Registration Dossier
Registration Dossier
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EC number: 684-597-9 | CAS number: 1072005-10-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No data on the test item is available. With the read-across substances the following results were obtained:
CAS 68583-51-7: negative in Ames test with 4 strains
CAS 91031-31-1: negative in Ames test with 5 strains
CAS 853947-59-8: negative in Chromosome aberration test
CAS 91031-31-1: negative in mouse lymphoma test
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30.06.1995 - 17.07.1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May 1983
- Principles of method if other than guideline:
- Only 4 bacterial strains used.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : CCR (Cytotest Cell Research GmbH & Co. KG), Roßdorf, Germany
- method of preparation of S9 mix: according to Ames et al., 1975 (obtained from the livers of male Wistar rats which received a single i.p. injection of Aroclor 1254 (500 mg/kg body weight) in olive oil 5 days prior to the preparation of the S9 fraction. The livers were removed from the animals, homogenized, and the supernatant of the 9000 x g centrifugation step (the "59 fraction") was frozen immediately. Protein content, sterility and activity of the preparation in the S. typhimurium gene mutation assay were certified by CCR.) - Test concentrations with justification for top dose:
- 50, 160, 500, 1600 and 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene (with S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h at 37°C
SELECTION AGENT: - Salmonella typhimurium: L-Histidine
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduced growth of the bacterial background lawn - 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- A reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity, was not detectable with any of the tester strains. In both tests with and without metabolic activation by S9 mix, the test item formed a precipitate at the highest concentration tested (5000 µg/plate). The precipitate did not interfere with colony counting and obviously did not effect the mutant frequency of any of the S. typhimurium strains. All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. In both experiments, no indication of test compound induced mutagenicity was observed with either one of the four tester strains TA 98, TA 100, TA 1535, and TA 1537, with or without metabolic activation.
- Conclusions:
- The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 in the presence and absence of a metabolizing system.
- Executive summary:
The mutagenicity of the test substance was studied with the mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) according to OECD guideline 471. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 50 to 5000 µg per plate in the presence and absence of S9. Precipitation of the test compound an the plates was observed at 5000 µg/plate. All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that under the experimental conditions described, the test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 in the presence and absence of a metabolizing system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13.03.1991 - 22.03.1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May 1983
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : CCR (Cytotest Cell Research GmbH & Co. KG), Roßdorf, Germany
- method of preparation of S9 mix: Male Wistar rats received a single intraperitoneal injection of Aroclor 1254 dissolved in olive oil at a dosage of 500 milligrams per kilogram b.w. five days previously to sacrifice to induce liver microsomal enzyme activity. The rats were sacrificed on the fifth day after application. Livers were removed under aseptic conditions and homogenized with a potassium chloride solution. After centrifugation, the supernatant fraction is the S9 fraction. It is labelled with lot numbers. The S9 fractions were transported frozen on dry ice and stored at -80° C. - Test concentrations with justification for top dose:
- 8, 40, 200, 1000 and 5000 µg/plate
- Vehicle / solvent:
- Tween 80/H2O
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2-aminoanthracene (with S9) for all strains, 4-ni tro-o-phenylendiamine TA 98 and TA 1538 without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h at 37°C
SELECTION AGENT: - Salmonella typhimurium: L-Histidine
NUMBER OF REPLICATIONS: 3 replicates each in two tests
DETERMINATION OF CYTOTOXICITY
- Method: reduced growth of the bacterial background lawn - 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test item did not induce any reverse mutations in the absence of rat liver enzymes (without S9-mix), nor did occur any induced reverse mutations in the presence of Aroclor 1254-induced S9-mix. Toxie effects up to 5000 pg/plate were not noted. Precipitations were noted at the concentration of
5000 pg/plate. - Conclusions:
- The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of a metabolizing system.
