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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions: only 4 strains tested

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, no E.coli WP2 or S. typhimurium TA 102 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from liver homogenates of Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
initial testing (plate incorporation; with and without S9 mix): 19.2-12000 µg/plate
repeat (plate incorporation; with and without S9 mix): 19-9600 µg/plate;
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Na-azide (NaN3, only TA 1535), nitrofurantoin (NF, only TA 100), 4-nitro-1,2-phenylene diamine (4-NPDA, TA 1537 and TA 98), 2-aminoanthracene (2-AA, all strains)
Remarks:
The positive controls NaN3, NF, and 4-NPDA were used without S9 mix; the positive control 2-AA was used with S9 mix.
Details on test system and experimental conditions:
Strains were obtained from Prof. B. Ames in 1982 and maintained properly.

METHOD OF APPLICATION: According to Ames et al. (Proc. nat. Acad. Sci. 70, 2281-2285, 1973; Mutation Res. 31, 347-364, 1975).
Plate incorporation for initial testing and independent repeat; for each strain and dose four plates were used in parallel, which was also valid for solvent and positive controls.

DETERMINATION OF CYTOTOXICITY
- mutant count: A toxic effect was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. This increase should be about twice that of negative controls.

Results and discussion

Test results
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 applicable
Positive controls validity:
valid
Additional information on results:
Evidence of mutagenic activity was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed.
The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance precipitation occurred at and above 1200 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses up to and including 19.2 µg/plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a very limited extent up to 12000 µg/plate for evaluation purposes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

A bacterial reverse mutation assay according to the protocol of Ames et al. (Proc. nat. Acad. Sci. 70, 2281-2285, 1973; Mutation Res. 31, 347-364, 1975) was conducted for the evaluation of point mutagenic effects. In this assay 4 strains of Salmonella typhimurium were used (TA 1535, TA 100, TA 1537, and TA 98). The initial plate incorporation test used test substance doses of 19.2-12000 µg/plate. Due to bacteriotoxic effects doses for independent repeat were lowered to a maximum of 9600 µg/plate.

Substance precipitation occurred at and above 1200 µg/plate. Doses up to and including 19.2 µg/plate did not cause any bacteriotoxic effect. The total bacteria counts remained unchanged and thus no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a very limited extent for evaluation purposes.

Evidence of mutagenic activity was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.