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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: OPPTS 799.9510
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: Commission Directive 92/69/EEC, EEC Method B.12
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the 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: 99.99 %

Method

Species / strainopen allclose all
Species / strain / cell type:
other: TA97a, TA98, TA100, TA1535
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9
Test concentrations with justification for top dose:
Nominal Concentrations: 20, 30, 40, 50, and 75%
Actual Mean Concentrations (first trial; -S9): 20.4, 33.5, 42.0, 55.5, and 82.7%
Actual Mean Concentrations (first trial; +S9): 21.7, 33.4, 47.6, 56.1, and 84.4%
Actual Mean Concentrations (second trial; -S9): 20.5, 30.9, 41.4, 53.9, and 85.1%
Actual Mean Concentrations (second trial; +S9): 20.8, 31.7, 41.9, 55.3, and 84.1%
Vehicle / solvent:
Filtered house-line air
Controls
Untreated negative controls:
yes
Remarks:
Filtered house-line air
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (TA98 -S9), N-ethyl-N-nitro-N-nitroguanidine (WP2urvA -S9), sodium azide (TA100 and TA1535 -S9), ICR 191 acridine (TA97a -S9), 9,10-dimethyl-1,2-benzanthracene (TA97a +S9), and 2-aminoanthracene (TA98, TA100, TA1535, and WP2urvA -S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Treatments with activation were conducted by adding S-9 mix and 0.1 mL of an overnight culture containing approximately 1E8 bacteria to top agar supplemented with L-histidine, biotin (for Salmonella strains) or L-tryptophan (for E. coli). Treatments in the absence of the metabolic activation system were identical to those with activation with the exception that 0.5 mL of sterile buffer was used as a replacement for the S-9.

Plates were exposed to dilutions of the test gas after being placed on stainless steel racks designed to fit in 6-L glass chambers. Chambers were equipped with stopcocks and o-ring gaskets. As gases, the test substance and filtered air flows were regulated using individual rotameters, and mixed prior to entry into the chambers. A flow rate of approximately 6 L/minute for approximately 5 minutes was used to create 5 volume changes to occur within the chambers to ensure homogenous concentrations. Chambers were closed and 2-3 samplings of each chamber were taken and analyzed by gas chromatography to determine the initial concentration of the test substance. Chamber concentrations were determined by comparison of integrated peak areas to gas standards. Chambers were placed into an incubator at 37ºC for approximately 48 hours. Chambers were again sampled and analyzed to determine the ending test substance concentration. Gas chromatographic analysis was used to confirm the concentration of test atmospheres. The chambers were flushed with at least 5 chamber volumes of filtered air. The plates were then removed and refrigerated until evaluation.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 trials with 3 replicates were plated for each tester strain, test concentration, and condition

OTHER: Bacterial background lawns were evaluated for evidence of test substance toxicity and precipitation. Evidence of toxicity observed in the microcolony background lawns was scored relative to the concurrent negative control plates and recorded with the mean revertant count for the strain, condition, and concentration. Revertant colonies for a given tester strain and condition were counted by an automated colony counter.
Evaluation criteria:
A test substance was classified as positive if the mean number of revertants in any strain (except S. typhimurium TA1535) at any concentration was at least 2 times greater than the mean number of revertants of the concurrent negative control, and there was a concentration-related increase in the mean number of revertants per plate in that same strain. For S. typhimurium TA1535, there must be no test substance concentration with a mean number of revertants that is at least 3 times greater than the mean number of revertants of its concurrent negative control and a concentration-related increase in the mean number of revertants per plate. A test substance was classified as negative if all positive classification criteria for all strains were not met.
Statistics:
Data for each tester strain were evaluated independently. For each tester strain, the mean number of revertants and the standard deviation at each concentration in the presence and absence of the exogenous metabolic system were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA97a, TA98, TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA97a (-S9; both trials), TA1535 (+S9; first trial), TA98 (+S9 and -S9; second trial)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S9 and -S9; both trials
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The actual concentrations for the first trial were 0, 20.4, 33.5, 42.0, 55.5, and 82.7% in the absence of S-9 and 0, 21.7, 33.4, 43.6, 56.1, and 84.4% in the presence of S-9. No test substance-related precipitate was observed at any concentration level. Test substance-related toxicity, evidenced by the concentration-dependent reduction in the mean number of revertants per plate, was observed in the first trial in S. typhimurium strains TA97a (-S-9) and TA1535 (+S-9), and in the E. coli strain (+S-9, -S-9). The actual concentrations for the second trial were 0, 20.5, 30.9, 41.4, 53.9, and 85.1% in the absence of S-9 and 0, 20.8, 31.7, 41.9, 55.3, and 81.4% in the presence of S-9. Test substance-related toxicity, evidenced by the concentration-dependent reduction in the mean number of revertants per plate, was observed in the second trial in S. typhimurium strains TA97a (-S-9) and TA98 (+S-9, -S-9), and in the E. coli strain (+S-9, -S-9).

All acceptability criteria were met in this test. All tester strains exhibited appropriate phenotypic characteristics. No test substance-related precipitate was observed. The mean number of revertants in the negative control for each strain was within the prescribed acceptable historical control range. Mean positive control values for the tester strains exhibited greater than a 3-fold increase over the means of the respective negative controls in both trials. Differences between targeted and actual doses in both analyses were acceptable for the purposes of this assay and in no way impacted the integrity or validity of this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). Under the conditions of the study, no evidence of mutagenic activity was detected in either trial with the test substance. Based on the findings, the test substance is concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli tester strains, in the presence or absence of an exogenous S9 metabolic activation system.
Executive summary:

The test substance was evaluated in the bacterial reverse mutation test using Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and Escherichia coli strain WP2uvrA (pKM101) in the presence and absence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The target concentrations for the first trial were 0, 20, 30, 40, 50, and 75%. In comparison to the negative control (air), tester strains were exposed to the test substance at actual mean concentrations of 20.4, 33.5, 42.0, 55.5, and 82.7% in the absence of S9, and 21.7, 33.4, 47.6, 56.1, and 84.4% in the presence of S9 for approximately 48 hours. The target concentrations for the second trial were also 0, 20, 30, 40, 50, and 75%. In comparison to the negative control (air), tester strains were exposed to the test substance at actual mean concentrations of 20.5, 30.9, 41.4, 53.9, and 85.1% in the absence of S9 and 20.8, 31.7, 41.9, 55.3, and 81.4% in the presence of S9. For the test substance, the mean number of revertants observed in the negative controls for each strain in both trials was within the acceptable historical negative control ranges. All tester strains exhibited appropriate phenotypic characteristics in both assays. No test substance-related precipitate was observed at any concentration of the test substance with any strain. Test substance-related toxicity, as evidenced by a concentration-related reduction in the mean number of revertants per plate, was observed with S. typhimurium strains TA97a, TA98, and TA1535 in at least one trial with or without S9 and E. coli  WP2uvrA (pKM101) in both trials with and without the metabolic activation system. No evidence of mutagenicity was detected with any tester strains. Under the conditions of this study, no evidence of mutagenic activity was detected in either trial with the test substance. Based on the findings, the test substance is concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli.