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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:
21/08/1996 to 04/11/1996
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 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:
liquid

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
Toxicity pre-screen 50, 167, 500, 1670, 5000 ug/plateInitial study for Salmonella and E.coli 16.7, 50, 167, 500, 1670, 5000 ug/plateConfirmatory study for Salmonella and E. coli 16.7, 50, 167, 500, 1670, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: none given
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate-incorporation and preincubationDURATION- Preincubation period: 30 minutes at 37 C- Exposure duration: 48 hoursSELECTION AGENT (mutation assays): absence of histidine or tryptophan in agarNUMBER OF REPLICATIONS: triplicate platesDETERMINATION OF CYTOTOXICITY- Method: growth of background lawn of non-revertant bacteria and size and number of revertant colonies
Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants.
Statistics:
Statistical analyses were performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon normal, spontaneous variation observed among replicate negative control cultures, as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test articles judged to be negative in this assay.

Results and discussion

Test results
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA100, TA102, WP2uvrA-
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:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: precipitation was observed at 1670 ug/plate and above- Other confounding effects:RANGE-FINDING/SCREENING STUDIES: A toxicity screening test using both the plate-incorporation and pre-incubation methods, was perforem in TA1537, TA100 and WP2uvrA-, in the absence of S9 only.COMPARISON WITH HISTORICAL CONTROL DATA: All control values were comparable to historical ranges
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The results for OS#114461 were negative in the Ames/Salmonella-E.coli Liquid reverse mutation assay under the conditions, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.
Executive summary:

Test Guidance

OECD Guideline No. 471

Method

The test material was evaluated in the Ames/Salmonella-E.coli bacterial reverse mutation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA102, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2uvrA-), in the presence and absence of an exogenous metabolic activation system (S9).

Results

Toxicity of the test material was first evaluated in a prescreen by treating duplicate cultures of strains TA1537, TA100 and WP2uvrA- with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9, using both the pre-incubation and plate-incorporation methods. Results of the prescreen indicated the test material was toxic to all three tester strains at 5000 ug/plate in the pre-cubation treatment conditions only. In addition, the test article was found to be incompletely soluble at doses => 500 ug/plate.

Based upon these findings, the test material was evaluated in triplicate cultures in all five strains of Salmonella strain WP2uvrA- at dose of 16.7, 50, 167, 500, 1670 and 5000 ug/plate, in the presence and absence of S9, using the plate-incorporation method. Six dose levels of the test material were evaluated with and without S9 in the event of unacceptable insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors.The test article again precipitated from solution =>167 ug/plate. No toxicity was again observed in any of the tester strains, with or without S9. Revertant frequencies for all doses of the test material in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative control cultures except for strain TA102 in the absence of S9. In this case a statistically significant increase in revertant frequencies, to approximately 1.1- to 1.5 -fold control values, were observed at doses of 50 to 1670 ug/plate. However, these increases were not linear.

The test material was re-evaluated in the confirmatory assay using the same dose levels under pre-incubation treatment conditions and also re-evaluated in TA102 without S9 under plate incorporation conditions. The test article again precipitated from solution at doses =>167 ug/plate. Inhibited growth again was observed in all tester strains at 5000 ug/plate without S9 Revertant frequencies for all dose of the test material in all tester strains with and without S9 approximated or were less than control value. All positive and negative control values in both assays were within acceptable limits.

Conclusion

Therefore, the results for the test material were negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.