Vitamin H; Coenzyme R (Wenninger, Canterbery, and

McEwen 2000; Lewis 1993; Budavari 1989; Informat­ics, Inc. 1974)

• FactorS; FactorS (Vitamin) (Lewis 1993) • Bioepiderm; and Bios II (Lewis 1993; Budavari 1989)

Physical and Chemical Properties The physical and chemical properties of Biotin are described

in Table 1.

Manufacture and Production d/-Biotin has been synthesized from 4-benzamido-3-

ketotetrahydrothiophene and methyl y-formylbutyrate (Harris et al. 1945) and from 3,4-diamino-2-carbomethoxythiophene (Rossy et al. 1981).

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2 COSMETIC INGREDIENT REVIEW

FIGURE 1 Formula for Biotin.

Natural Occurrence Biotin occurs naturally as the D-isomer and is present in

minute amounts in every living cell (Informatics, Inc. 1974). Biotin occurs in animal and plant tissues primarily in com­bined forms that are liberated by enzymatic hydrolysis during digestion (Gennaro 1990). Food sources of Biotin include organ meats, egg yolk, milk, fish, and nuts (Gilman 1990).

Analytical Methods Gennaro (1990) stated that microbiological methods are the

only feasible methods for the quantitative assay of Biotin be­cause they can detect low concentrations. After simple aqueous or acid extraction combined with heating, a microbiological as­say using growth of test organisms as the criterion is usually carried out. The microbiological assay using Lactobacillus plan­tarum is the most common analytical assay for Biotin (Hudson, Subramanian, and Allen 1984). However, microbiological as­says detect free but not bound Biotin, and oleic acid can partially substitute for Biotin in certain microorganisms (LSRO 1978).

Biotin has been determined using a radiometric­microbiological assay, liquid chromatography with fiuorimet­ric detection, chemiluminescence energy transfer, polarographic methods (Bonjour 1991), a high-performance liquid chromato­graphy/avidin-binding assay (Zempleni et al. 1996), fiuorometic methods (Lin and Kirsch 1979), an isotope dilution assay us­ing 3H-Biotin (Dakshinamurti and Allan 1979), an isotope di­lution assay using 14C-Biotin (Hood 1979), enzymatic methods (Haarasilta 1978), and colorimetric procedures (Plinton et al. 1969). Thin-layer chromatography has been used to determine Biotin in the presence of water-soluble vitamins (Groningsson and Jansson 1979). Liquid chromatography has been used to de­termine Biotin in pharmaceutical preparations, and it was quanti­tated using low ultraviolet (UV) wavelength detection (Hudson, Subramanian, and Allen 1984). A gas chromatography method is applicable for the detection and determination of d-Biotin in pharmaceutical injectable preparations and agricultural pre­mixes (Viswanthan et al. 1970). Biotin in physiological fluids can be determined using a protein-binding assay (Horsburgh and Gompertz 1978).

Impurities One company requires that Biotin contain <0.25% diamino

derivative, biotinylbiotin, and monobenzyl biotin (Cosmetic, Toiletry, and Fragrance Association [CTFA], personal commu­nication). Biotin, which is to be not less than 97.5% and more than 100.5% by assay, may contain <3 ppm arsenic and< 10ppm heavy metals (as Pb) (CTFA 1999a); Schweizerhall, Inc. (1998) reports that Biotin contains <10 mg/kg heavy metals (as Pb). According to the Japanese Standards of Cosmetic Ingredients, Biotin is to contain <0.047% chloride, <0.032% sulfate, <20 ppm heavy metals, and <4 ppm arsenic.

Ultraviolet Absorbance Biotin had low nonspecific absorption in the UVA and UVB

range and into the visible spectrum (CTFA 1998). Biotin is grad­ually destroyed by UV radiation (Bonjour 1991).

USE

Cosmetic Biotin functions as a hair-conditioning agent and a skin­

conditioning agent-miscellaneous (Wenninger, Canterbery, and McEwen 2000). The product formulation data submitted to the Food and Drug Administration (FDA) in 1998 stated that Biotin was contained in a total of 71 cosmetic product formu­lations (FDA 1998). Table 2 presents the distribution of Biotin uses as a function of the type of cosmetic product. For exam­ple, of 200 total bubble bath preparations reported to be on the market, only 1 was reported to contain Biotin.

Concentration of use values are no longer reported to the FDA by the cosmetic industry (FDA 1992). Information received from industry that describes the current concentrations of use as a function of type of cosmetic product is included in Table 2. In­dustry reported concentrations of use in mascara and nail polish and enamel products for which FDA had not received reports of use (CTFA 1999b).

International Biotin is listed in the Japanese Comprehensive Licensing

Standards of Cosmetics by Category (CLS) (Rempe and Santucci 1997). Biotin, which conforms to the specifications of the Japanese Standards of Cosmetic Ingredients, has precedent for use without restriction in all CLS categories, except eyeliner, lip, and oral preparations, for which there is no precedent for use. Biotin does not appear in Annex II (list of substances which must not form part of the composition of cosmetic products) or Annex III (list of substances which cosmetic products must not contain except subject to the restrictions and conditions laid down) of the Cosmetics Directive of the European Union (European Economic Community 1995).

Distributed for comment only -- do not cite or quote

Property

Physical characteristics

Molecular formula

Molecular weight Melting point

Biotin from liver or milk pH of 0.01% aqueous solution Isoelectric point

Biotin from liver or milk Solubility

Specific rotation [af5~

[a]2I~

[a]22~

[a]25~

Stability

Reactivity

BIOTIN

TABLE 1 Physical and chemical properties of Biotin

Description

White to off-white fine crystalline powder Practically white, crystalline powder

Needles obtained with water as the solvent Colorless, crystalline, monocarboxylic acid Practically white, crystalline powder in the form of fine,

long needles Fine long needles (from liver or milk) CwH16N203S

244.31 Decomposes, 229-232°C Decomposes, 232°C 232-233°C 4.5 pH3.5 pH 3.5 Soluble in hot water and dilute alkali, practically

insoluble in water and alcohol, and insoluble in other common organic solvents

More soluble in hot water and dilute alkali, insoluble in other common organic solvents

Soluble in hot water and dilute alkali, sparingly soluble in dilute acid, cold water, and alcohol, and insoluble in most organic solvents

