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Registration Dossier
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EC number: 438-930-8 | CAS number: 2550-52-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2 uvr A with and without metabolic activation
Chromosome aberration test (OECD 473): negative in V79 cells with and without metabolic activation
HPRT (OECD 476): negative in V79 cells with and without metabolic activation
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:
- 03 Mar - 06 Apr 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Commission Directive 92/69/EEC
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- MINISTERIUM FÜR RAUMORDNUNG UND UMWELT DES LANDES SACHSEN-ANHALT, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium strains)
trp operon (E. coli strain) - 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:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital and ß-Naphthoflavone
- Test concentrations with justification for top dose:
- Range-finding study:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in TA 100 and WP2 uvr A
Experiment 1:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in TA 1537, TA 1535 and TA 98
Experiment 2:
10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation in all strains
Top dose in experiment 1 and 2 was selected based on the precipitation of the test substance at 5000 µg/plate, which interfered with the scoring in the range-finding study. - Vehicle / solvent:
- - Vehicle/Solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: experiment 1: in agar (plate incorporation); experiment 2: preincubation
DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h (experiment 1 and 2)
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: determination of bacterial background lawn - Evaluation criteria:
- A test is considered to be positive if the test item induces dose related increases in numbers of revertants scored in two separate experiments and these increases are deemed to be of biological relevance. Reproducible increases at one experimental point may also indicate a positive response. For a biologically relevant response the number of revertants is expected to be at least double the spontaneous reversion rate in the S. typhimurium strains TA 98, TA 100 and the E. coli strain. In the S. typhimurium strains TA 1535 and TA 1537 the number of revertants is expected to be at least the triple of the spontaneous reversion rate. A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any experimental point is considered to be non-mutagenic in this test system.
- Statistics:
- Mean values and standard deviations were calculated.
- 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
- 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
- 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
- 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, but tested up to precipitating concentrations
- Remarks:
- (at 5000 µg/plate in range-finding study, reported as experiment 1)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (at 5000 µg/plate in range-finding study, reported as experiment 1)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the toxicity range-finding study at a concentration of 5 mg/mL the test material precipitated in the cell culture medium.
RANGE-FINDING/SCREENING STUDIES: A toxicity range-finding study was carried out to determine the maximum concentration of the test substance in the main experiments. Therefore, S. typhimurium strain TA 100 and E. coli WP2 uvr A were tested at concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate with and without metabolic activation. A precipitation of the test substance, which interfered with the scoring, was observerd at 5000 µg/plate. Based on this result concentrations of 10, 50, 100, 500 and 1000 µg/plate were chosen for the main experiments. The results of the range-finding study for TA 100 and WP2 uvr A are used for experiment 1.
HISTORICAL CONTROL DATA
In both independent experiments controls gave counts of spontaneous revertants within the normal ranges obtained in this laboratory, except of one untreated control of WP2 uvr A. However, these value is in the normal range of spontaneous revertants of WP2 uvr A (30 - 60) according to literature*. The spontaneous revertants of the vehicle control was within the normal range. *(Green and Muriel (1976), Mutation Res. 38, 3-32)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Bacterial background lawn indicated sufficient growth conditions in all experiments. - Conclusions:
- Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation.
- Executive summary:
A bacterial gene mutation assay (2001) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP. In two independent experiments, the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the E. coli strain WP2 uvr A were exposed to the test substance using the plate incorporation method (experiment 1) and pre-incubation method (experiment 2). In a toxicity range-finding study, in which 6 concentrations up to 5000 µg/plate were tested in TA 100 and E. coli WP2 uvr A, precipitation of the test substance, which interfered with scoring, was observed at 5000 µg/plate. Therefore concentrations of 10, 50, 100, 500 and 1000 µg/plate were selected for the main experiments. The test substance showed no bacterial toxicity at any dose in both experiments with or without metabolic activation. No biologically relevant increase in revertant numbers was observed after treatment with the test substance in any bacterial strain and at any concentration tested in the presence and absence of metabolic activation. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered non-mutagenic in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 5 Mar - 11 May 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- (1997)
- Deviations:
- yes
- Remarks:
- acceptance citeria vary from current test guideline (2016), no historical control data, no proficiency proof
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Commission Directive 92/69/EEC of 31 July 1992
- Deviations:
- yes
- Remarks:
- acceptance citeria vary from current test guideline (2016), no historical control data, no proficiency proof
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- MINISTERIUM FÜR RAUMORDNUNG UND UMWELT DES LANDES SACHSEN-ANHALT, Germany
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany
MEDIA USED
- Type and identity of media: Minimum Essential Medium (MEM) containing HANKS´ salts, 10% FCS and Penicillin/Streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- 0.6 mg/mL cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1: 6*, 13*, 25*, 50*, 100 and 200 µg/mL with an without metabolic activation
Experiment 2: 6*, 13* and 25* mg/mL without metabolic activation; 13*, 25*, and 50* mg/mL with metabolic activation
* concentrations chosen for cytogenetic analysis - Vehicle / solvent:
- Vehicle used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (10 µL/mL)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: Exp 1: 2 x 10E06; Exp 2: approx. 1 x 10E06
DURATION
- Exposure duration: Exp 1: 4 h; Exp 2: 4 h (+ S9), 18 h (-S9)
- Expression time (cells in growth medium):
- Fixation time: Exp 1: 18 h; Exp 2: 26 h
SPINDLE INHIBITOR: colcemid 0.2 µg/mL medium
STAIN: Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After cells were fixed with icecold glacial acid/methanol (1:4) solution, several drops of cell suspension were dropped onto clean, cooled microscope slides. Slides were dried and stained with Giemsa solution.
NUMBER OF CELLS EVALUATED: 200 per experiment
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100
DETERMINATION OF CYTOTOXICITY
- Method: other: confluency, damage of cell integrity
- Any supplementary information relevant to cytotoxicity:
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- not specified
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp 1: -S9: at and above 25 µg/mL, +S9: at and above 50 µg/mL; Exp 2: -S9: at 25 µg/mL, +S9: at 50 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Definition of acceptable cells for analysis: Only metaphases with a chromosome number of 22 ± 2 were analysed for scoring aberrations. Aberrations were not scored in polyploid, hyperploid or endoreduplicated cells. However, the incidence of such cells was recorded.
RANGE-FINDING/SCREENING STUDIES:
Cells treated with the vehicle showed no depression in growth in relation to an untreated control. Following 4 h treatment a slight depression in cell growth was observed at a concentration of 16 μg/mL and in a concentration of 30 μg/mL the inhibition was greater than 50%. Severe inhibition of > 77% were measured in concentrations exceeding 60 μg/mL. After 18 and 26 h continuous treatment similar results were obtained. Inhibition in cell growth of ≥ 50% was observed for concentrations starting at 30 µg/mL. Based on these results the 1. experiment included the following concentrations: 6, 13, 25, 50, 100 and 200 µg/mL. For the treatment without metabolic activation of the experiment 2 (continuous treatment) concentrations of 6, 13 and 25 µg/mL were evaluated. In presence of metabolic activation concentrations of 13, 25, and 50 µg/mL were evaluated. For the late sampling time only the highest concentration was analysed. - Conclusions:
- Under conditions of the in vitro chromosome aberration test the test substance did not induce chromosome aberrations in V79 cells.
- Executive summary:
The potential of the test substance to induce chromosome aberrations was evaluated in an in vitro mammalian cell chromosome aberrations test in V79 cells of the Chinese hamster according to OECD Guideline 473 and in compliance with GLP (2001). A pre-experiment in the absence of metabolic activation was performed in order to determine the concentration range for the cytogenetic experiments. Test substance concentrations of 8, 16, 30, 60, 130, 250 and 500 µg/mL were used. Reduction of cell growth of at least 50% occurred at 30 µg/mL after 4 h treatment and 30 µg/mL after 18 and 26 h treatment. Based on the results of the pre-experiment, in the first experiment V79 cells were exposed to the test substance for 4 h at 6, 13 and 25 µg/mL without metabolic activation and at 13, 25 and 50 µg/mL with metabolic activation and were harvested 18 h after treatment. For the second experiment V79 cells were exposed to the test substance for 18 h at concentrations of 6, 13 and 25 µg/mL without metabolic activation and for 4 h at 13, 25 and 50 µg/mL with metabolic activation and were harvested 18 h after treatment. Additionally, cells were harvested at a late sampling time of 26 h at concentrations of 25 µg/mL (26 h treatment) and 50 µg/mL (4 h treatment). Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred at a concentration of 25 µg/mL without metabolic activation and at 50 µg/mL with metabolic activation after 4 h treatment. After continuous treatment cytotoxicity was observed at concentrations exceeding 13 μg/mL. No relevant and reproducible increase in chromosomal aberrations was observed in the main experiments at any concentration in the absence or presence of metabolic activation. The positive control substances induced a sufficient number of aberrations to confirm effectiveness of the test procedures and the number of cells with aberrations (exclusive gaps) found in the negative control did not exceed 4.0%. In conclusion, the test substance did not induce chromosome aberrations in V79 cells under the experimental conditions reported and therefore is considered to be non-clastogenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 Aug - 07 Dec 2016