- Executive summary:
The mutagenicity of the test substance was studied in two independent experiments with the mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA 1538) according to OECD guideline 471. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in Tween 80/H2O and tested in concentrations of 8 to 5000 µg per plate in the presence and absence of S9. Precipitation of the test compound an the plates was observed at 5000 µg/plate. All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that under the experimental conditions described, the test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 1538 in the presence and absence of a metabolizing system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July - October 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- adopted May 1983
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- May 1992
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : ICN Biomedicals, Eschwege, Germany
- method of preparation of S9 mix: It was obtained from the livers of 8 - 10 weeks old male Sprague Dawley rats which received a single i.p. injection of 500 mg/kg b.w. Arochlor 1254 in corn oil 5 days prior to the preparation of the S9 fraction. The livers were removed from the animals, homogenized, and the supernatant of the 9000 x g centrifugation step (the "S9 fraction") was frozen immediately.
- quality controls of S9: Protein content, sterility and activity of the preparation in the S. typhimurium gene mutation assay were certified by Biomedicals and ICN. Additionally, the ethoxyresorufin O-dealkylase activity is determined bimonthly to assure a sufficient activity of the S9 fraction - Test concentrations with justification for top dose:
- - S9 mix: 0,10,40 and 80 µg/mL (18 h sampling time test #1 a, #2)
80 µg/mL (28 h sampling time test #2)
+S9 mix: 0, 10, 60 and 100 µg/mL (18 h sampling time test #1 a, #2)
100 µg/mL (28 h sampling time test #2)
100 µg/mL is the solubility limit - Vehicle / solvent:
- MEM4 medium containing 1 % ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 18 hours (Exp I), 28 h (Exp II)
- Fixation time: Hypotonic treatment was terminated after 8 min by fixation in methanol/glacial acetic acid.
NUMBER OF CELLS EVALUATED: Per experimental point, at least 2,000 cells (1,000 per slide) were scored. Where possible, 100 metaphases from each culture (i.e. 200 metaphases per experimental point) were analyzed for chromosomal aberrations.
DETERMINATION OF CYTOTOXICITY: The mitotic indices of representative cultures, as a measure for cytotoxicity, were determined. - Evaluation criteria:
- The test chemical is to be considered clastogenic in this assay if
1. it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
2. the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system (i.e. » 5%)
3. positive results can be varified in an independent experiment.
Increases in the proportion of cells with gaps or increases in the numbers of cells with structural aberrations not exceeding the normal range are considered on a case by case basis. The possible influence of pH, S9 mix or osmolality on the occurance of
chromosomal aberrations will also be considered. As this assay is not designed to detect numerical aberrations, polyploidy and endoreduplications are reported when seen, but these data are not used for any interpretation. - Statistics:
- From the proportion of cells treated with the test substance and harboured, structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- In chromosome aberration studies with metabolic activation, cells were treated with 3 concentrations ranging from 10 to 100 µg/ml. In the corresponding experiments without metabolic activation, 3 concentrations ranging from 10 to 80 µg/mL were applied. In both experiments, at the 18 h (test #1 a, #2) as well as at the 28 h (test #2) sampling time, the test item did not induce significant increase in the proportion of cells with chromosom aberration (excluding gaps).
The negative controls revealed chromosom aberration frequencies (without gaps) of 0 to 4.0 % which is consistent in the range of spontaneous aberration frequency for V79 celiline (in this test laboratory, maximum accepted spontaneous aberration frequency should be < 5 %).
The positive controls, Mitomycin C and Cyclophosphamide, led to biologically significant increase in the frequency of aberrations; the metabolic activation system was satisfactory and the test method worked..
The test item +/- S9 at both tested incubation times did not result in statistically or biological meaningful increase in frequency of cells with chromosome aberration. The frequency of polyploid cells was within the expected range (< 10 %). - Conclusions:
- In the in vitro cytogenetic assay as described in this report, the test item did not induce significant increase in the chromosomal aberration frequency of V79 Chinese hamster cells and is therefore judged to be not clastogenic in vitro.
