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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:
From March 17, 2009 to April 14, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidelines S2A and S2B (ICH, 1996, 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9
Vehicle / solvent:
The vehicle control article was deionized water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Sterility Controls
The most concentrated test substance dilution and the S9 mix (50 and 500 µL, respectively; the same volumes used in the assay) were checked for sterility by plating on selective agar.

Liver Homogenate
Liver homogenate (S9) was purchased from Molecular Toxicology, Inc. (Lot Nos. 2354 and 2376, containing 33.0 and 39.8 mg/mL protein, respectively). The homogenate was prepared from male Sprague-Dawley rats that had been injected (intraperitoneally) with Aroclor™ 1254 (200 mg/mL in corn oil) at 500 mg/kg, 5 days before sacrifice (Ames, et al., 1975).

S9 Mix
S9 mix was prepared on the day of use, maintained on ice, and contained the components indicated below (Table II; the mix and metabolic activation system are referred to as S9 interchangeably).

Tester Strains
The tester strains used were the Salmonella histidine auxotrophs TA98, TA100, TA1535, and TA1537 (Ames et al. 1975) and the E. coli tryptophan auxotroph WP2uvrA (Green and Muriel 1976). In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds.

Source of Tester Strains
Salmonella tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California (Berkeley, CA). E. coli tester strain WP2uvrA was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

Preparation of Overnight Cultures
Inoculation
Overnight cultures were inoculated into flasks containing culturing broth and the flasks were placed in a shaker/incubator programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 2°C) so that overnight cultures were in late log phase when optical density (OD) monitoring began.

Harvest
To ensure cultures are harvested at the appropriate phase, the growth of the culture was monitored using a spectrophotometer. An aliquot of each culture was diluted 1:4 and its OD determined at 650 nm (OD650(1:4)). Cultures were harvested once a predetermined OD was reached which ensured that cultures had reached late exponential or early stationary phase (representative of cultures with ≥109 cells/mL) and had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Once the target OD was reached (OD650(1:4) = 0.4 to 0.6), the cultures were removed from incubation and held at >0 to 10°C until used in the assay.

Test Design
The test article was evaluated in the initial mutagenicity assay at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with and without S9. Positive and vehicle controls were evaluated concurrently. All test and control articles were evaluated in duplicate plates. An independent confirmatory assay subsequently was performed at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. Positive and vehicle controls were evaluated concurrently and all test and control articles were evaluated in triplicate plates in the confirmatory assay. Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983).
Rationale for test conditions:
The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his–) or tryptophan (trp–) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his– or trp– cells.
Evaluation criteria:
Criteria for a Positive Response
A test substance is considered to have produced a positive response if it induces a dose dependent increase in revertant frequency that is ≥2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.
Criteria for a Negative Response
A test substance is considered to have produced a negative response if no dose-dependent, ≥2.0-fold or ≥3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.
Criteria for an Equivocal Response
Even after repeated trials, a test substance may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test Article Handling
Deionized water was the vehicle for this study. In solubility testing, the test substance was observed to form an opaque, white, homogeneous suspension at approximately 100, 50, and 25 mg/mL, and a translucent, white, homogeneous suspension at approximately 12.5 mg/mL, in deionized water. At 100 mg/mL, which was the most concentrated dose formulation prepared for treatment; the test substance was observed to form an off-white, opaque, non-viscous, homogeneous suspension. The test substance diluted to a solution at 3.2 mg/mL and remained freely soluble at all succeeding lower dilutions prepared.

Mutagenicity Assay
The test substance was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence and absence of S9 mix. All doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in duplicate plates. No positive increases in the mean number of revertants/plate were observed with any of the tester strains in the presence or absence of S9 mix. No dose-related decreases in revertant frequency were observed with any of the tester strains in the presence or absence of S9. Reduced bacterial background lawns were observed at ≥1600 μg/plate in the absence of S9 mix with tester strains TA100, TA1535, and TA1537.

Based on the results of the initial assay, the test substance was re-evaluated in the confirmatory mutagenicity assay at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. All doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. No positive increases in the mean number of revertants/plate were observed with any of the tester strains in the presence or absence of S9 mix. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was negative in the Bacterial Reverse Mutation Assay with a confirmatory assay.
Executive summary:

A study was conducted to determine the potential of the test substance and its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) according to OECD Guideline 471, EPA OPPTS Method 870.5100 and ICH Guidelines S2A and S2B, in compliance with GLP. The test substance was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. Based on the results of the initial assay, the test substance was re-evaluated in the confirmatory mutagenicity assay at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. All positive and vehicle control values were within acceptable ranges and all criteria for a valid study were met. Under the study conditions, the test substance was negative in the Bacterial Reverse Mutation Assay with a confirmatory assay (Farabaugh, 2009).