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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:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Not specified in report
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material: Purity of the test material was listed as approximately 99%. Half-log dilutions of test material were made in dimethylsulphoxide vehicle. Prior to use, the solvent was dried with molecular sieves. Concentrations of test material were 50, 150, 500, 1500 and 5000 micrograms/plate. Vehicle and positive controls (2, 3 or 5 micrograms/plate N-ethyl-N'-nitro-N-nitrosoguanidine for WP2uvrA-, TA100 and TA1535 (respectively), 80 micrograms/plate 9-aminoacridine for TA1537, 0.2 micrograms/plate 4-nitroquinoline-1-oxide for TA98, and 0.5, 1, 2, or 10 micrograms/plate 2-aminoanthacene for TA98, TA100, TA1535 and TA1537, and WP2uvrA- in the presence of S-9, respectively.

Batch # 3I15-XT7B

Method

Target gene:
TA98- hisD3052, TA100- hisG46, TA1535- hisG46 and TA1537- hisC3076
Species / strain
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2uvrA-
Details on mammalian cell lines (if applicable):
Test strains: The S. typhiurium strains were obtained from the Univerity of California Berkely and E. coli WP2uvrA- was obtained from the British Industrial Biological Research Association. All strains were stored at -196 degrees C. Characterization checks were carried out to determine the amino acid requirement, presence of rFa. R factors, uvrB mutation and the spontaneous reversion rate. Overnight subcultures were prepared in nutrient broth and incubated at 37 degrees C for approximately 10 hours.
Metabolic activation:
with and without
Metabolic activation system:
S-9 was prepared from the livers of male Sprague-Dawley rats that had recevied a single i.p. injection of 500 mg/kg Aroclor 1254 five days before S-9 preparation.
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 micrograms/plate
Vehicle:
dimethylsulphoxide
Controls
Negative controls:
yes
Remarks:
dimethylsulphoxide
Solvent controls:
yes
Remarks:
dimethylsulphoxide
Positive controls:
yes
Positive control substance:
other: Vehicle and positive controls: N-ethyl-N'-nitro-N-nitrosoguanidine for WP2uvrA-, TA100 and TA1535, 9-aminoacridine for TA1537,4-nitroquinoline-1-oxide for TA98, and 2-aminoanthacene for TA98, TA100, TA1535 and TA1537, and WP2uvrA- in the presence of S-9.
Details on test system and conditions:
Test conduct : A preliminary test performed with strains TA100 and WP2uvra- showed that concentrations of test material up to 5000 micrograms (the maxiumum recommended dose) were not toxic to bacteria. Two mutation studies were then conducted as follows. A mixture of 0.1 ml of bacterial suspension, 0.1 ml of test material (or negative or positive control), 0.5 ml of S-9 mix (or phosphate buffer) and 2 ml of molten, trace histidine/tryptophan-supplemented media was overlaid onto sterile plates of Vogel-Bonner minimal agar (30 ml/plate). Each condition was tested in triplicate. Due to the volatility of the test material, agar plates from each dose level were placed in individual, sealed stainless steel containers immediately after treatment. All plates were incubated at 37 degrees C for approximately 48 hours and the frequency of revertant colonies was assessed using a colony counter.

Test material: Half-log dilutions of test material were made in dimethylsulphoxide vehicle. Prior to use, the solvent was dried with molecular sieves. Concentrations of test material were 50, 150, 500, 1500 and 5000 micrograms/plate. Vehicle and positive controls (2, 3 or 5 micrograms/plate N-ethyl-N'-nitro-N-nitrosoguanidine for WP2uvrA-, TA100 and TA1535 (respectively), 80 micrograms/plate 9-aminoacridine for TA1537, 0.2 micrograms/plate 4-nitroquinoline-1-oxide for TA98, and 0.5, 1, 2, or 10 micrograms/plate 2-aminoanthacene for TA98, TA100, TA1535 and TA1537, and WP2uvrA- in the presence of S-9, respectively.

S-9: S-9 was prepared from the livers of male Sprague-Dawley rats that had recevied a single i.p. injection of 500 mg/kg Aroclor 1254 five days before S-9 preparation. The S-9 was stored at -196 degrees C until use. Prior to use, all bataches of S-9 were checked for the ability to induce a positive response with 2-aminoanthacene. S-9 mix was prepared according to conventional methods. Sterility checks were done on the day of each experiment.
Evaluation criteria:
Interpretation of results: A test was positive if there was at least a 2-fold, dose-dependent increase in the mutation rate (with respect to the spontaneous rate) in one or more strains in the presence and/or absence of S-9 in both experiments. In the event the two experiments gave equivocal or conflicting results, a third study was recommended.
Statistics:
None

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and WP2uvrA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
A preliminary test performed with strains TA100 and WP2uvra- showed that concentrations of test material up to 5000 micrograms (the maxiumum recommended dose) were not toxic to bacteria.
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Results of a plate incorporation test performed in the same strains by the same laboratory (SPL Project number 630/2) in 1994 also were negative.

First study: There was no increase in the frequency of revertant colonies with any dose of test material in the absence or presence of S-9. The average numbers of revertants in control cultures TA98, TA100, TA1535, TA1537 and WP2uvrA- without activation were 28, 116, 16, 11 and 28, respectively. The numbers of revertants in treated strains TA98, TA100, TA1535, TA1537 and WP2uvrA- without activation ranged from 21-30, 97-122, 12-22, 7-12 and 18-36, respectively. The average numbers of revertants in control cultures TA98, TA100, TA1535, TA1537 and WP2uvrA- with activation were 31, 127, 18, 9 and 28, respectively. The numbers of revertants in treated strains TA98, TA100, TA1535, TA1537 and WP2uvrA- with activation ranged from 22-33, 111-133, 11-23, 6-16 and 23-32, respectively.


Second study: There was no increase in the frequency of revertant colonies with any dose of test material in the absence or presence of S-9. The average numbers of revertants in control cultures TA98, TA100, TA1535, TA1537 and WP2uvrA- without activation were 22, 106, 21, 12 and 22, respectively. The numbers of revertants in treated strains TA98, TA100, TA1535, TA1537 and WP2uvrA- without activation ranged from 17-27, 83-123, 18-30, 7-16 and 15-29, respectively. The average numbers of revertants in control cultures TA98, TA100, TA1535, TA1537 and WP2uvrA- with activation were 30, 93, 17, 12 and 22, respectively. The numbers of revertants in treated strains TA98, TA100, TA1535, TA1537 and WP2uvrA- with activation ranged from 16-35, 85-180, 10-21, 8-16 and 16-29, respectively. For strain TA100 in the presence of S-9, 180 revertants were observed in one of the three replicates treated with 1500 micrograms/plate. This was close to being 2 times that of the control value. The other two values at this concentration were 94 and 104, which were in line with values obtained at other concentrations. Therefore, the value of 180 appears to be an outlier.

Both tests were valid, as the positive controls induced at least a 4-fold increase in the number of revertant colonies in both studies.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was considered to be nonmutagenic under the conditions of this test.
Executive summary:

None