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Diss Factsheets

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)

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)
Version / remarks:
according to EU method B.13/14
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate is included in the Study Report attached
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6-methylbenzotriazole
EC Number:
205-265-8
EC Name:
6-methylbenzotriazole
Cas Number:
136-85-6
Molecular formula:
C7H7N3
IUPAC Name:
6-methylbenzotriazole
Test material form:
solid
Specific details on test material used for the study:
Batch n°: N6A3I
Expiry date: 04 August 2022

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium LT2: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: S9 was obtained from a specialized com-pany Trinova Biochem GmbH, Gießen, Germany and stored at < - 75 °C. Batch numbers: 3850, 3913 and 3833.

Specification S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.

- method of preparation of S9 mix: S9-Mix is a liver enzyme mixture used for the test with metabolic activation. It consists of S9 (liver enzyme mixture), buffer, salts, NADP and Glucose-6-phosphate:
Phosphate buffer: 22.5 mL
0.1M NAPD solution : 1.0 mL
1M G6P solution 0.125 mL
Salt solution: 0.5 mL
Rat liver S9: 1.0 mL

- concentration or volume of S9 mix and S9 in the final culture medium : 500 uL

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
Sterility control: Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37°C +/-1°C, four replicates.
Test concentrations with justification for top dose:
Experiment 1a:
The following concentrations will be tested in the experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.
Signs of toxicity towards all bacteria strains could be observed (decrease in the number of reverstants) in the treatment with and without metabolic activation in the highest concentration (5000 µg/plate) only. Due to the toxicity results a further experiment was performed with lower concentrations.

Experiment 1b:
The following concentrations will be tested in the experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate, 50 µg/plate and 15 µg/plate.

Experiment 2:
he following concentrations will be tested in the first experiment:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 312 µg/plate, 156µg/plate and 78 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
In a non-GLP pre-test, the solubility of the test item was determined in demineralised water, acetone and dimethyl sulfoxide (DMSO). The test item is soluble in a concentration of 50 g/L in DMSO and acetone.
Based on these results, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
The following mutagenic substances were used as positive controls: Nitrophenylendiamine, 20 µg/plate: strains TA97a, TA98 and TA102 without S9 mix Sodium azide, 1 µg/plate: strains TA100 and TA1535 without S9 mix Benzo-a-pyrene, 20 µg/plate: strain TA9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3
- Number of independent experiments: 3

TREATMENT:
For the plate incorporation method, 2 mL liquid top-agar is added to 0.1 mL of the appro-priate solution of the test item, 0.1 mL of the overnight culture of the respective strain and 0.5 mL phosphate buffer or S9 mix.
For the pre-incubation method, 0.1 mL of the solution of the test item are incubated 20 to 30 min. at 37 ± 1 °C with 0.1 mL of the overnight culture of each strain and 0.5 mL of the phosphate buffer or S9 mix at 37 ± 1 °C in the incubation chamber before adding 2 mL liquid top-agar.
The mixture is vortexed gently, then poured on a minimal glucose plate and evenly distributed. The plates are closed, left to solidify for a few minutes, then inverted and placed in a dark incubator at 37 ± 1 °C.
After incubation for 48 – 72 hours, the revertants are counted and the numbers for each plate are recorded.

Evaluation criteria:
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and positive controls. Additionally the absolute number of revertants (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Signs of toxicity was observed (decrease in the number of revertants) in the treatment with and without metabolic activation at highest concentration (5000 µg/plate) only.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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
Remarks:
Signs of toxicity was observed (decrease in the number of revertants) in the treatment with and without metabolic activation at highest concentration (5000 µg/plate) only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Signs of toxicity was observed (decrease in the number of revertants) in the treatment with and without metabolic activation at highest concentration (5000 µg/plate) only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Signs of toxicity was observed (decrease in the number of revertants) in the treatment with and without metabolic activation at highest concentration (5000 µg/plate) only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Signs of toxicity was observed (decrease in the number of revertants) in the treatment with and without metabolic activation at highest concentration (5000 µg/plate) only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Any other information on results incl. tables

In all experiments, no precipitation of the test item 5-Methyl-1H-benzotriazole was observed at any of the tested concentrations up to 5000 µg/plate.

In the all experiments, the test item caused cytotoxicity towards all bacteria strains in the highest concentration (5000 µg/plate), only.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.

All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory see chapter 16, page 47) and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:
The test item 5-Methyl-1H-benzotriazole showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and all strain-specific positive control values were within the laboratory his-torical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that 5-Methyl-1H-benzotriazole is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.