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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:
1987-06-16 to 1987-06-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

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 instead of five
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-119717-AAA (T001036)
- Physical state: solid
Specific details on test material used for the study:
- Stability of the test article: known
- Storage condition of test material: at room temperature in closed containers
- Other: The test substance was synthesized in the Janssen Pharmaceutica Research Laboratories and passed the specifications of the chemical quality department.

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
other: TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9 at 20 and 50 µg/plate
Test concentrations with justification for top dose:
- Preliminary dose range finding study: 0, 10, 100, 250, 500, 1000, 2500 and 5000 µg/plate
- Initial and repeat experiments: 0, 10, 100, 250, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- 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. The test substance was still not soluble upon warming up to 45 °C, so a new solution was prepared in absolute ethanol following the same procedure. Since the test substance was not more soluble in ethanol than water, DMSO was chosen as the vehicle.
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 metabolic activation; TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 1.0 µg/plate dissolved in DMSO
Remarks:
with metabolic activation; for TA98 and TA100
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in agar (plate incorporation)
The following procedure was used for the plate incorporation assay: 0.1 mL of an overnight culture in nutrient broth (Oxoid) is introduced into test tubes. This corresponds to an average of 1-6 x 10^8 viable bacteria per tube. The histidine-biotine (0.05 mM) or tryptophane (5 µg) supplemented top agar (2 mL) is then added, followed by the substrate dilution (0.1 mL). When specified S-9 mix is then added (0.5 mL) and the content of the test tube well mixed. This mixture is then layered on minimal glucose agar (Vogel Bonner E medium) containing petri dishes following the method of Ames et al. The minimal glucose containing petri dishes are used within the week that they were prepared.

DURATION
- Exposure duration: 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Method: reduction of the background lawn

OTHER EXAMINATIONS:
Before use of any test culture, the growth requirements and the genetic identity of the strains, their sensitivity to UV radiation and crystal violet and their resistance to ampicillin were checked. Each strain must present a spontaneous reversion rate within the historical control data of the testing facility.
After incubation of the plates the number of revertant are counted manually. The sensitivity of the strains and the activity of the metabolizing system are confirmed by testing specific mutagens. Historical control data on spontaneous revertants and on revertants in the positive and solvent control grops of each strain used in the study were included.
Evaluation criteria:
Reversion values were considered as positive if:
1) there was a significant increase in the number of revertant colonies (i.e., at least a two-fold increase compared to the solvent control;
2) a dose effect relationship was observed; and
3) these effects could be reproduced.

When equivocal results are obtained, more assays may be required, in order to evaluate the mutagenic potential of the test substance.
Statistics:
Based on the evaluation criteria, no statistical analysis was required.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: In the preliminary toxicity test, low precipitation of the test substance occurred at 5000 µg/plate both with and without S9. No precipitation was reported in the main experiment.


RANGE-FINDING/SCREENING STUDIES:
A preliminary plate incorporation test was performed to determine the non-inhibitory concentrations of the test substance. In this pre-test, strain TA100 was tested with 6 concentrations of the test substance ranging from 100 - 5000 µg/plate with and without S9 metabolic activation. Toxic effects were evident by a clear reduction of the background lawn. The highest concentration that does not precipitate and that is not toxic for the bacteria, or the lowest concentration that gives visible toxic effects, was used as the highest one in the main assay.


COMPARISON WITH HISTORICAL CONTROL DATA:
Vehicle and positive control values were within historical data control range. The positive control substances induced marked increases in revertant colony numbers with all strains.
The test substance was toxic to the strain TA100 at 5000 µg/plate as the number of revertant colonies was found to be strongly reduced (pin-point colonies). Toxic action of the test substance to TA100 was also detected by a thinning of the bacterial lawn at 5000 µg/plate. Considering the reversion rate of TA100 to prototrophy with the test substance, a significant increase in the number of revertant colonies could be evidenced from dose levels of 250 µg/plate in the absence or in the presence of rat metabolic activation system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the preliminary dose range finding study toxicity to the strain TA100 was observed at 5000 µg/plate as the number of revertant colonies was found to be stronglyl reduced (pin-point colonies). Toxic action was also detected by a thinning of the bacterial background lawn at 5000 µg/plate.
- In the initial test in the absence and presence of S9, moderate toxicity was observed at 5000 µg/plate for TA98. For TA100, moderate and low toxicity were observed at 5000 µg/plate in the absence and presence of S9, respectively.
- In the repeat test in the absence of S9, low and moderate toxicity was observed for TA98 at 2500 and 5000 µg/plate, respectively. For TA100, moderate toxicity was observed at 5000 µg/plate. In the presence of S9, low toxicity was observed at 5000 µg/plate for both TA98 and TA100.

Any other information on results incl. tables

TA100 showed increased reversion to prototrophy at doses of 250 µg/plate and higher both in the presence and absence of S9. These findings were confirmed in the repeat test. The increased amount of S9 used (20 µL/plate-initial test increased to 50 µL/plate-repeat test) resulted in an increased reversion to prototrophy.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive in strain TA100

The test substance was evaluated for mutagenic potential using the Ames Assay. Based on the increase of the reversion rates, it can be concluded that the test substance has mutagenic properties towards Salmonella typhimurium TA100 in the absence and in the presence of rat metabolic activation system. The test substance was negative towards TA98.