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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
Study period:
1995-09-28 until 1996-01-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983 and revised draft document 1994
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Specific details on test material used for the study:
Stabilizer: Hydrocarbon

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
With S9 mix: 3, 10, 30, 100, 300 µg/plate
Without S9 mix: 30, 100, 300, 1000, 3000 µg/plate
except for the TA 102 : 10, 30, 100, 300, 1000 µg/plate
Vehicle / solvent:
- Vehicle used: acetone
- Justification for choice of vehicle: the test substance was not soluble either in distilled water or in dimethylsulfoxide.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
in strains TA 1535, TA 100 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In TA 1537 strain in strain TA 98 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
in strain TA 98 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
in strain TA 102 without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: anthramine
Remarks:
in strains TA 1535, TA 1537, TA 98, TA 100, TA 102 with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation; in suspension

Treatment:
- direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test substance solution (0.05 to 0.1 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium
- preincubation method (second experiment with S9 mix): test substance solution (0.05 to 0.1 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter.

Preliminary toxicity test:
To assess the toxicity of the test substance to the bacteria, six dose-levels (one plate/ dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with or without S9 mix. The sterility of the test substance was checked during this test and was found to be satisfactory.

Mutagenicity experiments:
In two independent experiments, five dose-levels of the test substance (three plates/dose-level) were tested on each strain, with or without S9 mix.
In each experiment, the following controls were included using triplicate plates:
. vehicle controls: each bacterial tester strain treated with the vehicle,
. positive controls: each bacterial tester strain treated with appropriate reference mutagens.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle) were calculated.
Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
not applicable

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was freely soluble in the vehicle (acetone) at 50 mg/mL, whereas it was not soluble in distilled water or in dimethylsulfoxyde.
Consequently, with a maximum dose-volume of 100 µL/plate, the dose-levels were: 15, 50, 150, 500, 1500 and 5000 µg/plate. No precipitate was observed in the Petri plate when scoring the revertants. Without S9 mix, slight to considerable toxicity was noted at 5000 µg/plate in the TA 98 and TA 100 strains whereas with S9 mix toxicity was noted at dose-levels higher than 150 µg/plate: clearing of the bacterial lawn and/or reduction of the number of revertants.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.
Executive summary:

The test substance (in acetone as vehicle) was examined for its potential to induce reverse mutation in Salmonella typhimurium according to OECD No. 471 guideline and EC method B.14. Selection of concentrations was based on a preliminary toxicity test, thus, the selected treatment-levels were: without S9 mix: 30, 100, 300, 1000, 3000 µg/plate, with S9 mix: 3, 10, 30, 100, 300 µg/plate except for the TA 102: 10, 30, 100, 300, 1000 µg/plate.

The test substance induced significant and reproducible increases in the number of revertants, with and without S9 mix, in any of the five strains, except in the TA 1535 strain with S9 mix. Under the experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.