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Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

semi-solid (amorphous): gel

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Any other information on results incl. tables

In the first experiment, the maximum dose level of the test item was selected as 5000 μg/plate (the maximum recommended concentration) for all of the bacterial strains dosed in the presence of S9-mix (except TA100) and WP2uvrA dosed in the absence of S9-mix. Due to excessive test item toxicity, the toxic limit was set for all of the Salmonella bacterial tester strains dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains dosed in the absence of S9-mix from 50 μg/plate (TA100 and TA1537) and 150 μg/plate (TA1535 and TA98). Escherichia coli strain WP2uvrA exhibited substantial reductions in colony frequency at 5000 μg/plate. In the presence of S9-mix, weakened background lawns were initially noted from 150 μg/plate for Salmonella strains TA100 and TA1537 and 500 μg/plate for TA98 and TA1535. Escherichia coli strain WP2uvrA exhibited substantial reductions in colony frequency at 5000 μg/plate.

In the second experiment, the maximum recommended dose level or the toxic limit was employed as the maximum concentration, depending on bacterial strain type and absence or presence of S9-mix. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns initially noted at 50 μg/plate in both the presence and absence of S9-mix.

A test item precipitate was observed under a low power microscope at 1500 μg/plate and by eye at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small statistical value was noted in Experiment 1 (WP2uvrA at 15 μg/plate in the presence of S9-mix), however this response was within the in-house historical vehicle/untreated control value for the strain and was, therefore considered of no biological relevance.

Applicant's summary and conclusion

Conclusions:

Test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

1.1 Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

1.2 Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was initially 1.5 to 5000 μg/plate. However, the test item exhibited excessive toxicity and consequently, there were an insufficient number of non-toxic dose levels for all of the Salmonella bacterial tester strains dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix. Therefore, these strains were repeated using an amended dose range of 0.05 to 150 μg/plate. Experiment 2 was perormed on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 (and repeat) and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and absence or presence of S9-mix. Up to eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non-toxic dose levels.

1.3 Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment, the maximum dose level of the test item was selected as 5000 μg/plate (the maximum recommended concentration) for all of the bacterial strains dosed in the presence of S9-mix (except TA100) and WP2uvrA dosed in the absence of S9-mix. Due to excessive test item toxicity, the toxic limit was set for all of the Salmonella bacterial tester strains dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains dosed in the absence of S9-mix from 50 μg/plate (TA100 and TA1537) and 150 μg/plate (TA1535 and TA98). Escherichia coli strain WP2uvrA exhibited substantial reductions in colony frequency at 5000 μg/plate. In the presence of S9-mix, weakened background lawns were initially noted from 150 μg/plate for Salmonella strains TA100 and TA1537 and 500 μg/plate for TA98 and TA1535. Escherichia coli strain WP2uvrA exhibited substantial reductions in colony frequency at 5000 μg/plate.

In the second experiment, the maximum recommended dose level or the toxic limit was employed as the maximum concentration, depending on bacterial strain type and absence or presence of S9-mix. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns initially noted at 50 μg/plate in both the presence and absence of S9-mix.

A test item precipitate was observed under a low power microscope at 1500 μg/plate and by eye at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small statistical value was noted in Experiment 1 (WP2uvrA at 15 μg/plate in the presence of S9-mix), however this response was within the in-house historical vehicle/untreated control value for the strain and was, therefore considered of no biological relevance.