- 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 29 July 2016)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008 of 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: in vitro mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University Darmstadt, Germany
MEDIA USED
- Type and identity of media: MEM medium containing HANKS´ salts, 10% FBS, neomycin (5 µg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-Experiment 1:
4 h treatment 15.6, 31.3, 62, 125, 250, 500, 1000 and 2000 µg/mL with and without metabolic activation
Pre-experiment 2:
4 h treatment: 0.1, 0.3, 0.5, 1, 2, 4, 8 and 16 µg/mL without metabolic activation
Experiment 1:
4 h treatment: 0.5, 1*, 2*, 4*, 6*, 8*, 12 and 16 µg/mL without metabolic activation
4 h treatment: 2, 4*, 8*, 16*, 32*, 48 and 64 µg/mL with metabolic activation
Experiment 2:
4 h treatment: 16, 32*, 36*, 40*, 44* and 48* µg/mL with metabolic activation
*concentrations chosen for mutation rate analysis - Vehicle / solvent:
- - Vehicle/solvent used: ethanol 0.5% (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen based on solubility properties and compatibility with the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h (experiment 1 and 2 with and without metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)
STAINING: 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: duplicates in three independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency I or cell density below 50%
CALCULATION AND PROCESSING OF DATA
cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity.
cloning efficiency II (viability): cloning efficiency determined after the expression period to measure viability of the cells without selective agent.
cloning efficiency I (survival, absolute): mean number of colonies per flask divided by the number of cells seeded per test point
cloning efficiency I (survival, relative): cloning efficiency I absolute divided by the cloning efficiency I absolute of the corresponding control × 100
cloning efficiency II (viability, absolute): mean number of colonies per flask divided by the number of cells seeded
cloning efficiency II (viability, relative): cloning efficiency II absolute divided by the cloning efficiency II absolute of the corresponding control × 100 - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range. A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
A t-test was performed to evaluate a possible significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp 1: -S9: at ≥ 1 µg/mL; Exp 2: at 48 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test substance.
- Precipitation: The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 h) prior to removal of test substance. Precipitation occurred at 250.0 µg/mL and above after 4 h treatment with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
Two pre-experiments were performed in order to determine the concentration range of the mutagenicity experiments. The highest applied concentration (2000 μg/mL) was chosen with respect to the current OECD guideline. The first pre-experiment, performed in the concentration range from 15.6 to 2000 μg/mL, was not analysable in the absence of metabolic activation following 4 hours treatment due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, the pre-test was repeated in a concentration range from 0.13 and 16.0 μg/mL without metabolic activation (4 hours treatment). - Conclusions:
- Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.
- Executive summary:
