[No public or meaningful name is available]

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:

August 23, 2018 to September 28, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic Acids, C18-alkane hydroxyl and C18-alkene, potassium salts

Cas Number:

2210238-56-3

IUPAC Name:

Sulfonic Acids, C18-alkane hydroxyl and C18-alkene, potassium salts

Test material form:

cream / paste

Details on test material:

Stability: Stable under storage condition
Storage condition: Room temperature, tight container, dark place

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

To set the dose levels for the main tests, 50 mg/mL solution was diluted 4 times at a common ratio of 4 and a total of 5 dose levels were selected (19.5, 78.1, 313, 1250 and 5000 µg/plate) in the dose-finding test.
From the result, precipitation of the test article on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation.
The details of result shown in Table1 of attachments.

Vehicle / solvent:

Distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

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:

For the test article treatment group, negative control group and positive control group, the number of plates used at each dose level was 2 in the dose-finding test, while it was 3 in the two main tests.


1. The test article solution, vehicle or positive control article (0.1 mL of each) was placed into a sterilized small test tube, 0.5 mL of 0.1 mol/L phosphate buffer(pH 7.4) for the system without metabolic activation or 0.5 mL of S9 Mix for the system with metabolic activation was added, and then 0.1 mL of bacterial suspension was added to each tube.
2. Each mixture was pre-incubated while shaking (80 rpm) at 37°C for 20 minutes. After pre incubation, 2.0 mL of top agar kept at 45°C was added to each tube, and this mixture was shaken and overlaid uniformly on the minimal glucose agar plate medium.
3. For a sterility test, 0.1 mL of the test article solution at the highest dose or 0.5 mL of S9 Mix was measured in a small test tube, and after 2.0 mL of top agar was added, it was overlaid uniformly on the minimal glucose agar plate medium. These processes, 1 to 3, were performed under fluorescent lamps with ultraviolet-absorbing films.
4. After verifying that the overlaid top agar was solidified, the minimal glucose agar plate medium was put upside down in an incubator and incubated at 37°C for 48 hours for the dose-finding test and the first main test, and for 48.5 hours for the second main test.
5. After incubation, the culture was observed for the presence or absence of precipitation of the test article on the plate. The number of revertant colonies was counted with an automatic colony counters (Colony Analyzer CA-11D systems, System Science Co., Ltd.) (Area correction, correction factor: 1.21). The presence or absence of growth inhibition of the bacteria was observed using a stereoscopic microscope.

Evaluation criteria:

If a two-fold or more dose-dependent increase in the number of revertant colonies compared to that of spontaneous revertant colonies (the negative control value) and reproducibility were noted, or even if no clear dose-response was observed but there were at least two-fold increase in the number of revertant colonies in comparison with that of spontaneous revertant colonies and reproducibility in the two main tests or additional confirmation test, the test article was judged to be positive. For the results of the main tests, the mean ± S.D. was also recorded.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The detail of results shown in table2 -7 of attachments.

Applicant's summary and conclusion

Conclusions:

In conclusion the test substance was judged to have no potential to induce gene mutations (negative) under the conditions of this study.

Executive summary:

In order to examine the mutagenic potential of Sulfonic acids, C18-alkane hydroxy and C18-alkene, potassium salts, a reverse mutation assay 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. Distilled water was used as the vehicle for the test article.

A dose-finding test was conducted at dose levels between 19.5 and 5000 µg/plate. From the result, the minimum dose which showed growth inhibition was selected as the maximum dose for the main test, and the main test was conducted at 8 dose levels between 0.15 and 19.5 µg/plate in S. typhimurium TA100, TA1535 and TA1537 without metabolic activation, at 6 dose levels between 2.44 and 78.1 µg/plate in S. typhimurium TA98 without metabolic activation, at 6 dose levels between 9.77 and 313 µg/plate in S. typhimurium TA100, TA1535 and TA1537 with metabolic activation, and at 6 dose levels between 39.1 and 1250 µg/plate in S. typhimurium TA98 with metabolic activation. In the case of E. coli WP2 uvrA with or without metabolic activation, the main test were conducted at 5 dose levels between 313 and 5000 µg/plate, because growth inhibition was not observed. The main test was conducted twice at the same dose levels.

 

-Precipitation by Test Article

Precipitation of the test article on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation.

 

-Growth Inhibition

In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 2.44 µg/plate or more in S. typhimurium TA1535 and TA1537 without metabolic activation, at 9.77 µg/plate or more in S. typhimurium TA100 without metabolic activation, at 39.1 µg/plate or more in S. typhimurium TA98 without metabolic activation, at 156 µg/plate or more in S. typhimurium TA100, TA1535 and TA1537 with metabolic activation and at 625 µg/plate or more in S. typhimurium TA98 with metabolic activation.

 

-Number of Revertant Colonies

In the two main tests, there was no dose-dependent increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group in any strains irrespective of the presence/absence of metabolic activation.

 

In conclusion, Sulfonic acids, C18-alkane hydroxy and C18-alkene, potassium salts was judged to have no potential to induce gene mutations (negative) under the conditions of this study.