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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative Ames test

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr - Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD471, version 21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0015064167
- Expiration date of the lot/batch: March 01, 2018
- Purity test date: Aug 02, 2017 (date of report 17L00118)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Target gene:
histidine (S. typhimurium), tryptophane (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Wistar rats, having received phenobarbital i.p. and b-naphtoflavone orally
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate in standard plate and preincubation test with and without S9 mix
Vehicle / solvent:
water (ultrapure)
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water with and without S9 mix
Positive controls:
yes
Positive control substance:
other: With S9 mix: 2-aminoanthracene (2-AA). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): TA 1535, TA 100 ; 4-nitro-o-phenylenediamine (NOPD): TA 98; 9-aminoacridine (AAC): TA 1537; 4-nitroquinoline-N-oxide (4-NQO): E. coli WP2 uvrA.
Details on test system and experimental conditions:
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 (up to maximum test dose)
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd 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: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

METHOD OF APPLICATION:
SPT: in agar (plate incorporation method); based on Ames et al. 1975 and Maron et al., 1983
PIT: addition of soft agar after 20 min in suspension on a shaker; based on Yahagi et al., 1977 and Matsushima et al., 1980

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates per dose or per control

DETERMINATION OF
- MUTAGENICITY: individual plate count, mean number of revertant colonies per plate and standard deviation
- TOXICITY: decrease in numhber of revertantns (factor >= 0.6); clearing/diminution of the background lawn (=reduced his- or trp- background growth)
Evaluation criteria:
Acceptance criteria for a valid test
- Number of revertant colonies in negative controls within the range of historical negative control data for each tester strain
- Sterility controls without indication of bacterial contamination
- Distinct increase in the number of revertant colonies in all positive controls within the range of the historical positive control data
- Fresh bacterial culture containing approximately 10e9 cells per mL.

Assessment criteria for a positive result: 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 with or without S9 mix.
Assessment criteria for a negative result: Number of revertants for all tester strains within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
bacteria, other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
SPT: A slight decrease in the number of trp+ revertants in E.coli with S9 mix at 5000 μg/plate. PIA: A slight decrease in the number of his+ revertants in TA 1537 and TA 98 both with S9 mix at 5000 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No test substance precipitation was found with and without S9 mix.
Conclusions:
non-mutagenic in Ames test
Executive summary:

The test substance was tested in 2017 in a reverse mutation assay (GLP, OECD471) using several bacterial strains, i.e. S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvr. The dose range was 33 μg – 5000 μg/plate (SPT and PIT); both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or preincubation test with or without S9 mix. Therefore, under the experimental conditions of this study, the test substance is not mutagenic in the S. typhimurium/E. coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance was tested in 2017 in a reverse mutation assay (GLP, OECD471) using several bacterial strains, i.e. S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvr. The dose range was 33 μg – 5000 μg/plate (Standard Plate Test and PreIncubation Test); both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the SPT or PIT with or without S9 mix. Therefore, under the experimental conditions of this study, the test substance is not mutagenic in the S. typhimurium/E. coli reverse mutation assay in the absence and the presence of metabolic activation.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The S. typhimurium/E. coli reverse mutation assay was negative with and without metabolic activation up to a concentration of 5000 µg/plate. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.