(carboxylatomethyl)dodecyldimethylammonium

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

(carboxylatomethyl)dodecyldimethylammonium

EC Number:

211-669-5

EC Name:

(carboxylatomethyl)dodecyldimethylammonium

Cas Number:

683-10-3

Molecular formula:

C16H33NO2

IUPAC Name:

(carboxylatomethyl)dodecyldimethylammonium

Test material form:

solid

Details on test material:

Chemical Name Lauryl betaine
Physical State liquid - The sample was dried under a nitrogen purge to remove water prior to analysis.
CAS # 683-10-3
EU # 211-669-5
Manufacturer Colonial Chemical
Batch/lot # 53715J16
Received on November 22, 2016
Tested on January 31, 2017

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.

Method

Species / strain

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Applicant's summary and conclusion

Conclusions:

Acceptance Criteria of Test System
It was judged that this test was conducted appropriately since two-fold or more increase in the number of revertant colonies in comparison with the negative control was observed in the plate treated with the positive control article for each tester strain, the number of revertant colonies in the plates in the negative control group and positive control group were within the range of the control limit of background data (Attached Data), and no contaminants such as other bacteria were seen in this test system.

Discussion on results.
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.