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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the dose-finding test and the two main tests, there was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.

Since two-fold or more increase in the number of revertant colonies in comparison with the negative control group was observed in the positive control group for each tester strain, it was judged that the reactions of the bacterial strains to the mutagenic agents were suitable and thus the study was conducted appropriately. In conclusion, lauryl betaine (C12) had no bacterial gene mutation inducibility (negative) under the conditions of this study.

Cetyl betaine (C16) is essentially the same molecule as lauryl betaine but with an extra C4 in the alkyl chain so it is likely that the toxicology of the two species will be very similar. Hence, it is assessed that the AMES test response for cetyl would be similar to lauryl.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
• “Standards Established by the Minister of Labour in Accordance with the Industrial Safety and Health Law, Article 57-4-1”, (Notification No. 208, December 25, 2016 Ministry of Health, Labour, Japan)
• “Guidelines for Toxicity Testing of New Chemical Articles”, (Notification 1221 No. 1 of Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Heisei 20151209 No. 1 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 1512211 of Environmental Policy Bureau, Ministry of the Environment, Japan, on December 21,2015)
Deviations:
no
Principles of method if other than guideline:
A bacterial reverse mutation test was conducted in Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA100, TA1535, TA98 and TA1537, and Escherichia coli (hereinafter referred to as E. coli) WP2 uvrA with or without metabolic activation by the pre-incubation method to evaluate the presence or absence of gene mutation inducibility of LAURYL BETAINE. Distilled water was used as the vehicle for the test article.
A dose-finding test was conducted with dose levels between 1.22 and 5000 µg/plate to set the dose levels for the main test. From the result of the dose-finding test, the minimum dose which showed growth inhibition was selected as the maximum dose for the main test, and the main tests were conducted at 6 dose levels between 2.44 and 78.1 µg/plate in all S. typhimurium TA strains without metabolic activation, between 9.77 and 313 µg/plate in E. coli WP2 uvrA without metabolic activation, between 9.77 and 313 µg/plate in all S. typhimurium TA strains with metabolic activation and between 39.1 and 1250 µg/plate in E. coli WP2 uvrA with metabolic activation. The main test was conducted twice at the same dose levels for all S. typhimurium TA strains without metabolic activation, because the number of dose levels at which growth inhibition was not observed was less than 4.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Date of Receipt: April 19, 2017
Name: Lauryl dimethylamino acetic betain
Another Name: LAURYL BETAIN
CAS Number: 683-10-3
Lot Number: 53217I16
Purity: 34 wt%
Name and concentration of Impurity:
66 wt% (NaCl: 6-8%, water: about 60%)
Molecular Weight: 271.44
Melting Point: >100°C
Vapor Pressure: 20 mmHg (25°C)
Description at Ordinary Temperature: Transparent liquid (specific gravity: 1.05 (25°C), pH:5-9)
Stability: Fine
Not reactive to light, and not affected by air.
Solubility: Water: 100%
Storage Conditions: Room temperature
Storage Place: The test article storage room at Tokyo Laboratory
Storage Temperature: Measured temperature during the storage period (from April 19, 2017 to May 30, 2017) was between 18.6 and 20.5°C.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
The EColi strain was E. coli WP2 uvrA. These strains were selected since they have high sensitivity to mutagens and are used widely in bacterial mutagenicity studies
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
This test article solution was diluted 6 times using a common ratio of 4 to prepare the test article solution at a total of 7 concentrations: 50, 12.5, 3.13, 0.781, 0.195, 0.0488 and 0.0122 mg/mL.
Vehicle / solvent:
Distilled water
Untreated negative controls:
yes
Remarks:
Distilled water, which was used for preparation of the test solution, was selected as the negative control article.
Negative solvent / vehicle controls:
no
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)- aminopropylamino]acridine・2HCl ; 2-Aminoanthracene
Details on test system and experimental conditions:
Test Methods
Pre-culturing
1) After the frozen bacterial strains were thawed, the bacterial suspensions, 20 µL of each S. typhimurium TA strain or 10 µL of E. coli strain bacteria, were seeded to a sterilized L-shaped tube (48-mL capacity) containing 10 mL of Nutrient Broth No. 2. The culture was set in a thermostatically controlled bath shaker (ML-10 Cool Bath Shaker with PU-6, Taitec Corporation).
2) The culture remained program-controlled at 4°C until the start of pre-culturing (for 6 hours and 30 minutes), and was then pre-cultured while shaking (100 rpm) for 9 hours at 37°C. The bacterial suspensions were discarded after use.
3) At the end of pre-culturing, absorbance was measured by a spectrophotometer (Mini photo 518R, Taitec Corporation) and the level of acceptance for the converted number of bacteria was set at 1×109 cells/mL or higher. The bacterial suspensions were allowed to stand at room temperature until use. Each viable cell count of bacteria is shown in Text Table 3.