Slightly soluble in water or alcohol Soluble in hot water and dilute ethanol, sparingly soluble

in cold water, insoluble in chloroform, benzene and ether +89° to +93° (2%, NaOH 0.1 mol/l) +89 to +93°

+91° (1 g dissolved in 0.1 N NaOH) 92° (0.1 N NaOH, 1 %) +91 o (from liver or milk) Dry crystalline D-Biotin is fairly stable to air, daylight,

and heat, but is gradually destroyed by UV; aqueous solutions are relatively stable if weakly acidic or alkaline, but the biological activity is destroyed by heating in strongly acidic or alkaline solutions

Water solutions are stable at 100°C; the dry substance is thermo- and photostable; unstable in strong acids and alkaline solutions and in oxidizing agents

Pure compound is stable to air and temperature, moderately acid and neutral solutions are stable for several months, alkaline solutions are less stable, but appear reasonably stable up to pH 9

Emits toxic fumes of NOx and SOx when heated to decomposition

Incompatible with nitrous acid, oxidizing agents, formaldehyde, strong acid, or alkali

Reference

CTFA 1999a National Academy of

Sciences (NAS) 1996 Lide 1993 Gennaro 1990 Informatics, Inc. 1974

Budavari 1989 Wenninger, Canterbery,

and McEwen 2000 NAS 1996; Lide 1993 CTFA 1999a; NAS 1996 Lide 1993 Budavari 1989 Budavari 1989 Budavari 1989 Informatics, Inc. 1974 CTFA 1999a

NAS 1996

Bonjour 1991

Gennaro 1990 LSRO 1978

CTFA 1999a NAS 1996; Informatics,

Inc. 1974 Budavari 1989

3

Bonjour 1991; Grasselli 1973 Informatics, Inc. 1974 Bonjour 1991

Gennaro 1990

Informatics, Inc. 1974

Lewis 1993

Budavari 1989; Informatics, Inc. 1974

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4 COSMETIC INGREDIENT REVIEW

TABLE2 Product formulation data on Biotin

Product category (number of formulations in the category)

(FDA 1998)

Bubble baths (200) Mascara (167) Other eye makeup preparations (120) Hair conditioners (636) Shampoos-noncoloring (860) Tonics, dressings, and other hair-grooming aids (549) Other hair preparations (276) Foundations (287) Nail polish and enamel (80) Bath soaps and detergents (385) Cleansing preparations (653) Face and neck preparations-excluding shaving (263) Body and hand preparations-excluding shaving (796) Moisturizing preparations (769) Night preparations (188) Paste masks/mud packs (255) Skin fresheners (184) Other skin care preparations (692) Indoor tanning preparations (62) Other suntan preparations (38) 1998 total for Biotin

Noncosmetic Biotin is generally recognized as safe as a dietary supple­

ment, as a nutrient, and in animal feed (FDA 1997). Its func­tional use in food is as a nutrient and dietary supplement (NAS 1996). The adult daily requirement has been assigned a provi­sional value of 30 to 100 {Lg Biotin by the Committee on Dietary Allowances, and the average daily diet provides 100 to 300 {Lg (Gilman 1990).

GENERAL BIOLOGY

Absorption, Distribution, Metabolism, and Excretion A group of 11 male humans were given twice daily for 90 days

a vitamin supplement that contained 396 {Lg Biotin (as analyzed) (Singh, Moses, and Deuster 1992). A second group was given a placebo and served as the control. For 4 days prior to dosing and at weeks 6 and 13, baseline dietary records, fasting blood samples, and 24 hour urine samples were collected. This infor­mation and these values were also collected from nine test and eight control subjects 13.5 weeks after dose termination. In test subjects, a significant increase in the Biotin concentration in the blood and urine was observed after 6 and 13 weeks of dos­ing. These values returned to baseline values when measured 13.5 weeks after dose termination.

Number of formulations containing Biotin

(FDA 1998)

2 20 11 7 2 2

2

1 5 4 2 2 6

1 71

Current concentration of use

(CTFA 1999b)

0.0001% 0.01%

0.0001%-0.01% 0.0001%-0.001%

0.001%

0.0001%-0.001%

0.002%-0.6%

0.005%

Male Sprague-Dawley rats that had been fed a Biotin­sufficient diet containing 1640 pmol/g Biotin for at least 1 week were used to study Biotin excretion (Wang, Patel, and Mock 1996). In a dose-range study, rats were dosed with 6.6 to 1025 pmol/g D-carbonyl-14C-Biotin. Urine was collected after 3, 6, 9, and 24 hours, and daily for 5 days. At all doses, at least 50% of the radioactivity was excreted in the urine within 72 hours. At the 1025-pmol/g dose, approximately 80% of the radioactivity was excreted within 24 hours. Based on the re­sults of the dose-range study, 57 pmol/g was used in the primary study.

In the primary study using six animals, >98% of the to­tal radioactivity was excreted in the urine; Biotin, bisnorbiotin, and biotin sulfoxide accounted for >90% of the total urinary radioactivity. After 24 hours, unchanged Biotin accounted for 51% of the radioactivity, bisnorbiotin accounted for 29%, and biotin sulfoxide for 10%. On day 2, bisnorbiotin was the prin­cipal metabolite; the metabolite profiles were then relatively stable. Of the radioactivity extracted from the feces, 93% was unchanged Biotin, 6% was biotin sulfoxide, and 1% was bis­norbiotin. The researchers compared the metabolic profile of the rats with that obtained using 10 human subjects. During the first 24 hours, the human and rat profiles were similar; however, significant differences were observed between days 2 to 5.

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BIOTIN 5

Rats were dosed by intraperitoneal (IP) injection with 4 to 5 ml solution containing 13.5 J.tg ureido carbonyl-labeled 14C-Biotin (0.063 J.tC) (Fraenkel-Conrat and Fraenkel-Conrat 1952). Urine, feces, and expired carbon dioxide were collected. Approximately 85% of the radioactivity was excreted in the urine within 24 hours, and 87% was excreted over 3 days. Ap­proximately 7% of the radioactivity was recovered in the feces over 3 days. Approximately 5% to 8% of the radioactivity re­mained in the liver after 3 to 6 days, considerably less was found in the kidneys and spleen, and no radioactivity was detected in the adrenal glands, mesentery, lungs, blood, thyroid gland, or skeletal muscle.

