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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2014 to 23 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: There were no deviations (unplanned changes) from the study plan.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
No
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Clear colourless liquid
- Storage conditions: Room temperature in the dark

Method

Target gene:
TA1537: his C 3076; rfa-; uvrB-
TA98: his D 3052; rfa-;uvrB-; R-factor
TA1535: his G 46; rfa-; uvrB-
TA100: his G 46; rfa-; uvrB-; R-factor
WP2uvrA: trp-; uvrA-
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/beta-Naphthoflavone
Test concentrations with justification for top dose:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
The test item was miscible in dimethyl sulphoxide (after an extended period) and acetone at 50 mg/mL in solubility checks performed in-house
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following theaddition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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.
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, initially from 150 and 500 μg/plate in the absence and presence of 89-mix respectively. These results meant that the test item was tested up to the toxic limit in Experiment 2. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns initially noted from 150 and 500 μg/plate in the absence and presence of 89-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without 89-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of 89-mix.
There were no 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 in the first mutation test. 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 in Experiment 2.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the 89-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

GUIDELINE

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.

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 1.5 to 5000 µg/plate. The experiment was repeated 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 ranged between 0.5 and 1500 µg/plate, depending on presence or absence of S9-mix.

Up to seven test item dose levels were selected in Experiment 2 in arder to achieve both four non-toxic dose levels and the toxic limit of the test item.

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.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. These results meant that the test item was tested up to the taxie limit in Experiment 2. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns initially noted from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. 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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no 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 in the first mutation test. 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 in Experiment 2.

CONCLUSION

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