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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:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
other: U. S. Environmental Protection Agency (40 CFR 798.5265)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 98.7%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No data
- Expiration date of the lot/batch: No data
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under test conditions: Stable during the study period as indicated by an absence of change in test substance peak areas relative to total peak areas in gas chromatography analyses.

Method

Target gene:
histidine operon, tryptophan operon
Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA97, TA98, TA100, TA1535
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 fraction
Test concentrations with justification for top dose:
0.1, 0.2, 0.3, 0.4 and 0.5%
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, ICR-191 Acridine methylmethanesulfonate
Details on test system and experimental conditions:
The substance was tested as a gas, with precautions to avoid generating an explosive atmosphere according to recommendations in OECD 403 and EPA 40 CFR 798.1150.

METHOD OF APPLICATION: The test substance was introduced into a sealed chamber containing cultured agar plates. Treatments without activation were conducted by adding 0.1 mL of an overnight culture containing approximately 1 x 10e8 bacteria to 2 mL top agar (0.6% agar (w/v) and 0.6% NaCl (w/v)) supplemented with 0.05 mM L-histidine, 0.05 mM biotin for Salmonella strains or 0.05 mM L-tryptophan for E. coli. These components were mixed and poured into a plate containing 25 mL of Davis minimal agar. Plates were then placed onto stainless steel racks and inserted into sterile 6-liter glass exposure chambers. Separate chambers were used for each concentration and for activated and nonactivated treatments. The test substance and diluent gas were metered through Matheson rotameters, mixed in 1/4 inch stainless steel tubing and introduced into the exposure chambers at a flow rate of approximately 8 L/minute. The chambers were then sealed by shutting the chamber stopcocks after flushing with at least five volume changes of the appropriate test substance-air mixture. Treatments with activation were conducted exactly as described for the non-activated treatments except that 0.5 mL S9 Mix was added to the bacteria/top agar mixture before it was poured onto a Davis minimal agar plate.

DURATION
- Exposure duration: incubated at approximately 37°C for approximately 48 hours.

NUMBER OF REPLICATIONS: two trials, 3 replicates per dose

OTHER: The lowest explosive limit (LEL) for the test substance is 1%. Thus, the maximum concentration of test substance evaluated was 0.5% in air.
Evaluation criteria:
A test substance is classified as POSITIVE when:
1. The average number of revertants in any strain at any test substance concentration studied is at least two times greater than the average number of revertants in the negative control.
AND
2. There is a positive dose-relationship in the same strain.

A test substance is classified as NEGATIVE when:
1. There are no test substance concentrations with an average number of revertants which is at least two times greater than the average number of revertants in the solvent control.
OR
2. There is no positive dose-relationship.

A test substance is classified as EQUIVOCAL when the test substance is not clearly negative yet does not meet the criteria of a positive response.
Statistics:
Trials were evaluated independently. For each selected tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA97, TA98, TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No precipitation of the test article or background lawn death was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the test article was negative in the Bacterial Reverse Mutation assay in the presence and absence of metabolic activation (S9 mix).
Executive summary:

The mutagenic activity of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium (strains: TA1535, TA97, TA98, and TA100) and E. coli (strain: WP2uvrA) in the presence or absence of a metabolic activation system (S9-mix: Aroclor 1254-induced rat liver). This study was performed in accordance with EPA GLP 40CFR792 and OECD GLP C(81)30(Final), Annex 2. The study design was based on OECD 471 (1997). The maximum concentration was limited to one half of the lower exposure limit (1%) because of the volatility of the test substance. The test article was exposed to the cells at 0.1, 0.2, 0.3, 0.4, and 0.5% in the presence or absence of S9-mix. Dry compressed air was used for the negative control. Strain specific positive controls were tested in parallel. Two independent trials were performed for each concentration. Positive and negative controls performed as expected indicating that all criteria for a valid study were met. No substantial increases in revertant colonies were observed in test article-treated cultures in the presence or absence of S9-mix. Based on the results of the study, the test article was negative in the Bacterial Reverse Mutation assay in the presence and absence of metabolic activation (S9 mix).