Groups of six male Sprague-Dawley rats that had been fed Biotin-sufficient or Biotin-free diets were dosed by IP injection with 0.5 J.tg, 4 J.tg, or 1 mg/100 g body weight ureido carbonyl­labeled 14C-Biotin (Lee, Wright, and McCormick 1972). Urine, feces, and expired carbon dioxide were collected. For the rats fed the Biotin-sufficient diet and then dosed with 1 mg Biotin, >90% of the dose was excreted in the urine within 12 hours. Significant radioactivity was not expired in carbon dioxide, and only a trace was excreted in the feces. The initial excretion rate decreased with decreasing doses of Biotin; this decrease was more marked with Biotin-deficient rats. For the animals fed a Biotin-sufficient diet and dosed with 0.5 or 4 J.tg Biotin, 47% of the dose was excreted after 12 hours and 83% was excreted after 8 hours, respectively; for the animals fed a Biotin-free diet and dosed with 0.5 J.tg, 29% of the dose was excreted 12 hours after dosing.

Groups of five male Sprague-Dawley rats that had been fed Biotin-sufficient or Biotin-deficient diets were dosed by IP in­jection with 0.5 mg/100 g body weight ureido carbonyl-labeled 14C-Biotin (Lee et al. 1973). The excretion rate was similar for both groups. Approximately 95% of the dose was excreted in the urine within 24 hours, and 84% was excreted within the first 3 hours. Significant radioactivity was not found in the feces or expired carbon dioxide.

Fasted rats and chicks fed a Biotin-deficient diet for 6 and 5 weeks, respectively, were dosed intramuscularly with 1 ftC

(1 0 J.tg) Biotin-C1400H/100 g body weight (Dakshinamurti and Mistry 1963). Approximately 81% of the dose was recovered for rats after 4 hours, with approximately 30%, 16%, and 20% of the dose recovered in the excreta, liver, and injected muscle, respectively. Approximately 82% of the dose was recovered for chicks after 3 hours, with approximately 31%, 17%, and 14% of the dose recovered in the excreta, liver, and kidneys, respectively.

The uptake of d-[8,9-3H(N)] Biotin by isolated hepatocytes from male Sprague-Dawley rats fed chow or a Biotin-deficient diet was studied (Rose et al. 1986). Uptake of Biotin was less by cells from rats fed a Biotin-deficient diet. Biotin uptake was markedly sensitive to temperature. 3H-Biotin was Na+ depen­dent and facilitated by an acid-anion carrier.

Rose ( 1996) stated that, in both humans and rats, total excre­tion of Biotin is greater than dietary intake and postulated that Biotin synthesis occurs by microorganisms in the colon.

Biochemistry Intestinal synthesis is an important factor in the supply of

Biotin to the body (Gennaro 1990). Biotin-producing microor­ganisms exist in the intestinal tract (Bonjour 1991).

Biotin functions in carbon dioxide fixation reactions in inter­mediate metabolism, transferring the carboxyl group to acceptor molecules (Gennaro 1990). It acts similarly in decarboxylation reactions. Biotin is essential in human metabolism for its part in the previously described enzymatic steps, in catalyzing deami­nation of amino acids, and in oleic acid synthesis. Biotin is a co­factor for the enzymatic carboxylation of pyruvate, acetyl coen­zyme A (CoA), propionyl CoA, and ,8-methylcrotonyl CoA, and, therefore, plays an important role in carbohydrate and fat metabolism (Gilman 1990).

The effect of Biotin on free-radical generation was examined in a spectrophotometric assay of cytochrome c reduction and de­termination of the 2-methyl-6-phenyl-3,7-dihydroimidazo[l,3-a]-pyrazin-3-one (CLA)-dependent chemiluminescence res­ponse of human neutrophils or a hypoxanthine-xanthine oxidase (XOD) system (Sekiguchi and Nagarnine 1994). In the cytochrome c reduction assay, two Biotin and two non-Biotin groups were used, and one of each was either stimulated or not stimulated with N -formylmethionylleucylphenylalanine (f-MLP). Superoxide anion (02) generation from neutrophils stimulated by f-MLP was reduced significantly by the addition of Biotin. Also, Biotin significantly reduced the generation of free-radical species (including 02) in a concentration-dependent manner in the CLA-dependent chemiluminescence test of neu­trophils stimulated by f-MLP. The IC50 was 1.12 x w-7 moles. Using the hypoxanthine-XOD system, Biotin did not have an in­hibitory effect on oxidative metabolism by directly scavenging superoxide anion.

The mechanism of Biotin uptake was examined using cul­tured Hep G2 cells (Said, Ma, and Kamanna 1994 ). Biotin up­take was mediated through aNa+ -dependent, carrier-mediated mechanism; it was dependent on incubation temperature and intracellular energy.

BIOTIN DEFICIENCY

Dietary Impact Male Long-Evans rats were fed a Biotin-deficient diet, a diet

supplemented with 0.2 mg/kg Biotin, or the diet supplemented with 0.2 mg/kg Biotin and 5% cellulose for 3 weeks (Rader et al. 1986). Rats were weighed daily, feed consumption was measured weekly, and the animals were killed on day 20 or 21. The animals fed the Biotin-deficient diet had significantly de­creased growth and their concentrations of some trace minerals were significantly increased as compared to animals of the other two groups.

Male inbred-BHE kine 3 rats were fed a semipurified diet with or without 5 mg/kg Biotin and controls were fed chow for 6 weeks (Marshall et al. 1972). Feed consumption and body

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6 COSMETIC INGREDIENT REVIEW

weights were significantly decreased and all relative organ-to­body weights, except relative liver weights, were increased for the animals fed the semipurified diet without Biotin when com­pared to those fed the Biotin-supplemented diet and those fed chow. Animals fed the Biotin-supplemented diet had decreased serum cholesterol concentrations compared to the group fed the Biotin-deficient diet.

