Radish Root Ferment Extract

Safety Assessment of Radish Root – Derived Ingredients
as Used in Cosmetics
Status: Draft Report for Panel Review

Release Date: November 10, 2021
Panel Meeting Date: December 6-7, 2021
The Expert Panel for Cosmetic Ingredient Safety members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito,
M.D.; David E. Cohen, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Lisa A. Peterson, Ph.D.; Ronald C. Shank,
Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The Cosmetic Ingredient Review (CIR) Executive
Director is Bart Heldreth, Ph.D. This safety assessment was prepared by Preethi Raj, Senior Scientific Analyst/Writer, CIR.
© Cosmetic Ingredient Review

1620 L Street, NW, Suite 1200 ♢ Washington, DC 20036-4702 ♢ ph 202.331.0651 ♢ fax 202.331.0088 ♢
[email protected]
Distributed for Comment Only -- Do Not Cite or Quote
SAFETY ASSESSMENT FLOW CHART

INGREDIENT/FAMILY Radish Root-Derived Ingredients ___________________
MEETING ___December 2021 ___________________________________________________
Public Comment CIR Expert Panel Report Status
2021 Priority List

INGREDIENT PRIORITY LIST
Notice to Proceed
without an SLR
April 14, 2021
DRAFT REPORT
December 2021
Draft Report
60 day public comment period
Table
Table IDA TR
IDA Notice IDA

DRAFT TENTATIVE
REPORT
Draft TR
Table
Table
Issue TR
Tentative Report
Draft FR DRAFT FINAL REPORT
60 day Public comment period
Table
Table Different Conclusion
Issue
PUBLISH Final Report FR
Commitment & Credibility since 1976
Memorandum
To: Expert Panel for Cosmetic Ingredient Safety Members and Liaisons
From: Preethi S. Raj, M.Sc.
Senior Scientific Analyst, CIR
Date: November 10, 2021
Subject: Safety Assessment of Radish Root-Derived Ingredients as Used in Cosmetics
Enclosed is the Draft Report of the Safety Assessment of Radish Root-Derived Ingredients as Used in Cosmetics (identified
as report_RadishRoot_122021 in the pdf). This is the first time the Panel is seeing a safety assessment of these 7 cosmetic
ingredients. Due to a dearth of published data found upon search of these ingredients, a Scientific Literature Review Notice
to Proceed (SLR NTP) was announced on April 14, 2021. Information sought included chemistry, manufacturing,
composition, impurities, toxicity, and dermal irritation/sensitization data.
Concentration of use data and 2021 VCRP data were received from the Council and the FDA, respectively, in January 2021
(data1_RadishRoot_122021; VCRP_RadishRoot_122021). The following unpublished data were also received, and have
been incorporated in the report:
data2_RadishRoot_122021
• Anonymous. (2019) Repeated insult patch test (eyebrow gel containing 0.04% Leuconostoc/Radish Root Ferment
Filtrate)
data3_RadishRoot_122021 (all for Leuconostoc/Radish Root Ferment Filtrate)

• Active Micro Technologies. (2018) Bacterial reverse mutation test
• Active Micro Technologies. (2017) Dermal and ocular irritation tests
• Active Micro Technologies. (2017). Kow statement
• Active Micro Technologies. (2017). OECD TG 442C: In chemico skin sensitization
• Active Micro Technologies. (2017). OECD TG 442D: In vitro skin sensitization
• Active Micro Technologies. (2017). Phototoxicity assay analysis
• AMA Laboratories. (2008). HRIPT in 50 subjects (was cited to Active Micro Technologies on the memo)
data4_RadishRoot_122021
• Active Micro Technologies. (2021). Composition for Leuconostoc/Radish Root Ferment Filtrate
• Active Micro Technologies. (2020). Specifications for Leuconostoc/Radish Root Ferment Filtrate
Also included in this package, for your review, are a flow chart (flow_RadishRoot_122021), literature search strategy
(search_RadishRoot_122021), ingredient data profile (dataprofile_RadishRoot_122021), and ingredient history
(history_RadishRoot_122021).
After reviewing these documents, if the available data are deemed sufficient to make a determination of safety, the Panel
should issue a Tentative Report with a safe as used, safe with qualifications, or unsafe conclusion, and Discussion items
should be identified. If the available data are insufficient, the Panel should issue an Insufficient Data Announcement
(IDA), specifying the data needs therein.
__________________________________________________________________________________________
1620 L Street, NW Suite 1200, Washington, DC 20036
(Main) 202-331-0651 (Fax) 202-331-0088
(email) [email protected] (website) www.cir-safety.org
CIR History of:
Radish Root-Derived Ingredients
January 2021
-Frequency of use data obtained
-Concentration of use data submitted by Council
April 2021
-SLR Notice to Proceed was issued
Data received (for Leuconostoc/Radish Root Ferment Filtrate):
• April 29, 2021: HRIPT of eyebrow gel containing 0.04% Leuconostoc/Radish Root Ferment Filtrate
• May 6, 2021: Kow statement, bacterial reverse mutation assay, dermal and ocular irritation tests, In
Chemico skin sensitization, in vitro skin sensitization, phototoxicity, and HRIPT data for
Leuconostoc/Radish Root Ferment Filtrate
• May 7, 2021: Composition and Specifications information for Leuconostoc/Radish Root Ferment
Filtrate
•
December 2021
-A Draft Report is being presented to the Panel.

Radish Root -Derived Ingredients Profile* – December 6-7, 2021 – Writer, Preethi Raj
Repeated Dermal Dermal Ocular Clinical
Toxicokinetics Acute Tox DART Genotox Carci
Dose Tox Irritation Sensitization Irritation Studies
Method of Mfg
Retrospective/
Reported Use
Case Reports
Phototoxicity
log P/log Kow
Multicenter
Penetration
Impurities
Inhalation
Inhalation
In Vitro
In Vitro
In Vitro
In Vitro
Human
Human
Dermal
Dermal
Dermal
Dermal
Dermal
In Vivo
Animal
Animal
Animal
ADME
Oral
Oral
Oral
Oral
Lactobacillus/Radish Root Ferment
X
Filtrate
Lactobacillus/Radish Root Ferment
X X
Extract Filtrate
Leuconostoc/Radish Root Ferment
X X X X X X X X X X
Filtrate
Leuconostoc/Radish Root Ferment
X
Lysate Filtrate
Raphanus Sativus (Radish) Root Extract X X X
Raphanus Sativus (Radish) Root Juice X X X
Raphanus Sativus (Radish) Root Powder X
* “X” indicates that data were available in a category for the ingredient
1
[Radish Root-Derived Ingredients]
Ingredient CAS # PubMed FDA HPVIS NIOSH NTIS NTP FEMA EU ECHA ECETOC SIDS SCCS AICIS FAO WHO Web
Lactobacillus/Radish NR  NR NR NR NR NR NR NR NR NR NR NR NR NR NR 
Root Ferment Extract
Filtrate
Lactobacillus/Radish NR  NR NR NR NR NR NR * NR NR NR NR NR NR NR
Root Ferment Filtrate
Leuconostoc/Radish NR NR NR NR NR NR NR NR * NR NR NR NR NR NR NR
Root Ferment Filtrate
Leuconostoc/Radish 1686112-10-6 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Root Ferment Lysate
Filtrate
Raphanus Sativus 84775-94-0 * NR NR NR * NR NR NR * NR NR NR NR NR NR
(Radish) Root Extract
Raphanus Sativus NR * NR NR NR NR NR NR NR NR NR NR NR NR NR NR
(Radish) Root Juice
Raphanus Sativus NR  NR NR NR NR NR NR NR NR NR NR NR NR NR NR
(Radish) Root Powder
Botanical and/or Fragrance Websites (if applicable)

Ingredient CAS # Dr. Duke’s Taxonomy GRIN Sigma-Aldrich AHPA AGRICOLA IFRA RIFM
Lactobacillus/Radish Root NR NR NA NA NA NR NA NR NR
Ferment Extract Filtrate
Lactobacillus/Radish Root NR NR NA NA NA NR NA NR NR
Ferment Filtrate
Leuconostoc/Radish Root NR NR NA NA NA NR NA NR NR
Ferment Filtrate
Leuconostoc/Radish Root 1686112-10-6 NR NA NA NA NR NA NR NR
Ferment Lystate Filtrate
Raphanus Sativus (Radish) 84775-94-0   30857 * NR * NR NR
Root Extract
Raphanus Sativus (Radish) NR   30857 * NR * NR NR
Root Juice
Raphanus Sativus (Radish) NR   30857 * NR * NR NR
Root Powder
 - data is available
* - data is available, but not relevant
Search Strategy (PubMed)
Fermentation cosmetics – 2738 hits/
Lactobacillus/Radish Root Ferment Extract Filtrate -1 hit/1 useful
((Lactobacillus Ferment Extract Filtrate) OR (Radish Root Ferment Extract Filtrate)) OR (Lactobacillus/Radish Root Ferment Extract Filtrate) – 22 hits/1 useful
General Web Search (Google)

Fermented radish atopic dermatitis – 11,000,000/2
Fermented radish dermal safety – 3,460,000/2

Probiotic radish dermal safety – 193,000/4
Lactobacillus/Radish Root Ferment Extract Filtrate – 74,300 hits/0 useful

Lactobacillus/Radish Root Ferment Extract Filtrate cosmetics – 59,500 hits/4 useful
Lactobacillus/Radish Root Ferment Extract Filtrate chemical properties - 63,000 hits/ 2 useful
Lactobacillus/Radish Root Ferment Extract Filtrate method of manufacture – 396,00 hits/ 1 useful
Lactobacillus/Radish Root Ferment Extract Filtrate composition – 80 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate impurities – 25,300 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate dermal penetration – 262,000 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate penetration enhancement – 244,000 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate toxicokinetics – 10 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate acute toxicity – 22 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate short term toxicity – 34 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate subchronic toxicity - 18 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate chronic toxicity – 24 hits/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate developmental toxicity – 26 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate reproductive toxicity- 25 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate genotoxicity – 19 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate carcinogenicity – 24 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate mutagenicity – 19 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate dermal irritation – 259,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate dermal sensitization – 120,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate phototoxicity – 25 results/ 0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate ocular irritation – 250,000 results/0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate mucous membrane irritation – 32 results/0 useful
Lactobacillus/Radish Root Ferment Extract Filtrate clinical studies – 255,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate chemical properties – 9 results/ 0 useful

Lactobacillus/Radish Root Ferment Filtrate method of manufacture – 109 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate composition – 258,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate impurities – 25, 900 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate dermal penetration – 259,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate penetration enhancement – 246,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate toxicokinetics- 11 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate acute toxicity – 23 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate short term toxicity – 40 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate subchronic toxicity – 20 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate chronic toxicity – 28 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate developmental toxicity – 29 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate reproductive toxicity – 33 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate genotoxicity – 30 results/ 0 useful

Lactobacillus/Radish Root Ferment Filtrate carcinogenicity- 36 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate mutagenicity – 40 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate dermal irritation – 86 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate dermal sensitization – 122,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate phototoxicity – 23 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate ocular irritation – 130,000 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate mucous membrane irritation – 25 results/ 0 useful
Lactobacillus/Radish Root Ferment Filtrate clinical studies - 187,700 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate– 289, 000 results/ 0 useful

Leuconostoc/Radish Root Ferment Filtrate chemical properties – 12,100 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate method of manufacture – 40,000 results/0 useful
Leuconostoc/Radish Root Ferment Filtrate composition – 19,800 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate impurities – 74,500 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate dermal penetration – 64,900 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate penetration enhancement – 26,900 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate toxicokinetics – 10 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate acute toxicity – 23 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate short term toxicity – 234,000 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate subchronic toxicity – 11 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate chronic toxicity – 27 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate developmental toxicity – 30,700 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate reproductive toxicity – 44 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate genotoxicity – 37 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate carcinogenicity – 47 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate mutagenicity – 35 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate dermal irritation – 36,900 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate dermal sensitization – 7, 080 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate phototoxicity – 12 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate ocular irritation – 29, 200 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate mucous membrane irritation – 32 results/ 0 useful
Leuconostoc/Radish Root Ferment Filtrate clinical studies – 10,700 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate – 25, 900 results/ 0 useful

Leuconostoc/Radish Root Ferment Lysate Filtrate chemical properties – 26 results/0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate method of manufacture – 35 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate composition – 38 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate impurities – 28 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate dermal penetration – 9,320 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate penetration enhancement – 4,880 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate toxicokinetics – 8 results/ 0 useful

Leuconostoc/Radish Root Ferment Lysate Filtrate acute toxicity – 24 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate short term toxicity – 15 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate subchronic toxicity – 11 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate chronic toxicity – 20 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate developmental toxicity – 17 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate reproductive toxicity – 15 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate genotoxicity – 11 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate carcinogenicity – 11 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate mutagenicity – 12 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate dermal irritation – 34 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate dermal sensitization – 29 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate phototoxicity – 11 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate ocular irritation – 24 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate mucous membrane irritation – 9 results/ 0 useful
Leuconostoc/Radish Root Ferment Lysate Filtrate clinical studies – 27 results/ 0 useful
Raphanus Sativus (Radish) Extract – 200,000 results/ 5 useful

Raphanus Sativus (Radish) Extract chemical properties – 132, 000 results/ 6 useful
Raphanus Sativus (Radish) Extract method of manufacture – 117 results/ 0 useful
Raphanus Sativus (Radish) Extract composition – 122, 000 results/ 4 useful
Raphanus Sativus (Radish) Extract impurities – 53,200 results/ 2 useful
Raphanus Sativus (Radish) Extract dermal penetration – 84, 700 results/ 0 useful
Raphanus Sativus (Radish) Extract penetration enhancement – 57,100 results/ 0 useful
Raphanus Sativus (Radish) Extract toxicokinetics – 12 results/ 0 useful
Raphanus Sativus (Radish) Extract acute toxicity – 46 results/ 0 useful
Raphanus Sativus (Radish) Extract short term toxicity – 102,000 results/ 0 useful
Raphanus Sativus (Radish) Extract subchronic toxicity – 39 results/ 0 useful
Raphanus Sativus (Radish) Extract chronic toxicity – 41,700 results/ 0 useful
Raphanus Sativus (Radish) Extract developmental toxicity – 96,200 results/ 0 useful
Raphanus Sativus (Radish) Extract reproductive toxicity – 102,000 results/ 0 useful
Raphanus Sativus (Radish) Extract genotoxicity – 51 results/ 0 useful
Raphanus Sativus (Radish) Extract carcinogenicity – 59 results/ 0 useful
Raphanus Sativus (Radish) Extract mutagenicity – 60 results/ 0 useful
Raphanus Sativus (Radish) Extract dermal irritation – 18,800 results/ 2 useful
Raphanus Sativus (Radish) Extract dermal sensitization – 46 results/ 0 useful
Raphanus Sativus (Radish) Extract phototoxicity – 23 results/ 0 useful
Raphanus Sativus (Radish) Extract ocular irritation – 24 results/ 0 useful
Raphanus Sativus (Radish) Extract mucous membrane irritation – 35 results/ 0 useful
Raphanus Satibus (Radish) Extract clinical studies – 78 results/ 1 useful
Raphanus Sativus (Radish) Root Juice – 67,100 results/ 4 useful

