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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, 2016 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay usingS.typhimuriumstrains TA98, TA100, TA1535, TA1537 andE.colistrainWP2uvrA-in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre-incubation methods 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 1.5 to 5000 µg/plate,two bacterial strains (TA100 and TA1535 dosed in the absence of S9-mix) exhibited excessive toxicity and required a repeat experiment employing an amended dose range of 0.05 to 150 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 1500 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.  All 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. The maximum dose level of the test recommended in the first experiment was selected as the maximum recommended dose level of 5000 µg/platealthough two bacterial strains (TA100 and TA1535 dosed in the absence of S9-mix) exhibited excessive toxicity and a repeat experiment was performed using the toxicity of the test item as the maximum dose. Inthe first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all the tester strains dosed in the absence of S9-mix, initially from 50 μg/plate (TA100 and TA1535) and 150 μg/plate (TA98, TA1537 and WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted to all the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item was employed as the maximum dose in the second mutation test. In the second mutation test, the test item induced an identical toxic response with weakened bacterial background lawns initially noted from50 μg/plate (absence of S9-mix) and 500 μg/plate (presence S9-mix). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix. A light, globular precipitate was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies. There were no toxicologically significant 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 significant 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 (pre-incubation method). Small but statistically significant increase in TA98 revertant colony frequency were observed in the first mutation test at 15 and 50 μg/plate in the absence of S9-mix, this was considered to be of non biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle control.It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response inS.typhimuriumstrains TA98, TA100, TA1535, TA1537 and E.coli strainWP2uvrA-in the presence and absence of S-9 mix.

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:
10 August 2016 to 20 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines (OECD 471) and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Physical state / Appearance: Colorless liquid
Storage Conditions: Room temperature in the dark under nitrogen
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. Two of the bacterial tester strains (TA100 and TA1535 dosed in the absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, and150 μg/plate.
Experiment 2:
TA100 and TA1535 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
TA100 and TA1535 (with S9-mix), WP2uvrA, TA98 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
TA98, WP2uvrA and TA1537 (with S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
Vehicle / solvent:
Vehicle (s)/solvent (s) used: Dimethyl sulphoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study - (positive result);
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations. Page 16
Report Envigo Study Number: GP71PC
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
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
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Additional information on results:
All positive, negative and vehicle controls were within laboratory historical values.
Conclusions:
Under the condition of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay usingS.typhimuriumstrains TA98, TA100, TA1535, TA1537 andE.colistrainWP2uvrA-in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre-incubation methods 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 1.5 to 5000 µg/plate,two bacterial strains (TA100 and TA1535 dosed in the absence of S9-mix) exhibited excessive toxicity and required a repeat experiment employing an amended dose range of 0.05 to 150 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 1500 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.  All 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. The maximum dose level of the test recommended in the first experiment was selected as the maximum recommended dose level of 5000 µg/platealthough two bacterial strains (TA100 and TA1535 dosed in the absence of S9-mix) exhibited excessive toxicity and a repeat experiment was performed using the toxicity of the test item as the maximum dose. Inthe first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all the tester strains dosed in the absence of S9-mix, initially from 50 μg/plate (TA100 and TA1535) and 150 μg/plate (TA98, TA1537 and WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted to all the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item was employed as the maximum dose in the second mutation test. In the second mutation test, the test item induced an identical toxic response with weakened bacterial background lawns initially noted from50 μg/plate (absence of S9-mix) and 500 μg/plate (presence S9-mix). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix. A light, globular precipitate was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies. There were no toxicologically significant 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 significant 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 (pre-incubation method). Small but statistically significant increase in TA98 revertant colony frequency were observed in the first mutation test at 15 and 50 μg/plate in the absence of S9-mix, this was considered to be of non biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle control.It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response inS.typhimuriumstrains TA98, TA100, TA1535, TA1537 and E.coli strainWP2uvrA-in the presence and absence of S-9 mix.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The negative results observed in the OECD 471 bacterial reverse mutation assay (Anon. 2016) indicates that based on available data, the substance does not meet the criteria for classification as mutagenic in accordance with the GHS and CLP criteria.