Rats were fed a diet containing crystalline egg albumin with or without 25 f.Lg Biotin or a diet with casein instead of egg white for 6 weeks (Tuszynska 1970a). After 6 weeks, the rats fed the diet without Biotin had poor growth, loss of body fat, "bald patches" on the back, neck, and head with "dull and sparse hair" elsewhere, purulent conjunctivitis, "circles around the eyes," and a "kangaroo-like posture." Half of the animals died when the Biotin-deficient diet was continued for an ad­ditional 2 weeks. Biotin supplementation reversed the adverse effects. Tuszynska (1970b) administered 500 f.lg Biotin orally and dermally to Biotin-deficient rats every other day for 2 weeks, giving a total dose of 4 mg. Oral administration of Biotin pro­duced faster improvement and cessation of the Biotin-deficient signs. Three times as much Biotin was needed when applied to the skin to exert the same affect as Biotin given orally.

Specific-pathogen-free Sprague-Dawley rats were fed for 7 weeks a Biotin-deficient basal diet that contained 30% egg white solids; some of the animals were given 150 f.Lg Biotin orally once weekly (Britton 1980). Animals fed a Biotin­deficient diet and not given Biotin had significantly decreased body weight gains, feed consumption, and feed efficiency ra­tios compared to those given Biotin. Seven of eight animals fed the Biotin-deficient diet had severe hair loss. Blood lipids and plasma cholesterol were greater for animals given Biotin, whereas lactate concentrations were greater for the rats fed a Biotin-deficient diet.

Immunological Effects Female Lewis rats were fed a Biotin-deficient diet, a Biotin­

adequate diet, or commercial feed; the animals fed the Biotin­deficient or -adequate diets were pair-fed to ensure they were given the same amount of feed (by weight), whereas the com­mercial feed was given ad libitum (Rabin 1983). Some of the animals were immunized with purified guinea pig myelin basic protein (MBP) followed by evaluation for experimental allergic encephalomyelitis (EAE); some of the animals were given IP injections of 1 ml of a 2% suspension of sheep erythrocytes, and the hemagglutination antibody and plaque-forming cell re­sponses were determined after 4 days. Groups of 14 animals were not immunized and were fed their respective diets for 20 weeks.

The body weights and thymus gland weights of the animals fed the Biotin-deficient diet were significantly decreased after 20 weeks, and four of the animals had hair loss around the nose while five had eczematous-appearing skin with diffuse hair loss. The 14 animals in each of the other groups appeared normal. All of the animals fed the Biotin-adequate diet or commer­cial feed and immunized with MBP at I 0 weeks had marked

clinical changes, including hindquarter weakness or paralysis, and the spinal cords had extensive perivascular lymphocytic in­filtration. Animals fed the Biotin-deficient diet did not have signs of EAE and the spinal cords had only a few perivascular lym­phocytic infiltrates. Transfer of lymphocytes from animals fed a Biotin-adequate diet and immunized to MBP determined that the afferent limb of immune response is altered by Biotin defi­ciency. In examining the immune response in animals injected with sheep erythrocytes, it was found that the total number of plaque-forming cells in the spleen and the number per million lymphocytes were significantly decreased for animals fed the Biotin-deficient diet compared to the other animals.

Teratogenic Effects To determine the teratogenic effects of Biotin deficiency on

different species and strains of animals, groups of II Jcl:ICR mice, 14 C57BL/6N/Jcl mice, 11 A/Jax sci mice, 10 Jcl:Wistar rats, and 11 Std:Syrian hamsters were fed a Biotin-deficient diet starting on day 0 of gestation (Watanabe and En do 1989). Groups of 10 to 15 animals per species and strain were fed a control diet that contained 5.0 mg/kg Biotin. Maternal weight gain and general appearance of the animals were observed every other day. The mice, rats, and hamsters were killed on days 18, 20, and 15 of gestation, respectively, and the live and dead fetuses and number of resorptions were counted. The hepatic Biotin concentration was determined in some of the dams and their fetuses.

Maternal body weight gains were decreased 60% to 90% for the ICR and C57BL mice, the rats, and the hamsters fed the Biotin-deficient diet as compared to those animals of the con­trol groups, and daily feed consumption was decreased during the second half of gestation. Significant differences in maternal body weights and feed consumption were not observed for A/Jax mice, and these dams did not have any signs of Biotin deficiency. The frequency of dams with implants did not differ between the Biotin-deficient and the control groups, but the number of dams with live fetuses/number of dams mated was decreased for the C57BL mice and the hamsters fed the Biotin-deficient diets. For all groups except the A/Jax mice, fetal body weights were significantly decreased as compared to the respective control groups. Malformed fetuses were found in all ICR and C57BL mice fed the Biotin-deficient diet, and greater than 90% of the live fetuses were grossly malformed. The most common malfor­mations were cleft palate, micrognathia, and micromelia in both strains; open eyelid was also observed for a number of C57BL mouse fetuses. In the Biotin-deficient A/Jax mouse group, cleft palate and micrognathia occurred at an incidence of 14%, with 31% of the fetuses having malformations. No malformations were observed in the fetuses of rats fed the Biotin-deficient diet. In hamsters, teratogenicity was "rather equivocal" because embryonic lethality was much greater for the animals fed the Biotin-deficient diet, and an insufficient number of fetuses were examined. A large difference in hepatic Biotin concentrations for the control animals was not seen among the species, but the

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BIOTIN 7

concentrations of the dams fed the Biotin-deficient diets were about half that of the control animals. A large difference was observed in hepatic Biotin concentrations for the fetuses of the control ICR mice, rats, and hamsters, but the concentrations were similar for fetuses of the Biotin-deficient dams.

Clinical Effect Biotin deficiency, which can occur by the feeding of un­

cooked egg whites or by the omission of Biotin from the diet, can cause alopecia and a characteristic scaly, erythematous der­matitis around body orifices in infants, children, and adults (Mock 1991). For adults, prolonged Biotin deficiency can re­sult in depression, lethargy, hallucinations, and paresthesias of the extremities.

ANIMAL TOXICOLOGY

Acute Toxicity Oral

The oral LD50 of Biotin for mice was reported as > 10 g/kg (Informatics, Inc. 1974), and the oral LD50 for rats was reported as > 1.45 mmol/kg (Bonjour 1991 ), but no details of study pro­cedures were given.

Parenteral Crittenden (1948) reported that single intravenous (IV) in­

jections to mice of 1 g/kg dl-Biotin were tolerated without signs of toxic effects, but details were not provided. Likewise with­out giving details, Bonjour (1991) reported that the IV LD5o of Biotin for mice was >4.1 mmol/kg, and the IP LD5o values for rats and cats were >0.12 and >0.001 mmol/kg, respectively.