The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2017). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Two pre-experiments were performed in order to determine the concentration range for the mutagenicity experiments. In the first pre-experiment a 4 h treatment with test substance concentrations between 15.6 and 2000 µg/mL with and without metabolic activation induced severe toxicity, therefore a second pre-experiment with concentrations between 0.1 and 16 µg/mL without metabolic activation was conducted. Relevant toxic effects occurred at 2 µg/mL and above in the absence of metabolic activation after 4 h treatment. Based on the results of the pre-experiment, cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation up to concentrations of 16 and 64 µg/mL, respectively. The second experiment was performed with a treatment time of 4 h with and without metabolic activation up to concentrations of 48 µg/mL. Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred in the first experiment at 1 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects occurred at 48 μg/mL. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The 95% confidence interval was exceeded in the second experiment at all endpoints, but t-test showed no significant increase in mutation frequency compared to the solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture II of the second experiment with metabolic activation. Since this effect was not reproducible in parallel culture and t-test remained not-significant, the result was judged as biologically irrelevant. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Mutant colonies of solvent controls were in the acceptable historical range of controls. In conclusion, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
Referenceopen allclose all
Table 1: Test Results of Experiment 1
EXPERIMENT 1 (plate incubation method) |
|||||
S9-Mix |
Without
|
||||
Test item (mg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvr A |
NC |
16.0±5.3 |
11.3±3.2 |
22.0±3.0 |
239.0±31.4 |
39.0±4.2 |
SC |
16.0±6.6 |
13.7±4.2 |
22.0±5.6 |
207.0±12.0 |
23.0±1.0 |
0.01 |
12.7±4.9 |
13.3±2.3 |
16.3±3.2 |
251.0±11.3 |
27.3±5.7 |
0.05 |
13.0±1.7 |
11.0±4.4 |
18.0±4.6 |
213.0±8.9 |
25.3±5.7 |
0.1 |
8.7±2.5 |
13.0±1.7 |
20.3±3.5 |
191.0±6.2 |
26.7±4.9 |
0.5 |
10.3±3.1 |
8.0±1.7 |
15.0±1.7 |
221.7±25.2 |
31.7±6.5 |
1 |
10.0±3.5 |
8.3±2.5 |
13.7±3.2 |
209.0±10.4 |
26.3±9.1 |
5 |
|
|
|
226.0±28.7 |
21.7±5.7 |
NaN3 |
1818.7±546.0 |
|
|
2490.7±187.5 |
|
9-AA |
|
676.0±511.6 |
|
|
|
2-NF |
|
|
585.3±102.1 |
|
|
MMS |
|
|
|
|
118.0±23.3 |
S9-Mix |
With
|
||||
Test item (mg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvr A |
NC |
16.3±5.5 |
13.0±7.0 |
22.3±5.7 |
255.0±18.7 |
36.7±2.5 |
SC |
16.0±2.6 |
14.0±1.0 |
20.7±8.1 |
221.3±22.5 |
31.3±4.0 |
0.01 |
14.7±9.0 |
15.7±5.9 |
16.3±2.1 |
234.0±19.3 |
29.7±11.6 |
0.05 |
14.3±3.2 |
11.7±3.5 |
21.7±2.1 |
246.0±9.2 |
34.7±2.5 |
0.1 |
12.3±4.0 |
12.7±3.2 |
16.7±5.5 |
222.3±27.1 |
33.0±3.5 |
0.5 |
13.0±4.4 |
10.3±2.3 |
16.0±8.2 |
257.7±34.0 |
30.0±5.3 |
1 |
9.3±1.5 |
12.0±3.6 |
17.7±3.2 |
226.7±16.5 |
30.00±5.3 |
5 |
|
|
|
212.0±16.5 |
44.7±10.6 |
2-AA |
127.0±18.2 |
210.3±53.3 |
1736.0±138.8 |
1560.0±52.5 |
77.3±8.0 |
NC = Negative Control (untreated) SC = Solvent Control (DMSO) NaN3: sodium acide; 9-AA: 9-aminoacridine; 2-NF: 2-nitrofluorene; MMS: methyl methanesulphonate; 2-AA: 2-aminoanthracene |
|||||
Table 2: Test Results of Experiment 2
EXPERIMENT 2 (pre-incubation method) |
|||||
S9-Mix |
Without
|
||||
Test item (mg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvr A |
NC |
12.0±3.0 |
17.7±2.1 |
20.7±8.6 |
229.3±32.3 |
22.0±2.6 |
SC |
11.7±2.5 |
15.7±2.1 |
15.3±7.8 |
199.0±39.0 |
27.7±4.6 |
0.01 |
10.3±5.7 |
6.0±1.7 |
14.7±3.5 |
225.3±9.6 |
24.0±5.0 |
0.05 |