- Executive summary:
The test item was tested for its ability to induce chromosomal aberrations in an in vitro mammalian cell system. V79 cells (Chinese hamster lung cells) were exposed in presence and absence of rat liver S9. 16 and 26 h after the start of exposure, cells were arrested in metaphase by 2 h treatment with Colcemid. After hypotonic treatment cells were fixed and Giemsa stained. The mitotic indices of representative cultures were determined, as a measure for cytotoxicity. For each experimental point in duplicate 100 metaphases per culture were evaluated. The sensitivity of the test system was demonstrated with positive controls (Mitomycin C without S9 mix and Cyclophosphamide with S9 mix). Results were confirmed in a second, independent experiment. First 10 concentrations were tested in the range of 1 to 100 µg/mL (solubility limit) in presence and absence of metabolic activation. Cytotoxicity was evaluated to determine dose range for further testing. With metabolic activation cells were treated with 3 test item concentrations ranging from 10 to 100 µg/mL test item. Without metabolic activation 3 concentrations ranging from 10 to 80 µg/mL were applied. In both experiments, at 18 h or 28 h sampling time, the test item did not induce significant increase in chromosomal aberration. From the experiments performed, it is concluded that under the conditions of the test system the test item was not clastogenic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June - August 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted July 21, 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 2008
- Qualifier:
- according to guideline
- Guideline:
- other: The recommendations of the 'International Workshop on Genotoxicity Tests Workgroup' (the IWGT), published in the literature (Clive et ai., 1995, Moore et al, 1999. 2000, 2002, 2003, 2006 and 2007).
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- method of preparation of S9 mix: Rat liver microsomal enzymes were routinely prepared from adut maie Wistar rats, which were obtained from Charles River, Sulzfeld, Germany. The rats were oralty dosed at three consecutive days with a suspension of phenobarbital (Bufa B.V., Usselstein, The Nethedands; 80 mg/kg body weight) and 13-naphthoflavone (Sigma Aldrich Chemie; 100 mg/kg body weight) in com oit (Roth, Karlsruhe, Germany) food access denied 3 to 4 hours befor dosing. One day afler the final exposure (24 h), the rats were sedated and then killed. The rats received a limited quantity of food during the night before sacrification. Livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4, Merck) containing 0.1 mM Na2-EDTA (Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer wfth a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supematant (S9-fraction) was transferred into sterile ampules, which were stored in liquid nitfagen (-196°C) for a maximum of 1 year. - Test concentrations with justification for top dose:
- First test: 0.1. 0.3. 1, 3. 10, 33, 100 and 333 µg/mL (absence and presence of 8% (v/v) S9-mix)
Second test: 3, 10, 33, 100, 125, 140, 175, 200, 225, 250 and 275 µg/mL (without S9-mix)
0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL (with 12 % (v/v) S9-mix) - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h (presence of S9-mix), 3 and 24 hours (absence of S9-mix)
- Expression time (cells in growth medium): 2 days
SELECTION AGENT: fluorothymidine
Second mutagenicity test: To test the mutagen potential of the test item, a second mutation experinent was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12% (v1v) S9-mix with a 3 hour incubation.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The mutation assay was considered acceptable if following criteria were met:
a) The absolute cloning efficiency of the solvent controls (CEday2) is, between 65 and 120%. An acceptable number of surviving cells 10^6 could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent controi is ≥ 50 x 10^-6 and ≤ 170 x 10^-6.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10^-6 and for CP not below 700 x 10^-6. - Statistics:
- The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(contols) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative resut can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency (MF) of controls + 126. b) The resuits are conflrmed in an indepen repeated test.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- First mutagenicity test: No severe toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item cultures were comparable to the numbers of small and large colonies of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the dose levels of 200 to 275 µg/ml were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 3, 10, 33, 100, 125, 140 and 175 µg/ml. In the absence of S9-mix, the relative total growth of the highest test substance concentration 175 µg/mL was reduced by 91% compared solvent control. The relative total growth at 140 µg/mL was reduced by 80% compared to the total growth of the solvent controls
In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. - Conclusions:
- The evaluation of the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat), no mutagenicity was detected.