Raphanus Sativus (Radish) Root Juice chemical properties – 50,2000 results/8 useful
Raphanus Sativus (Radish) Root Juice method of manufacture – 43,900 results/ 6 useful
Raphanus Sativus (Radish) Root Juice composition – 78,400 results/ 3 useful
Raphanus Sativus (Radish) Root Juice impurities – 39 results/ 0 useful
Raphanus Sativus (Radish) Root Juice dermal penetration – 42,500 results/ 0 useful
Raphanus Sativus (Radish) Root Juice penetration enhancement – 24, 700 results/ 0 useful
Raphanus Sativus (Radish) Root Juice toxicokinetics – 21 results/ 0 useful
Raphanus Sativus (Radish) Root Juice acute toxicity – 24,100 results/ 0 useful
Raphanus Sativus (Radish) Root Juice short term toxicity – 69 results/ 0 useful
Raphanus Sativus (Radish) Root Juice subchronic toxicity – 12,200 results/ 0 useful
Raphanus Sativus (Radish) Root Juice chronic toxicity – 22,100 results/ 0 useful
Raphanus Sativus (Radish) Root Juice developmental toxicity – 29,100 results/ 0 useful
Raphanus Sativus (Radish) Root Juice reproductive toxicity – 47,100 results/ 0 useful
Raphanus Sativus (Radish) Root Juice genotoxicity – 39 results/ 0 useful
Raphanus Sativus (Radish) Root Juice carcinogenicity – 45 results/ 0 useful
Raphanus Sativus (Radish) Root Juice mutagenicity – 41 results/ 1 useful
Raphanus Sativus (Radish) Root Juice dermal irritation – 7,540 results/ 0 useful
Raphanus Sativus (Radish) Root Juice dermal sensitization – 13,300 results/ 0 useful
Raphanus Sativus (Radish) Root Juice phototoxicity – 2,150 results/ 0 useful
Raphanus Sativus (Radish) Root Juice ocular irritation – 60 results/ 0 useful
Raphanus Sativus (Radish) Root Juice mucous membrane irritation – 50 results/ 0 useful
Raphanus Sativus (Radish) Root Juice clinical studies – 30,100 results/ 6 useful
NIOSH Raphanus Sativus (Radish) Root Juice – 8 results/5 useful
Raphanus Sativus (Radish) Root Powder – 81,800 results/ 4 useful

Raphanus Sativus (Radish) Root Powder chemical properties – 98,900 results/ 0 useful
Raphanus Sativus (Radish) Root Powder composition – 98,700 results// 5 useful
Raphanus Sativus (Radish) Root Powder impurities – 40,300 results/ 0 useful
Raphanus Sativus (Radish) Root Powder method of manufacture – 194,000 results/ 1 useful
Raphanus Sativus (Radish) Root Powder dermal penetration – 98 results/ 0 useful
Raphanus Sativus (Radish) Root Powder penetration enhancement – 69,200 results/ 0 useful
Raphanus Sativus (Radish) Root Powder toxicokinetics – 10 results/ 0 useful
Raphanus Sativus (Radish) Root Powder acute toxicity – 32 results/ 0 useful
Raphanus Sativus (Radish) Root Powder short term toxicity – 98,000 results/ 0 useful
Raphanus Sativus (Radish) Root Powder subchronic toxicity – 36 results/ 0 useful
Raphanus Sativus (Radish) Root Powder chronic toxicity - 33 results/ 0 useful
Raphanus Sativus (Radish) Root Powder developmental toxicity – 57 results/ 0 useful
Raphanus Sativus (Radish) Root Powder reproductive toxicity – 85 results/ 0 useful
Raphanus Sativus (Radish) Root Powder genotoxicity – 33 results/ 1 useful
Raphanus Sativus (Radish) Root Powder carcinogenicity – 42 results/ 0 useful

Raphanus Sativus (Radish) Root Powder mutagenicity – 48 results/ 1 useful
Raphanus Sativus (Radish) Root Powder dermal irritation – 55 results/ 0 useful
Raphanus Sativus (Radish) Root Powder dermal sensitization – 24 results/ 0 useful
Raphanus Sativus (Radish) Root Powder phototoxicity – 33 results/ 0 useful
Raphanus Sativus (Radish) Root Powder ocular irritation – 41 results/ 0 useful
Raphanus Sativus (Radish) Root Powder mucous membrane irritation – 22 results/ 0 useful
Raphanus Sativus (Radish) Root Powder clinical studies – 50 results/ 0 useful
LINKS
Search Engines
 Pubmed - http://www.ncbi.nlm.nih.gov/pubmed
 Connected Papers - https://www.connectedpapers.com/
appropriate qualifiers are used as necessary

search results are reviewed to identify relevant documents
Pertinent Websites
 wINCI - http://webdictionary.personalcarecouncil.org
 FDA databases http://www.ecfr.gov/cgi-bin/ECFR?page=browse
 FDA search databases: http://www.fda.gov/ForIndustry/FDABasicsforIndustry/ucm234631.htm;,
 Substances Added to Food (formerly, EAFUS): https://www.fda.gov/food/food-additives-petitions/substances-added-food-formerly-eafus
 GRAS listing: http://www.fda.gov/food/ingredientspackaginglabeling/gras/default.htm
 SCOGS database: http://www.fda.gov/food/ingredientspackaginglabeling/gras/scogs/ucm2006852.htm
 Indirect Food Additives: http://www.accessdata.fda.gov/scripts/fdcc/?set=IndirectAdditives
 Drug Approvals and Database: http://www.fda.gov/Drugs/InformationOnDrugs/default.htm
 FDA Orange Book: https://www.fda.gov/Drugs/InformationOnDrugs/ucm129662.htm
 (inactive ingredients approved for drugs: http://www.accessdata.fda.gov/scripts/cder/iig/
 HPVIS (EPA High-Production Volume Info Systems) - https://iaspub.epa.gov/oppthpv/public_search.html_page
 NIOSH (National Institute for Occupational Safety and Health) - http://www.cdc.gov/niosh/
 NTIS (National Technical Information Service) - http://www.ntis.gov/
o technical reports search page: https://ntrl.ntis.gov/NTRL/
 NTP (National Toxicology Program ) - http://ntp.niehs.nih.gov/
 Office of Dietary Supplements https://ods.od.nih.gov/
 FEMA (Flavor & Extract Manufacturers Association) GRAS: https://www.femaflavor.org/fema-gras
 EU CosIng database: http://ec.europa.eu/growth/tools-databases/cosing/
 ECHA (European Chemicals Agency – REACH dossiers) – http://echa.europa.eu/information-on-
chemicals;jsessionid=A978100B4E4CC39C78C93A851EB3E3C7.live1
 ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) - http://www.ecetoc.org
 European Medicines Agency (EMA) - http://www.ema.europa.eu/ema/
 OECD SIDS (Organisation for Economic Co-operation and Development Screening Info Data Sets)- http://webnet.oecd.org/hpv/ui/Search.aspx
 SCCS (Scientific Committee for Consumer Safety) opinions: http://ec.europa.eu/health/scientific_committees/consumer_safety/opinions/index_en.htm
 AICIS (Australian Industrial Chemicals Introduction Scheme)- https://www.industrialchemicals.gov.au/
 International Programme on Chemical Safety http://www.inchem.org/

 FAO (Food and Agriculture Organization of the United Nations) - http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfa-additives/en/
 WHO (World Health Organization) technical reports - http://www.who.int/biologicals/technical_report_series/en/
 www.google.com - a general Google search should be performed for additional background information, to identify references that are available, and for other general
information
Botanical Websites, if applicable

 Dr. Duke’s - https://phytochem.nal.usda.gov/phytochem/search
 Taxonomy database - http://www.ncbi.nlm.nih.gov/taxonomy
 GRIN (U.S. National Plant Germplasm System) - https://npgsweb.ars-grin.gov/gringlobal/taxon/taxonomysimple.aspx
 Sigma Aldrich plant profiler- http://www.sigmaaldrich.com/life-science/nutrition-research/learning-center/plant-profiler.html
 American Herbal Products Association Botanical Safety Handbook (database) - http://www.ahpa.org/Resources/BotanicalSafetyHandbook.aspx
 National Agricultural Library NAL Catalog (AGRICOLA) https://agricola.nal.usda.gov/
 The Seasoning and Spice Association List of Culinary Herbs and Spices
 http://www.seasoningandspice.org.uk/ssa/background_culinary-herbs-spices.aspx
Fragrance Websites, if applicable

 IFRA (International Fragrance Association) – https://ifrafragrance.org/
 Research Institute for Fragrance Materials (RIFM) - https://www.rifm.org/#gsc.tab=0
Safety Assessment of Radish Root – Derived Ingredients

as Used in Cosmetics
Status: Draft Report for Panel Review

Release Date: November 10, 2021
Panel Meeting Date: December 6-7, 2021
The Expert Panel for Cosmetic Ingredient Safety members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito,
M.D.; David E. Cohen, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Lisa A. Peterson, Ph.D.; Ronald C. Shank,
Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The Cosmetic Ingredient Review (CIR) Executive
Director is Bart Heldreth, Ph.D. This safety assessment was prepared by Preethi Raj, Senior Scientific Analyst/Writer, CIR.
© Cosmetic Ingredient Review

