Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not reported
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 from rats induced with Aroclor 1254
Test concentrations with justification for top dose:
20 - 2000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard solvent
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2-anthramine, 2-nitrofluorene, sodium azide, and 9-aminoacridine
Positive control substance:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 10-hr
- Exposure duration: 72 hours
- Expression time (cells in growth medium): 10e8 to 10e9 cells per mL
- Selection time (if incubation with a selection agent): 72 hours



SELECTION AGENT (mutation assays): not reported
SPINDLE INHIBITOR (cytogenetic assays): not reported
STAIN (for cytogenetic assays): not reported


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: elimination of a uniform background lawn


Evaluation criteria:
A mutagenicity assay is considered valid if the following conditions are met. First, the spontaneous reversion rate, with and without metabolic activation, must be reasonably consistent with the expected range for the strain being used. Second, the positive control materials must elicit a positive response. And third, strains must maintain characteristics, i.e., nutritional requirements, crystal violet sensitivity and ampicillin resistance. A test article is considered positive (mutagenic) if it elicits in independent assays a number of revertants per plate at least 2 times that observed in the solvent control (background). A response that does not meet this criteria but elicits a potential biologically significant response (e.g., a dose related increase in revertants per plate over 3 concentrations) is considered as equivocal response and requires further evaluation. A test article is considered negative (non-mutagenic) if the criteria for a positive assay were not met and the test article was tested up to 2,000 mg/plate or the limit of solubility or toxicity, whichever was lower. Toxicity is defined as the elimination of a uniform background lawn.
Statistics:
Statistical methods beyond the calculation of the mean and standard deviation are not considered necessary for the onterpretation of this study.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test article did precipitate
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: In 1984 (see IUCLID section 7.6.1, record IUC4#1), the test material was reported to be mutagenic in strain 1535. However, in that study, the test agent did not induce a dose related increase in mutation frequency. Rather, the sporadic positive response observed was based upon a statistical increase in mutation frequency compared to the negative controls. In contrast, our current criteria for a mutagenic response is based upon a two-fold or greater increase in mutation frequency compared to the negative controls. Because the marginal response observed in the 1984 study may not be biologically meaningful, the assay was repeated according to our current test system.


COMPARISON WITH HISTORICAL CONTROL DATA: not reported


ADDITIONAL INFORMATION ON CYTOTOXICITY: none
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537

Any other information on results incl. tables

FREQUENCY OF EFFECTS: The test article did not induce an increase in revertants when compared to solvent controls.


Applicant's summary and conclusion

Conclusions:
Negative
Executive summary:

The test material was evaluated for mutagenic activity in the Salmonella typhimurium gene mutation assay (Ames test). Tester strains were TA98, TA100, TA1535 and TA1537 in the presence and absence of a metabolic activation system (S-9 liver fraction from Aroclor 1254 induced rats). Dimethyl sulfoxide (DMSO) was used as the solvent for the test article and as the solvent control. In the presence of metabolic activation, 2 -anthramine was used as the positive control for all strains. In the absence of metabolic activation, the positive controls used were: 2 -nitrofluorene (TA98), sodium azide (TA100 and TA1535), and 9 -aminoacridine (TA1537). The test article was evaluated at concentrations ranging from 20 to 2000 ug/plate (all concentrations were based in the compound as received) and the number of revertants was determined.

The test article did not induce an increase in revertants when compared to solvent controls. This was true for all tester strains both with and without metabolic activation. Toxicity was observed in all strains, both without and with metabolic activation at 2000 ug/plate.

Under the conditions of this study, the test material is not mutagenic in the Salmonella gene mutation assay.