- Executive summary:
The effects of the test item on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells was assessed. The test was performed in two independent experiments in the absence and presence of S9 -mix (rat liver S9-mix induced by a combination of phenobarbital and & β-naphthoflavone). The study procedures described in this report were based on the OECD guideline 476. The test item was suspended in dimethyl sulfoxide (DMSO). In the first experiment, the test item was tested up to concentrations of 333 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. However the test item precipitated already in the exposure medium at 100 mg/mL. In the second experiment, the test item was tested up to a concentrations of 333 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubation in absence and presence of S9-mix, respectively. The test item was tested up to cytotoxic level of 91% in the absence of S9--mix. No toxicity was observed in the presence of S9-mix. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical controls and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 17-fold for MMS in the absence of S9-mix, and by 16- and 14-fold for CIP in the presence of S9-mix. It was therefore concluded that the test conditions, both in absence and presence of S9-mix were appropriate and that the metabolic activation system (S9-mix) worked properly. In absence of S9-mix, the test item did not induce a significant increase in mutation frequency in the first experiment. The result was confirmed in an independently repeated experiment with modification in duration of treatment time. In presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independently repeated experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please refer to Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
Referenceopen allclose all
Table 1: Mean number of revertants with S9 mix
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
|
Deionised water |
- |
13 |
16 |
42 |
125 |
acetone |
- |
9 |
19 |
44 |
125 |
Test substance |
50 |
13 |
19 |
47 |
130 |
160 |
11 |
15 |
57 |
120 |
|
500 |
9 |
15 |
60 |
117 |
|
1600 |
9 |
15 |
59 |
117 |
|
5000 |
10 |
18 |
64 |
125 |
|
Positive control 1 |
2.5 |
197 |
113 |
1396 |
1617 |
Positive Control 1: 2 –Aminoanthracene
Table 2: Mean number of revertants without S9 mix
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
|
Deionised water |
- |
8 |
11 |
20 |
108 |
acetone |
|
9 |
11 |
22 |
98 |
Test substance |
50 |
5 |
17 |
17 |
108 |
160 |
7 |
16 |
20 |
122 |
|
500 |
7 |
16 |
21 |
105 |
|
1600 |
5 |
15 |
23 |
89 |
|
5000 |
6 |
15 |
20 |
113 |
|
Positive control 1 |
2.5 |
|
|
115 |
|
Positive control 2 |
2.5 |
330 |
|
|
372 |
Positive control 3 |
25 |
|
47 |
|
|
Positive control 1:2-Nitronuorene
Positive control 2:Sodium azide
Positive control 3:9-Aminoacridine
Table 1: Mean number of revertants with S9 mix (first test)
