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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:
weight of evidence
Study period:
23 January 1995 to 1 May 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted by an internationally recognized test procedure. The stability and purity of the test article were not reported.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
also 21 CFR 58, 40 CFR 792, nad 40 CFR 160
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: EC95-0200, 5-SSIPA
Lot No.: 45795
Description: white powder
Date received: 01/17/95

Method

Target gene:
Mutations in the histidine operon of Salmonella typhimurium including hisG46, hisC3076 and hisD3052.
Species / strain
Species / strain / cell type:
other: Salmonella TA1535, TA100, TA1537 and TA98; and E. coli WP2uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9 rat liver homogenate; Molecular Toxicology, Inc., Annapolis, MD
Test concentrations with justification for top dose:
In a range-finidng study, ten doses of 6.67 to 5,000 micrograms/plate were tested with TA100 and WP2uvrA.

Definitive studies (in duplicate) were conducted at dose levels of 100, 333, 667, 1000, 3330 and 5000 micrograms/plate both with or without S9 mix.
Vehicle / solvent:
Deionized water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle only
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-nitrofluorene; sodium azide; ICR-191; and 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Source of Tester Strains

Salmonella typhimurium strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California. The E. coli strain WP2uvrA was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland.

Inoculum

Overnight cultures for use in all testing procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Overnight cultures (shaking at 125 +/- 25 rpm; incubation at 37 +/- 2 deg. C) in log phase or late log phase of growth were harvested.

Plate Incorporation Method

The test article, tester strain and S9 mix (where appropriate) were combined in molten agar and overlaid onto a minimal agar plate. After incubation of plates at 37 +/- 2 deg C for 48 +/- 8 hr, revertant colonies were counted. All doses, vehicle and positive controls were plated in triplicate.

Plate Counts

The numbers of revertant colonies per plate for vehicle controls and all plates containing test article were counted manually. Plate counts for positive controls were counted by automated colony counter.
Evaluation criteria:
A minimum of 3 non-toxic doses were required to evaluate assay data.

For tester strains TA98, TA100 and WP2uvrA, at least a 2-fold increase in the mean revertants per plate over the vehicle control was required for a positive response. This increase in mean number of revertants per plate had to be accompanied by a dose response to increase concentrations of the test article.

For tester strains TA1535 and TA1537, at least a 3-fold increase in the mean revertants per plate over the vehicle control was required for a positive response. This increase in mean number of revertants per plate had to be accompanied by a dose response to increase concentrations of the test article.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

Results and discussion

Test results
Species / strain:
other: Salmonella TA1535, TA100, TA1537 and TA98; and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with tester strain TA100 without S9 and at 3330 and 5000 micrograms/plate - slight reduction in bacterial lawn
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Salmonella-Escherichia coli/mammalian-microsome reverse mutation assay indicated that under the test conditions, the test article did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-treated rat liver.