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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:
August 8, 1990 to September 10, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to the EEC Directive dated September 19, 1984, No. L251, Vol. 27 pp. 137-139 and EPA Toxic Substances Control Act; Good Laboratory Practice Regulations 1:0 CFR Part 792 Thursday August 17, 1989.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1984
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: Genetic identity: RFA mutation using crystal violet; R factor (ampicillin resistance) using ampicillin disc's, UVR B deletion by exposing the strains to ultraviolet light and the histidine requirement by culturing the strains with and without histidines.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian activation mixture (S-9 mix).
Test concentrations with justification for top dose:
Five concentrations of the test material (312.5, 625, 1250, 2500 and 5000 µg/plate), separated by half-log intervals, were evaluated with and without metabolic activation.
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: N-methyl-N-nitro-N-nitrosoguanidine; 2-anthramin
Details on test system and experimental conditions:
Strains details:
TA-1535, TA-98, TA-1537 and TA-100 from Department of Biochemistry, University of California, Berkeley, USA
Escherichia coli WP2 from MRC Cell Mutation Unit, University of Sussex, Falmer. Brighton, England

The genetic identity of each bacterial tester strain is verified at the time that the master plates are made. Genetic identity is verified by testing for the following: RFA mutation using crystal violet; R factor (ampicillin resistance) using ampicillin disc's, UVR B deletion by exposing the strains to ultraviolet light and the histidine requirement by culturing the strains with and without histidines.

The bacterial strains are cultured in Oxoid media #2. The selective medium is Minimal Agar Davis (Difco) with 22 glucose and the overlay agar is 0.6% purified agar with either 0.05 mM histidine for the Salmonella strains or 0.05 mM tryptophan for the E, coli, 0.05 mM biotin and 0.1 M sodium chloride.

Activation System:
Bacteria were exposed to the test substance both in the presence and absence of a mammalian activation mixture (S-9 mix). S-9 mix is prepared in accordance with published procedures (Ames, et al, 1975; Matsushima, et al, 1976), using a 9,000 x g supernatant prepared from Sprague-Dawley adult male rat liver induced by AROCLOR® 1254 five days prior to kill (Organon Teknika Corporation, Durham, NC).

Study System:
Five concentrations of the test material, separated by half-log intervals, were evaluated with and without metabolic activation. Concentration of test chemical (see above) and appropriate tester strain were added to 2 ml top agar held at 45˚C, which was then pour-plated immediately on the surface of hardened minimal agar. In the nonactivation assay, 0.5 ml phosphate buffer was added just prior to plating while 0.5 ml S-9 activation mix was added for the activation assay.

Positive and negative control assays were conducted with each experiment and consisted of direct-acting mutagens for nonactivation assays and mutagens that require metabolic biotransformation in activation assays. Negative controls consisted of the test article solvent in the overlay agar together with the other essential components. Plates were incubated for 72 hours and counted. All testing was done in triplicate.

Evaluation criteria:
The data are presented as the number of revertant colonies per plate. The number of revertant colonies on both negative (solvent) and positive control plates are also presented. The mean (X) number of revertants per plate and standard deviation are also given.
Statistics:
No data.

Results and discussion

Test results
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA98, TA100, WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxicity or precipitate was noted. There was no evidence of genetic activity observed in either of the tests.

Applicant's summary and conclusion

Conclusions:
The test material was evaluated, in two independent tests, for genetic activity in the Salmonella typhimurium and Escherichia coli Reverse Mutation Assays. No toxicity or precipitate was noted. There was no evidence of genetic activity observed in either of the tests. The positive controls gave a vaild result.