Test substance |
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
Buffer |
- |
9.3 |
10.0 |
26.0 |
67.7 |
18.7 |
Solvent |
- |
12.0 |
9.3 |
25.0 |
64.3 |
17.0 |
Test substance |
5000 |
9.3 |
9.0 |
35.0 |
74.7 |
19.0 |
1000 |
9.7 |
11.0 |
37.7 |
76.0 |
23.7 |
|
200 |
12.3 |
10.7 |
36.3 |
67.3 |
24.3 |
|
40 |
9.0 |
8.7 |
29.7 |
74.3 |
15.7 |
|
8 |
9.7 |
7.3 |
28.7 |
72.3 |
19.0 |
|
Positive control 1 |
2.5 |
274 |
124.7 |
|
|
|
5 |
|
|
1218.0 |
936.7 |
1499.7 |
Positive Control 1: 2 –Aminoanthracene
Table 2: Mean number of revertants without S9 mix (first test)
Test substance |
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
Buffer |
- |
9.7 |
8.7 |
19 |
55.7 |
8.3 |
Solvent |
|
10 |
6.7 |
22 |
61.3 |
7.7 |
Test substance |
5000 |
8.3 |
6.0 |
22.7 |
61.3 |
7.7 |
1000 |
8 |
11.3 |
19.3 |
62.0 |
9.7 |
|
200 |
8.3 |
8.3 |
18.7 |
70.3 |
9.3 |
|
40 |
6.3 |
9 |
23.3 |
67.3 |
10.7 |
|
8 |
6 |
10 |
18.7 |
51.0 |
8.3 |
|
Positive control 1 |
2.5 |
|
|
565.7 |
|
1678 |
Positive control 2 |
2 |
384 |
|
|
285 |
|
Positive control 3 |
80 |
|
955.3 |
|
|
|
Positivecontrol 1: 4-nitro-o-phenylendiamine
Positive control 2:Sodium azide
Positive control 3:9-Aminoacridine
Table 3: Mean number of revertants with S9 mix (second test)
Test substance |
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
Buffer |
- |
15.0 |
11.3 |
37.7 |
132.7 |
18.3 |
Solvent |
- |
19.0 |
7.7 |
45.0 |
111.3 |
17.7 |
Test substance |
5000 |
11.7 |
5.3 |
35.7 |
113.3 |
13.7 |
1000 |
15.0 |
8.3 |
48.7 |
122.3 |
18.0 |
|
200 |
13.0 |
13.3 |
45.0 |
121.0 |
19.7 |
|
40 |
12.7 |
7.7 |
36.7 |
117.7 |
21.3 |
|
8 |
14.0 |
9.3 |
31.3 |
113.7 |
15.7 |
|
Positive control 1 |
2.5 |
162.3 |
80.7 |
|
|
|
5 |
|
|
806.3 |
906.3 |
1318.0 |
Positive Control 1: 2 –Aminoanthracene
Table 4: Mean number of revertants without S9 mix (second test)
Test substance |
Dose/Plate (µg) |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
TA 1538 |
Buffer |
- |
11.0 |
9.3 |
21.0 |
103 |
7.3 |
Solvent |
|
12.3 |
9.0 |
25.0 |
111 |
9.0 |
Test substance |
5000 |
7.0 |
5.3 |
20.3 |
108 |
8.0 |
1000 |
11.3 |
7.7 |
24.3 |
115 |
9.3 |
|
200 |
13.0 |
9.3 |
24.3 |
103.3 |
8.0 |
|
40 |
11.7 |
10.7 |
24.0 |
109. |
7.0 |
|
8 |
13.0 |
10.0 |
22.7 |
94.0 |
10.0 |
|
Positive control 1 |
2.5 |
|
|
643.0 |
|
1500.7 |
Positive control 2 |
2 |
350.3 |
|
|
318 |
|
Positive control 3 |
80 |
|
678.7 |
|
|
|
Positivecontrol 1: 4-nitro-o-phenylendiamine
Positive control 2:Sodium azide
Positive control 3:9-Aminoacridine
Table 1: Summary of data obtained in chromosomal aberration test #1 a
Treatment |
Mitotic index [% mean] |
Total of mitotic cells scored |
Proportion of cells with exchanges [%] |
Proportions of cells with aberrations incl. gaps [%] |
Proportions of cells with aberrations excl. gaps [%] |
significance |
||
µg/mL |
Time [h] |
S9 mix |
||||||
Solvent control |
18 |
- |
10.7 |