Short-Term Toxicity Oral

Groups of 15 and 10 mice approximately 7 and 14 weeks of age, respectively, were dosed orally with 1 mg of dl-Biotin or d-Biotin in 0.2 ml saline for 60 days; 10 7-week-old and 10 14-week-old mice, the control group, were given 0.2 ml of saline (Crittenden 1948). Signs of toxicity attributable to Biotin were not observed, and no significant differences in mortality were observed among the groups.

Five male rats were dosed orally with 50 mg of d-Biotin for 10 days (Crittenden 1948). Signs of toxicity were not observed, and changes in the number of erythrocytes or leukocytes or in the amount of hemoglobin were not seen. Gross lesions were not observed at necropsy.

Bonjour (1991) reported that the short-term LD50 was > 1450 JLmol/kg in a study in which rats were dosed orally with Biotin for 10 days. No further study details were provided.

Groups of Mongolian gerbils (number per group not stated) fed a normal or hypercholesterolemic diet were dosed orally with 1 mg/kg Biotin for 3 or 6 weeks (Informatics, Inc. 1974). At study termination, body weights, adrenal gland weight, liver

weight, serum lipids, adrenal gland and hepatic cholesterol, and fecal lipid excretion were determined. Consistently significant changes in serum free fatty acids, triglycerides, or cholesterol were not observed, and little change in adrenal glands or hep­atic cholesterol was observed. Body weights of the animals fed normal diet and dosed for 6 weeks were significantly decreased as compared to control values.

Parenteral Groups of Mongolian gerbils (number per group not stated)

fed a basal or hypercholesterolemic diet were dosed subcuta­neously (SC) with 1 mg/kg Biotin for 3 or 6 weeks (Informatics, Inc. 1974). At study termination, body weights, adrenal gland weight, liver weight, serum lipids, adrenal gland and hepatic cholesterol, and fecal lipid excretion were determined. Consis­tently significant changes in serum free fatty acids, triglycerides, or cholesterol were not observed, and little change in adrenal gland or hepatic cholesterol was observed. For animals fed a basal diet, body weights of those dosed for 6 weeks were signif­icantly decreased and liver weights of those dosed for 3 weeks were significantly increased as compared to control values.

Bonjour ( 1991) reported that the short-term LDso values were >0.41 and >6.15 JLmol/kg, respectively, in rabbits and dogs dosed IV with Biotin for 30 and 10 days, respectively, but further details were not provided.

Four dogs were dosed with 10 mg of d-Biotin by IV injection for 10 days (Crittenden 1948). Changes in physical, urinary, or blood parameters were not observed. One animal was killed, and gross lesions were not observed.

Subchronic Toxicity Oral

Groups of five male rats were dosed orally with 5 mg of dl-Biotin or d-Biotin for 120 days; the control group was given saline (Crittenden 1948). The average body weight of the control group was greater than that of the test groups at study termina­tion. The animals were clinically normal throughout the study. Significant changes in the number of blood cells, the amount of hemoglobin, or in hepatic or renal function were not observed.

Bonjour (1991) reported that the subchronic LDso values were >0.82 and >0.41 JLmol/kg, respectively, in rabbits and piglets dosed orally with Biotin for 102 and 122 days, respec­tively, but further details were not provided.

Parenteral Bonjour (1991) reported that the subchronic LD5o was

>0.82 JLmol/kg in rabbits dosed SC with Biotin for 102 days, but further study details were not provided.

Chronic Parenteral Toxicity Bonjour (1991) reported that the chronic LD50 was

>0.41 JLmol/kg in rabbits dosed SC with Biotin for 180 days, but details of the study were not provided.

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8 COSMETIC INGREDIENT REVIEW

Dermal Irritation Intradermal injection of 0.1 cc Biotin into the abdominal skin

of guinea pigs did not produce irritation at the site of the injection (Crittenden 1948). Intramuscular injection of 0.5 or 1.0 ml of 0.1% d-Biotin into rabbits did not produce irritation.

Dermal Sensitization

Published data on the sensitization potential of Biotin using animals were not found.

Ocular Irritation Application of 0.1% d-Biotin, pH 7.3, to the eye of rabbits

was reported to produce only slight and transient irritation, but details of the study were not provided (Crittenden 1948).

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Three groups of six female Holtzman rats, after having a nor­

mal estrus for three cycles, were dosed SC with 50 mglkg Biotin in 0.2 ml ofO.l N NaOH in morning and evening injections dur­ing the diestrus stage (Paul, Duttagupta, and Agarwal 1973a). The animals were killed 7, 14, or 21 days after dosing. A con­trol group of six rats was given SC an equal volume of 0.1 N NaOH during the diestrus stage and killed after 7 days. The estrus cycle was studied daily, and the leukocyte concentration of vaginal smears was determined. The ovaries were examined microscopically and the hepatic and uterine glycogen concentra­tions were estimated. The Biotin-treated animals had irregular estrus cycles, and the number of vaginal leukocytes progres­sively increased until day 14, and then declined sharply. Biotin did not significantly alter ovarian, uterine, or liver weights, and body weight gain was normal. At microscopic examination of the ovaries, it was observed that the process of formation of the corpora lutea was enhanced, but several of the corpora lutea were atrophic. Hepatic glycogen, concentration and total, was signif­icantly reduced after 14 days and returned to control amounts after 21 days. Uterine glycogen, concentration and total, was slightly reduced at 21 days after Biotin treatment.

Groups of 12 female Holtzman rats were dosed with 50 mglkg Biotin dissolved in 0.5 ml of 0.1 N NaOH in two SC injec­tions (Paul, Duttagupta, and Agarwall973b ). The rats were then mated with untreated males 7, 14, or 21 days after dosing. An untreated group of 12 rats was the negative-control group. Six rats of each group were killed on day 14 of gestation, and the remaining six were killed on day 21 of gestation. A group of six rats was treated with Biotin and mated, and the animals in this group were given 1 f.1,g 17 ,B-estradiol in 0.1 ml olive oil on days 6 to 21 of gestation. Another group of six rats was dosed SC with 100 mg/kg Biotin in 1 ml of 0.1 N NaOH in four injections for 2 days. The animals of these two groups were killed on day 21 of gestation.

