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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:
28 Jun 2016 - 07 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Batch identification: 001-150606
- Date of production: 26 May 2015
- Physical state, appearance: solid, black
- Content: Approx. 98%
- Test substance No.: 16/0074-1

Method

Target gene:
his for S. typhimurium
trp for E. Coli
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:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix

2nd Experiment
Strains: TA 100, TA 1537 and TA 98
Doses: 0; 0.3; 1; 3.3; 10; 33 and 100 μg/plate (TA 100)
0; 0.1; 0.3; 1; 3.3; 10 and 33 μg/plate (TA 1537 and TA 98)
Type of test: Standard plate test with and without S9 mix

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
Vehicle / solvent:
- Vehicle used: The test substance was dissolved in dimethyl sulfoxide (DMSO).
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Please refer to 'any other information on materials and methods incl. tables'
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours at 37°C in the dark
NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

NUMBER OF CELLS EVALUATED:
all revertants / colonies counted

DETERMINATION OF CYTOTOXICITY
Following incubation, the bacterial colonies (his+ revertants for Salmonella typhimurium, trp+ revertants for E. coli) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
- Toxicity: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
- Solubility: If precipitation of the test material was observed, it would be recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
Rationale for test conditions:
Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Test substance precipitation was found from about 100 μg/plate onward with and without S9 mix.

CYTOTOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was only observed using the tester strains TA 1535 and E. coli WP2 uvrA, depending on the strain and test conditions about 1000 μg/plate onward.

ADDITIONAL INFORMATION ON MUTAGENICITY
TA 100
- without S9 mix: Increase of revertants at concentrations of 100 up to 5000 μg/plate in the 1st Experiment and at a concentration of 100 μg/plate in the 2nd Experiment.
- with S9 mix: Increase of revertants at concentrations of 100 up to 5000 μg/plate in the 1st Experiment and at concentrations of 33 and 100 μg/plate in the 2nd Experiment.
TA 98
- without S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 0.3 up to 33 μg/plate in the 2nd Experiment.
- with S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 1.0 up to 33 μg/plate in the 2nd Experiment.
TA 1537
- without S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 10 and 33 μg/plate in the 2nd Experiment.
- with S9 mix:
Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 3.3, 10 and 33 μg/plate in the 2nd Experiment.

Applicant's summary and conclusion