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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity in vitro - Gene Mutation in Bacteria (Bacterial Reverse Mutation Assay/Ames): negative with and without metabolic activation (equivalent to OECD471) - In vitro micronucleus test (in human lymphocytes and hepatocytes): negative with and without metabolic activation (similar to OECD474) - Gene Mutation in Mammalian Cells (L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay): negative with and without metabolic activation (according to OECD476)
Link to relevant study records
Reference
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:
June 21, 2005 - August 26, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted equivalent to OECD guideline 471 and under GLP conditions.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble at 50 mg/mL in distilled water and dissolved in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL with DMSO was considered to be stable and therefore preferably selected as a solvent in this study.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2uvrA); sodium azide (TA1535); Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HC1 (TA1537). +S9: 2-aminoanthracene (all test strains)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min.
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups.

NUMBER OF CELLS EVALUATED: S. typhimurium and E. coli strain were in the range of 2.3-2.7x109 cells/mL and 3.8-4.2 x 109 cells/mL resp.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibiton

OTHER EXAMINATIONS:
- Other: RANGE-FINDING/SCREENING STUDIES: Dose finding Test-1 and Test-2: Two dose-finding tests were performed to determine test concentrations in the main test. Based on observed growth inhibition at more than 78.1 µg/plate without S9 mix and at more than 313 µg/plate with S9 mix in all test strains, these concentrations were used as highest doses in the main test.
- Other: The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
The test compound was judged positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner and a reproducibility of the test results was also assured. In all other cases, it was judged negative.
Statistics:
No statistical methods were used.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no confounding effects found.

RANGE-FINDING/SCREENING STUDIES:
DOSE FINDING TEST-1: Bacterial growth inhibition was observed at more than 78.1 µg/plate without S9 mix, and at more than 313 µg/plate with S9 mix in all test strains. The test compound precipitates were observed at 5,000 µg/plate without S9 mix.
DOSE FINDING TEST-2: Bacterial growth inhibition was observed at 78.1 µg/plate without S9 mix in all test strains, and at more than 156 µg/plate in TA100, TA1535 and TA1537, 313 µg/plate in WP2 uvrA and TA98 with S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacterial growth inhibition was observed at 78.1 µg/plate without S9 mix in all test strains, and at more than 156 µg/plate in TA100, TA1535 and TA1537, and at 313 µg/plate in WP2uvrA and TA98 with S9 mix.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The mutagenicity of the test compound Cashmeran was tested in a Bacterial Reverse Mutation Assay (Ames). The number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and the number of revertant (Trp+) colonies in test strain WP2uvrA, both in the absence and presence of S9-metabolic activation, was less than twice that of each negative control in all test strains. As the test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory, the test was considered valid. It is concluded that Cashmeran has no ability to induce mutations in bacteria under the present test conditions.
Executive summary:

The ability of Cashmeran to induce mutations was investigated in the Bacterial Reverse Mutation Assay (Ames) by using 4 histidine-requiring strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537), and a tryptophan-requiring strain of Escherichia coli (WP2 uvrA) with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The study procedures described in this reverse mutation assay were equivalent to OECD guideline 471. Test concentrations used in the main test were as follows: - S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate; + S9 mix: 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate.

The mutagenicity of the test compound was judged negative because the number of revertant colonies in the test compound treatment groups was less than twice that of each negative control in all test strains. The number of revertant colonies in the positive control groups were more than twice that of negative control groups. The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory, indicating that the test can be considered valid.

It is concluded that Cashmeran has no ability to induce mutations in bacteria under the present test conditions.

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

Additional information

Additional information from genetic toxicity in vitro:

The ability of Cashmeran to induce mutations in bacteria was investigated in the Bacterial Reverse Mutation Assays (Ames test) performed equivalent to OECD 471. In absence as well as in presence of a metabolic activation system, Cashmeran did not induce gene mutations in bacteria, and was considered negative in the Ames test.

Furthermore, Cashmeran was examined for its cytogenicity in the micronucleus test (similar to OECD474) with human lymphocytes and hepatocytes in vitro in the presence and absence of metabolic activation. Cashmeran did not induce a significant increase in the frequency of micronuclei in both human lymphocytes and human hepatocytes. It is concluded that Cashmeran is not cytogenic in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.

The test article, Cashmeran, was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay (in accordance with OECD 476), both in the presence and absence of exogenous metabolic activation, to investigate its potential to induce gene mutations in mammalian cells. All of the cloned cultures treated with Cashmeran in both the Definitive and Confirmatory Mutation Assays had Mutant Frequencies that were similar to that of their corresponding solvent control cultures. Therefore, under the test conditions, Cashmeran is negative in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay.


Justification for selection of genetic toxicity endpoint
The Ames test is one of the three genotoxicity assays needed to assess this endpoint.

Justification for classification or non-classification

Based on the available information in the dossier, Cashmeran does not need to be classified for genotoxicity when considering the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.