1620 L Street, NW, Suite 1200 ♢ Washington, DC 20036-4702 ♢ ph 202.331.0651 ♢ fax 202.331.0088 ♢
[email protected]
ABBREVIATIONS
ACP acid phosphatase
AD atopic dermatitis
ALT alanine transaminase
ARE-Nrf2 antioxidants response elements – transcription factor Nrf2
AST aspartate aminotransferase
CAS Chemical Abstracts Service
CIR Cosmetic Ingredient Review
Council Personal Care Products Council
Dictionary International Cosmetic Ingredient Dictionary and Handbook
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DPPH 2,2-diphenyl-1-picrylhydrazyl
DPRA direct reactivity peptide assay
EC European Commission
ECHA European Chemicals Agency
FDA Food and Drug Administration
GRAS generally recognized as safe
HaCaT human keratinocyte cell line
HRIPT human repeated insult patch test
IC50 half-maximal inhibitory concentration
IgE immunoglobulin E
LD lethal dose
LDH lactate dehydrogenase
MIC minimum inhibitory concentration
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide
N/A not applicable
NR not reported/none reported
OD odds ratio
OECD Organisation for Economic Co-operation and Development
Panel Expert Panel for Cosmetic Ingredient Safety
PBS phosphate buffer solution
TG test guideline
US United States
VCRP Voluntary Cosmetic Registration Program
INTRODUCTION
This assessment reviews the safety of the following 7 radish root-derived ingredients as used in cosmetic formulations:
Lactobacillus/Radish Root Ferment Extract Filtrate Raphanus Sativus (Radish) Root Extract
Lactobacillus/Radish Root Ferment Filtrate Raphanus Sativus (Radish) Root Juice
Leuconostoc/Radish Root Ferment Filtrate Raphanus Sativus (Radish) Root Powder
Leuconostoc/Radish Root Ferment Lysate Filtrate
According to the web-based International Cosmetic Ingredient Dictionary and Handbook (wINCI; Dictionary), these
ingredients are mostly reported to function in cosmetics as hair and skin conditioning agents (Table 1).1 These ingredients
are also reported to function as antimicrobial agents, a preservative, an anti-dandruff agent, an antifungal agent, and an
antioxidant. It should be noted that most of these other functions are not considered cosmetic functions in the United States
(US), and therefore, use as such does not fall under the purview of the Expert Panel for Cosmetic Ingredient Safety (Panel).
Botanicals, such as radish root-derived ingredients, may contain hundreds of constituents. Thus, in this assessment, the
Panel will assess the safety of each of the Raphanus sativus-derived ingredients as a whole, complex mixture; toxicity from
single components may not predict the potential toxicity of botanical ingredients. Some of the ingredients reviewed in this
safety assessment may be consumed as food, and daily exposure from food use would result in much larger systemic
exposures than those from use in cosmetic products. Therefore, the primary focus in this assessment of these ingredients is to
evaluate the potential for effects from topical exposures.
This safety assessment includes relevant published and unpublished data that are available for each endpoint that is
evaluated. Published data are identified by conducting an exhaustive search of the world’s literature. A listing of the search
engines and websites that are used and the sources that are typically explored, as well as the endpoints that the Panel typically
evaluates, is provided on the Cosmetic Ingredient Review (CIR) website (https://www.cir-
safety.org/supplementaldoc/preliminary-search-engines-and-websites; https://www.cir-safety.org/supplementaldoc/cir-report-
format-outline). Unpublished data are provided by the cosmetics industry, as well as by other interested parties.
The cosmetic ingredient names, according to the Dictionary, are written as listed above, without italics. In many of the
published studies, it is not known how the substance being tested compares to the ingredient as used in cosmetics. Therefore,
if it is not known whether the ingredients being discussed are cosmetic ingredients, the test substances will be identified by
the standard taxonomic practice of using italics to identify genus and species (i.e., “ Lactobacillus/radish root…”,
“Leuconostoc/radish root…”, or “Raphanus sativus (radish)….” However, if it is known that the substance is a cosmetic
ingredient, the terminology the International Nomenclature Committee (INC) terminology will be used (e.g., Raphanus
Sativus (Radish) Root Extract).
CHEMISTRY
Definition and Plant Identification
The ingredients in this report are related as derivatives from the same species, Raphanus sativus. Additionally, only
ingredients made from the root portion of the Raphanus sativus plant are being reviewed. The definitions of these radish
root-derived ingredients are presented in Table 1.1 Leuconostoc/Radish Root Ferment Lysate Filtrate and Raphanus Sativus
(Radish) Root Extract have the CAS Nos. 1686112-10-6 and 84775-94-0, respectively.
Raphanus sativus is a tap root from the Brassicaceae family, which has been historically cultivated in Asia and Europe.2
It grows in temperate climates at altitudes between 190 and 1240 m, is 30 - 90 cm high, and has thick edible roots which have
a pungent taste and are of various sizes, forms, and colors.3 Generically, the root is defined as the organ of a plant that
absorbs and transports water and nutrients, lacks leaves and nodes, and is usually underground.1
Four of these ingredients are filtrates of Raphanus sativus fermented with either the Lactobacillus or Leuconostoc
microorganism. Both strains are gram-positive and anaerobic, occurring as non-spore forming rods and cocci, and are
considered lactic acid bacteria because they consume carbohydrates to produce lactic acid.4 A lysate is obtained by breaking
down cell outer membranes via chemical or physical processes.5 The filtrate ingredients in this report are made by removing
the bacterial cells (alive or dead), potentially along with other molecules (like peptides), from the fermented products.5
Chemical Properties
A supplier has indicated that 1 g of Leuconostoc/Radish Root Ferment Filtrate is specified to contain 48 – 52% solids
(when observed for 1 h at 105° C).6 The log Kow of Leuconostoc/Radish Root Ferment Filtrate is -1.92.7 Additional physical
and chemical properties are presented in Table 2.
Method of Manufacture
The methods below are general to the processing of the radish root-derived ingredients, and it is unknown if they apply
to cosmetic ingredient manufacturing. In some cases, the definition of the ingredients, as given in the Dictionary, provides
insight as to the method of manufacture.
Lactobacillus/Radish Root Ferment Extract Filtrate
Lactobacillus/Radish Root Ferment Extract Filtrate is a filtrate of the extract of the product obtained by the fermentation
of the roots of Raphanus sativus (radish) by the microorganism, Lactobacillus.1
Lactobacillus/Radish Root Ferment Filtrate
Lactobacillus/Radish Root Ferment Filtrate is a filtrate of the product obtained by the fermentation of the roots of
Raphanus sativus (radish) by the microorganism, Lactobacillus.1
Leuconostoc/Radish Root Ferment Filtrate is a filtrate of the product obtained by the fermentation of Raphanus sativus
roots by the microorganism, Leuconostoc.1
Leuconostoc/Radish Root Ferment Lysate Filtrate is a filtrate of a lysate of the product obtained by the fermentation of
the roots of Raphanus sativus (radish) by the microorganism, Leuconostoc.1
Raphanus Sativus (Radish) Root Extract
Radish roots, sized 30 cm each, were made into powder by washing, cutting into ∼ 3 mm pieces, being dried at 60 °C
for 21 h, and then being blended and sieved with a 60 mesh sifter.8 The resulting powder was macerated at a 1:10 ratio at
∼24 °C using 3 different solvents (hexane, ethyl acetate, and ethanol) for 8, 16, and 24 h. The resulting suspensions were
filtered and evaporated at 45 °C.
One gram of powdered black Raphanus sativus roots was used to make an ethanolic radish root extract.9 Aqueous
ethanol, 50 ml, 50% (v/v) was used to extract the powder on a magnetic stirrer for 120 min at room temperature, and then
centrifuged at 5000 rpm for 10 min at 4 °C.
Raphanus Sativus (Radish) Root Juice
Fresh Raphanus sativus roots were washed well and processed in an electric blender to obtain 2 l of fresh root juice.10
The Raphanus sativus root juice was then filtered and concentrated in a rotary evaporator at 35 ± 5 °C under reduced
pressure. The resulting material was freeze dried to obtain a semisolid mass of 40 g, 11.3% w/w, which was then dissolved
in distilled water.
Raphanus Sativus (Radish) Root Powder
White radish roots were washed with water, sliced, and dried at 50 °C.11 The dried slices of white radish were ground to
a powder and sieved through a 40 mesh sifter. The resulting product was stored in a sealed bag and frozen at -20 °C until
further extraction. In another study, peeled and unpeeled black radish roots were sliced and freeze-dried before being ground
to a fine powder and sifted through a 0.5 mm mesh sieve; the powdered samples were stored in air-tight containers at 4 °C.9
Composition and Impurities
A supplier has reported that a sample of Leuconostoc/Radish Root Ferment Filtrate, with a pH 4.0 – 6.0, comprises
48.80% water, 30.60% protein, 20.10% phenolics (tested as salicylic acid), and 0.50% polysaccharide content.12
Specifications for this ingredient provide the following parameters: < 20 ppm heavy metals, < 10 ppm lead, < 2 ppm arsenic,
and < 1 ppm cadmium.6 Additionally, the ingredient was specified to be positive to ninhydrin, and potentially contain 18-
22% phenolics (tested as salicylic acid), and 0.10 - 0.50% bacteriocins (quantified via high-performance liquid
chromatography).
In one study, a 16-h, crude ethyl acetate Raphanus sativus root extract contained the highest total phenolic and
flavonoid content at 37.37 mg gallic acid equivalents (GAE)/g, and 5.74 mg quercetin equivalents (QE)/g, respectively.8 A
compositional analysis of fresh radish root extracts yielded a flavonoid content of 267.47 ± 6.38 mg quercetin/100 g, total
phenolic content of 371.59 mg/100 g, and 380 ± 0.87 g/100g potassium (highest mineral content).13 Silica gel
chromatography of a dichloromethane extract of Raphanus sativus roots yielded the following constituents: 3-(E)-
(methylthio)methylene-2-pyrrolidinethione, a mixture of 4-methylthio-3-butenyl isothiocyanate and 4-(methylthio)butyl
isothiocyanate, β-sitosterol, β-sitosteryl-3β-glucopyranoside-6'-O-palmitate, monoacylglycerols, and a mixture of α-linolenic
acid and linoleic acid.14 A methanolic extract of Daikon (vegetable; a Raphanus sativus var.) was the most constituent-rich,
compared to extracts made with water, petrolatum, ethanol, and chloroform; phytochemical screening showed the presence of
alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, and glycosides.15
USE
Cosmetic
The safety of the cosmetic ingredients addressed in this assessment is evaluated based on data received from the US
Food and Drug Administration (FDA) and the cosmetics industry on the expected use of these ingredients in cosmetics. Use
frequencies of individual ingredients in cosmetics are collected from manufacturers and reported by cosmetic product
category in the FDA Voluntary Cosmetic Registration Program (VCRP) database. Use concentration data are submitted by
the cosmetic industry in response to a survey, conducted by the Personal Care Products Council (Council), of maximum
reported use concentrations by product category.
According to 2021 VCRP survey data, Leuconostoc/Radish Root Ferment Filtrate is reported to be used in 255
formulations, 104 of which are leave-on moisturizing products; Lactobacillus/Radish Root Ferment Filtrate and Raphanus
Sativus (Radish) Root Extract are reported to have 2 uses each, in moisturizing and face and neck products, respectively
(Table 3).16 The results of the concentration of use survey conducted by the Council indicate that Raphanus Sativus (Radish)
Root Extract has the highest reported maximum concentration of use in leave-on products, at up to 6% in lipstick;
Leuconostoc/Radish Root Ferment Filtrate is used at up to 1.1% in skin cleansing products.17 The highest concentration of
use reported for products resulting in leave-on dermal exposure is 0.03% Leuconostoc/Radish Root Ferment Filtrate in face
and neck spray formulations. Use concentration data were not reported for Lactobacillus/Radish Root Ferment Filtrate, but
uses were reported in the VCRP. Lactobacillus/Radish Root Ferment Extract Filtrate, Leuconostoc/Radish Root Ferment
Lysate Filtrate, Raphanus Sativus (Radish) Root Juice, and Raphanus Sativus (Radish) Root Powder are not reported to be in
use in the VCRP or industry survey (Table 4).
Raphanus sativus- derived ingredients have been reported to be used in products that may lead to incidental ingestion
and exposure to mucous membranes; for example, Raphanus Sativus (Radish) Root Extract is reported to be used in a
lipstick at up to 6%,17 and Leuconostoc/Radish Root Ferment Filtrate is reported to be used at up to 0.01% in other eye
makeup preparations.17 Additionally, Leuconostoc/Radish Root Ferment Filtrate is reported to be used in products that could
be potentially inhaled, e.g., Leuconostoc/Radish Root Ferment Filtrate is used in spray face and neck products at up to
0.03%.17 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.18,19 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.20,21
All of the radish root-derived ingredients named in the report are not restricted from use in any way under the rules
governing cosmetic products in the European Union.22
Non-Cosmetic
According to the US FDA, commercially-produced products of carbohydrates, such as glucose, sucrose, or lactose, which
undergo lactic acid fermentation, are generally recognized as safe (GRAS) for their intended use in foods [21CFR § 184.1016].
Leuconostoc is an approved bacterial strain used to produce a butter starter distillate [21CFR § 184.1848].
Furthermore, Raphanus sativus roots are consumed as cruciferous vegetables worldwide, both raw and cooked, in pickles,
salads, and curries.23 Of note, Raphanus sativus fermented with Lactobacillus strains is consumed as a non-salted dish called
Sinki in South Asia.24 The Korean dish, kimchi, comprises variations of a mixed vegetable brine fermentation (achieved with
lactic acid bacteria, such as Lactobacillus or Leuconostoc), and often includes radish roots.25 Generally, Lactobacillus and
Leuconostoc strains are used in the lactic acid fermentation of dairy, sauerkraut, and various food products.26,27
TOXICOKINETIC STUDIES
No relevant toxicokinetics studies on radish root-derived ingredients were found in the published literature, and
unpublished data were not submitted. In general, toxicokinetics data are not expected to be found on botanical ingredients
because each botanical ingredient is a complex mixture of constituents.
TOXICOLOGICAL STUDIES
Subchronic Toxicity Studies
Oral
Groups of albino rats were dosed with 0, 150, 250, 350, 450, or 550 mg/kg bw of methanolic Daikon (vegetable; a
Raphanus sativus var.) extract, in the diet, for 90 d.15 Body weight, as well as various hematological parameters and
enzymes, including red blood cell count, hemoglobin, white blood cell count, aspartate aminotransferase (AST), alanine
transaminase (ALT), acid phosphatase (ACP), urea, uric acid, and protein were measured and compared at 30 and 90 d of
treatment. Upon sacrifice, heart, kidney, liver, spleen, and brain weights were also measured, and those of treated animals
were compared to controls. No statistically significant differences were observed between the mean body weights, organ
weights, and measured hematological parameters in treated animals, compared to controls, throughout the experiment.
DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES

Developmental and reproductive toxicity studies were not found in the published literature, and unpublished data were
not submitted.
GENOTOXICITY STUDIES
The genotoxicity potential of Leuconostoc/Radish Root Ferment Filtrate was evaluated in a bacterial reverse mutation
assay (Ames test) at concentrations of 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate, in distilled water, using the
following strains: Salmonella typhimurium TA98, TA100, TA1535, TA 1537, and Escherichia coli WP2 uvrA.28 Distilled
water served as the negative control and appropriate positive controls were used. The test substance did not induce a
mutagenic effect in the presence or absence of metabolic activation.
In a Comet assay, the genotoxic potential of radish juice made from white, red, and large red Raphanus sativus tubers,
as well as dichloromethane extracts of hydrolyzed Raphanus sativus white and cherry belle, red tubers, was tested in breast
adenocarcinoma (MCF-7), chronic myelogenous leukemia (K562), and colorectal cancer (HT-29) cell lines.29 Each cell line
was incubated with 500 µl of the root juice and 50 µg/ml of the dichloromethane juice extract; porcine aortic endothelial
(PAE) cell lines were used as the negative control and immortalized cell lines exposed to 0.01% hydrogen peroxide for 20
min were used as positive controls. Tail length, percent deoxyribonucleic acid (DNA), and tail moment measurements were
used to evaluate the extent of DNA damage. Juices from all 3 tubers exhibited significantly lower DNA damage in the
porcine aortic endothelial cells, compared to positive controls. The breast adenocarcinoma cell line, MCF-7, showed the
greatest amount of genetic fragmentation among all cancer cells, and the white tuber root juice was the most genotoxic
towards aberrant cell lines.
CARCINOGENICITY STUDIES
Carcinogenicity studies were not found in the published literature, and unpublished data were not submitted.
OTHER RELEVANT STUDIES

Radical Scavenging and Skin Lightening Potential
Raphanus Sativus (Radish) Root Extract and Raphanus Sativus (Radish) Root Juice
A freeze-dried juice and methanolic extract of white Raphanus sativus roots were evaluated for tyrosinase inhibition,
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, cytotoxicity, and L-ascorbic acid content.30 The ability of
Raphanus sativus root extract and root juice to scavenge DPPH, superoxide anion, and singlet oxygen was measured in
triplicates, which were used to calculate average half-maximal inhibitory concentration (IC50) values, compared to L-ascorbic
acid and Trolox, a water soluble analog of Vitamin E. Various concentrations of the Raphanus sativus root extract and root
juice in 20% v/v propylene glycol (in water) were tested, in tandem with L-ascorbic acid and licorice extract as reference
tyrosinase inhibitors, using the DOPAchrome method. Lactate dehydrogenase (LDH) activity in fibroblasts treated with the
root extract and root juice, compared to L-ascorbic acid and sinapic acid, was used to measure cytotoxic activity. Five
replicates of the root extract and root juice were titrated with 0.1 N iodine to determine the L-ascorbic acid or vitamin C
content. The freeze-dried juice showed higher potential for tyrosinase inhibition compared to the methanolic extract (IC50 =
3.09 mg/ml vs. IC50 = 9.62 mg/ml). The radical scavenging activity of the freeze-dried juice on DPPH radical, superoxide
anion radical, and singlet oxygen were also greater compared to the methanolic extract (IC50 = 0.64, 4.20, 1.42 mg/ml vs. IC50
= 1.25, 6.28, 2.40 mg/ml). Although a dose-dependent release of LDH was observed for both the root extract and root juice,
the observed cytotoxicity was relatively lower than in the reference antioxidants. The authors surmised that the higher L-
ascorbic content of 1 mg of freeze-dried Raphanus sativus root juice compared to the root extract (24.11 µg vs. 8.28 µg), as
well as higher phenolic content, may be responsible for greater anti-tyrosinase and radical scavenging activity, possibly
lending to skin lightening.
Raphanus sativus radish root extracts were eluted using 3 solvents with varying polarities (hexane (non-polar), ethyl
acetate (semi-polar), and ethanol (polar)) for 8, 16, and 24 h each, to determine which extract would have the highest
phenolic or flavonoid content.8 The radish root extract extracted with ethyl acetate for 16 h was found to have the highest
flavonoid content, and was used for further testing. The ethyl acetate radish root extract was tested for phenolic and
flavonoid content stability based on changes in pH (4, 5, 6, and 7) and heating temperature (70, 80, 90 °C). In conjunction,
the IC50 value of the ethyl acetate root extract was measured in a DPPH assay. Overall, decreases in total phenolic and
flavonoid content, as well as antioxidant activity, were observed when the radish root extract was exposed to increasing heat
and pH. Statistically significant interactions between change in pH and heating temperature with antioxidant activity were
observed. The radish root extract with a pH of 4 at a temperature of 70° C had an IC50 value (1071.93±45.71 mg/l ) closest to
that of the control extract (770.78±99.91 mg/l) which was not exposed to pH or temperature changes).
Antimicrobial Activity
According to specifications provided by a supplier, a sample of Leuconostoc/Radish Root Ferment Filtrate is expected
to have a minimum inhibitory concentration (MIC) of 1- 4% against Pseudomonas aeruginosa , 0.50 – 4% against
Escherichia coli, and 0.25 – 2% against Aspergillus brasiliensis, Candida albicans, and Staphylococcus aeruginosa.6
The antimicrobial potential of Raphanus sativus root juice was compared to that of ampicillin in strains of
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pnuemoniae, and Enterococcus faecalis.10
Upon incubation with 0.078 - 2.5 mg/ml of the root juice for 24 h, the highest MIC values were against P. aeruginosa at
0.625 ± 0.4 mg/ml and S.aureus at 0.312 ± 0.2 mg/ml (significantly greater than the corresponding ampicillin MIC values of
0.156 ± 0.8 mg/ml and 0.156 ± 0.07 mg/ml) and the Raphanus sativus root juice MIC values against E.coli and E. faecalis
were equivalent to ampicillin MIC values.
DERMAL IRRITATION AND SENSITIZATION STUDIES

