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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October 2016 - 17 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day.
Test concentrations with justification for top dose:
- Experiment 1:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2, without S9:
TA 100 and TA 1535: 1.5, 5, 15, 50, 150, 300, 500 and 1500 μg/plate
TA 98: 1.5, 5, 15, 50, 150, 200, 250 and 500 μg/plate
TA 1537: 15, 50, 150, 250, 500, 750, 1500 and 5000 μg/plate
WP2uvrA: 5, 15, 50, 150, 500, 750, 1500 and 5000 μg/plate
- Experiment 2, with S9:
TA 100 and TA 1535: 1.5, 5, 15, 50, 150, 300, 500 and 1500 μg/plate
TA 98 and TA 1537: 15, 50, 150, 200, 250, 500, 750, 1500 and 5000 μg/plate
WP2uvrA: 5, 15, 50, 150, 500, 750, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene (2AA) in the presence of S9-mix, 1 μg/plate in DMSO for TA100, 2 μg/plate in DMSO for TA1535 and TA1537, 10 μg/plate in DMSO for WP2uvrA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION:
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 0.9 to 9 x 10^9 bacteria per mL

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
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.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
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.

Results and discussion

Test resultsopen allclose all
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
In experiment 1 only
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle (dimethyl sulphoxide) 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 in excess of the minimum positive control values over the previous two years, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Negative (solvent/vehicle) historical control data:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 500 μg/plate in both the absence and presence S9-mix.
- In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 300 μg/plate (TA1535), 500 μg/plate (TA100 and TA98), 750 μg/plate (TA1537) and at 5000 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted from 500 μg/plate (TA100 and TA1535), 1500 μg/plate (TA98 and TA1537) and at 5000 μg/plate (WP2uvrA).

Applicant's summary and conclusion

Conclusions:
The substance is mutagenic (in the absence of S9-mix only) in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments in the absence and presence of S9 -mix. Adequate negative and positive controls were included. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 500 μg/plate in both the absence and presence S9-mix. Consequently, the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item was employed as the maximum dose in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the first mutation test, the test item induced statistically significant increases in the revertant colony frequency of tester strains TA100, WP2uvrA, TA98 and TA1537 at sub-toxic dose levels in the absence of S9-mix only. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains dosed in the presence of metabolic activation (S9-mix) or for TA1535 dosed in the absence of S9-mix. Results for the second mutation test showed that the test item again induced statistically significant increases in the revertant colony frequency of tester strains TA100, TA98 and TA1537 at sub-toxic dose levels in the absence of S9-mix only. Once again, there were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains dosed in the presence of metabolic activation (S9-mix) or for TA1535 (and WP2uvrA in this experiment) dosed in the absence of S9-mix.

The test item was considered, therefore, to have induced substantial, dose-related and reproducible increases in the frequency of revertant colonies for Salmonella strains TA100, TA1537 and TA98 dosed in the absence of S9-mix at sub-toxic dose levels of the test item. The individual revertant counts at the non-toxic, statistically significant dose levels for TA98 and TA1537 in particular, exceeded the in-house untreated/vehicle control counts for the strains. Based on the results, it is concluded that the substance is mutagenic (in the absence of S9-mix only) in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.