8.7±0.6 |
7.7±3.2 |
17.7±6.5 |
201.0±36.6 |
18.0±4.0 |
0.1 |
10.0±1.0 |
6.3±1.2 |
25.0±1.7 |
218.0±28.0 |
20.3±7.0 |
0.5 |
11.0±4.4 |
3.3±1.5 |
14.3±3.8 |
185.3±21.1 |
22.0±4.0 |
1 |
10.7±2.1 |
2.7±1.5 |
15.3±5.1 |
203.3±10.0 |
23.7±5.7 |
NaN3 |
1712.0±40.0 |
|
|
1400.0±172.1 |
|
9-AA |
|
1164.0±1.4 |
|
|
|
2-NF |
|
|
1400.0±172.1 |
|
|
MMS |
|
|
|
|
256.3±37.7 |
S9-Mix |
With
|
||||
Test item (mg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvr A |
NC |
14.7±9.2 |
7.3±0.6 |
46.3±6.0 |
214.3±9.0 |
15.3±4.5 |
SC |
14.0±3.5 |
7.3±4.5 |
47.3±3.1 |
216.7±25.2 |
20.7±1.5 |
0.01 |
14.0±3.0 |
8.3±1.2 |
44.0±5.6 |
206.3±25.5 |
17.0±3.6 |
0.05 |
13.7±5.5 |
11.7±4.2 |
49.7±8.4 |
199.3±15.6 |
16.7±5.1 |
0.1 |
12.3±4.9 |
4.7±0.6 |
39.7±9.0 |
193.0±14.0 |
22.3±4.5 |
0.5 |
14.7±3.2 |
6.3±1.5 |
41.3±2.9 |
188.3±4.7 |
19.0±4.0 |
1 |
14.3±2.5 |
4.7±1.5 |
32.7±3.8 |
180.3±6.7 |
13.3±1.2 |
2-AA |
118.3±14.4 |
188.3±7.8 |
1501.3±287.4 |
1464.0±68.4 |
206.3±29.7 |
NC = Negative Control (untreated) SC = Solvent Control (DMSO) NaN3: sodium acide; 9-AA: 9-aminoacridine; 2-NF: 2-nitrofluorene; MMS: methyl methanesulphonate; 2-AA: 2-aminoanthracene |
|||||
Table1: Results of Experiment 1
Dose (µg/mL) |
No. of metaphases scored |
Aberrations |
Aberrant metaphases |
|||
|
|
Total no. including gaps |
Total no. excluding gaps |
Total no. excluding gaps |
% excluding gaps |
% mean of 2 replicates |
Exposure period 4 h, fixation time 18 h, without S9 mix |
||||||
DMSO |
100 |
1 |
0 |
0 |
0 |
0.5 |
DMSO |
100 |
1 |
1 |
1 |
1 |
|
6 |
100 |
2 |
1 |
1 |
1 |
1.0 |
6 |
100 |
1 |
1 |
1 |
1 |
|
13 |
100 |
1 |
1 |
1 |
1 |
0.5 |
13 |
100 |
1 |
0 |
0 |
0 |
|
25 |
100 |
1 |
1 |
1 |
1 |
1.0 |
25 |
100 |
1 |
1 |
1 |
1 |
|
EMS |
100 |
34 |
33 |
25 |
25 |
27.5 |
EMS |
30 |
15 |
15 |
9 |
30 |
|
Exposure period 4 h, fixation time 18 h, with S9 mix |
||||||
DMSO |
100 |
2 |
1 |
1 |
1 |
1.0 |
DMSO |
100 |
1 |
1 |
1 |
1 |
|
13 |
100 |
2 |
0 |
0 |
0 |
1.0 |
13 |
100 |
3 |
3 |
2 |
2 |
|
25 |
100 |
0 |
0 |
0 |
0 |
1.5 |
25 |
100 |
4 |
4 |
3 |
3 |
|
50 |
100 |
2 |
1 |
1 |
1 |
1.5 |
50 |
100 |
4 |
2 |
2 |
2 |
|
CP |
25 |
14 |
14 |
9 |
36 |
38.0 |
CP |
25 |
21 |
20 |
10 |
40 |
|
Table 2. Results of Experiment 2
Dose (µg/mL) |
No. of metaphases scored |
Aberrations |
Aberrant metaphases |
|||
|
|
Total no. including gaps |
Total no. excluding gaps |
Total no. excluding gaps |
% excluding gaps |
% mean of 2 replicates |
Exposure period 18 h, fixation time 18 h, without S9 mix |
||||||
DMSO |
100 |
2 |
1 |
1 |
1 |
1.0 |
DMSO |
100 |
1 |
1 |
1 |
1 |
|
6 |
100 |
1 |
0 |
0 |
0 |
0.5 |
6 |
100 |
1 |
1 |
1 |
1 |
|
13 |
100 |
2 |
2 |
2 |
2 |
1.5 |
13 |
100 |
1 |
1 |
1 |
1 |
|
25 |
100 |
0 |
0 |
0 |
0 |
1.0 |
25 |
100 |
3 |
3 |
2 |
2 |
|
EMS |
26 |
16 |
15 |
10 |
38 |
39.0 |
EMS |
25 |
10 |
10 |
10 |
40 |
|
Exposure period 26 h, fixation time 26 h, without S9 mix |
||||||
DMSO |
100 |
2 |
1 |
1 |
1 |
1.0 |
DMSO |
100 |
2 |
1 |
1 |
1 |
|
25 |
100 |
1 |
1 |
1 |
1 |
2.0 |
25 |
100 |
5 |
3 |
3 |
3 |
|
Exposure period 4 h, fixation time 18 h, with S9 mix |
||||||
DMSO |
100 |
0 |
0 |
0 |
0 |
0 |
DMSO |
100 |
0 |
0 |
0 |
0 |
|
13 |
100 |
4 |
2 |
2 |
2 |
2.0 |
13 |
100 |
3 |
3 |
2 |
2 |
|
25 |
100 |
2 |
2 |
2 |
2 |
1.0 |
25 |
100 |
1 |
0 |
0 |
0 |
|
50 |
100 |
0 |
0 |
0 |
0 |
1.0 |
50 |
100 |
2 |
2 |
2 |
2 |
|
CP |
100 |
53 |
51 |
23 |
23 |
23.5 |
CP |
100 |
50 |
49 |
24 |
24 |
|
Exposure period 4 h, fixation time 26 h, with S9 mix |
||||||
DMSO |
100 |
1 |
0 |
0 |
0 |
0 |
DMSO |
100 |
0 |
0 |
0 |
0 |
|
50 |
100 |
5 |
2 |
2 |
2 |
2.0 |
50 |
100 |
3 |
2 |
2 |
2 |
|
EMS: Ethylmethansulfonate; CP: Cyclophosphamide (positive controls)
The following acceptance criteria were met for the present study:
- The number of cells with aberrations (exclusive gaps) found in the negative control did not
exceed 4.0%.