Irritation
In Vitro
The dermal irritancy of Leuconostoc/Radish Root Ferment Filtrate was evaluated in a reconstructed three-dimensional
human epidermis model (EpiDerm).31 A single application of 30 µl of the test article was applied to the epidermis model
(3 tissue samples). Sterile phosphate-buffered saline (PBS) was used as the negative control and chloropromazine, ranging
from concentrations of 0.001-0.1%, were used as positive controls. The tissues were washed with sterile PBS 1 h after the
application, post-incubated under normal medium and culture conditions for 2 h, and then measured for cell viability via a 3-
(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay. Under the conditions of this study, the test article
was considered non-irritating.
Sensitization
In Chemico/In Vitro
The sensitization potential of Leuconostoc/Radish Root Ferment Filtrate (in acetonitrile) was evaluated in a direct
peptide reactivity assay (DPRA), in accordance with Organisation for Economic Cooperation and Development (OECD) test
guideline (TG) 442C.32 This assay is designed to mimic the covalent binding of electrophilic chemicals to nucleophilic
centers in skin proteins by quantifying the reactivity of test chemicals towards the model synthetic peptides containing
cysteine and lysine. The mean percent depletion of cysteine and lysine was 2.89%, which was interpreted as minimal
reactivity in the assay, with a prediction of no sensitization.
Leuconostoc/Radish Root Ferment Filtrate was also evaluated for sensitization potential in an antioxidants response
elements - transcription factor Nrf2 (ARE-Nrf2) luciferase assay utilizing the KeratinoSens™ cell line, in accordance with
OECD TG 442D.33 In this assay, transfection with the luciferase gene allows for measurement of the activation of the
Keap1-Nrf2-ARE complex, a proxy for sensitization. Fifty µl, each, of 12 concentrations of the test article prepared in
DMSO (ranging from 0.98 – 2000 µM) were added to human keratinocyte (HaCaT) cell lines seeded for 24 h, as per the
KeratinoSens method, and were incubated for 48 h. No significant increases in luciferase expression were observed; the
test article was predicted to be a non-sensitizer.
Human
In a human repeated insult patch test (HRIPT), Leuconostoc/Radish Root Ferment Filtrate, as a 10% dilution in water,
was tested in 50 subjects.34 The test material was applied to the back via 9, occlusive, 24- h induction applications (0.2 g
applied to an unspecified area), made over a 3-wk induction period. Induction sites were scored 24 or 48 h after patch
removal. After a 2-wk non-treatment period, a 24-h challenge application was made to a previously untreated site in the
same manner as the induction applications, and the reactions were scored on a scale of 0 - 4 at 24 and 48 h after application.
No adverse reactions occurred during the course of the study; the researchers concluded that the test material was not an
irritant or sensitizer.
An eyebrow gel formulation containing 0.04% Leuconostoc/Radish Root Ferment Filtrate was tested neat in an HRIPT
in 105 subjects.35 The test material was applied to the back via 9, occlusive, 24- h induction applications (0.2 g applied to
approximately 0.75 in2), made over a 3-wk induction period. Induction sites were scored 24 or 48 h after patch removal.
After a 2-wk non-treatment period, a 24-h challenge application was made to a previously untreated site in the same manner
as the induction applications, and the reactions were scored on a scale of 0 - 4, at 24 and 72 h after application. No signs of
irritation or sensitization were observed during induction or challenge; the researchers concluded that the test material did not
cause dermal sensitization.
Photosensitization/Phototoxicity
In Vitro
The phototoxicity of Leuconostoc/Radish Root Ferment Filtrate was tested using a reconstructed three-dimensional
human epidermis model (EpiDerm).36 Five concentrations of the test article 0, 0.4%, 1.2%, 3.7%, and 11%, diluted in
Dulbecco’s modified Eagle medium were used. Sterile deionized water and 0.001-0.1% chloropromazine were used as
negative and positive controls, respectively. After the EpiDerm model was incubated in growth media for 1 h, 50 µl of
each test article concentration was applied to tissue inserts and allowed to incubate overnight at 37 °C. The tissue inserts
were either irradiated with 6 J/cm2 UVA(ultraviolet), or incubated without irradiation, for 1 h at room temperature and were
tested in an MTT assay. As per the definition of a potential photoirritant reducing cell viability by ≥ 20%, when comparing
irradiated to non-irradiated controls, significant reduction was only seen in the 11% concentration, with and without radiation
(51.1% and 72.6%, respectively). Leuconostoc/Radish Root Ferment Filtrate was therefore not considered a photosensitizer
at the 0.4, 1.2, or 3.7% concentrations.
OCULAR IRRITATION STUDIES

In Vitro
The ocular irritation potential of Leuconostoc/ Radish Root Ferment Filtrate to cause eye irritation was evaluated in a
reconstructed human cornea-like epithelium test, using an EpiOcular™ three-dimensional human cornea model.31 Fifty μl of
the undiluted test article were applied to 2 tissue samples. The treated tissues were incubated for 90 min, washed out with
PBS, post-incubated under normal medium and culture conditions for 2 h, and then measured for cell viability via an MTT
assay. The negative control tissues received applications of de-ionized water. The test article was considered to be non-
irritating.
CLINICAL STUDIES
Occupational Exposures
A 46-yr-old kitchen porter, with metal allergy and no prior food allergies, presented to the emergency room with
dizziness, generalized eruptions on the skin, and gastrointestinal upset.37 During recent employment in a Korean kitchen, she
had been exposed to Raphanus sativus roots while chopping fresh young radish, 3 and 1 d prior to her hospital treatment.
Upon initial exposure, she experienced immediate urticaria with pruritus and burning sensation (which spontaneously
disappeared); however, upon second exposure, the pruritus presented more severely with generalized erythematous eruption
and dizziness. Systemic anaphylatic symptoms manifested within 12 h. Upon hospital admission, total serum
immunoglobulin E (IgE) level was measured at 30 IU/l; she received subcutaneous epinephrine (0.3 ml) followed by
intravenous saline and antihistamine. Three wk post-recovery, she tested positive to a skin prick test with young radish
extract; 5 controls tested with a skin prick test using young radish extract and 55 common allergens did not exhibit positive
reactions. The allergic reaction was attributed to biphasic, IgE-mediated anaphylaxis to physical contact with young radish.
A 38-yr-old waitress, with no prior history of dermatological illness, developed an acute vesiculo-bullous dermatitis of
both palms, 3 wk after chopping tomatoes, cabbage, endive, and radishes for the salad bar.38 She sought medical attention 2
wk after the dermatitis appeared; findings were normal, with the exception of the sides of her fingers, which were more
severely affected. Patch tests were performed with the neat application of Raphanus sativus root juice, cabbage leaf, tomato
fruit, and endive leaf. Additionally, patch tests were performed with 0.1% allyl isothiocyanate, 0.1% benzyl isothiocyanate,
0.05% phenyl isothiocyanate, 1% sinigrin, and 1% myrosinase (all in petrolatum). Samples of the thioglucoside, sinigrin,
which yields allyl isothiocyanate, and of the enzyme, myrosinase, were mixed together and either applied to the skin
immediately after mixture or 1 wk later; a positive reaction to the previously mixed test article was observed. Positive
reactions to allyl isothiocyanate, and benzyl isothiocyanate were also observed. There was no reaction to freshly mixed
sinigrin and myrosinase. No further details were provided.
SUMMARY
This assessment reviews the safety of the following 7 radish root-derived ingredients. According to the Dictionary,
various functions are reported for these ingredients, with hair and skin conditioning agents being the most common. Most of
the other reported are not considered cosmetic functions in the US, and therefore, use as such does not fall under the purview
of the Panel. Leuconostoc/Radish Root Ferment Filtrate is reported to have the greatest frequency of use, in 255
formulations, 104 of which are in leave-on moisturizing products. The highest reported concentration of use amongst these
ingredients is for Raphanus Sativus (Radish) Root Extract, at up to 6% in lipstick formulations. Raphanus sativus roots are
widely consumed in raw, cooked, and fermented forms; in the US, foods that are commercially produced using lactic acid
fermentation are considered to have GRAS status.
Groups of albino rats were administered up to 550 mg/kg bw/d of methanolic Daikon (vegetable) extract, in the diet, for
90 d. Throughout the course of the experiment, no statistically significant differences were seen between controls and treated
animals for mean body weights, organ weights, and hematological parameters such as red blood cell count, hemoglobin,
white blood cell count, AST, ALT, ACP, urea, uric acid, and protein levels.
Leuconostoc/Radish Root Ferment Filtrate was not genotoxic when tested at concentrations up to 5000 µg/plate in an
Ames test. In a study evaluating the genotoxic potential of several Raphanus sativus root juices against cancerous cell lines,
500 µl of the white tuber root juice caused the most DNA damage in all aberrant cell lines; the breast cancer adenoma cell
line was the most highly affected.
Raphanus sativus root juice exhibited a higher potential for tyrosinase inhibition (IC50=3.09 mg/ml vs. 9.62 mg/ml),
radical scavenging, and had a higher content of L-ascorbic acid than a methanolic Raphanus sativus root extract. In another
study, ethyl acetate Raphanus sativus root extract exposed to pH and temperature changes exhibited an IC50 value that was
closest to an unexposed control extract at a pH of 4 and temperature of 70 °C. A sample of Leuconostoc/Radish Root Ferment
Filtrate exhibited MIC values of 1 - 4% against P. aeruginosa, 0.50 - 4% against E.coli, and 0.25 - 2% against A.brasiliensis,
C.albicans, and S.aeruginosa. The highest MIC values for a Raphanus sativus root juice, which were greater than the
corresponding ampicillin MIC values, were against P.aeruginosa and S.aureus at 0.625± 0.4 mg/ml and 0.312±0.2 mg/ml,
respectively.
A single 30 µl application of Leuconostoc/Radish Root Ferment Filtrate did not cause irritation in a triplicate series of
EpiDerm model epidermis tests. In a DPRA assay testing the sensitizing potential of 100 mM Leuconostoc Ferment Filtrate,
the mean percent depletion for cysteine and lysine was 2.89%; the test article was predicted to be a non-sensitizer.
Leuconostoc/Radish Root Ferment Filtrate, tested at concentrations of up to 2000 µM in DMSO (50 µl), was found to be non-
sensitizing in an ARE-Nrf2 luciferase assay. Leuconostoc/Radish Root Ferment Filtrate, as a 10% dilution in water, did not
cause sensitization in an occlusive HRIPT using 50 subjects. An eyebrow gel formulation containing 0.04%
Leuconostoc/Radish Root Ferment Filtrate also was found to be non-sensitizing in an occlusive HRIPT using 105 subjects.
Leuconostoc/Radish Root Ferment Filtrate was tested at 0, 0.4, 1.2, 3.7, and 11% (in Dulbecco’s modified Eagle
medium) for phototoxicity in an irradiated EpiDerm reconstructed epidermis model. Significant reduction in cell viability
(≥20 %) when compared to non-radiated controls, was seen at the 11% concentration, both with and without radiation; the
test article was not considered a photosensitizer. Leuconostoc/Radish Root Ferment Filtrate was not considered an ocular
irritant when tested in 2 EpiOcular human cornea-like epithelium tissue samples.
A 46-yr old female kitchen porter, with pre-existing metal allergy, presented to the emergency room with dizziness,
generalized eruptions on the skin, and gastrointestinal upset after chopping fresh young radish 3 and 1 d prior to
hospitalization. Systemic anaphylactic symptoms manifested within 12 h. Three wk post-recovery the subject tested positive
to a skin prick test with young radish extract, which was attributed to biphasic, IgE-mediated anaphylaxis upon physical
contact. A 38-yr old female waitress developed an acute vesiculo-bullous dermatitis of both palms 3 wk after chopping
tomatoes, cabbage, endive, and radishes for the salad bar. Patch tests were performed with the neat application of all plant
substances, plus, 0.1% each of allyl isothiocyanate, benzyl isothiocyanate, sinigrin, myrosinase, and 1% sinigrin, either
mixed with 1% myrosinase 1 wk prior to application, or mixed with 1% myrosinase immediately prior to application.
Positive reactions were observed for Raphanus sativus root juice, allyl isothiocyanate, benzyl isothiocyanate, and to the
sinigrin previously mixed with myrosinase.
DISCUSSION
To be developed.
CONCLUSION
To be determined.
TABLES
Table 1. INCI names, definitions, and functions of Raphanus sativus (root)-derived ingredients in this safety assessment1
Ingredient/ CAS Number Definition Function(s)
Lactobacillus/Radish Root is a filtrate of the extract of the product obtained by the fermentation Preservative
Ferment Extract Filtrate of the roots of Raphanus sativus (radish) by the microorganism,
Lactobacillus.
Lactobacillus/Radish Root is a filtrate of the product obtained by the fermentation of the roots Antimicrobial agent;
Ferment Filtrate of Raphanus sativus (radish) by the microorganism, Lactobacillus. hair conditioning agent;
skin- conditioning agent -
miscellaneous
Leuconostoc/Radish Root is a filtrate of the product obtained by the fermentation of Raphanus Anti-dandruff agent;
Ferment Filtrate sativus roots by the microorganism, Leuconostoc. antifungal agent;
antimicrobial agent;
hair conditioning agent;
skin-conditioning agent -
miscellaneous
Leuconostoc/Radish Root is a filtrate of a lysate of the product obtained by the fermentation of Hair conditioning agent;
Ferment Lysate Filtrate the roots of Raphanus sativus (radish) by the microorganism, skin-conditioning agent-
1686112-10-6 Leuconostoc. miscellaneous
Raphanus Sativus (Radish) is the extract of the roots of Raphanus sativus. Antioxidant;
Root Extract skin-conditioning agents -
84775-94-0 (generic) miscellaneous
Raphanus Sativus (Radish) is the juice expressed from the roots of Raphanus sativus. Skin-conditioning agents -
Root Juice miscellaneous
Raphanus Sativus (Radish) is the powder obtained from the dried, ground roots of Raphanus Skin – conditioning agents –
Root Powder sativus. emollient; skin – conditioning
agents – humectant
Table 2. Chemical properties of Leuconostoc/Radish Root Ferment Filtrate

Property Value Reference
Physical Form Clear to slightly hazy liquid 6
Color Yellow to light amber 6
Odor Characteristic 6
Specific Gravity (@ 25 ºC) 1.140 – 1.180 6
pH 4.0 - 6.0 6
log Kow; Kow -1.92; 0.013 7

Table 3. Frequency (2021) and concentration (2020)17 of use according to duration and exposure
16
# of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%)
Lactobacillus/Radish Root Ferment Leuconostoc/Radish Root Ferment Raphanus Sativus (Radish) Root
Filtrate Filtrate Extract
Totals* 2 NR 255 0.0001 – 1.1 2 6
Duration of Use
Leave-On 2 NR 185 0.0001 - 0.03 2 6
Rinse-Off NR NR 70 0.0001 – 1.1 NR NR
Diluted for (Bath) Use NR NR NR NR NR NR
Exposure Type
Eye Area NR NR 3 0-0.002 – 0.01 NR NR
Incidental Ingestion NR NR 9 0.0002 NR 6
Incidental Inhalation-Spray 2a NR 5; 111a; 40b 0.0001-0.03; 0.001a NR NR
Incidental Inhalation-Powder NR NR 40b; 1c 0.0002 - 0.002c 2b NR
Dermal Contact 2 NR 220 0.0002 – 1.1 2 NR
Deodorant (underarm) NR NR 1a NR NR NR
Hair - Non-Coloring NR NR 26 0.0001 – 0.002 NR NR
Hair-Coloring NR NR NR NR NR NR
Nail NR NR NR 0.0022 – 0.01 NR NR
Mucous Membrane NR NR 39 0.0002 NR 6
Baby Products NR NR 1 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.
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 – not reported
Table 4. Raphanus sativus – derived ingredients not reported to be in use16,17

