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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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: Ames et al. (1975)
Version / remarks:
Ames, B.N., J. McCann and E. Yamasaki. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research 31:347-364 (1975)
Qualifier:
according to
Guideline:
other: Green and Muriel (1976)
Version / remarks:
Green, M.H.L. and W.J. Muriel. Mutagen testing using trff reversion in Escherichia coli.
Mutation Research 38:3-32. (1976).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored frozen at - Solubility and stability of the test substance in the solvent/vehicle: At 100 mg per ml, which was the most concentrated stock dilution prepared, the test article formed a clear colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test material was applied in DMSO.

Method

Target gene:
Histidine locus (Salmonella typhimurium) and tryptophan locus (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Metabolic activation:
with and without
Metabolic activation system:
An exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9).
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
Metabolic activation:
with and without
Metabolic activation system:
An exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9).
Test concentrations with justification for top dose:
33.3, 100, 333, 1000, 3330, and 5000 ug/plate
Vehicle:
DMSO- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene: TA100, TA1535, TA1537 with S9; ICR-191: TA1537 without S9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): At least 0.5 x 10^9 cells/mL

DURATION
- Preincubation period: None
- Exposure duration: 52 +/- 4 hours
- Expression time (cells in growth medium): 52 +/- 4 hours
- Selection time (if incubation with a selection agent): 52 +/- 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 52 +/- 4 hours

SELECTION AGENT (mutation assays): Histidine and tryptophan-minimal agar.

NUMBER OF CELLS EVALUATED: All colonies were counted with an automatic colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn growth
Rationale for test conditions:
Based on the method of Ames et al. (1975).
Evaluation criteria:
TA98, TA100, WP2uvrA: For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose reponse to increasing concentrations of the test article.

TA1535 and TA1537: For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the test article was positive in the Ames assay in the presence and absence of metabolic activation.
Executive summary:

The Ames mutagenicity potential of the test article was evaluated in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Eschericia coli WP2uvrA in the presence and absence of metabolic activation (S9 -mix). The study was conducted according to a method equivalent to OECD 471 and was conducted in compliance with GLP regulations. The tester strains were exposed to the test article dissolved in DMSO via the plate incorporation method at concentrations of 33.3, 100, 333, 1000, 3330, and 5000 ug/plate. Following incubation with the test article for 52 +/- 4 hours, the colonies were counted with an automatic colony counter. Cytotoxicity was observed in all S. typhimurium strains at 3300 and 5000 ug/plate. No cytotoxicity was observed in the E. coli strain up to the limit concentration. The test article caused positive increases in the number of revertants per plate with tester strains TA98 (2.9 and 2.8 -fold), TA100 (18.6 and 17.6 -fold), TA1535 (211.8 and 136.4 -fold), and WP2uvrA (24.2 and 22.9 -fold) in the presence of S9 -mix and with tester strains TA100 (11.4 and 8.4 -fold), TA1535 (78.7 and 62.1 -fold), and WP2uvrA (12.0 and 6.6 -fold) in the absence of S9 -mis. With tester strain TA98 in the absence of S9 mix, a 3.0 -fold increase was observed in the initial experiment and a 1.9 -fold increase was observed in the confirmatory assay. No positive increases were observed with any of the remaining tester strain/activation condition combinations. Based on the results of the study, the test article was positive in the Ames assay in the presence and absence of metabolic activation.