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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was considered to be non-mutagenic under the conditions of the Ames test (OECD 471 and EU Method B.13/14).

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:
01 February 2018 to 19 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Vehicle / solvent:
Dimethyl sulfoxide
Untreated negative controls:
yes
Remarks:
dimethyl sulfoxide
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
5 μg/plate for TA1535; solvent saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191
Remarks:
2.5 μg/plate for TA1537; solvent DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
10 μg/plate for TA98; solvent DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
650 μg/plate for TA100; solvent DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 μg/plate for WP2uvrA; solvent DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.5 μg/plate for TA1535 (5% and 10% S9) and TA1537 (5% S9); 5 μg/plate for TA1537 (10% S9); 1 μg/plate for TA98 (5% and 10% S() and TA100 (5% S9); 2 μg/plate for TA100 (10% S9); 15 μg/plate for WP2uvrA (5% and 10% S9); solvent DMSO in all cases
Details on test system and experimental conditions:
- Range-finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1 without metabolic activation: 0.55, 1.7, 5.4, 17, 52, 164 μg/plate
- Experiment 1 with metabolic activation: 5.4, 17, 52, 164, 512, 1600 μg/plate
- Experiment 2 without metabolic activation: 3.1, 6.3, 13, 25, 50, 100 μg/plate
- Experiment 2 with metabolic activation: 63, 125, 250, 500, 1000, 2000 μg/plate
Evaluation criteria:
ACCEPTABILITY CRITERIA
- A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
- All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Statistics:
INTERPRETATION
- No formal hypothesis testing was done.
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
- A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Additional information on results:
DOSE-RANGE FINDING AND FIRST MUTATION EXPERIMENT
- The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
- Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98:
(i) Absence of S9-mix: 0.55, 1.7, 5.4, 17, 52, 164 μg/plate.
(ii) Presence of S9-mix: 5.4, 17, 52, 164, 512, 1600 μ/plate.
- The results are shown in Table 1 and Table 2 (attached).
- Precipitation of test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards.
- To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated in Appendix 2 (attached).
- Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. Except in tester train TA98 in the presence of S9-mix and in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed.

SECOND MUTATION EXPERIMENT
- To obtain more information about the possible mutagenicity of test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix.
- Based on the results of the first mutation assay, the test item was tested up to the dose level of 100 and 1500 μg/plate in the absence and presence of S9-mix, respectively, for tester strains TA1535, TA1537, TA98 and TA100. The test item was tested up to the dose level of 2000 μg/plate in tester strain WP2uvrA in the absence and presence of S9-mix.
- The results are shown in Table 3 (attached).
- Precipitation of test item on the plates was observed at the start of the incubation period at the concentration of 1000 μg/plate and upwards and at the end of the incubation period at the top dose of 100 µg/plate in the absence of S9-mix and at the top dose of 1500 µg/plate in the presence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at dose levels of 250 μg/plate and upwards in the absence and presence of S9-mix in tester strain WP2uvrA.
- Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. Except in tester train TA98 in the presence of S9-mix and in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed.
- In the second mutation assay, no increase in the number of revertants was observed upon treatment with test item under all conditions tested.
Conclusions:
Based on the results of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in theEscherichia colireverse mutation assay.
Executive summary:

GUIDELINE

The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains ofSalmonella typhimurium(S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9). The investigation was performed in accordance with OECD Guideline 471.Genetic Toxicology: Bacterial Reverse Mutation Test (adopted July 21, 1997) and EC Guideline No. 440/2008 Part B: Methods for the Determination of Toxicity andother health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”:Official Journal of the European Union No. L142, 31 May 2008.

 

METHODS

The test material was a clear colourless liquid. The vehicle of the test item was dimethyl sulfoxide. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 1600μg/plate and upwards in the absence and presence of S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay. Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence of S9-mix and at a concentration range of 5.4 to 1600 μg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose level of 1600 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix except in tester strain TA98 in the presence of S9-mix where no toxicity was observed. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 3.1 to 100 μg/plate in the absence of S9-mix and at a concentration range of 47 to 1500 μg/plate in the presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and TA100. The test item was tested at a concentration range of 63 to 2000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strain WP2uvrA.The test item precipitated on the plates at the top dose of 100μg/platein the absence of S9-mix and at the top dose of 1500μg/platein the presence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100. The test item precipitated at dose levels of 250 μg/plate and upwards in the absence and presence of S9-mix in tester strain WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix except in tester strain TA98 in the presence of S9-mix and in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed.

 

RESULTS

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly.

 

CONCLUSION

Based on the results of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in theEscherichia colireverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

GUIDELINE

The objective of the study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus ofEscherichia coli(E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9). The investigation was performed in accordance with OECD Guideline 471.Genetic Toxicology: Bacterial Reverse Mutation Test(adopted July 21, 1997) and EC Guideline No. 440/2008 Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”: Official Journal of the European Union No. L142, 31 May 2008.

METHODS

The test material was a clear colourless liquid. The vehicle of the test item was dimethyl sulfoxide. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test material precipitated on the plates at dose levels of 1600μg/plate and upwardsin the absence and presence of S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay. Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence of S9-mix and at a concentration range of 5.4 to 1600 μg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98.The test itemprecipitated on the plates at the top dose level of 1600 μg/plate. Cytotoxicity, as evidenced by adecrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix except in tester strain TA98 in the presence of S9-mix where no toxicity was observed. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 3.1 to 100 μg/plate in the absence of S9-mix and at a concentration range of 47 to 1500 μg/plate in the presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and TA100.The test item was tested at a concentration range of 63 to 2000 μg/plate in the absence andpresence of 10% (v/v) S9-mix in the tester strain WP2uvrA.The test item precipitated on the plates at the top dose of 100μg/platein the absence of S9-mix and at the top dose of 1500μg/platein the presence of S9-mix in testerstrains TA1535, TA1537, TA98 and TA100. The test item precipitated at dose levels of 250 μg/plate and upwards in the absence and presence of S9-mix in tester strain WP2uvrA.Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of thebacterial background lawn and/or the presence of microcolonies, was observed in all testerstrains in the absence and presence of S9-mix except in tester strain TA98 in the presence of S9-mix and in tester strain WP2uvrAin the absence and presence of S9-mix, where no toxicity was observed.

RESULTS

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrAboth in the absence and

presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate,and that the metabolic activation system functioned properly.

 

CONCLUSION

Based on the results of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

The test material was considered to be non-mutagenic under the conditions of the Ames test andclassification under the terms of Regulation (EC) No 1272/2008 is not required.