- The positive control chemicals produced marked increases in cells with chromosomal
aberrations.
Experiment 1: Cultures exposed to the test item in the absence of metabolic activation in concentrations of 50 μg/mL and higher were completely damaged. The cells did not adhere anymore to the culture flasks. These cultures couldn't be harvested and analysed. At a dose level of 25 mg/mL the degree of confluency was decreased in relation to the control. Cultures treated with lower concentrations showed no cytotoxic effect. Thus concentrations of 6, 13 and 25 µg/mL were analysed. At dose levels of 100 and 200 µg/mL in presence of metabolic activation the cells were detached. At a concentration of 50 μg/mL marked cytotoxicity was observed. Concentrations of 13, 25 and 50 μg/mL were evaluated. Aberration scores with and without S9 metabolic activation did not exceed 1.5% metaphases with aberrations in negative controls and cultures treated with the test item.
Experiment 2: Cultures treated with the test item continuously in the absence of metabolic activation at concentrations of 25 μg/mL showed marked reduction in degree of confluency and damage of cell integrity and at 13 µg/mL a slight effect. Following 4 h treatment with metabolic activation depression in confluency was observed at concentration of 50 μg/mL. The maximum aberration score determined in negative controls and cultures treated with the test item was 2%.
Marked increases in the frequencies of aberrant cells compared with controls were seen in cultures treated with the positive control chemicals ethylmethane sulfonate and cyclophosphamide both in Experiment 1 and 2.
Table 1. Experiment 1 – 4 h exposure – without metabolic activation
Concentration [µg/mL] |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies per 106cells |
95% confidence interval |
culture I |
|||||
0 (ethanol) |
100 |
100 |
100 |
17.5 |
0.2 – 29.7 |
0.5 |
81.4 |
61.2 |
49.9 |
# |
|
1 |
76.2 |
63.1 |
48.1 |
17.2 |
0.2 – 29.7 |
2 |
53.7 |
71.1 |
38.2 |
21.7 |
0.2 – 29.7 |
4 |
54.9 |
65.7 |
36.1 |
23.7 |
0.2 – 29.7 |
6 |
41.9 |
11.2 |
4.7 |
15.6 |
0.2 – 29.7 |
8 |
8.4 |
11.3 |
1.0 |
6.6 |
0.2 – 29.7 |
12 |
culture was not continued## |
||||
16 |
culture was not continued## |
||||
EMS, 300 |
81.7 |
86.5 |
70.6 |
289.1 |
0.2 – 29.7 |
culture II |
|||||
0 (ethanol) |
100 |
100 |
100 |
12.6 |
0.2 – 29.7 |
0.5 |
68.2 |
77.6 |
52.9 |
# |
|
1 |
68.0 |
70.5 |
48.0 |
24.5 |
0.2 – 29.7 |
2 |
79.0 |
88.6 |
70.0 |
27.2 |
0.2 – 29.7 |
4 |
47.6 |
79.3 |
37.8 |
19.5 |
0.2 – 29.7 |
6 |
28.5 |
95.2 |
27.2 |
19.4 |
0.2 – 29.7 |
8 |
8.3 |
65.5 |
5.4 |
18.2 |
0.2 – 29.7 |
12 |
## |
6.1 |
# |
||
16 |
culture was not continued## |
||||
EMS, 300 |
77.5 |
92.2 |
71.4 |
271.4 |
0.2 – 29.7 |
EMS: Ethylmethanesulphonate
