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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:
2002-03-29 to 2002-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Isocarb 24
- Substance type: Product
- Lot/batch No.: ISC2403, 04058/MA
- Physical state: crystalline at ambient conditions

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
5000, 2500, 1250, 625 and 313 µg/plate.
Vehicle / solvent:
acetone
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Deficiencies/Proficiencies: histidine auxotroph
- Metabolic activation system: rat liver S9 of Phenobarbital/beta-Naphthoflavone induced male Sprague-Dawley rats
ADMINISTRATION:
- Dosing:
experiment I and II:
313, 625, 1250, 2500 and 5000 µg/plate
- Number of replicates: 3
- Application:
Experiment I (plate-incorporation method): The first experiment was perfonned using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was
then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.
The overlay mixture was composed as follows:
(i) Overlay agar (held at 45°C) 2 ml
(ii) Test or control item solution 0.1 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) O.S ml
(iv) Bacterial suspension 0.1 ml

Experiment II and III (pre-incubation method): Since acetone is known to be toxic at 50 Ill/plate when used for the pre-incubation method, 25
Ill/plate of solvent or test item solution were used.
The components were added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 ml
(ii) Test item solution or its solvent 0.025ml
Positive control solution or its solvent O.OSOml
(iii) S9 mix or phosphate buffer (PH 7.4, 0.1 M) O.S ml

The incubate was vortexed and placed at 37°C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Evaluation criteria:
The test item is considered as a mutagen if at least a two-fold increases in mean revertant numbers are observed at two consecutive dose-levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels.
Statistics:
regression analysis

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of result:
negative

The test item does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

The test item was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium. The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using acetone. In the toxicity test, the test item was assayed at a maximum dose-level of 5000 flg/plate and at four lower dose-levels spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 flg/plate. No signs of toxicity were observed at any doselevel tested, in any tester strain, in the absence or presence of S9 metabolic activation. Two main experiments were performed. In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 flg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500,1250,625 and 313 flg/plate. As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step and used the same dose-levels as Main Assay I. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.