Lactobacillus/Radish Root Ferment Extract Filtrate
Raphanus Sativus (Root) Powder
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http://webdictionary.personalcarecouncil.org/jsp/IngredientSearchPage.jsp. Last Updated 2021. Accessed October
20, 2021.
2. Sabishruthi S, K Rajan A, Sai C, Arshath A, Benita S. A disquisition on Raphanus sativus Linn- a propitious medicinal
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Unpublished data submitted by Personal Care Products Council on May 7, 2021.
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Unpublished work submitted by Personal Care Products Council on May 6, 2021.
8. Eveline E, Pasau R. Antioxidant activity and stability of radish bulbs (Raphanus sativus L.) crude extract. IOP
Conference Series: Earth and Environmental Science. 2019;292:012036.
9. Enkhtuya E, Tsend M. The effect of peeling on antioxidant capacity of Black Radish Root. Ital J Food Sci.
2020;32:701-711.
10. Shukla S, Chatterji S, Yadav DK, Watal G. Antimicrobial efficacy of Raphanus sativus root juice. Int J Pharm Pharm
Sci. 2011;3:89-92.
11. Duy H, Ngoc P, Anh L, Dong D, Nguyen DC, Than VT. In vitro antifungal efficacy of white radish (Raphanus sativus
L.) root extract and application as a natural preservative in sponge cake. Processes. 2019;7:549.
12. Active Micro Technologies. 2021. Composition Leucidal® Liquid (Leuconotoc/Radish Root Ferment Filtrate).
Unpublished data submitted by Personal Care Products Council on May 7, 2021.
13. Goyeneche R, Roura S, Ponce AG, et al. Chemical characterization and antioxidant capacity of red radish (Raphanus
sativus L.) leaves and roots. J Func Foods. 2015;16:256-264.
14. Ragasa C, Ebajo Jr V, Tan MC, Brkljača R, Urban S. Chemical constituents of Raphanus sativus. Der Pharma
Chemica. 2015;7:354-357.
15. Baranidharan B, Shamina S. Subacute toxicity study of Daikon (vegetable) extract on albino rats. World J Pharm Res.
2018;7(6):725-731.
16. U.S. Food and Drug Administration Center for Food Safety & Applied Nutrition (CFSAN). 2021. Voluntary
Cosmetic Registration Program - Frequency of Use of Cosmetic Ingredients. Obtained under the Freedom of
Information Act from CFSAN; requested as "Frequency of Use Data" January 4, 2021; received January 21, 2021.
17. Personal Care Products Council. 2020. Concentration of Use by FDA Product Category: Leuconostoc/Radish Root
Ferment Filtrate and Related Ingredients. Unpublished data submitted by Personal Care Products Council on
January 6, 2021.
18. Johnsen M. The influence of particle size. Spray Technol Marketing. 2004;14(11):24-27.
19. Rothe H. 2011 2011. Special aspects of cosmetic spray safety evaluations: Principles on inhalation risk assessment.
Unpublished data presented at the 26 September Expert Panel meeting. Washington D.C.
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Updated version for ConsExpo4. 2006. Pages 1-77. http://www.rivm.nl/bibliotheek/rapporten/320104001.pdf.
Accessed June 25, 2019.
21. Rothe H, Fautz R, Gerber E, et al. Special aspects of cosmetic spray safety evaluations: Principles on inhalation risk
assessment. Toxicol Lett. 2011;205(2):97-104.
22. European Commission. CosIng database; following Cosmetic Regulation No. 1223/2009.
http://ec.europa.eu/growth/tools-databases/cosing/. Last Updated 2020. Accessed 04/21/2021.
23. Manivannan A, Kim J-H, Kim D-S, Lee E-S, Lee H-E. Deciphering the nutraceutical potential of Raphanus sativus-A
comprehensive overview. Nutrients. 2019;11(2):402.
24. Tamang J, Sarkar P. Sinki: A traditional lactic acid fermented radish tap root product. J Gen Appl Microbiology.
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25. Patra JK, Das G, Paramithiotis S, Shin H-S. Kimchi and other widely consumed traditional fermented foods of Korea:
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26. Vedamuthu ER. The dairy Leuconostoc: Use in dairy products. J Dairy Sci. 1994;77(9):2725-2737.
27. Ashaolu TJ, Reale A. A holistic review on Euro-Asian Lactic acid bacteria fermented cereals and vegetables.
Microorganisms. 2020;8(8).
28. Active Micro Technologies. 2018. Bacterial reverse mutation test Leucidal® Liquid (Leuconostoc/Radish Root
Ferment Filtrate). Unpublished data submitted by Personal Care Products Council on May 6, 2021.
29. Tan MCS, Enriquez MLD, Arcilla RG, Noel MG. Determining the apoptotic-inducing property of isothiocyanates
extracted from three cultivars of Raphanus sativus Linn. Using the comet assay. J Appl Pharm Sci. 2017;7(09):044-
051.
30. Jakmatakul R, Suttisri R, Tengamnuay P. Evaluation of antityrosinase and antioxidant activities of Raphanus sativus
root: Comparison between freeze-dried juice and methanolic extract. Thai J Pharm Sci. 2009;33:22-30.
31. Active Micro Technologies. 2017. Dermal and ocular irritation tests Leucidal® Liquid (Leuconostoc/Radish Root
32. Active Micro Technologies. 2017. OECD TG 442C: In Chemico skin sensitization Leucidal® Liquid
(Leuconostoc/Radish Root Ferment Filtrate). Unpublished data submitted by Personal Care Products Council on
May 6, 2021.
33. Active Micro Technologies. 2017. OECD TG 442D: In Vitro skin sensitization Leucidal® Liquid
(Leuconostoc/Radish Root Ferment Filtrate). Unpublished data submitted by Personal Care Products Council on
May 6, 2021.
34. AMA Laboratories. 2008. 50 Human subject repeat insult patch test skin irritation/sensitization evaluation (occlusive
patch) Leucidal® Liquid (Leuconostoc/Radish Root Ferment Filtrate). Unpublished data submitted by Personal
Care Products Council on May 6, 2021.
35. Personal Care Products Council. 2021. Repeated insult patch test (eyebrow gel containing 0.04%Leuconostoc/Radish
Root Ferment Filtrate) Unpublished data submitted by Personal Care Products Council on April 29, 2021.
36. Active Micro Technologies. 2017. Phototoxicity Assay Analysis Leucidal® Liquid (Leuconostoc/Radish Root
37. Lee YH, Lee JH, Kang HR, Ha JH, Lee BH, Kim SH. A case of anaphylaxis induced by contact with young radish
(Raphanus sativus L). Allergy Asthma Immunol Res. 2015;7(1):95-97.
38. Mitchell JC, Jordan WP. Allergic contact dermatitis from the radish, Raphanus sativus. Br J Dermatol.
1974;91(2):183-189.
Concentration of Use by FDA Product Category - Leuconostoc/Radish Root Ferment Filtrate and
Related Ingredients*
Leuconostoc/Radish Root Ferment Filtrate Lactobacillus/Radish Root Ferment Extract

Filtrate Raphanus Sativus (Radish) Root Extract
Lactobacillus/Radish Root Ferment Filtrate Raphanus Sativus (Radish) Root Juice
Raphanus Sativus (Radish) Root Powder
Ingredient Product Category Maximum

Concentration of Use
Leuconostoc/Radish Root Ferment Filtrate Eye makeup removers 0.002%
Leuconostoc/Radish Root Ferment Filtrate Mascaras 0.002%
Leuconostoc/Radish Root Ferment Filtrate Other eye makeup 0.01%
preparations
Leuconostoc/Radish Root Ferment Filtrate Hair conditioners 0.0001%
Leuconostoc/Radish Root Ferment Filtrate Hair sprays
Pump spray 0.0001%
Leuconostoc/Radish Root Ferment Filtrate Shampoos (noncoloring) 0.002%
Leuconostoc/Radish Root Ferment Filtrate Tonics, dressings, and other 0.001%
hair grooming aids
Leuconostoc/Radish Root Ferment Filtrate Lipstick 0.0002%
Leuconostoc/Radish Root Ferment Filtrate Other makeup preparations 0.024%
Leuconostoc/Radish Root Ferment Filtrate Basecoats and undercoats 0.0022%
Leuconostoc/Radish Root Ferment Filtrate Nail creams and lotions 0.01%
Leuconostoc/Radish Root Ferment Filtrate Other manicuring preparations 0.0022%
Leuconostoc/Radish Root Ferment Filtrate Bath soaps and detergents 0.0002%
Leuconostoc/Radish Root Ferment Filtrate Other personal cleanliness 0.0002%
products
Leuconostoc/Radish Root Ferment Filtrate Skin cleansing (cold creams, 0.04-1.1%
cleansing lotions, liquids, and
pads)
Leuconostoc/Radish Root Ferment Filtrate Face and neck products
Not spray 0.0002%
Spray 0.03%
Leuconostoc/Radish Root Ferment Filtrate Body and hand products
Not spray 0.002%
Leuconostoc/Radish Root Ferment Filtrate Moisturizing products
Not spray 0.01%
Leuconostoc/Radish Root Ferment Filtrate Other skin care preparations 0.001%
Raphanus Sativus (Radish) Root Extract Lipstick 6%
*Ingredients included in the title of the table but not found in the table were included in the
concentration of use survey, but no uses were reported.
Information collected in 2020

Table prepared: January 5, 2021
Memorandum
TO: Bart Heldreth, Ph.D.

Executive Director - Cosmetic Ingredient Review
FROM: Carol Eisenmann, Ph.D.

Personal Care Products Council
DATE: April 29, 2021
SUBJECT: Leuconostoc/Radish Root Ferment Filtrate
Anonymous. 2019. Repeated insult patch test (eyebrow gel containing 0.04% Leuconostoc/Radish
Root Ferment Filtrate).
eyebrow gel containing 0.04%

Leuconostoc/Radish Root
Ferment Filtrate
Memorandum


DATE: May 6, 2021
Active Micro Technologies. 2018. Bacterial reverse mutation test Leucidal® Liquid
(Leuconostoc/Radish Root Ferment Filtrate).
Active Micro Technologies. 2017. Dermal and ocular irritation tests Leucidal® Liquid
Active Micro Technologies. 2017. Kow statement Leucidal® Liquid (Leuconostoc/Radish Root
Ferment Filtrate).
Active Micro Technologies. 2017. OECD TG 442C: In Chemico skin sensitization Leucidal® Liquid
Active Micro Technologies. 2017. OECD TG 442D: In Vitro skin sensitization Leucidal® Liquid
Active Micro Technologies. 2017. Phototoxicity Assay Analysis Leucidal® Liquid

Active Micro Technologies. 2008. 50 Human subject repeat insult patch test skin irritation
/sensitization evaluation (occlusive patch) Leucidal® Liquid (Leuconostoc/Radish Root
Ferment Filtrate).
Bacterial Reverse Mutation Test
107 Technology Drive  Lincolnton, (AMES
NC 28092
(704) 276-7100  Fax (704) 276-7101
Test Article: Leucidal® Liquid Sponsor:

Code Number: M15008 Active Micro Technologies, LLC
CAS #: 84775-94-0 107 Technology Drive
Lincolnton, NC 28092
Study Director: Erica Segura

Principle Investigator: Monica Beltran
Test Performed: Reference:

Genotoxicity: Bacterial Reverse Mutation Test OECD471/ISO10993.Part 3
Test Request Number: 1004
SUMMARY
A Salmonella typhimurium/Escherichia coli reverse mutation standard plate incorporation study described by Ames et
al. (1975) was conducted to evaluate whether a test article solution Leucidal® Liquid would cause mutagenic
changes in the average number of reveratants for histidine-dependent Salmonella typhimurium strains TA98, TA100,
TA1537, TA1535 and tryptophan-dependent Escherichia coli strain WP2uvrA in the presence and absence of Aroclor-
induced rat liver S9. This study was conducted to satisfy, in part, the Genotoxicity requirement of the International
Organization for Standardization: Biological Evaluation of Medical Devices, Part 3: Tests for Genotoxicity,
Carcinogenicity and Reproductive Toxicity.
The stock test article was tested at eight doses levels along with appropriate vehicle control and positive controls with
overnight cultures of tester strains. The test article solution was found to be noninhibitory to growth of tester strain
TA98, TA100, TA1537, TA1535 and WP2uvrA after Sport Inhibition Screen.
Separate tubes containing 2 ml of molten top agar at 450C supplemented with histidine-biotin solution for the
Salmonella typhimurium strains and supplemented with tryptophan for Escherichia coli strain were inoculated with 100
µl of tester strains, 100 µl of vehicle or test article dilution were added and 500 µl aliquot of S9 homogenate,
simulating metabolic activation, was added when necessary. After vortexing, the mixture was poured across the
Minimal Glucose Agar (GMA) plates. Parallel testing was also conducted with positive control correspond to each
strain, replacing the test article aliquot with 50µl aliquot of appropriate positive control. After the overlay had solidified,
the plates were inverted and incubated for 48 hours at 37 0C. The mean numbers of revertants of the test plates were
compared to the mean number of revertants of the negative control plates for each of the strains tested. The means
obtained for the positive controls were used as points of reference.
Under the conditions of this assay, the test article solution was considered to be Non-Mutagenic to Salmonella
typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli tester strain WP2uvrA. The negative
and positive controls performed as anticipated. The results of this study should be evaluated in conjunction with other
required tests as listed in ISO 100993, Part 3: Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicology.
All Salmonella tester strain cultures demonstrated the presence of the deep rough mutation (rfa) and the deletion in
the uvrB gene. Cultures of tester strains TA98 and TA100 demonstrated the presence of the Pkm101 plasmid R-
factor. All WP2 uvrA cultures demonstrated the deletion in the uvrA gene. All cultures demonstrated the characteristic
mean number of spontaneous revertants in the vehicle controls as follows: TA98, 10-50; TA100, 80-240; TA1535, 5-
45; TA1537, 3-21, WP2uvrA, 10-60.
This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied.
This information is offered solely for your investigation, verification, and consideration.
Page 1 of 9 Version#6/08-06-18/Form#55
NC 28092
(704) 276-7100  Fax (704) 276-7101
I. Introduction
A. Purpose
A Salmonella typhimurium/Escherichia coli reverse mutation standard plate incorporation study was conducted to
evaluate whether a test article solution would cause mutagenic changes in the average number of revertants for
Salmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli WP2uvrA in the presence
and absences of the S9 metabolic activation. Bacterial reverse mutation tests have been widely used as rapid
screening procedures for the determination of mutagenic and potential carcinogenic hazards.
II. Materials
A. Storage Conditions: Room temperature (23-25C).

B. Vehicle: Sterile DI Water.
C. Preparation: Eight different doses level were prepared immediately before use with sterile DI water.
D. Solubility/Stability: 100% Soluble and Stable.
E. Toxicity: No significant inhibition was observed.
III. Test System
A. Test System
Each Salmonella typhimurium and Escherichia coli tester strain contains a specific deep rough mutation (rfa), the
deletion of uvrB gene and the deletion in the uvrA gene that increase their ability to detect mutagens, respectively.
These genetically altered Salmonella typhimurium strains (TA98, TA100, TA1537 and TA1535) and Escherichia coli
strain (WP2uvrA) cannot grow in the absence of histidine and tryptophan, respectively. When placed in a histidine-
tryptophan free medium, only those cells which mutate spontaneously back to their wild type states are able to form
colonies. The spontaneous mutation rate (or reversion rate) for any one strain is relatively constant, but if a mutagen
is added to the test system, the mutation rate is significantly increased.
Tester strain Mutations/Genotypic Relevance

TA98 hisD3052, Dgal chlD bio uvrB rfa pKM101
TA100 hisG46, Dgal chlD BIO uvrB rfa pKM101
TA1537 hisC3076, rfa, Dgal chlD bio uvrB
TA 1535 hisG46, Dgal chlD bio uvrB rfa
WP2uvrA trpE, uvrA
rfa = causes partial loss of the lip polysaccharide wall which increases
permeability of the cell to large molecules.
uvrB = deficient DNA excision-repair system (i.e., ultraviolet sensitivity)
pKM101 = plasmid confers ampicillin resistance (R-factor) and enhances
sensitivity to mutagens.
uvrA = All possible transitions and transversions, small deletions.
B. Metabolic Activation
Aroclor induced rat liver (S9) homogenate was used as metabolic activation. The S9 homogenate is prepared from
male Sprague Dawley rats. Material is supplied by MOLTOX, Molecular Toxicology, Inc.
C. Preparation of Tester strains

Cultures of Salmonella typhimurium TA98, TA100,TA1537, TA1535 and Escherichia coli WP2uvrA were inoculated to
individual flasks containing Oxoid broth No.2. The inoculated broth cultures were incubated at 37°C in an incubator
shaker operating at 140-150 rpm for 12-16 hours.
NC 28092
(704) 276-7100  Fax (704) 276-7101
D. Negative Control
Sterile DI water (vehicle without test material) was tested with each tester strain to determine the spontaneous
reversion rate. Each strain was tested with and without S9 activation. These data represented a base rate to which
the number of reveratants colonies that developed in each test plate were compared to determine whether the test
material had significant mutagenic properties.
E. Positive Control
A known mutagen for each strain was used as a positive control to demonstrate that tester strains were sensitive to
mutation to the wild type state. The positive controls are tested with and without the presence of S9 homogenate.
F. Titer of the Strain Cultures:

Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth; to confirm this,
serial dilutions from each strain were conducted, indicating that the initial population was in the range of 1 to 2x109/ml.
IV. Method
A. Standard Plate Incorporation Assay:

Separate tubes containing 2 ml of molten top agar supplemented with histidine-biotin solution for the Salmonella
typhimurium and tryptophan for Escherichia coli were inoculated with 100 µl of culture for each strain and 100 µl of
testing solution or vehicle without test material. A 500 µl aliquot of S9 homogenate, simulating metabolic activation,
was added when necessary. The mixture was poured across Minimal Glucose Agar plates labeled with strain number
and S9 activation (+/-). When plating the positive controls, the test article aliquot was replaced by 50µl aliquot of
appropriate positive control. The test was conducted per duplicate. The plates were incubated for 37°C for 2 days.
Following the incubation period, the revertant colonies on each plate were recorded. The mean number of reveratnts
was determined. The mean numbers of revertants of the test plates were compared to the mean number of reverants
of the negative control of each strain used.
V. Criteria for a Valid Test
For the test solution to be evaluated as a test failure or “potential mutagen” there must have been a 2-fold or greater
increase in the number of mean revertants over the means obtained from the negative control for any or all strains.
Each positive control mean must have exhibited at least a 3-fold increase over the respective negative control mean
of the Salmonella and Escherichia coli tester strains used.
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion
in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid
R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the
characteristic mean number of spontaneous revertants in the vehicle controls as follows: TA98, 10-50; TA100, 80-
240; TA1535, 5-45; TA1537, 3-21, WP2uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated,
tester strain culture titers must be greater than or equal to 0.3x10 9 cells/ml. The mean of each positive control must
exhibit at least 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A
minimum of three non-toxicdose levels is required to evaluate assay data. A dose level is considered toxic if one of
both of the following criteria are met: (1). A >50% reduction in the mean number of revertants per plate as compared
to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the
revertant count. (2). At least a moderate reduction in the background lawn.
VI. Results and Discussion
A. Solubility:
Water was used as a solvent. Solutions from the test article were made from 0.015 to 50mg/ml.
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B. Dose levels tested:

The maximum dose tested was 5000 µg per plate. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500
and 5000 µg per plate.
C. Titer (Organisms/ml):
5 x 108 UFC/ml plate count indicates that the initial population was in the range of 1 to 2 x 10 9 UFC/ml.
D. Standard Plate Incorporation Assay

In no case was there a 2-fold or greater increase in the mean number of revertant testing strains TA98, TA100,
TA1537, TA1535 and WP2uvrA in the presence of the test solution compared with the mean of vehicle control value.
The positive controls mean exhibited at least a 3-fold increase over the respective mean of the Salmonella
typhimurium and Escherichia coli tester strains used. The results are summarized in Appendix 2.
All Salmonella tester strain cultures demonstrated the presence of the deep rough mutation (rfa) and the deletion in
the uvrB gene. Cultures of tester strains TA98 and TA100 demonstrated the presence of the Pkm101 plasmid R-
factor. All WP2 uvrA cultures demonstrated the deletion in the uvrA gene. All cultures demonstrated the characteristic
mean number of spontaneous revertants in the vehicle controls as follows: TA98, 10-50; TA100, 80-240; TA1535, 5-
45; TA1537, 3-21, WP2uvrA, 10-60.
VII. Conclusion
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay
indicate that under the conditions of this assay, the test article solution was considered to be Non-Mutagenic to
Salmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli WP2uvrA.The negative
and positive controls performed as anticipated. The results of this study should be evaluated in conjunction with other
required tests as listed in ISO 100993, Part 3: Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicology.
NC 28092
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Appendix 2:
Bacterial Mutation Assay

Plate Incorporation Assay Results
TA98
Concentration µg
per Plate Revertants per plate
Mean
(CFU)
5000 32 34 33
1500 15 17 16
500 28 32 30
150 26 36 31
Test Solution w/ S9
50 28 18 23
15 14 20 17
5.0 24 21 23
1.5 26 26 26
5000 18 16 17
1500 33 45 39
500 15 19 17
150 21 35 28
Test Solution w/o S9
50 18 23 21
15 25 27 26
5.0 21 21 21
1.5 25 15 20
DI Water w/S9 36 36 36
DI Water w/o S9 28 32 30
2-aminoanthracen w/ S9 410 398 404
2-nitrofluorene w/o S9 257 225 241
Historical Count Positive w/S9 43-1893
Historical Count Positive w/o S9 39-1871
Historical Count Negative w/S9 4-69
Historical Count Negative w/o S9 3-59
*CFU = Colony Forming Units

*Mean = Average of duplicate plates
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TA100
Concentration µg
Mean
(CFU)
5000 112 110 111
1500 108 144 126

500 115 117 116
150 114 132 123
Test Solution w/ S9
50 128 156 142
15 144 162 153
5.0 132 146 139
1.5 168 134 151
5000 132 148 140
1500 112 124 118
500 152 126 139
150 112 68 90
50 102 44 73
15 116 125 121
5.0 136 112 124
1.5 126 124 125
DI Water w/S9 154 185 170
DI Water w/o S9 194 210 202
Sodium azide w/o S9 398 410 404

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TA1537
Concentration µg
Mean
(CFU)
5000 10 8 9
1500 16 22 19
500 14 12 13
150 24 16 20
Test Solution w/ S9
50 22 24 23
15 14 14 14
5.0 12 32 22
1.5 19 25 22
5000 42 22 32
1500 12 12 12
500 10 8 9
150 10 12 11
50 14 18 16
15 22 14 18
5.0 16 22 19
1.5 16 11 14
2-aminoacridine w/o S9 348 306 327

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TA1535
Concentration µg
Mean
(CFU)
5000 24 35 25
1500 25 28 27
500 42 31 37
150 22 16 19
Test Solution w/ S9
50 21 24 23
15 18 15 17
5.0 17 17 17
1.5 14 22 18
5000 45 61 53
1500 48 33 41
500 82 81 82
150 65 42 54
50 15 28 22
15 12 25 19
5.0 44 36 40
1.5 22 24 23
Sodium azide w/o S9 416 475 446

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WP2uvrA
Concentration µg
Mean
(CFU)
5000 20 32 26
1500 21 11 16
500 26 24 25
150 25 42 34
Test Solution w/ S9
50 29 36 33
15 20 12 16
5.0 45 47 46
1.5 51 55 53
5000 62 36 49
1500 44 62 53
500 26 38 32
150 16 16 16
50 35 52 44
15 61 47 54
5.0 52 37 45
1.5 40 60 50
Methylmethanesulfonate w/o S9 385 363 374

Dermal and Ocular Irritation Tests

107 Technology Drive  Lincolnton, NC 28092
(704) 276-7100  Fax (704) 276-7101
Tradename: Leucidal® Liquid
Code: M15008
CAS #: 1686112-10-6 (or) 84775-94-0
Test Request Form #: 23
Lot #: 26051
Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092

Principle Investigator: Meghan Darley
Test Performed:
In Vitro EpiDerm™ Dermal Irritation Test (EPI-200-SIT)
EpiOcular™ Eye Irritation Test (OCL-200-EIT)
SUMMARY
In vitro dermal and ocular irritation studies were conducted to evaluate whether Leucidal® Liquid would induce
dermal or ocular irritation in the EpiDerm™ and EpiOcular™ model assays.
The product was tested according to the manufacture’s protocol. The test article solution was found to be a non-
irritant. Reconstructed human epidermis and cornea epithelial model were incubated in growth media overnight to
allow for tissue equilibration after shipping from MatTek Corporation, Ashland, MA. Test substances were applied to
the tissue inserts and incubated for 60 minutes for liquid and solid substances in the EpiDerm™ assay and 30
minutes for liquid substances and 90 minutes for solid substances in the EpiOcular™ assay at 37°C, 5% CO2, and
95% relative humidity (RH). Tissue inserts were thoroughly washed and transferred to fresh plates with growth
media. After post substance dosing incubation is complete, the cell viability test begins. Cell viability is measured by
dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl)], present in the cell mitochondria, into blue formazan
salt that is measured after extraction from the tissue. The irritation potential of the test chemical is dictated by the
reduction in tissue viability of exposed tissues compared to the negative control.
Under the conditions of this assay, the test article was considered to be non-irritating. The negative and positive
controls performed as anticipated.
I. Introduction
A. Purpose
In vitro dermal and ocular irritation studies were conducted to evaluate whether a test article would induce dermal or
ocular irritation in the EpiDerm™ and EpiOcular™ model assays. MatTek Corporation’s reconstructed human
epidermal and human ocular models are becoming a standard in determining the irritancy potential of test substances.
They are able to discriminate between irritants and non-irritants. The EpiDerm™ assay has accuracy for the
prediction of UN GHS R38 skin irritating and no-label (non-skin irritating) test substances. The EpiOcular™ assay can
differentiate chemicals that have been classified as R36 or R41 from the EU classifications based on Dangerous
Substances Directive (DSD) or between the UN GHS Cat 1 and Cat 2 classifications.

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II. Materials
A. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidity
B. Equipment: Forma humidified incubator, ESCO biosafety laminar flow hood, Synergy HT
Microplate reader; Pipettes
C. Media/Buffers: DMEM based medium; DPBS; sterile deionized H2O
D. Preparation: Pre-incubate (37°C) tissue inserts in assay medium; Place assay medium and MTT
diluent at 4°C, MTT concentrate at -20°C, and record lot numbers of kit components
E. Tissue Culture Plates: Falcon flat bottom 96-well, 24-well, 12-well, and 6-well tissue culture plates
F. Reagents: MTT (1.0mg/mL); Extraction Solution (Isopropanol); SDS (5%); Methyl Acetate
G. Other: Nylon Mesh Circles (EPI-MESH); Cotton tip swabs; 1mL tuberculin syringes; Ted
Pella micro-spatula; 220mL specimen containers; sterile disposable pipette tips;
Parafilm
III. Test Assay

A. Test System
The reconstructed human epidermal model, EpiDerm™, and cornea epithelial model, EpiOcular™, consist of normal
human-derived epidermal keratinocytes which have been cultured to form a multilayer, highly differentiated model of
the human epidermis and cornea epithelium. These models consist of organized basal, spinous, and granular layers,
and the EpiDerm™ systems also contains a multilayer stratum corneum containing intercellular lamellar lipid layers
that the EpiOcular™ system is lacking. Both the EpiDerm™ and EpiOcular™ tissues are cultured on specially
prepared cell culture inserts.
B. Negative Control
Sterile DPBS and sterile deionized water are used as negative controls for the EpiDerm™ and EpiOcular™ assays,
respectfully.
C. Positive Control
Known dermal and eye irritants, 5% SDS solution and Methyl Acetate, were used as positive controls for the
EpiDerm™ and EpiOcular™ assays, respectfully.
D. Data Interpretation Procedure

a. EpiDerm™
An irritant is predicted if the mean relative tissue viability of the 3 tissues exposed to the test substance is
reduced by 50% of the mean viability of the negative controls and a non-irritant’s viability is ˃ 50%.
b. EpiOcular™
An irritant is predicted if the mean relative tissue viability of the 2 tissues exposed to the test substance is
reduced by 60% of the mean viability of the negative controls and a non-irritant’s viability is ˃ 40%.
IV. Method
A. Tissue Conditioning
Upon MatTek kit arrival at Active Micro Technologies, LLC the tissue inserts are removed from their shipping medium
and transferred into fresh media and tissue culture plates and incubated at 37°C at 5% CO2 and 95% relative
humidity for 60 minutes. After those 60 minutes the inserts are transferred into fresh media and tissue culture plates
and incubated at 37°C at 5% CO2 and 95% relative humidity for an additional 18 to 21 hours.

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B. Test Substance Exposure

a. EpiDerm™
30µL (liquid) or 25mg (solid) of the undiluted test substance is applied to 3 tissue inserts and allowed to
incubate for 60 minutes in a humidified incubator (37°C, 5% CO2, 95% RH).
b. EpiOcular™
Each tissue is dosed with 20µL DPBS prior to test substance dosing. 50µL (liquid) or 50mg (solid) of the
undiluted test substance is applied to 2 tissue inserts and allowed to incubate for 90 minutes in a humidified
incubator (37°C, 5% CO2, 95% RH).
C. Tissue Washing and Post Incubation

a. EpiDerm™
All tissue inserts are washed with DPBS, dried with cotton tipped swab, and transferred to fresh media and
culture plates. After 24 hours the inserts are again transferred into fresh media and culture plates for an
additional 18 to 20 hours.
b. EpiOcular™
Tissue inserts are washed with DPBS and immediately transferred into 5mL of assay medium for 12 to 14
minutes. After this soak the inserts are transferred into fresh media and tissue culture plates for 120 minutes
for liquid substances and 18 hours for solid substances.
D. MTT Assay
Tissue inserts are transferred into 300µL MTT media in pre-filled plates and incubated for 3 hours at 37°C, 5% CO2,
and 95% RH. Inserts are then removed from the MTT medium and placed in 2mL of the extraction solution. The
plate is sealed and incubated at room temperature in the dark for 24 hours. After extraction is complete the tissue
inserts are pierced with forceps and 2 x 200µL aliquots of the blue formazan solution is transferred into a 96 well plate
for Optical Density reading. The spectrophotometer reads the 96-well plate using a wavelength of 570 nm.
V. Acceptance Criterion
A. Negative Control
The results of this assay are acceptable if the mean negative control Optical Density (OD570) is ≥ 1.0 and ≤ 2.5
(EpiDerm™) or ≥ 1.0 and ≤ 2.3 (EpiOcular™).
B. Positive Control
a. EpiDerm™
The assay meets the acceptance criterion if the mean viability of positive control tissues expressed as a % of
the negative control is ≤ 20%.
b. EpiOcular™
The assay meets the acceptance criterion if the mean viability of positive control tissues is < 60% of control
viability.
C. Standard Deviation
Since each irritancy potential is predicted from the mean viability of 3 tissues for EpiDerm™ and 2 tissues for
EpiOcular™, the variability of the replicates should be < 18% for EpiDerm™ and < 20% EpiOcular™.
VI. Results
A. Tissue Characteristics
The tissue inserts included in the MatTek EpiDerm™ and EpiOcular™ assay kits were in good condition, intact, and
viable.