#culture was not continued as a minimum of only four analysable concentrations is required
##culture was not continued due to exceedingly severe cytotoxic effects
Table 2. Experiment 1 – 4 h exposure – with metabolic activation
Concentration [µg/mL] |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies per 106 cells |
95% confidence interval |
culture I |
|||||
0 (ethanol) |
100 |
100 |
100 |
13.7 |
0.6 – 28.7 |
2 |
culture was not continued## |
||||
4 |
91.0 |
107.5 |
97.8 |
24.5 |
0.6 – 28.7 |
8 |
71.5 |
130.7 |
93.4 |
14.7 |
0.6 – 28.7 |
16 |
85.1 |
112.5 |
95.7 |
25.1 |
0.6 – 28.7 |
32 |
54.2 |
118.3 |
64.1 |
27.4 |
0.6 – 28.7 |
48 |
1.9 |
104.7 |
2.0 |
## |
|
64 |
1.5 |
66.4 |
1.0 |
## |
|
DMBA, 2.3 |
95.6 |
108.6 |
103.8 |
139.1 |
0.6 – 28.7 |
culture II |
|||||
0 (ethanol) |
100 |
100 |
100 |
17.3 |
0.6 – 28.7 |
2 |
culture was not continued## |
||||
4 |
72.6 |
116.4 |
84.6 |
13.2 |
0.6 – 28.7 |
8 |
85.6 |
102.6 |
87.8 |
28.3 |
0.6 – 28.7 |
16 |
62.5 |
111.0 |
69.3 |
23.2 |
0.6 – 28.7 |
32 |
57.2 |
102.1 |
58.4 |
26.3 |
0.6 – 28.7 |
48 |
5.0 |
50.6 |
2.5 |
## |
|
64 |
1.2 |
24.8 |
0.3 |
## |
|
DMBA, 2.3 |
68.6 |
105.5 |
72.4 |
164.9 |
0.6 – 28.7 |
DMBA: 7,12-dimethylbenzanthracen
##culture was not continued due to exceedingly severe cytotoxic effects
Table 3. Experiment 2 – 4h exposure – with metabolic activation
Concentration [µg/mL] |
Rel. cloning efficiency I [%] |
Rel. cell density [%] |
Rel. cloning efficiency II [%] |
Mutant colonies per 106 cells |
95% confidence interval |
culture I |
|||||
0 (ethanol) |
100 |
100 |
100 |
36.3 |
0.6 – 28.7 |
16 |
95.6 |
101.3 |
96.9 |
# |
|
32 |
94.5 |
85.2 |
80.5 |
35.3 |
0.6 – 28.7 |
36 |
94.7 |
88.3 |
83.6 |
34.9 |
0.6 – 28.7 |
40 |
102.7 |
76.8 |
78.8 |
38.1 |
0.6 – 28.7 |
44 |
73.5 |
83.3 |
61.2 |
36.3 |
0.6 – 28.7 |
48 |
58.2 |
58.5 |
34.0 |
33.2 |
0.6 – 28.7 |
DMBA, 2.3 |
73.4 |
110.4 |
81.1 |
211.9 |
0.6 – 28.7 |
culture II |
|||||
0 (ethanol) |
100 |
100 |
100 |
24.9 |
0.6 – 28.7 |
16 |
99.4 |
112.3 |
111.6 |
# |
|
32 |
104.8 |
88.4 |
92.7 |
32.7 |
0.6 – 28.7 |
36 |
98.3 |
91.4 |
89.9 |
36.5 |
0.6 – 28.7 |
40 |
93.7 |
84.2 |
78.9 |
39.9 |
0.6 – 28.7 |
44 |
74.5 |
51.3 |
38.2 |
39.2 |
0.6 – 28.7 |
48 |
51.5 |
62.4 |
32.1 |
41.5 |
0.6 – 28.7 |
DMBA, 2.3 |
72.1 |
136.9 |
98.6 |
226.7 |
0.6 – 28.7 |
DMBA: 7,12-dimethylbenzanthracene
#culture was not continued as a minimum of only four analysable concentrations is required
No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation.
In the first experiment the 95% confidence interval was not exceeded. In the second experiment the 95% confidence interval was exceeded at all experimental points. However, the t-test run at every one of the experimental points exceeding the 95% confidence interval showed no-significant increase of the mutation frequency compared to the corresponding solvent control.