200 |
0.0 |
2.0 |
0.0 |
- |
10 |
- |
8.0 |
200 |
1.0 |
3.5 |
1.5 |
x |
|
40 |
- |
6.7 |
200 |
0.5 |
3.0 |
0.5 |
x |
|
80 |
- |
5.3 |
200 |
1.5 |
5.0 |
0.0 |
x |
|
0.03 Pos. control |
- |
2.1 |
65 |
12.3 |
24.6 |
18.5 |
s |
|
0.04 Pos. control |
- |
2.9 |
135 |
14.8 |
40.0 |
31.9 |
s |
|
Solvent control |
18 |
+ |
7.0 |
200 |
3.5 |
6.5 |
4.0 |
- |
10 |
+ |
6.7 |
200 |
1.0 |
3.5 |
1.5 |
x |
|
80 |
+ |
6.1 |
200 |
1.0 |
4.5 |
1.0 |
x |
|
100 |
+ |
5.7 |
200 |
1.5 |
4.0 |
2.0 |
x |
|
3 Pos. control |
+ |
2.7 |
200 |
24.0 |
45.0 |
34.0 |
s |
|
x: not significant
s: significant
Table 2: Summary of data obtained in chromosomal aberration test #2
Treatment |
Mitotic index [% mean] |
Total of mitotic cells scored |
Proportion of cells with exchanges [%] |
Proportions of cells with aberrations incl. gaps [%] |
Proportions of cells with aberrations excl. gaps [%] |
significance |
||
µg/mL |
Time [h] |
S9 mix |
||||||
Solvent control |
18 |
- |
7.9 |
200 |
1.0 |
6.0 |
3.5 |
- |
10 |
- |
7.5 |
200 |
0.0 |
4.0 |
0.5 |
x |
|
40 |
- |
8.0 |
200 |
0.0 |
9.5 |
3.0 |
x |
|
80 |
- |
5.8 |
200 |
0.0 |
5.5 |
0.5 |
x |
|
0.03 Pos. control |
- |
3.4 |
200 |
7.5 |
23.5 |
14.0 |
s |
|
Solvent control |
18 |
+ |
6.3 |
200 |
1.0 |
8.0 |
2.0 |
- |
10 |
+ |
6.5 |
200 |
0.0 |
5.5 |
0.0 |
x |
|
80 |
+ |
5.7 |
200 |
1.0 |
4.5 |
1.5 |
x |
|
100 |
+ |
6.7 |
200 |
0.0 |
6.5 |
1.5 |
x |
|
3 Pos. control |
+ |
1.6 |
174 |
20.1 |
42.0 |
33.3 |
s |
|
Solvent control |
28 |
- |
7.9 |
200 |
0.0 |
3.5 |
0.0 |
- |
80 |
- |
5.8 |
200 |
0.0 |
4.5 |
0.5 |
x |
|
0.03 Pos. control |
- |
4.4 |
200 |
17.0 |
43.5 |
32.5 |
s |
|
Solvent control |
28 |
+ |
9.2 |
200 |
0.5 |
6.5 |
1.5 |
- |
100 |
+ |
8.4 |
200 |
0.0 |
3.5 |
1.0 |
x |
|
3 Pos. control |
+ |
6.0 |
200 |
19.5 |
36.5 |
27.5 |
s |
|
x: not significant
s: significant
Table 1: Experiment 1: Cytotoxic and mutagenic response of C-SAT 100024 in the mouse lymphoma L5178Y test system
Dose (µg/ml) |
RSG (%) |
CE day2 (%) |
RS day2 (%) |
RTG (%) |
Mutation frequency x 10-5 |
||
total |
( small |
large ) |
|||||
Without metabolic activation 3 hours treatment |
|||||||
SC1 |
100 |
70 |
100 |
100 |
55 |
( 35 |
20 ) |
SC2 |
101 |
67 |
( 48 |
18 ) |
|||
0.1 |
97 |
101 |
118 |
114 |
56 |
( 29 |
25 ) |
0.3 |
92 |
101 |
118 |
108 |
52 |
( 28 |
23 ) |
1 |
102 |
118 |
138 |
140 |
66 |
( 48 |
16 ) |
3 |
102 |
91 |
107 |
109 |
63 |
( 46 |
15 ) |
10 |
97 |
95 |
111 |
108 |
66 |
( 32 |
33 ) |
33 |
92 |
97 |
113 |
104 |
76 |
( 46 |
28 ) |
100(1) |
90 |
91 |
107 |
97 |
81 |
( 32 |
46 ) |
333(1) |
64 |
102 |
120 |
77 |
64 |
( 48 |
14 ) |
MMS |
64 |
60 |
70 |
45 |
685 |
( 521 |
118 ) |
With 8% (v/v) metabolic activation 3 hours treatment |
|||||||
SC1 |
100 |
80 |
100 |
100 |
70 |
( 37 |
31 ) |
SC2 |
102 |
63 |
( 35 |
26 ) |
|||
0.1 |
93 |
108 |
119 |
111 |
74 |
( 47 |
25 ) |
0.3 |
97 |
89 |
97 |
94 |
71 |
( 45 |
24 ) |
1 |
95 |
108 |
119 |
113 |
