Registration Dossier

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:
2015-06-19 till 2015-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 471 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: US Food and Drug Administration, Center for Food Safety & Applied Nutrition, Redbook 2000 Toxicological Principles for the safety of Food Ingredients. IV.C.1.a. Bacterial Reverse Mutation Test, July 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl 2-methylbenzoate
EC Number:
201-932-2
Cas Number:
89-71-4
Molecular formula:
C9H10O2
IUPAC Name:
methyl 2-methylbenzoate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Ylanganate
- Storage condition of test material: ambient temperature (15-25°C)
- Appearance: clear, colourless liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate (S9-mix)
Test concentrations with justification for top dose:
First bacterial reverse mutation test:
Experiment 1: 0, 21, 62, 185, 556, 1667, 5000 µg/plate (all strains)
Experiment 2: 0, 21, 62, 185, 556, 1667, 5000 µg/plate (strain TA1535 and TA100 without S9-mix)
Experiment 3: 0, 21, 62, 185, 556, 1667, 5000 µg/plate (strain TA 1535 without S9-mix)

Second bacterial reverse mutation test
Experiment 1: 51, 128, 320, 800, 2000, 3500, 5000 µg/plate (all strains)
Experiment 2: 51, 128, 320, 800, 2000, 3500, 5000 µg/plate (strain TA1535, E. coli WP2 uvr A without S9-mix)
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
ethylnitrosurea
other: 2-aminoanthracene, N-ethyl-N-nitrosourea
Remarks:
In the absence of S9-mix: sodium azide: TA 1535 and TA 100; 9-aminoacridine: TA 1537; 2-nitrofluorene: TA 98; N-ethyl-N-nitrosourea: WP2uvrA. In the presence of S9-mix: 2-aminoanthracene: TA 1535, TA 98, TA 100, WP2uvrA; benzo(a)pyrene: TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours at ca. 37 °C

NUMBER OF REPLICATIONS: All determinations were made in triplicate.

DETERMINATION OF CYTOTOXICITY
- Toxicity was defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (solvent) control and/or the occurrence of pinpoint colonies.
Evaluation criteria:
The study was considered valid if the mean colony counts of the vehicle control values of the strains were within the acceptable ranges, if the results of the positive controls met the criteria for a positive response, if no more than 5 % of the plates was lost through contamination or other unforeseen events and if at least three concentrations were non-toxic.

A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner or if a two-fold or greater increase was observed compared to the negative control plates. A clear positive response did not need to be verified. Marginally or weakly positive results should be verified by additional testing.

A test substance was considered to be negative in the bacterial gene mutation test if it showed neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the concentrations tested.

Positive results from the bacterial reverse mutation test indicate that a test substance induces point mutations by base pair substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that, under the test conditions used, the test substance is not mutagenic in the tested strains. Although most studies give clearly positive or negative results, in rare cases the data set may preclude making a definite judgement about the mutagenic potential of the test substance. Results may remain equivocal in this case.

Both numerical significance and biological relevance were considered together in the evaluation.
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
The first test included three experiments. In the first experiment, for strain TA 1535 and TA 100, both in the absence of S9-mix, the negative control was outside the acceptable range. Therefore, the experiment was repeated for these strains. In the second experiment for strain TA 1535 (-S9-mix), the negative control was outside the historical range. Therefore the experiment was repeated for this strain The second test included two experiments. In the first experiment, for strains TA 1535 and WP2 uvrA, both in the absence of S9-mix, the negative control was outside the acceptable range. Therefore, the experiment was repeated for these strains.

In both the first and second test, negative controls (vehicle) and positive controls were run simultaneously with the test substance. In some of the experiments, the mean numbers of his+ and trp+ revertant colonies of the negative control in some of the strains appeared to be outside the acceptable range. Therefore, the experiments were repeated until the acceptance criteria were met. Finally, both tests of this study were considered valid.

In both the first and second test, the test substance was found toxic to all strains tested, both in the absence and presence of S9-mix. In the first test, toxicity was observed at and above 1667 μg/plate and at 5000 μg/plate for the Salmonella strains and WP2 uvrA, respectively. In the second test, toxicity was observed at and above 800 μg/plate for TA 1537, in the absence of S9-mix and at and above 2000 μg/plate for all other strains tested. In all experiments, toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls and/or pinpoint colonies.

In all experiments, a dose related precipitation of the test substance in the final treatment mix (top agar) was observed. Precipitation was observed in all mixtures at and above 1667 μg/plate (first test) and 2000 μg/plate (second test) with the unaided eye.

In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the test conditions (OECD 471, GLP) the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance is not mutagenic.
Executive summary:

According to OECD guideline 471 and GLP, the test substance, dissolved in DMSO, was examined for its possible mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). Two independent bacterial reverse mutation tests were performed. In the first test, three independent experiments were performed where six concentrations of the test substance ranging from 21 to 5000 μg/plate were tested. In the second test, two independent experiments were performed where seven concentrations of the test substance ranging from 51 to 5000 μg/plate were tested. In both the first and second test, negative controls (vehicle) and positive controls were run simultaneously with the test substance. In some of the experiments, the mean numbers of his+ and trp+ revertant colonies of the negative control in some of the strains appeared to be outside the acceptable range. Therefore, the experiments were repeated until the acceptance criteria were met. Finally, both tests of this study were considered valid. In both the first and second test, the test substance was found toxic to all strains tested, both in the absence and presence of S9-mix. In the first test toxicity was observed at and above 1667 μg/plate and at 5000 μg/plate for the Salmonella strains and WP2 uvrA, respectively. In the second test, toxicity was observed at and above 800 μg/plate for TA 1537, in the absence of S9-mix and at and above 2000 μg/plate for all other strains tested. In all experiments toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls and/or pinpoint colonies. In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and presence of the S9 mix, indicate that the test substance is not mutagenic under the conditions used in this study.