A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment II with metabolic activation. This trend however, was judged as irrelevant as it was not reproduced in the parallel culture and all of the t-tests remained not-significant.
In the main experiment with and without S9 mix the range of the solvent controls was from 12.6 up to 36.3 mutants per 10E6 cells; the range of the groups treated with the test item was from 6.6 up to 41.5 mutants per 10E6 cells. The highest solvent control value of 36.3 mutants per 10E6 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture (24.9 mutant colonies per 10E6 cells) was fully acceptable.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria
A bacterial gene mutation assay (2001) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP. In two independent experiments, the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the E. coli strain WP2 uvr A were exposed to the test substance using the plate incorporation method (experiment 1) and pre-incubation method (experiment 2). In a toxicity range-finding study, in which 6 concentrations up to 5000 µg/plate were tested in TA 100 and E. coli WP2 uvr A, precipitation of the test substance, which interfered with scoring, was observed at 5000 µg/plate. Therefore concentrations of 10, 50, 100, 500 and 1000 µg/plate were selected for the main experiments. The test substance showed no bacterial toxicity at any dose in both experiments with or without metabolic activation. No biologically relevant increase in revertant numbers was observed after treatment with the test substance in any bacterial strain and at any concentration tested in the presence and absence of metabolic activation. The revertant frequencies of the vehicle control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance is considered non-mutagenic in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.
Cytogenicity in mammalian cells
The potential of the test substance to induce chromosome aberrations was evaluated in an in vitro mammalian cell chromosome aberrations test in V79 cells of the Chinese hamster according to OECD Guideline 473 and in compliance with GLP (2001). A pre-experiment in the absence of metabolic activation was performed in order to determine the concentration range for the cytogenetic experiments. Test substance concentrations of 8, 16, 30, 60, 130, 250 and 500 µg/mL were used. Reduction of cell growth of at least 50% occurred at 30 µg/mL after 4 h treatment and 30 µg/mL after 18 and 26 h treatment. Based on the results of the pre-experiment, in the first experiment V79 cells were exposed to the test substance for 4 h at 6, 13 and 25 µg/mL without metabolic activation and at 13, 25 and 50 µg/mL with metabolic activation and were harvested 18 h after treatment. For the second experiment V79 cells were exposed to the test substance for 18 h at concentrations of 6, 13 and 25 µg/mL without metabolic activation and for 4 h at 13, 25 and 50 µg/mL with metabolic activation and were harvested 18 h after treatment. Additionally, cells were harvested at a late sampling time of 26 h at concentrations of 25 µg/mL (26 h treatment) and 50 µg/mL (4 h treatment). Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred at a concentration of 25 µg/mL without metabolic activation and at 50 µg/mL with metabolic activation after 4 h treatment. After continuous treatment cytotoxicity was observed at concentrations exceeding 13 μg/mL. No relevant and reproducible increase in chromosomal aberrations was observed in the main experiments at any concentration in the absence or presence of metabolic activation. The positive control substances induced a sufficient number of aberrations to confirm effectiveness of the test procedures and the number of cells with aberrations (exclusive gaps) found in the negative control did not exceed 4.0%. In conclusion, the test substance did not induce chromosome aberrations in V79 cells under the experimental conditions reported and therefore is considered to be non-clastogenic.
Gene mutation in mammalian cells
The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2017). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Two pre-experiments were performed in order to determine the concentration range for the mutagenicity experiments. In the first pre-experiment a 4 h treatment with test substance concentrations between 15.6 and 2000 µg/mL with and without metabolic activation induced severe toxicity, therefore a second pre-experiment with concentrations between 0.1 and 16 µg/mL without metabolic activation was conducted. Relevant toxic effects occurred at 2 µg/mL and above in the absence of metabolic activation after 4 h treatment. Based on the results of the pre-experiment, cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation up to concentrations of 16 and 64 µg/mL, respectively. The second experiment was performed with a treatment time of 4 h with and without metabolic activation up to concentrations of 48 µg/mL. Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred in the first experiment at 1 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects occurred at 48 μg/mL. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The 95% confidence interval was exceeded in the second experiment at all endpoints, but t-test showed no significant increase in mutation frequency compared to the solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture II of the second experiment with metabolic activation. Since this effect was not reproducible in parallel culture and t-test remained not-significant, the result was judged as biologically irrelevant. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Mutant colonies of solvent controls were in the acceptable historical range of controls. In conclusion, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