64 |
( 42 |
20 ) |
3 |
100 |
98 |
107 |
107 |
78 |
( 53 |
23 ) |
10 |
96 |
95 |
104 |
100 |
87 |
( 54 |
29 ) |
33 |
81 |
108 |
119 |
96 |
55 |
( 32 |
22 ) |
100(1) |
83 |
98 |
107 |
89 |
83 |
( 60 |
21 ) |
333(1) |
89 |
94 |
103 |
92 |
63 |
( 23 |
39 ) |
CP |
48 |
60 |
66 |
32 |
1074 |
( 829 |
144 ) |
Note:
all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative
Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS
= Methylmethanesulfonate; CP = Cyclophosphamide
(1) = C-SAT 100024 precipitated in the exposure medium
Table 2: Experiment 2: Cytotoxic and mutagenic response of C-SAT 100024 in the mouse lymphoma L5178Y test system
Dose (µg/ml) |
RSG (%) |
CE day2 (%) |
RS day2 (%) |
RTG (%) |
Mutation frequency x 10-5 |
||
total |
( small |
large ) |
|||||
Without metabolic activation 24 hours treatment |
|||||||
SC1 |
100 |
120 |
100 |
100 |
50 |
( 23 |
25 ) |
SC2 |
110 |
51 |
( 29 |
20 ) |
|||
3 |
79 |
135 |
117 |
92 |
58 |
( 42 |
14 ) |
10 |
91 |
137 |
119 |
109 |
51 |
( 39 |
11 ) |
33 |
74 |
133 |
115 |
85 |
71 |
( 32 |
35 ) |
100(1) |
31 |
110 |
96 |
30 |
100 |
( 35 |
60 ) |
125(1) |
30 |
118 |
103 |
31 |
70 |
( 31 |
36 ) |
140(1) |
20 |
118 |
103 |
20 |
103 |
( 51 |
47 ) |
175(1) |
10 |
102 |
89 |
9 |
134 |
( 53 |
72 ) |
MMS |
84 |
102 |
89 |
75 |
853 |
( 456 |
230 ) |
With 12% (v/v) metabolic activation 3 hours treatment |
|||||||
SC1 |
100 |
113 |
100 |
100 |
67 |
( 44 |
21 ) |
SC2 |
105 |
77 |
( 50 |
24 ) |
|||
0.1 |
99 |
110 |
101 |
100 |
73 |
( 49 |
21 ) |
0.3 |
103 |
123 |
113 |
117 |
72 |
( 47 |
23 ) |
1 |
111 |
104 |
95 |
105 |
82 |
( 52 |
27 ) |
3 |
117 |
97 |
89 |
104 |
94 |
( 62 |
29 ) |
10 |
120 |
115 |
105 |
126 |
87 |
( 57 |
26 ) |
33 |
111 |
107 |
98 |
108 |
85 |
( 59 |
23 ) |
100(1) |
102 |
131 |
120 |
123 |
80 |
( 51 |
26 ) |
333(1) |
94 |
97 |
89 |
83 |
92 |
( 64 |
24 ) |
CP |
91 |
70 |
64 |
59 |
979 |
( 621 |
221 ) |
Note:
all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloninq Efficiency; RS = Relative
Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS
= Methylmethanesulfonate; CP = Cyclophosphamide
(1) = C-SAT 100024 precipitated in the exposure medium
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
No data on the test item is available. With the read-across substances the following results were obtained:
The mutagenicity of the read across substance CAS 68583-51-7 was studied with the mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) according to OECD guideline 471. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 50 to 5000 µg per plate in the presence and absence of S9. Precipitation of the test compound and the plates was observed at 5000 µg/plate. All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that under the experimental conditions described, the test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 in the presence and absence of a metabolizing system.