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B. Tissue Viability Assay

The results are summarized in Figures 1 and 2. In no case was the tissue viability ≤ 50% for EpiDerm™ or ≤ 60% for
EpiOcular™ in the presence of the test substance. The negative control mean exhibited acceptable relative tissue
viability while the positive control exhibited substantial loss of tissue viability and cell death.
C. Test Validity
The data obtained from this study met criteria for a valid assay.
VII. Conclusion
Under the conditions of this assay, the test article substance was considered to be non-irritating. The negative and
positive controls performed as anticipated.
Figure 1: EpiDerm tissue viability
Figure 2: EpiOcular tissue viability

(704) 276-7100  Fax (704) 276-7101
December 21, 2017
To whom it may concern,
This letter is to certify that Leucidal® Liquid (M15008) manufactured by Active Micro Technologies, LLC
has an average octanol/water partition coefficient (Kow) of 0.013 and Log(Kow) of -1.92.
The aforementioned results were analyzed via EPA OPPTS 830.7570 testing at Jordi Labs, LLC.
Thank you for your interest in Active Micro Technologies’ products. If you have any further questions,
feel free to contact us at (704) 276-7100.
Best Regards,
Tomorrow's Vision... Today!®

Heather Ferguson |R&D Coordinator
107 Technology Drive | Lincolnton, NC 28092
Direct: 704.276.7083|Main: 704.276.7100|Fax: 704.276.7101
Email: [email protected]
www.activeconceptsllc.com
Version#1/12-21-17
OECD TG 442C:
In Chemico Skin Sensitization
(704) 276-7100  Fax (704) 276-7101
Code: M15008
CAS #: 1686112-10-6 (or) 84775-94-0
Lot #: 4786P
Sponsor: Active Micro Technologies, LLC; 107 Technology Drive Lincolnton, NC 28092
Test Performed:
OECD TG 442C: In Chemico Skin Sensitization
Direct Peptide Reactivity Assay (DPRA)
Introduction
A skin sensitizer is a substance that will lead to an allergic response following skin contact1. Haptenation is the
covalent binding of a hapten, or low-molecular weight substance or chemical, to proteins in the skin. This is
considered the prominent mechanism which defines a chemical as a sensitizer. Haptenation is described as a
"molecular initiating event" in the OECD Adverse Outcome Pathway (AOP) for skin sensitization which summarizes
the key events known to be involved in chemically-induced allergic contact dermatitis2. The direct peptide reactivity
assay (DPRA) is designed to mimic the covalent binding of electrophilic chemicals to nucleophilic centers in skin
proteins by quantifying the reactivity of chemicals towards the model synthetic peptides containing cysteine and lysine.
The DPRA is able to distinguish sensitizers from non-sensitizer with 82% accuracy (sensitivity of 76%; specificity of
92%)3.
This assay was conducted to determine skin sensitization hazard of Leucidal® Liquid in accordance with European
Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and OECD Test Guideline 442C.
Assay Principle
The DPRA is an in chemico method which addresses peptide reactivity by measuring depletion of synthetic
heptapeptides containing either cysteine or lysine following 24 hours incubation with the test substance. The peptide
is a custom material containing phenylalanine to aid in detection. Depletion of the peptide in the reaction mixture is
measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion
values are then calculated and used in a prediction model which allows assigning the test chemical to one of four
reactivity classes used to support the discrimination between sensitizers and non-sensitizers.
1. United Nations Economic Commission (UNECE) (2013) Global Harmonized System of Classification and Labelling of Chemicals (GHS) 5th Revised Edition
2. OECD (2012). The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168
3. EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report; pp 1 -74.
Page 1 of 4 Version#2/12-19-17
OECD TG 442C:
(704) 276-7100  Fax (704) 276-7101
Materials
A. Equipment: HPLC-UV (Waters Alliance 2695 - Waters 996 Photodiode Array);

Pipettes; Analytical balance
B. HPLC/Guard Columns: Agilent Zorbax SB-C18 2.1mm x 100mm x 3.5µm; Phenomenex
Security Guard C18 4mm x 2mm
C. Chemicals: Trifluoroacetic acid; Ammonium acetate; Ammonium hydroxide;
Acetonitrile; Cysteine peptide (Ac-RFAACAA-COOH); Lysine peptide
(Ac-RFAAKAA-COOH); Cinnamic aldehyde
D. Reagents/Buffers: Sodium phosphate buffer (100mM); Ammonium acetate buffer
(100mM)
E. Other: Sterile disposable pipette tips
Methods
Solution Preparation:
 0.667mM Cysteine Peptide in 100mM Phosphate Buffer (pH 7.5)

 0.667mM Lysine Peptide in 100mM Ammonium Acetate Buffer (pH 10.2)
 100mM Cinnamic Aldehyde in Acetonitrile
 100mM Leucidal® Liquid in Acetonitrile
Reference Controls:
 Reference Control A: For calibration curve accuracy

 Reference Control B: For peptide stability over analysis time of experiment
 Reference Control C: For verification that the solvent does not impact percent peptide depletion
Sample, Reference Control, and Co-Elution Control Preparation:
 Once these solutions have been made they should be incubated at room temperature, protected from light,
for 24±2 hours before running HPLC analysis.
 Each chemical should be analyzed in triplicate.
1:10 Ratio, Cysteine Peptide 1:50 Ratio, Lysine Peptide

0.5mM Peptide, 5mM Test Chemical 0.5mM Peptide, 25mM Test Chemical
 750µL Cysteine Peptide Solution  750µL Lysine Peptide Solution

(or 100mM Phosphate Buffer, pH 7.5, for Co-Elution (or 100mM Ammonium Acetate Buffer, pH 10.2,
Controls) for Co-Elution Controls)
 200µL Acetonitrile  250µL Test Chemical Solution
 50µL Test Chemical Solution (or Acetonitrile for Reference Controls)
(or Acetonitrile for Reference Controls)
OECD TG 442C:
(704) 276-7100  Fax (704) 276-7101
Calibration Curve:
 Standards are prepared in a solution of 20% Acetonitrile:Buffer

o For the Cysteine peptide using the phosphate buffer, pH 7.5
o For the Lysine peptide using the ammonium acetate buffer, pH 10.2
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7

mM Peptide 0.534 0.267 0.1335 0.0667 0.0334 0.0167 0.000
HPLC Analysis:
 HPLC-UV system should be equilibrated at 30°C with 50% Mobile Phase A (0.1% (v/v) trifluoroacetic acid
in water) and 50% Mobile Phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for 2 hours
 Absorbance is measured at 220nm
 Flow Conditions:
Time Flow %A %B
0 minutes 0.35 mL/min 90 10
13.5 minutes 0.35 mL/min 90 10
20 minutes End Run
Data and Reporting
Acceptance Criteria:
1. The following criteria must be met for a run to be considered valid:

a. Standard calibration curve should have an r2 > 0.99.
b. Mean percent peptide depletion values of three replicates for the positive control cinnamic aldehyde
should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69% for the
lysine peptide and the maximum standard deviation should be <14.9 for the percent cysteine
depletion and <11.6 for the percent lysine depletion.
c. Mean peptide concentration of reference controls A should be 0.50±0.05mM and the coefficient of
variable of the peptide peak areas for reference B and C in acetonitrile should be <15.0%.
2. The following criteria must be met for a test chemical’s results to be considered valid:
a. Maximum standard deviation should be <14.9 for percent cysteine depletion and <11.6 for percent
lysine depletion.
b. Mean peptide concentration of the three reference control C should be 0.50±0.05mM.
OECD TG 442C:
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Prediction Model:
Cysteine 1:10/Lysine 1:50 Prediction Model

Mean of Cysteine and Lysine % Depletion Reactivity Class Prediction
0% < Mean % Depletion < 6.38% Minimal Reactivity Non-sensitizer
6.38% < Mean % Depletion < 22.62% Low Reactivity Sensitizer
22.62% < Mean % Depletion < 42.47% Moderate Reactivity Sensitizer
42.47% < Mean % Depletion < 100% High Reactivity Sensitizer
If co-elution occurs with the lysine peptide, than the cysteine 1:10 prediction model can be used:
Cysteine 1:10 Prediction Model

Mean of Cysteine and Lysine % Depletion Reactivity Class Prediction
0% < Cys % Depletion < 13.89% Minimal Reactivity Non-sensitizer
13.89% < Cys % Depletion < 23.09% Low Reactivity Sensitizer
23.09% < Cys % Depletion < 98.24% Moderate Reactivity Sensitizer
98.24% < Cys % Depletion < 100% High Reactivity Sensitizer
Results and Discussion
The data obtained from this study met criteria for a valid assay and the controls performed as anticipated.
Percent peptide depletion is determined by the following equation:
𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎 𝑖𝑛 𝑅𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒 𝐼𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝐷𝑒𝑝𝑙𝑒𝑡𝑖𝑜𝑛 = [1 − (𝑀𝑒𝑎𝑛 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎 𝑖𝑛 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙𝑠 𝐶 )] × 100
Based on HPLC-UV analysis of Leucidal® Liquid (code M15008) we can determine that this product is not a
sensitizer and will not cause allergic contact dermatitis. The Mean Percent Depletion of Cysteine and Lysine was
2.89% causing minimal reactivity in the assay giving us the prediction of a non-sensitizer.
OECD TG 442D:
In Vitro Skin Sensitization
(704) 276-7100  Fax (704) 276-7101
Code: M15008
CAS #: 1686112-10-6 (or) 84775-94-0
Lot #: 4752P
Test Performed:
OECD TG 442D: In Vitro Skin Sensitization
ARE-Nrf2 Luciferase Test Method
Introduction
Skin sensitization refers to an allergic response following skin contact with the tested chemical, as defined by the
United Nations Globally Harmonized System of Classification and Labelling of Chemicals1. Substances are
classified as skin sensitizers if there is evidence in humans that the substance can lead to sensitization by skin
contact or positive results from appropriate tests, both in vivo and in vitro. Utilization of the KeratinoSens™ cell line
allows for valid in vitro testing for skin sensitization.
This assay was conducted to determine skin sensitization potential of Leucidal® Liquid in accordance with the UN
GHS.
Assay Principle
The ARE-Nrf2 luciferase test method addresses the induction of genes that are regulated by antioxidant response
elements (ARE) by skin sensitizers. The Keap1-Nrf2-ARE pathways have been shown to be major regulator of
cytoprotective responses to oxidative stress or electrophilic compounds. These pathways are also known to be
involved in the cellular processes in skin sensitization. Small electrophilic substances such as skin sensitizers can
act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by covalent modification of its cysteine
residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2).
The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying
enzymes.
The skin sensitization assay utilizes the KeratinoSens™ method which uses an immortalized adherent human
keratinocyte cell line (HaCaT cell line) that has been transfected with a selectable plasmid to quantify luciferase
gene induction as a measure of activation of Keap1-Nrf2-antioxidant/electrophile response element (ARE). This
test method has been validated by independent peer review by the EURL-ECVAM. The addition of a luciferin
containing reagent to the cells will react with the luciferase produced in the cell resulting in luminescence which can
be quantified with a luminometer.
1. United Nations (UN) (2013). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Fifth revised edition, UN New York and Geneva, 2013
OECD TG 442D:
(704) 276-7100  Fax (704) 276-7101
Materials
A. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidity (RH)

B. Equipment: Humidified incubator; Biosafety laminar flow hood; Microplate Reader;
Pipettes
C. Cell Line: KeratinoSens™ by Givaudan Schweiz AG
D. Media/Buffers: Dulbecco's Modified Eagle Medium (DMEM); Fetal Bovine Serum
(FBS); Phosphate Buffered Saline (PBS); Geneticin
E. Culture Plate: Flat bottom 96-well tissue culture treated plates
F. Reagents: Dimethyl Sulfoxide (DMSO); Cinnamic Aldehyde; ONE-Glo Reagent;
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT);
sodium lauryl sulfate (SLS)
G. Other: Sterile disposable pipette tips; wash bottles
Methods
KeratinoSens™ were into seeded four 96-well tissue culture plates and allowed to grow to 80 – 90% confluency in
DMEM containing 10% FBS and 500µg/mL G418 geneticin. Twelve test concentrations of Leucidal® Liquid were
prepared in DMSO with a concentration range from 0.98 – 2000 µM. These 12 concentrations were assayed in
triplicate in 2 independently performed experiments. The positive control was cinnamic aldehyde for which a series of
5 concentrations prepared in DMSO had final test concentrations of 4 – 64 µM. The negative control was a 1% test
concentration of DMSO.
24 hour post KeratinoSens™ seeding, the culture media was removed and replaced with fresh media containing 10%
FBS without G418 geneticin. 50 µL of the above described test concentrations was added to the appropriate wells.
The treated plates were then incubated for 48 hours at 37°C in the presence of 5% CO2 and 95% relative humidity.
After treatment incubation was complete the media was removed and the wells were washed with PBS 3 times.
One of the four plates was used for a cytotoxicity endpoint, where MTT was added to the wells and incubated for 4
hours at 37°C in the presence of 5% CO2. SLS was then added to the wells and incubated overnight at room
temperature. A spectrometer measured the absorbance at 570 nm. The absorbance values (optical density) were then
used to determine the viability of each well by comparing the optical density of each test material treated well to that of
the solvent control wells to determine the IC50 and IC30 values.
The remaining 3 plates were used in the luciferase induction endpoint of the assay. 100 µL of Promega’s ONE-Glo
Reagent was added to 100 µL of fresh media containing 10% FBS without geneticin. Cells were incubated for 5 minutes
to induce cell lysis and release luciferin into the media. Plates were read with a luminometer and EC1.5 and maximum
response (Imax) values were obtained.
Data and Reporting
Acceptance Criteria:
1. Gene induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above
the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM).
2. The EC1.5 value should be within two standard deviations of the historical mean and the average induction
in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8.
3. The average coefficient of variability of the luminescence reading for the negative (solvent) control DMSO
should be below 20% in each experiment.
OECD TG 442D:
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A KeratinoSens™ prediction is considered positive if the following conditions are met:
1. The Imax is higher than 1.5-fold and statistically significantly higher as compared to the solvent (negative)
control
2. The cellular viability is higher than 70% at the lowest concentration with a gene induction above 1.5 fold (i.e.,
at the EC1.5 determining concentration)
3. The EC1.5 value is less than 1000 µM (or < 200 µg/ml for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Results
Compound Classification EC1.5 (µM) IC50 Imax

Cinnamic aldehyde Sensitizer 19 289.19 µM 31.6
DMSO Non-Sensitizer No Induction 243.24 µM 1.2
Leucidal® Liquid Non-Sensitizer No Induction > 1000 µM 0.5
Table 1: Overview of KeratinoSens™ Assay Results
KeratinoSens™ Assay
M15008 Leucidal® Liquid
Fold Induction of Luciferase Activity
6
5
4
3
2
1
0
50 µm 90 µM 125 µM 50 µM 90 µM 125 µM 50 µM 90 µM 125 µM
Cinnamic aldehyde DMSO Leucidal® Liquid
Figure 1: Fold Induction of Luciferase
Discussion
As shown in the results, Leucidal® Liquid (code M15008) was not predicted to be a skin sensitizer based on the
KeratinoSens™ ARE-Nrf2 Luciferase Test Method as there was not a significant increase in luciferase expression. It
can be concluded that Leucidal® Liquid can be safely used in cosmetics and personal care products at typical use
levels.
Phototoxicity Assay Analysis

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Code: M15008
CAS #: 1686112-10-6 (or) 84775-94-0
Lot #: 24723
Test Performed:
In Vitro EpiDerm™ Model (EPI-200-SIT) Phototoxicity
SUMMARY
In vitro phototoxicity irritation studies were conducted to evaluate whether Leucidal® Liquid would induce phototoxic
irritation in the EpiDerm™ model assay.
The product was tested according to the manufacturer’s protocol. The test article solution was found to be a non-
photoirritant at concentrations of 0.4%, 1.2%, and 3.7%. Reconstructed human epidermis was incubated in growth
media for one hour to allow for tissue equilibration after shipping from MatTek Corporation, Ashland, MA. Test
substance was applied to the tissue inserts in five varying concentrations and incubated overnight at 37°C, 5% CO2,
and 95% relative humidity (RH). The following day, the appropriate tissue inserts were irradiated (UVA) for 60
minutes with 1.7 mW/cm2 (=6 J/cm2). After substance incubation, irradiation, and washing was completed, the cell
viability test was conducted. Cell viability was measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl
thiazole 2-yl)], present in the cell mitochondria, into blue formazan salt that was measured after extraction from the
tissue. The photoirritation potential of the test chemical was dictated by the reduction in tissue viability of UVA
exposed tissues compared to non-UVA exposed tissues.
Under the conditions of this assay, the test article was considered to be non-phototoxic at concentrations of 0.4%,
1.2%, and 3.7%. The negative and positive controls performed as anticipated.
I. Introduction
A. Purpose
In vitro dermal phototoxicity study was conducted to evaluate whether a test article would induce
photoirritation in the EpiDerm™ model assay. MatTek Corporation’s reconstructed human epidermal model
is becoming a standard in determining the phototoxicity potential of a test substance. This assay is able to
discriminate between photoirritants and non-photoirritants at varying concentrations.

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II. Materials
A. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidity

B. Equipment: Forma humidified incubator, ESCO biosafety laminar flow hood, Synergy
HT Microplate reader; UVA-vis Irradiation Equipment; UVA meter; Pipettes
C. Media/Buffers: Dulbecco’s Modified Eagle Medium (DMEM) based medium; Dulbecco’s
Phosphate-Buffered Saline (DPBS); sterile deionized H2O
D. Preparation: Pre-incubate (37°C) tissue inserts in assay medium; Place assay medium
and MTT diluent at 4°C, MTT concentrate at -20°C, and record lot numbers
of kit components
E. Tissue Culture Plates: Falcon flat bottom 96-well, 24-well, and 6-well tissue culture plates
F. Reagents: MTT (3-4,5-dimethyl thiazole 2-yl) (1.0mg/mL); Extraction Solution
(Isopropanol); Chlorpromazine; Triton X-100 (1%)
G. Other: Wash bottle; sterile disposable pipette tips; Parafilm; forceps
III. Test Assay
A. Test System
The reconstructed human epidermal model, EpiDerm™ consists of normal human-derived epidermal
keratinocytes which have been cultured to form a multilayer, highly differentiated model of the human
epidermis. This model consists of organized basal, spinous, and granular layers, and contains a multilayer
stratum corneum containing intercellular lamellar lipid layers. The EpiDerm™ tissues are cultured on
specially prepared cell culture inserts.
B. Negative Control
Sterile deionized water is used as the negative controls for the EpiDerm™ Phototoxicity assay.
C. Positive Control
Concentrations of chloropromazine, ranging from 0.001% to 0.1%, were used as positive controls for the
EpiDerm™ Phototoxicity assay.
D. Data Interpretation Procedure

A photoirritant is predicted if the mean relative tissue viability of the 2 tissues exposed to the test substance
and 60 minutes of 6 J/cm 2 is reduced by 20% compared to the non-irradiated control tissues.
IV. Method
A. Tissue Conditioning
Upon MatTek kit arrival at Active Micro Technologies, LLC the tissue inserts are removed from their shipping
medium and transferred into fresh media and tissue culture plates and incubated at 37°C at 5% CO2 and
95% relative humidity for 60 minutes. After those 60 minutes the inserts are transferred into fresh media and
tissue culture plates and tissue insert dosing begins.
B. Test Substance Exposure

50µL of the diluted test substance in their respective concentrations are applied to 2 tissue inserts and allowed
to incubate for overnight in a humidified incubator (37°C, 5% CO2, 95% RH).

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C. Tissue Irradiation
Tissue inserts in their 6-well plates are UVA-irradiated for 60 minutes with 6 J/cm 2 at room temperature. The
non-irradiated tissue inserts are incubated at room temperature in the dark.
D. Tissue Washing and Post Incubation

After UVA-irradiation and dark incubation is complete the tissue inserts are washed using sterile DPBS and
transferred to fresh 6-well plates and media for overnight incubation at 37°C, 5% CO2, 95% RH.
E. MTT Assay
Tissue inserts are transferred into 300µL MTT media in pre-filled plates and incubated for 3 hours at 37°C,
5% CO2, and 95% RH. Inserts are then removed from the MTT medium and placed in 2mL of the extraction
solution. The plate is sealed and incubated at room temperature in the dark for 24 hours. After extraction is
complete the tissue inserts are pierced with forceps and 2 x 200µL aliquots of the blue formazan solution is
transferred into a 96 well plate for Optical Density reading. The spectrophotometer reads the 96-well plate
using a wavelength of 570 nm.
V. Acceptance Criterion
A. Negative Control
The results of this assay are acceptable if the mean negative control Optical Density (OD 570) is ≥ 0.8.
B. Positive Control
The assay meets the acceptance criterion if a dose dependent reduction in cell viability in the UVA-irradiated
tissues is between 0.00316% and 0.0316%.
C. Standard Deviation
Since the phototoxicity potential is predicted from the mean viability of 2 tissues for the EpiDerm™
Phototoxicity Protocol, the variability of the replicates should not exceed 30%.
VI. Results
A. Tissue Characteristics
The tissue inserts included in the MatTek EpiDerm™ assay kit were in good condition, intact, and viable.
B. Tissue Viability Assay

The results are summarized in Figure 1. Cell viability is calculated for each tissue as a percentage of the
corresponding vehicle control either irradiated or non-irradiated. Tissue viability was not reduced by 20% in
the presence of the test substance and UVA-irradiation at concentrations of 0.4%, 1.23%, and 3.7%. The
negative control mean exhibited acceptable relative tissue viability while the positive control exhibited dose
dependent loss of tissue viability and cell death.
C. Test Validity
The data obtained from this study met criteria for a valid assay. The negative and positive controls performed
as anticipated.

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VII. Conclusion
Phototoxicity (photoirritation) is defined as an acute toxic response that is elicited after exposure of the skin to
certain chemicals and subsequent exposure to light. Under the conditions of this assay, the test article substance
was considered to be non-phototoxic at concentrations of 0.4%, 1.2%, and 3.7%. There is a decrease in
viability at the 11% test concentration with and without irradiation but this concentration is significantly higher
than the suggested use levels. We can safely say that Leucidal® Liquid is not a photoirritant when used at the
suggested use levels of 2 – 4%.
EpiDerm Phototoxicity Assay

M15008 Leucidal® Liquid Without Irradiation
120 With Irradiation
Tissue Viability (% Untreated Control)
100
80
60
40
20
0
0.4% 1.2% 3.7% 11%
Control Leucidal® Liquid
Without Irradiation 100 96.5 96.1 101.8 72.6
With Irradiation 100 101 94.1 98.5 51.1
Figure 1: EpiDerm Phototoxicity Graph

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The following report evaluates a sample of
Leucidal® Liquid (M15008)* – AMA Lab No. L-2090
Provided by Active Concepts, LLC to AMA Laboratories, Inc.
Utilizing the Repeat Insult Patch Test

Skin Irritation / Sensitization Evaluation (Occlusive Patch)
February 18, 2008
*The test material was received as a 10% Dilution in Water

Memorandum


DATE: May 7, 2021
Active Microtechnologies. 2021. Composition Leucidal® Liquid (Leuconostoc/Radish Root Ferment

Filtrate).
Active Microtechnologies. 2020. Specifications Leucidal® Liquid (Leuconostoc/Radish Root

Ferment Filtrate).

(704) 276-7100  Fax (704) 276-7101
May 6, 2021
To whom it may concern,
This letter is to certify that Leucidal® Liquid (M15008), manufactured by Active Micro Technologies, LLC,
located at 107 Technology Drive, Lincolnton, NC 28092, is standardized for 0.10 – 0.50% Bacteriocins, 18.0
– 22.0% Phenolics and 48.0 -52.0% Solids as noted on the product Specification.
Solids often refers to the amount of dry extract within a tested liquid. The typical chemical composition of
the solids and water content within Leucidal® Liquid is as follows:
Content Percentage (%)

Water Content 48.80%
Protein Content 30.60%
Phenolics Content* 20.10%
Polysaccharide Content 0.50%
Total 100.00%
*tested as salicylic acid
We further certify that Leucidal® Liquid does not contain detectable amounts of the five organic acids tested
for via HPLC analysis, such as Citric Acid, Malic Acid, Tartaric Acid, Lactic Acid, and Glycolic Acid.
Please note that the values above are based on testing of one particular lot. All other lots may not be an
exact match to these particular values. Leucidal® Liquid is not standardized for protein or polysaccharide
content at this time.
Thank you for your interest in Active Micro Technologies’ products. If you have any further questions, feel
free to contact us at (704) 276-7100.
Best Regards,
Tomorrow's Vision... Today!®

Heather Ferguson |R&D Coordinator
107 Technology Drive | Lincolnton, NC 28092
Direct: 704.276.7083|Main: 704.276.7100|Fax: 704.276.7101
Email: [email protected]
www.activeconceptsllc.com
Version#1/05-06-21

(704) 276-7100  Fax (704) 276-7101
US Patent Number 10,159,708
US Patent Application Number 16/205,883
Specification
Product Name: Leucidal® Liquid
Code Number: M15008
CAS #’s: 1686112-10-6 (or) 84775-94-0
EINECS #’s: N/A (or) 283-918-6
INCI Name: Leuconostoc/Radish Root Ferment Filtrate
Specification Parameter
Appearance Clear
FreetoFlowing
Slightly Powder
Hazy Liquid
Color Yellow to Light Amber
Odor Characteristic
Solids (1g-105°C-1hr) 48.0

94.0%
– 52.0%
Minimum
pH 4.04.0
– 6.0
- 6.0
Specific Gravity (25°C) 1.140 – 1.180
Ninhydrin Positive
Characteristic 2.0 - 5.0%
Phenolics (tested as Salicylic Acid)¹ 18.0 – 22.0%
Heavy Metals < 20 ppm

Lead < 10 ppm
Arsenic < 2 ppm
Cadmium < 1 ppm
Bacteriocins (HPLC) 0.10 – 0.50%
Minimum Inhibitory Concentration2

Organism (ATCC#)
E. coli (#8739) 0.50 – 4.00%
S. aureus (#6538) 0.25 – 2.00%
P. aeruginosa (#9027) 1.00 – 4.00%
C. albicans (#10231) 0.25 – 2.00%
A. brasiliensis (#16404) 0.25 – 2.00%
Version#23/06-08-20/Form#1

(704) 276-7100  Fax (704) 276-7101
US Patent Number 10,159,708
US Patent Application Number 16/205,883
DO NOT FREEZE; Store at or near room temperature;

Mix well prior to use; May Sediment upon Standing
Product may change appearance if exposed to cold temperatures during shipment or storage.
If this happens, please gently warm to 45-50°C and mix until normal appearance is restored.
Note:
1) Phenolic compounds of natural origin, tested as Salicylic acid via USP HPLC method.
2) Refer to Inhibition Activity Data
Version#23/06-08-20/Form#1
2021 VCRP Frequency of Use Data – Radish Root-Derived Ingredients

Ingredient Name Category code and Description CPIS
count
Lactobacillus/Radish Root Ferment Filtrate
Total: 2
Lactobacillus/Radish Root Ferment Filtrate 12F- Moisturizing 2

Total: 255
Leuconostoc/Radish Root Ferment Filtrate 01B - Baby Lotions, Oils, Powders, and 1
Creams
Leuconostoc/Radish Root Ferment Filtrate 03G - Other Eye Makeup Preparations 3
Leuconostoc/Radish Root Ferment Filtrate 04E - Other Fragrance Preparation 5
Leuconostoc/Radish Root Ferment Filtrate 05A - Hair Conditioner 14
Leuconostoc/Radish Root Ferment Filtrate 05E - Rinses (non-coloring) 1
Leuconostoc/Radish Root Ferment Filtrate 05F - Shampoos (non-coloring) 8
Leuconostoc/Radish Root Ferment Filtrate 05G - Tonics, Dressings, and Other Hair 3
Grooming Aids
Leuconostoc/Radish Root Ferment Filtrate 07A - Blushers (all types) 3
Leuconostoc/Radish Root Ferment Filtrate 07E - Lipstick 9
Leuconostoc/Radish Root Ferment Filtrate 07I - Other Makeup Preparations 7
Leuconostoc/Radish Root Ferment Filtrate 10A - Bath Soaps and Detergents 30
Leuconostoc/Radish Root Ferment Filtrate 10B - Deodorants (underarm) 1
Leuconostoc/Radish Root Ferment Filtrate 11F - Shaving Soap 3
Leuconostoc/Radish Root Ferment Filtrate 12A - Cleansing 11
Leuconostoc/Radish Root Ferment Filtrate 12C - Face and Neck (exc shave) 39
Leuconostoc/Radish Root Ferment Filtrate 12D - Body and Hand (exc shave) 1
Leuconostoc/Radish Root Ferment Filtrate 12F - Moisturizing 104
Leuconostoc/Radish Root Ferment Filtrate 12G - Night 2
Leuconostoc/Radish Root Ferment Filtrate 12H - Paste Masks (mud packs) 3
Leuconostoc/Radish Root Ferment Filtrate 12I - Skin Fresheners 2
Leuconostoc/Radish Root Ferment Filtrate 12J - Other Skin Care Preps 5

Total: 2
Raphanus Sativus (Garden Radish) Root 12C - Face and Neck (exc shave) 2
Extract

Radish Root Ferment Extract

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Safety Assessment of Radish Root – Derived Ingredients

Status: Draft Report for Panel Review

© Cosmetic Ingredient Review

SAFETY ASSESSMENT FLOW CHART

MEETING ___December 2021 ___________________________________________________

Public Comment CIR Expert Panel Report Status

2021 Priority List

IDA Notice IDA

Draft FR DRAFT FINAL REPORT

60 day Public comment period

Table Different Conclusion

Commitment & Credibility since 1976

data3_RadishRoot_122021 (all for Leuconostoc/Radish Root Ferment Filtrate)

CIR History of:

Radish Root-Derived Ingredients

-Frequency of use data obtained

-Concentration of use data submitted by Council

-SLR Notice to Proceed was issued

Data received (for Leuconostoc/Radish Root Ferment Filtrate):

-A Draft Report is being presented to the Panel.

[Radish Root-Derived Ingredients]

Botanical and/or Fragrance Websites (if applicable)

General Web Search (Google)

Fermented radish dermal safety – 3,460,000/2

Lactobacillus/Radish Root Ferment Extract Filtrate – 74,300 hits/0 useful

Lactobacillus/Radish Root Ferment Filtrate chemical properties – 9 results/ 0 useful

Lactobacillus/Radish Root Ferment Filtrate genotoxicity – 30 results/ 0 useful

Leuconostoc/Radish Root Ferment Filtrate– 289, 000 results/ 0 useful

Leuconostoc/Radish Root Ferment Lysate Filtrate – 25, 900 results/ 0 useful

Leuconostoc/Radish Root Ferment Lysate Filtrate toxicokinetics – 8 results/ 0 useful

Raphanus Sativus (Radish) Extract – 200,000 results/ 5 useful

Raphanus Sativus (Radish) Root Juice – 67,100 results/ 4 useful

Raphanus Sativus (Radish) Root Powder – 81,800 results/ 4 useful

Raphanus Sativus (Radish) Root Powder carcinogenicity – 42 results/ 0 useful

appropriate qualifiers are used as necessary

 AICIS (Australian Industrial Chemicals Introduction Scheme)- https://www.industrialchemicals.gov.au/

 International Programme on Chemical Safety http://www.inchem.org/

Botanical Websites, if applicable

Fragrance Websites, if applicable

Safety Assessment of Radish Root – Derived Ingredients

Status: Draft Report for Panel Review

© Cosmetic Ingredient Review

DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES

OTHER RELEVANT STUDIES

DERMAL IRRITATION AND SENSITIZATION STUDIES

OCULAR IRRITATION STUDIES

Table 2. Chemical properties of Leuconostoc/Radish Root Ferment Filtrate

Color Yellow to light amber 6

Specific Gravity (@ 25 ºC) 1.140 – 1.180 6

log Kow; Kow -1.92; 0.013 7

Table 4. Raphanus sativus – derived ingredients not reported to be in use16,17

Leuconostoc/Radish Root Ferment Filtrate Lactobacillus/Radish Root Ferment Extract

Ingredient Product Category Maximum

Information collected in 2020

TO: Bart Heldreth, Ph.D.

FROM: Carol Eisenmann, Ph.D.

DATE: April 29, 2021

SUBJECT: Leuconostoc/Radish Root Ferment Filtrate

eyebrow gel containing 0.04%

TO: Bart Heldreth, Ph.D.

MEETING _December 2021 _________________________________________________