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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Not specified in report
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:


Target gene:
uvrA (for WP2 uvrA cultures) and uvrB gene ( for Salmonella cultures), TA-98- his D, TA-100- his G, TA-1535- his G, TA-1537- his C
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli (tester strain WP2uvrA)
Metabolic activation:
with and without
Metabolic activation system:
Acoclor 1254-induced rat liver S9 was used for the metabolic activation system.
Test concentrations with justification for top dose:
100, 333, 1000, 3333, 5000 ug per plate
Vehicle / solvent:
Water was selected as the solvent of choice. The test material was soluble in water at approximately 100ug.mL, the maximum concentration tested.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: All strains, activated - 2-aminoanthracene ;WP2uvrA, nonactivated - methyl methanesulfonate; TA98, nonactivated - 2-nitrofluorene; TA100, TA1535, nonactivated - sodium azide; TA1537, nonactivated - 9-aminoacridine
Details on test system and experimental conditions:
Salmonella tester strains were recieved directly from Dr. Bruce Ames, University of California, Berkeley, and the E.coli was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester TA1535 is reverted by mutagens that cause basepair substitutions. Tester TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E.coli is sensitive to basepair substitution mutations, rather than frameshift mutations.

To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Each culture wasmonitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 10^9 cells per milliliter.

Acoclor 1254-induced rat liver S9 was used for the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single IP injection of Acroclor 1254, 500mg/kg, five days prior to sacrifice. Each batch was assayed for its ability to metabolize.

Positive controls, appropriate to the given tester strains, were used as follows:
All strains, activated - 2-aminoanthracene
WP2uvrA, nonactivated - methyl methanesulfonate
TA98, nonactivated - 2-nitrofluorene
TA100, TA1535, nonactivated - sodium azide
TA1537, nonactivated - 9-aminoacridine

The concentration varied according to positive control compound. The sterility of the test article was verified.

Preliminary Toxicity Assay
Ten dose levels of the test article were plated, one plate per dose, with overnight cultures of TA100 and WP2 uvrA on selective minimal agar in the presence and absence of rat liver S9 activation.

Mutagenicity Assay
An initial and confirmatory assay was used to evaluate the mutagenic potential of the test material. A minimum of 5 dose levels of the test article, with vehicle and positive controls, were plated with tester strains in the presence and absence of rat liver S9 activation. All dose levels of test article, vehicle controls, and positive controls were plated in triplicate. The test systems were exposed to the test article via the plate incorporation methodology described by Ames, (1975) and updated by Maron and Ames (1983). Plates were coded according to the testing laboratory's standard procedures. Plates were incubated for 48-72 hours at 35-39C. Plates not counted immediately following the incubation period were stored at 2-6C until the colonies could be counted.

Toxicity and degree of precipation were scored relative to the vehicle control plate using a well-defined 9-point scale. Colonies were counted either by an automated colony counter or by hand unless the assay was preliminary or the plate exhibited toxicity. Plates exhibiting test article precipitate that interferes with an automated colony counter were counted by hand.
Evaluation criteria:
For a positive finding, the test material must cause a dose-related increase in mean revertants per plate of at least one tester strain with a minimum of 2 increasing concentrations of test article. Positive finding for TA1535 and TA1537 is an increase in mean revertants at the peak of dose-response greater than, or equal to, 3x mean control value. Positive finding for TA98, TA100, WP2 uvrA is an increase in mean revertants at the peak of dose-response greater than, or equal to, 2x mean control value.
Mean and standard deviation of the number of revertants per plate were calculated and reported.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium (tester strains TA98, TA100, TA1535, TA1537) and E.coli (tester strain WP2 uvrA)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Preliminary Toxicity Assay
The maximum dose tested was 5000ug/plate, achieved by using a concentration of 100mg/mL and a 50uL plating aliquot. Based on the findings, the maximum dose plated in the mutagenicity assay was 5000ug/plate. Neither precipitate nor appreciable toxicity was observed.

Mutagenicity Assay
Neither precipitate nor appreciable toxicity was observed in the initial or confirmatory assays. In neither the initial nor confirmatory assay was there a positive response with any of the tester strains at any dose level, in the presence or absence of S9 activation. The mean of each positive control exhibited at least a 3-fold increase in the number of revertants over the mean value of the respective vehicle control, indicating a valid test.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

The results of this study indicate that, under the conditions of this study, the test material did not cause a positive response with any of the tester strains in the presence and absence of Acrclor-induced rat liver S9.

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