Of the animals killed on day 14 of gestation, six, four, and three mated 7, 14, and 21 days after dosing, respectively, had

fetuses. Of the animals killed on day 21 of gestation, two, three, and one mated 7, 14, and 21 days after dosing, respectively, had fetuses. All negative-control animals had fetuses at both 14 and 21 days of gestation, and all of the animals given 17 ,B-estradiol had fetuses. None of the animals dosed with 100 mglkg Biotin mated in the 2 months following dosing. The fetal and placental weights of the existing neonates of animals dosed with 50 mglkg Biotin were decreased as compared to negative control values. The fetal and placental weights of neonates of animals given 17 .B -estradiol were comparable to those of the negative controls.

SPF outbred rats (lbm:ROROt) were dosed SC with 5 or 50 mglkg d-Biotin in 0.1 N NaOH, pH 12, given in two doses 5 hours apart on the day of vaginal estrus (Mittelholzer 1976). One control group of eight animals was given SC 0.1 N NaOH, and an untreated control group of seven animals was not dosed. Groups of eight animals were mated 7, 14, and 21 days after dosing and killed and examined on day 21 of gestation.

Significant differences were not observed in the number of implantations per gravid animal, the number of resorption sites, fetal weight, placental weight, ovary weight, or in the number of animals with irregular cycles or without evidence of mating between treated animals and animals of the control groups. The only difference observed was in the number of gravid animals. For the animals mated 7 days after dosing, 6/7, 8/8, and 7/8 animals of the 5-mg/k.g, 50-mg/k.g, or untreated-control groups, respectively, mated and 88%,71%, and 67%, respectively, of the animals that mated had fetuses on day 21 of gestation. Of the animals mated 14 or 21 days after dosing, all of the animals of each group mated, and 63%, 88%, and 100% of the animals in the 5-mg/k.g, 50-mg/k.g, or untreated-control groups, respectively, had fetuses on day 21 of gestation. Lesions were not observed in the ovaries at microscopic examination.

Groups of Holtzman rats were mated and the gravid females were dosed SC with 100 mg D(+)-Biotin in 0.2 ml of 0.1 N NaOH/kg body weight on days 0 and 1 of gestation (Paul and Duttagupta 1975). Nine animals were dosed with Biotin only, seven were given Biotin and 0.1 f.1,g 17 ,B-estradiol in 0.05 ml olive oil SC on days 5 to 20 of gestation, and seven were given Biotin and 4 mg progesterone in 0.2 ml olive oil SC on days 5 to 20 of gestation. Nine gravid animals were untreated and used as a negative-control group. Three groups of six nongravid animals were dosed in the same manner as the gravid animals and used as nonpregnant treated controls. The animals were killed and examined on day 21 of gestation.

Complete resorption of the fetuses occurred in eight of the nine rats dosed with Biotin only; dosing with estrogen or pro­gesterone prevented the resorptions. Fetal and placental weights from animals dosed with Biotin and estrogen or progesterone were decreased as compared to controls, but the decrease was not statistically significant. Biotin caused a decrease in body weights of gravid and nongravid animals; body weights of gravid animals given Biotin and progesterone were similar to gravid un­treated control, whereas body weights of gravid animals given Biotin and estrogen were increased. The uterine weights of

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BIOTIN 9

gravid animals given Biotin and estrogen were similar to that of gravid untreated controls, whereas the uterine weights of animals dosed with Biotin and progesterone were statistically significantly decreased.

The hepatic glycogen concentration of the gravid animals of all treatment groups and of the nongravid animals given Biotin only was similar to that of gravid controls; a decrease in the hep­atic glycogen concentration was observed for nongravid animals dosed with Biotin and estrogen or progesterone. A statistically significant decrease in uterine glycogen concentration was ob­served in the animals dosed with Biotin that had resorptions. The hepatic RNA concentration was statistically significantly reduced in gravid animals dosed with Biotin, but the uterine concentrations were not affected. Hepatic RNA concentrations of gravid animals dosed with Biotin and estrogen were simi­lar to those of untreated gravid controls, whereas uterine RNA concentrations of animals of this group were greater than con­trol values. A statistically significant increase in uterine DNA concentrations occurred in rats that were dosed with Biotin and had resorptions; uterine DNA concentrations of gravid animals dosed with Biotin and estrogen or progesterone were similar to control values. Hepatic and uterine protein concentrations were statistically significantly decreased in gravid animals dosed with Biotin; uterine protein concentrations of gravid animals dosed with Biotin and estrogen were similar to those of gravid con­trols. Glucose-6-phosphate dehydrogenase (G-6-PD) activity in the ovaries, adrenal glands, liver, and uterus of gravid animals dosed with Biotin were statistically significantly decreased as compared to gravid untreated controls; dosing with estrogen or progesterone in addition to Biotin significantly increased the en­zymic activity as compared to animals given Biotin only, but the values were still lower than those of controls.

In a similar study, groups of Holtzman rats were mated, and gravid females were dosed with 100 mg D(+)-Biotin in 0.2 ml of 0.1 N NaOH/kg body weight on days 13 and 14 of gestation (Paul and Duttagupta 1976). Eleven animals were dosed with Biotin only, seven were given Biotin and 0.1 {tg 17 ,8-estradiol in 0.05 ml olive oil SC until day 20 of gestation, and seven were given Biotin and 4 mg progesterone in 0.2 ml olive oil SC until day 20 of gestation. Nine gravid animals were untreated and used as a negative-control group. The animals were killed and examined on day 21 of gestation.

Resorptions occurred in 2 of the 11 animals dosed with Biotin only. The maternal body weights and the fetal, uterine, and placental weights of the remaining nine animals of this group were statistically significantly decreased as compared to con­trols. The maternal body weights and the fetal, uterine, and pla­cental weights of the animals dosed with Biotin and estrogen and the maternal body weights and uterine weights of the ani­mals dosed with Biotin and progesterone were similar to control values. Hepatic and ovarian weights were similar for animals of the test and control groups.

