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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No preincubation test performed
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his, trp operon
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Preliminary test: All strains: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 µg/plate

Main test:
Salmonella typhimurium strains: 0, 2.5, 7.5, 25, 75, 250, 750 µg/plate
Escherichia coli strain: 0, 25, 75, 250, 600, 1800, 5000 µg/plate
Doses were chosen based on the outcome of the preliminary toxicity test
Vehicle / solvent:
DMSO
Choice based on solubility of the test article and compatibility with the target cells.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test article. In the preliminary toxicity assay, the maximum dose tested was 5000 ug/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. No precipitate was observed. With all Salmonella tester strains, toxicity was observed at greater than or equal to 667 or at greater than or equal to 333 ug/plate, in the presence and absence of S9 activation, respectively. With the E. coli tester strain, toxicity was observed at greater than or equal to 3333 ug/plate. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay were 750 ug/plate for all Salmonella tester strains and 5000 ug/plate for the E. coli tester strain.


METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: two experiments (B1 and B2), triplicate plates


DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test article was soluble in DMSO at approximately 500 mg/mL, the maximum concentration tested.

Toxicity pretest: no precipitate was observed up to the maximum dose tested. With all Salmonella tester strains, toxicity was observed at 667 or at 333 µg/plate and above in the presence and absence of S9 activation, respectively. With the E. coli tester strain, toxicity was observed at 3333 µg/plate and above. Based on these findings, the maximum doses plated in the main test were 750 µg/plate for all Salmonella tester strains and 5000 µg/plate for E.coli tester strain.

Main test: no precipitation occurred at any concentration. Bacteriotoxic effects were noted in all Salmonella tester strains at 750 µg/plate (in the presence of S9 activation) or at ≥250 µg/plate (in the absence of S9 activation) as well as in in E. coli tester strain at 5000 µg/plate (± S9). The number of revertant colonies did not differ, in a biologically relevant manner, between plates containing the test substance and those containing the vehicle controls either with or without metabolic activation.

The positive controls induced adequate numbers of revertant colonies from each bacterial strain demonstrating the sensitivity and suitability of the test system.

Applicant's summary and conclusion