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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:
no data to 1987-04-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted with methods similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay) with acceptable deviations (only two tester strains were used, only brief information was on the test substance, and only a protocol with results were provided without a final report). The study was conducted in the presence and absence of metabolic activation with positive and vehicle controls. Since the results are positive, no further testing is considered relevant to cover this endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only two tester strains were used, only brief information was on the test substance, and only a protocol with results were provided without a final report
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T836
- Substance type: no data
- Physical state: no data
- Analytical purity: no data
- Impurities: no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: Batch C 1001
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: room temperature in closed containers
- Other: no data
Specific details on test material used for the study:
Storage: at room temperature in closed containers

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
other: TA98 and TA100
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 (20 and 50 µL)
Test concentrations with justification for top dose:
- Preliminary dose range finding assay (TA100 only): 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate
- Experiment 1 (without metabolic activation): 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate
- Experiment 1 (with metabolic activation): 25, 100, 250, 500, 1000, 2500 and 5000 µg/plate
- Experiment 2 (with and without metabolic activation): 25, 100, 250, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: In a preliminary solubility assay, the test substance (100 mg) was introduced in a test tube, with 2 mL distilled water and mixed vigorously. Since the test substance was still not soluble upon warming up to 45°C, a new solution was prepared in absolute ethanol following the same procedure. Based on the findings of this solubility test, ethanol was chosen as solvent.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9; at 0.5 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; at 0.1 µg/plate for TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9; at 1.0 µg/plate for TA98 and TA100
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

For each test, 0.1 mL of an overnight culture in nutrient broth (Oxoid) was introduced into test tubes. This corresponds to an average of 1 -6 x 10^8 viable bacteria per tube. The histidine-biotin (0.05 mM) supplemented top agar (2 mL) was then added, followed by the substrate dilution (0.1 mL). When specified S9 -mix was then added (0.5 mL) and the content of the test tube well mixed. This mixture was then layered on minimal glucose agar (Vogel Bonner E medium) containing petri dishes following the method of Ames. After incubation of the plates for 48 hours at 37°C in the dark, the number of revertant colonies were counted manually.

DURATION:
- Exposure duration: 48 hours
- Selection time: 48 hours, simultaneously with exposure

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: The background lawn of bacterial growth was checked as an indicator of the possible toxic effect of the test substance towards the strains.
Evaluation criteria:
- The test substance was considered positive if:
1) there was a significant increase in the number of revertant colonies (at least two-fold the spontaneous reversion of the corresponding solvent controls, when their spontaneous reversion was below 10);
2) a dose effect relationship was observed; and
3) these effects could be reproduced.
- The above mentioned criteria can be applied in most cases. When equivocal results are obtained more assays may be required, in order to evaluate the mutagenic potential of the test substance.
Statistics:
No statistical analyses were required for the given evaluation criteria.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no data
- Water solubility: not soluble at 50 mg/mL
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
- The results of the preliminary dose range finding study indicated that the test substance was non-toxic to TA100 at concentrations up to 5000 µg/plate as the number of revertant colonies was not found to be reduced.
- Toxic action of the test substance was also not detected by a thinning of the bacterial lawn at 5000 µg/plate or a significant reduction of the number of colonies. Considering the reversion rate of TA100 to prototrophy with the test substance, a significant increase in the number of revertant colonies could be evidenced at one dose level (2500 µg/plate) in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All positive and vehicle control mean values were within the range of historical data except for the vehicle control of TA100 in Experiment 2, which was only slightly above the max value in the historical data (176 versus 175).
- As expected, the appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiments 1 and 2 without S9 mix, and Experiment 2 with S9 mix, low toxic effects were observed at 5000 µg/plate in TA98.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

TA100 showed increased reversion to prototrophy at dose levels of 2500 and 5000 µg/plate both in the absence or presence of metabolic activation. These findings were confirmed by the repeated test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive in TA100 with and without metabolic activation

The test substance was evaluated for mutagenic potential in the Ames reversion mutation test using S. typhimurium tester strains TA98 and TA100 in the presence and absence of metabolic activation. Based on the results of the study, it was concluded that the test substance was mutagenic to TA100 in the absence and presence of metabolic activation.