Dosing with Biotin or Biotin and progesterone resulted in a statistically significant decrease in uterine and placental glyco-

gen concentrations; this decrease was not observed upon dosing with Biotin and estrogen. Hepatic glycogen and blood glucose concentrations were similar for animals of the treated and con­trol groups. Biotin and Biotin plus progesterone caused a sta­tistically significant decrease in the amount of hepatic protein. Hepatic RNA concentrations were statistically significantly in­creased by Biotin and estrogen. Ovarian, uterine, and hepatic, but not adrenal gland or placental, G-6-PD activity was statis­tically significantly decreased in animals dosed with Biotin; in animals dosed with Biotin and estrogen, adrenal gland G-6-PD activity was statistically significantly reduced.

ICR mice were dosed with Biotin orally or SC to determine the reproductive and developmental effects of Biotin (Watanabe 1996). Groups of gravid mice were fed 0 or 1000 mg/kg Biotin (0.1%) in commercial feed throughout gestation, dosed SC with 0 or 150 mg/kg Biotin in olive oil, or dosed SC with 0 or 50 mg/kg Biotin in 0.1 N NaOH, pH 11; the animals of the SC groups were dosed with 0.5 ml in three abdominal regions on days 0, 6, and 12 of gestation. An untreated control group was fed commercial diet. The animals were killed and examined on day 17 of gestation. The Biotin concentration of maternal and fe­tal organs was determined microbiologically, and the biotinidase activity of mice in the dietary group was measured on day 17 of gestation. All animals were observed daily for signs of toxicity and any deaths were recorded.

No signs of toxicity, behavioral changes, and mortality were observed. The number of resorbed or dead fetuses was slightly increased in the group dosed with Biotin in olive oil as compared to the group dosed with Biotin in NaOH; however, in none of the test groups was the increase statistically significant when compared to the untreated control group. Maternal body weight gain of all groups was statistically significantly decreased as compared to the untreated controls, but this difference was not attributed to Biotin. Fetal parameters were similar for all test groups, and a significant difference in external malformations was not observed between the Biotin-treated and untreated con­trol groups. No microscopic differences in the maternal liver, placenta, or ovaries were observed. In the treated groups, Biotin accumulation was observed in maternal and fetal organs. A sta­tistically significant increase in biotinidase activity was observed in the maternal serum and the placenta of rats fed Biotin as compared to controls, but no changes were observed in serum estradiol-E2 content between these groups. The researcher con­cluded that Biotin "did not disturb normal reproductive functions or embryonic development" in mice.

GENOTOXICITY The mutagenic potential of ::=:: 10,000 JLg/plate d-Biotin in

dimethyl sulfoxide was determined with and without metabolic activation in an Ames Salmonella/microsome assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and in Escherichia coli WP2 (uvrA) (SRI International 1979). Negative and positive controls were used,

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10 COSMETIC INGREDIENT REVIEW

and each test was performed in duplicate. d-Biotin was not mu­tagenic or toxic. A precipitate was observed on all plates at a concentration of 10,000 f,Lg.

In a Tradescantia-Micronucleus test, the mutagenic potential of 0.01 to 0.5 mM Biotin was determined with treatment made by absorption through the stem (Ma et al. 1984). Biotin was mutagenic, with a minimum effective dose of 0.1 mM.

nicotinic acid; and Biotin concentrate (Keller 1942). Rice and butter yellow produced no reaction, the vitamin B complex pro­duced erythema and central edema that lasted for 10 hours, the crystalline nicotinic acid produced erythema without edema that subsided after 1 hour, and the Biotin concentrate produced "a central, firm, raised, pale area with a surrounding zone of spread­ing erythema" within 2 hours.

The mutagenic potential of Biotin was also determined in the RK bacterial test using E. coli strain CHY832 (Hayes et al. 1984). Biotin, 1.0 mg/ml and pH 7.4, was not mutagenic. Clinical Studies

CARCINOGENICITY Published data on the carcinogenic potential of Biotin were

not found.

Biotin has been administered orally, intramuscularly, and in­travenously to children, patients, and normal subjects without adverse effects. These clinical studies are described briefly in Table 3.

CLINICAL ASSESSMENT OF SAFETY SUMMARY

Urticarial Reaction-Case Report A laboratory worker who reacted when mixing a "dry dusty

diet" was patch tested with four substances: rice and 3% "but­ter yellow" in olive oil; rice and a vitamin B complex including thiamine chloride, choline chloride, vitamin B2, riboflavin, pyri­doxine, nicotinic acid, pantothenic acid, and Biotin; crystalline

Biotin, also known as vitamin B7 or vitamin H, is a water­soluble vitamin used as a hair conditioner and as a skin­conditioning agent in cosmetics. In 1998, it was reported to the FDA that Biotin was used in 75 cosmetic formulations at concentrations of :::::0.6%.

Biotin occurs naturally as the D-isomer and has been synthe­sized from 4-benzamido-3-ketotetrahydrothiophene and methyl

TABLE3 Clinical use of Biotin

Subject

5-month-old infant with metabolic acidosis and ketosis

28 infants with Leiner's disease or other forms of dermatitis

30 infants with dermatitis seborrhoides

2-year-old with propionicacidemia and secondary ketotic hyperglycemia

Alopecic children 8 patients with seborrhea or other forms of dermatitis 211 patients with erythroderma and 192 with

seborrheic dermatitis 5 patients with onychodystrophy Normal subjects and patients with atherosclerosis

12 normal subjects and 140 patients with atherosclerosis and hypertensive disease

Women with diffuse alopecia 9 infants with seborrheic dermatitis and 2 infants

with Leiner's disease 4-month-old infant

11 hemodialysis patients

Dose

10 mglkg/day orally for 3 weeks

20 mg orally or 0.1 mg/day intramuscularly (IM) for 3-5 weeks

5 mg/day IM for 6-10 days, then orally until day 10 or 15

5 mg twice daily orally for 5 days

0.25 mg orally 2-3 times/day for 3 months 250 f,Lg/day for 4-8 weeks 1-4 tablets orally or 1-2 ampuls 1M­

containing 5 mg for 1-3 weeks 120 mg/day orally for 40 days 1, 3, or 5 mg every 24 h, internally, over 7

and 14 days 1, 3, or 5 mg/day for 7 or 14 days (route

not specified) 10 mg/day for 28 days (route not specified) Total dosage ranged from 3 mg over 2 weeks

to 10 mg injected over 4 days 40 mg injected over 2 months, 20 mg of

which were given in 4 days 50 mg intravenously for 2 months

Reference

Informatics, Inc. 1974

Informatics, Inc. 1974

Informatics, Inc. 1974

Informatics, Inc. 1974

Informatics, Inc. 1974 Informatics, Inc. 1974 Informatics, Inc. 1974

Informatics, Inc. 1974 Informatics, Inc. 1974

Informatics, Inc. 1974

Informatics, Inc. 1974 Informatics, Inc. 1974

Informatics, Inc. 1974

Koutsikos et al. 1996

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BIOTIN 11

y-formylbutyrate and from 3,4-diamino-2-carbomethoxythio­pene. Biotin had low nonspecific absorption in the UVA and UVB range and into the visible spectrum.