The mutagenicity of the read across substance CAS 91031-31-1 was studied in two independent experiments with the mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA 1538) according to OECD guideline 471. The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in Tween 80/H2O and tested in concentrations of 8 to 5000 µg per plate in the presence and absence of S9. Precipitation of the test compound an the plates was observed at 5000 µg/plate. All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that under the experimental conditions described, the test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 1538 in the presence and absence of a metabolizing system.
The read across substance CAS 853947-59-8 was tested for its ability to induce chromosomal aberrations in an in vitro mammalian cell system (V79 Chinese hamster lung cells). V79 cells were exposed both in the presence and absence of exogenous metabolic activation by Arochlor 1254 induced rat liver S9. 16 and 26 h after the start of exposure, cells were arrested in metaphase by 2 h treatment with Colcemid. After hypotonic treatment they were fixed and Giemsa stained. The mitotic indices of representative cultures, as a measure for cytotoxicity, were determined and metaphase cells were analysed for the presence of chromosomal aberrations. For each experimental point, at least duplicate cultures (100 metaphases per culture) were evaluated. To demonstrate the sensitivity of the test system, Mitomycin C (without S9 mix) and Cyclophosphamide (with S9 mix) were used as positive controls. Results were confirmed in a second, independent experiment. In test # 1, 10 concentrations of 1 to 100 µg/mL (solubility limit) test item in the presence and absence of exogenous metabolic activation were employed for screening of cytotoxicity in order to determine concentrations to be used in the chromosome aberration study. Test #1 without metabolic activation was completely repeated (named test #1 a), due to a very low mitotic index already in the negative controls. The experiments with and without S9 mix revealed a systematic influence of the test compound which led to a reduction in the mitotic index. In the chromosome aberration studies with metabolic activation cells were treated with 3 concentrations ranging from 10 to 100 µg/mL test item. In the corresponding experiments without metabolic activation 3 concentrations ranging from 10 to 80 µg/mL were applied. In both experiments, at the 18 h (test #1 a, #2) as well as the 28 h (test #2) sampling time, the test item did not induce significant increases in the proportion of cells with chromosomal aberrations (excluding gaps). The positive controls, Mitomycin C and Cyclophosphamide, did induce chromosomal aberrations, thus demonstrating the sensitivity of the test system against clastogenic agents. From the experiments performed, it is concluded that under the conditions of this in vitro test system the test item is not a clastogenic agent.
The effects of the read acoss substace CAS 91031-31-1 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells was assessed. The test was performed in two independent experiments in the absence and presence of S9 -mix (rat liver S9-mix induced by a combination of phenobarbital and & β-naphthoflavone). The study procedures described in this report were based on the OECD guideline 476. The test item was suspended in dimethyl suffoxide (DMSO). In the first experiment, the test item was tested up to concentrations of 333 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. However the test item precipitated already in the exposure medium at 100 mg/mL. In the second experiment, the test item was tested up to concentrations of 175 and 333 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. The test item was tested up to the cytotoxic level of 91% in the absence of S9--mix. No toxicity was observed in the presence of S9-mix. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 17-fold for MMS in the absence of S9-mix, and by 16- and 14-fotd for CIP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the test item did not induce a signfficant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Based on the results of the read across substance the test item is not genotoxic.
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is classified and labelled as not genetic toxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.
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