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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in any of the tests performed (Ames (2x), micronucleus assay, chromosome aberration test, SOS chromotest).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Test substance storage: At room temperature in the dark
- Description: Off white solid
- Expiry date: 19 January 1999
- Stability in vehicle: at least 96 hours in ethanol
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogenate
Test concentrations with justification for top dose:
Experiment 1a: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (TA100, WP2uvrA)
Experiment 1b: 10, 33, 100, 333, 1000 µg/plate (TA 1535, TA 1537, TA98)
Experiment 2: 10, 33, 100, 333, 1000 µg/plate (All strains)
Vehicle / solvent:
ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: Daunomycine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
Evaluation criteria:
Acceptability of the assay: A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Data evaluation: A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in any tester strain is not greater than two (2) times the solvent control, with or without metabolic activation b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if: a) It induces at least 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. b) The positive response should be reproducible in at least one independently repeated experiment.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
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:
Dose range finding test:
- The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards in tester strain TA100 and WP2uvrA.
- In tester strain WP2uvrA, no reduction of the bacterial background lawn and no decrease in the number of revertants was observed. In tester strain TA100, no reduction of the bacterial background lawn was observed. In the absence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 333 µg/plate and upwards, which was less than the minimal value of the historical control data range. In the presence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 1000 µg/plate and upwards.

Mutation assay:
- The test substance precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate in all tester strains.
- In tester strain TA100, a reduction in the number of revertant colonies was observed at the concentration of 1000 µg/plate, which was less than the minimal value of the historical control data range. In the other strains, no clear decrease in the number of revertants was observed. The bacterial background lawn was not reduced at all concentrations tested.

Number of revertants
- All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The negative control values were within our laboratory background historical control data ranges, except for WP2uvrA (first experiment). However, since these values were just outside the limit of the range, the validity of the test was considered not to be affected.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames

In a bacterial reverse mutation assay, performed according to OECD Guideline 471 and following GLP, the test substance was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). The test substance was dissolved in ethanol. In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in tester strain TA100 and WP2uvrA. The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards in tester strain TA100 and WP2uvrA. In tester strain WP2uvrA, no reduction of the bacterial background lawn and no decrease in the number of revertants was observed. In tester strain TA100, no reduction of the bacterial background lawn was observed. In the absence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 333 µg/plate and upwards, which was less than the minimal value of the historical control data range. In the presence of S9-mix, a reduction in the number of revertant colonies was observed at concentrations of 1000 µg/plate and upwards. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 10 to 1000 μg/plate in the absence and presence of S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay, the test substance was tested at the same concentration range as the first assay in the absence and presence of S9 -mix in all tester strains. The test substance precipitated in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate in all tester strains. In tester strain TA100, a reduction in the number of revertant colonies was observed at the concentration of 1000 µg/plate, which was less than the minimal value of the historical control data range. In the other strains, no clear decrease in the number of revertants was observed. The bacterial background lawn was not reduced at all concentrations tested. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In a publication by Mersch-Sundermanno (1998), the test substance was tested in the Salmonella typhimurium reverse mutation assay (plate-incorporation) with four strains of Salmonella typhimurium (TA97, TA98, TA100 and TA102). The test substance was dissolved in DMSO. The test was performed in triplicate in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). The strains were exposed to 0, 5, 16.6, 50, 166.6, and 500 µg/plate. The maximum concentration was chosen considering the limit of solubility of the compounds in the aqueous medium of the assays. In the present study the test substance did not show a mutagenic effect using the Salmonella/mammalian mutagenicity assay.

Chromosome aberration test

In a chromosome aberration test, performed in accordance with OECD Guideline 473 and following GLP, Chinese hamster lung (CHL) cells were exposed to the test substance in the presence and absence of a metabolic activation system. In the absence of S9-mix, the test substance was tested up to 42 µg/mL for a 24 h treatment/fixation time and up to 33 µg/mL for a 48 h treatment/fixation time in the first experiment. In the second experiment, the test substance was tested up to 33 µg/mL for a 24 h treatment/fixation time. In the presence of 8% (v/v) S9-fraction the test substance was tested up to 33 µg/mL for both a 3 h treatment time with a 24 h fixation time and a 3 h treatment time with a 48 h fixation time in the first experiment. In the second experiment, the test substance was tested up to 24 µg/mL for a 3 h treatment time with a 24 h fixation time. Positive control chemicals, mitomycin C and Cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In the absence of S9-mix statistically significant increases in the number of cells with chromosome aberrations were observed at concentrations of 18 and 33 µg/mL at the 24 h treatment/fixation time in the first experiment. Since, the types of aberrations observed were mainly breaks and gaps, the increases are not dose-related, the second experiment did not confirm this result and moreover the number of cells with chromosome aberrations are still within the historical control data range these increases are considered not biologically relevant. In the presence of S9-mix a statistically significant increase in the number of cells with chromosome aberrations was observed at the concentration of 33 µg/mL at the 3 h treatment time with a 24 h fixation time in the first experiment. Since, the types of aberrations observed were mainly breaks and gaps, the increase is not dose-related, the second experiment did not confirm this result and moreover the number of cells with chromosome aberrations are still within our historical control data range this increase is considered not biologically relevant. Finally, it is concluded that this test should be considered valid and that the test substance is not clastogenic under the experimental conditions of this test.

Micronucleus assay

In a publication by Kevekordes (1997), the test substance was examined in vitro in a micronucleus test using human lymphocytes in the presence and absence of rat liver S9 and using human hepatoma cell line Hep G2 (metabolic system not required). The test concentrations used were up to the toxic concentrations. No genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2 was observed.

SOS chromotest

In a publication by Mersch-Sundermanno (1998), the test substance was tested in the Escherichia coli PQ37 genotoxicity assay (SOS chromotest). The test substance was dissolved in DMSO. The test was performed in quadruplicate in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). The E. Coli was exposed to 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 µg/assay. The maximum concentration was chosen considering the limit of solubility of the compounds in the aqueous medium of the assays. In the present study the test substance did not show SOS inducing potency.

Justification for classification or non-classification

The test substance does not have to be classified for mutagenicity according to Regulation (EC) No 1272/2008.