Text Table 3 Viable Cell Count of Bacteria
Strains Cell Count of Bacteria (cells/mL)
Dose-finding Test First Main Test Second Main Test
S. typhimurium TA100 2.82 × 109 2.77 × 109 2.74 × 109
S. typhimurium TA1535 3.79 × 109 4.13 × 109 3.81 × 109
E. coli WP2 uvrA 5.50 × 109 5.81 × 109 -
S. typhimurium TA98 4.72 × 109 4.37 × 109 4.41 × 109
S. typhimurium TA1537 3.06 × 109 3.12 × 109 2.97 × 109

Number of Plates
The number of plates used at each dose level was two in both the dose-finding test and the two main tests.

Rationale for test conditions:
Observations of Dose-finding Test and Selection of Dose Levels for Main Test
To set the dose levels for the main test, the 50 mg/mL solution was diluted 6 times using a common ratio of 4 and a total of 7 dose levels were selected (1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate) in the dose-finding test.
In the dose-finding test, neither precipitation nor coloration by the test article on the plate was observed at any dose levels irrespective of the presence/absence of metabolic activation. In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 78.1 µg/plate or more for all S. typhimurium TA strains without metabolic activation, at 313 µg/plate or more for E. coli WP2 uvrA without metabolic activation, at 313 µg/plate or more for all S. typhimurium TA strains with metabolic activation and at 1250 µg/plate or more for E. coli WP2 uvrA with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.
Therefore, for the main tests, the minimum dose which showed growth inhibition was selected as the maximum dose, the maximum dose level was set at 78.1 µg/plate in all S. typhimurium TA strains without metabolic activation, at 313 µg/plate in E. coli WP2 uvrA without metabolic activation, at 313 µg/plate in all S. typhimurium TA strains with metabolic activation and at 1250 µg/plate in E. coli WP2 uvrA with metabolic activation, and a total of 6 dose levels were selected by diluting 5 times at a common ratio of 2. The main test was conducted twice at the same dose levels for all S. typhimurium TA strains without metabolic activation because the number of dose levels at which growth inhibition was not observed was less than 4.
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies compared to that of spontaneous revertant colonies (the negative control) and dose-response and reproducibility were noted, or even if no clear dose-response was observed but there was at least two-fold increase in comparison with the number of spontaneous revertant colonies and reproducibility was observed, the test article was judged to be positive. Statistical method was not applied to the judgment
Statistics:
Not applied
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations of First Main Test
In the First main tests, neither precipitation nor coloration by the test article on the plate was observed at any dose levels irrespective of the presence/absence of metabolic activation. In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 78.1 µg/plate in all S. typhimurium TA strains without metabolic activation, and at 156 µg/plate or more in E. coli WP2 uvrA without metabolic activation, at 313 µg/plate in all S. typhimurium TA strains with metabolic activation and at 625 µg/plate or more in E. coli WP2 uvrA with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.

Observations of Second Main Test
In the Second main tests, neither precipitation nor coloration by the test article on the plate was observed at any dose levels irrespective. In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 78.1 µg/plate in all strains.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response.

Conclusions:
In the dose-finding test and the two main tests, there was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.
Since two-fold or more increase in the number of revertant colonies in comparison with the negative control group was observed in the positive control group for each tester strain, it was judged that the reactions of the bacterial strains to the mutagenic agents were suitable and thus the study was conducted appropriately.
In conclusion, LAURYL BETAIN had no bacterial gene mutation inducibility (negative) under the conditions of this study.
Cetyl betaine is essentially the same molecule as lauryl betaine but with an extra C4 in the alkyl chain so it is likely that the toxicology of the two species will be very similar. Hence, it is assessed that the AMES test response for cetyl would be similar to lauryl.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Cetyl betaine (C16) and Lauryl betaine (C12) are very closely related chemical structures. The parent compound, betaine, is a naturally occurringN-trimethylated amino acid.

The individual betaines differ only in chain length. They are generally UVCB substances being manufactured from naturally occurring plant and animal sources.

 The Cosmetic Ingredient Review Expert Panel reviewed the product use, formulation and safety data of eleven alkyl betaines, as used in cosmetics. (Final report issued 2014-04-04)

The Panel concluded that the common core chemical structure, similar functions and concentrations in cosmetics, and the predicted physicochemical properties enabled grouping these ingredients and reading across the available toxicological data to support the safety assessment of each individual compound in the entire group.

Therefore it is assessed that it is acceptable to apply the results of this test to cetyl betaine.