Upon oral and IP administration, Biotin is excreted primarily in the urine.

The oral LD50 values of Biotin for mice and rats were > 10 gl kg and > 1.45 mmol/kg, respectively. The IV LD50 for mice and the IP LD50 values for rats and cats were >4.1, >0.12, and >0.001 mmol/kg, respectively. Biotin was not toxic in oral short-term or subchronic toxicity studies.

Intradermal injection of 0.1 cc Biotin into guinea pigs did not produce skin irritation. Biotin, 0.1% and pH 7.3, produced slight and transient ocular irritation in rabbits.

In a number of reproductive studies using rats, the number of resorptions in animals dosed with Biotin by SC injection was increased as compared to controls, and fetal, uterine, and placental weights were decreased. Dosing with estrogen gener­ally prevented the resorptions. In a reproductive study in which mice were dosed with Biotin orally and by SC injection, signifi­cant differences were not observed between treated and control groups.

Biotin was not mutagenic in an Ames test or an RK bacterial test, but was mutagenic in a Tradescantia-Micronucleus test.

In a case study, Biotin produced an urticarial reaction, but other clinical studies produced no adverse reactions.

DISCUSSION The Expert Panel recognized that data on the irritation and

sensitization potential of Biotin were absent from the report, but the Panel noted that a large number of people are exposed to Biotin daily. The Expert Panel was of the opinion that if Biotin had a strong potential for irritation or sensitization, case reports would be available in the published literature. The lack of such case reports was an indicator to the Expert Panel that Biotin did not have a strong potential for skin irritation or sensitization. UV absorption data demonstrated that phototoxicity would not be a concern.

Several of the available studies that assessed reproductive and developmental toxicity suggested effects of Biotin and one genotoxicity study was positive, but other studies suggested no effects. Assuming that a product containing 0.6% Biotin was applied at a rate of 15 g/day, and approximately 10% of the Biotin was absorbed, it was postulated that the daily exposure to Biotin would be approximately twelve-fold the total potential dietary intake. Based on the low toxic potential of Biotin, how­ever, and on Biotin's rapid metabolism and excretion, such that accumulation would not be a problem, the Expert Panel was of the opinion that no toxic effects would be expected.

CONCLUSION On the basis of the animal and clinical data included in this

report, the Cosmetic Ingredient Review Expert Panel concludes that Biotin is safe as used in cosmetic formulations.

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2 Available for review: Director, Cosmetic Ingredient Review, 1101 17th Street, N.W., Suite 310, Washington, DC 20036, USA.

Distributed for comment only -- do not cite or quote

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Distributed for comment only -- do not cite or quote

BIOTIN 1 02B - Bubble BathsBIOTIN 3 03B - EyelinerBIOTIN 23 03D - Eye LotionBIOTIN 7 03F - MascaraBIOTIN 21 03G - Other Eye Makeup PreparationsBIOTIN 44 05A - Hair ConditionerBIOTIN 3 05E - Rinses (non-coloring)BIOTIN 53 05F - Shampoos (non-coloring)BIOTIN 33 05G - Tonics, Dressings, and Other Hair Grooming AidsBIOTIN 1 05H - Wave SetsBIOTIN 32 05I - Other Hair PreparationsBIOTIN 4 06A - Hair Dyes and Colors (all types requiring caution statements and patch tests)BIOTIN 1 06D - Hair Shampoos (coloring)BIOTIN 9 07C - FoundationsBIOTIN 1 07H - Makeup FixativesBIOTIN 3 07I - Other Makeup PreparationsBIOTIN 1 08A - Basecoats and UndercoatsBIOTIN 5 08G - Other Manicuring PreparationsBIOTIN 1 11E - Shaving CreamBIOTIN 2 11G - Other Shaving Preparation ProductsBIOTIN 20 12A - CleansingBIOTIN 56 12C - Face and Neck (exc shave)BIOTIN 50 12D - Body and Hand (exc shave)BIOTIN 88 12F - MoisturizingBIOTIN 14 12G - NightBIOTIN 11 12H - Paste Masks (mud packs)BIOTIN 4 12I - Skin FreshenersBIOTIN 13 12J - Other Skin Care PrepsBIOTIN 1 13B - Indoor Tanning PreparationsBIOTIN 1 13C - Other Suntan Preparations

506

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Concentration of Use by FDA Product Category – Biotin

Product Category Maximum Concentration of Use Eyeliners 0.0000002-0.1% Eye lotions 0.000004-0.004% Mascara 0.0001-0.01% Other eye makeup preparations 0.001-0.01% Hair conditioners 0.000001-0.1% Hair sprays Aerosol Pump spray

0.001-0.009% 0.001-0.1%

Shampoos (noncoloring) 0.001-0.01% Tonics, dressings and other hair grooming aids Not spray

0.001-0.1% 0.0000002-0.001%

Other hair preparations (noncoloring) 0.0003% Face powders 0.1% Foundations 0.00004-0.0001% Cuticle softeners 0.1% Nail polish and enamel 0.0001-0.1% Other manicuring preparations 0.0001-0.01% Bath soaps and detergents 0.000006-0.001% Shaving cream 0.00004% Skin cleansing (cold creams, cleansing lotions, liquids and pads) 0.00002-0.0002% Face and neck products Not spray

0.00006-0.1%

Body and hand products Not spray

0.000004-0.0001%

Moisturizing products Not spray

0.0015-0.002%

Paste masks and mud packs 0.001% Skin fresheners 0.1% Other skin care preparations 0.000003-0.00006%

Information collected 2015-2016 Table prepared February 16, 2016

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