Registration Dossier
Registration Dossier
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EC number: 262-104-4 | CAS number: 60207-90-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No genotoxic activity of the test substance was found in any of the following studies:
- Ames test, +S9 negative, -S9 negative, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, OECD TG 471, Woods, 2017;
- In vitro micronucleus test in human lymphocytes, +S9 negative, -S9 negative, OECD TG 487, Gilby, 2017
- In vitro gene mutation in mouse lymphoma L5178Y cells, +S9 negative, -S9 negative, OECD TG 490, Gilby, 2017
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:
- 18 Apr 2017 to 08 May 2017
- 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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his- (S. typhimurium), trp- (E. coli)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone - Test concentrations with justification for top dose:
- Experiment I: 5; 15; 50; 150; 500; 1500; and 5000 µg/plate
Experiment II: 5; 15; 50; 150; 500; 1500; and 5000 µg/plate - Vehicle / solvent:
- - Solvent used: DMSO
- Justification for choice of solvent:
The solubility of test substance was assessed at 50 mg/mL in dimethyl sulfoxide (DMSO), in which it dissolved. DMSO was, therefore, used as the vehicle for this study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three
- Number of independent experiments : two
METHOD OF TREATMENT:
- Cell density at seeding: 10^9/mL
- Test substance added in medium (Experiment 1) and by preincubation (Experiment 2)
TREATMENT AND HARVEST SCHEDULE:
- Experiment 1 Plate incubation method: Aliquots of 0.1 mL of the test item solutions (seven concentrations up to 5000 µg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar.
- Experiment 2 Preincubation method: As a clear negative response was obtained in the first experiment, a variation to the test procedure was used for the second experiment. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 34 to 39°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 µg/plate.
- Exposure duration: 48 hours
FOR GENE MUTATION:
- All plates were incubated at approximately 34 to 39 °C at least 48 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. - Evaluation criteria:
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed additionally since this is not required by the guideline.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important - Statistics:
- The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- other: S. typhimurium
- Remarks:
- TA98, TA100, TA1535, TA1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Experiment 1 (Plate Incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA100, TA1537 and WP2 uvrA (pKM101) following exposure to test substance and in strains TA98 and TA1535 in the presence of metabolic activation only. Precipitate was observed on all plates containing test substance at 5000 µg/plate in the absence of S9 mix. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second experiment. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.
The revertant colony count seen in the vehicle control plates in strain WP2 uvrA (pKM101) in the presence of S9 mix were above the laboratory historical control data and therefore this was repeated.
Experiment 1 repeat (Plate Incubation Assay)
Toxicity, observed as a thin background lawn of non-revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to test substance. No precipitate was observed on plates containing test substance. No substantial increases in revertant colony numbers over control counts were obtained with
any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in the presence of S9 mix.
Experiment 2 (Pre-incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) following exposure to test substance. Precipitate was observed on all plates containing test substance at 5000 µg/plate in the absence of S9 mix.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. An insufficient number of non-toxic concentrations was achieved in strains TA100 and TA1537 in the absence of S9 mix therefore these were repeated with a modified concentration range.
Experiment 2 repeat (Pre-incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA100 and TA1537 following exposure to test substance in the absence of S9 mix. No precipitate was observed on plates containing test substance.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in the absence of S9 mix.
Please, see 'Any other information on results incl. tables' - Conclusions:
- In this in vitro assessment of the mutagenic potential of the test substance performed in compliance with GLP and following the OECD 471 guideline, it was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
- Executive summary:
In this in vitro assessment of the mutagenic potential of the test substance performed in compliance with GLP and following the OECD 471 guideline, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.
Toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance in both tests. Precipitate was observed on all plates containing the test substance at 5000 µg/plate in the absence of S9 mix in the first and second test. No evidence of mutagenic activity was seen at any concentration of the test substance in either experiment. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.
It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 Apr 2017 to 10 Jun 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- CELLS USED
Type and source of cells: human lymphocytes
For lymphocytes:
Sex, age and number of blood donors: Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media. Blood was pooled from one male and one female donor for the preliminary and first main tests and from two female donors for the additional main test.
Whether whole blood or separated lymphocytes were used: whole blood
Whether blood from different donors were pooled or not: pooled
Mitogen used for lymphocytes: phytohaemagglutinin
MEDIA USED
Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L-glutamine; 34 to 39°C - Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
Source of S9 : S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/β-naphtoflavone to stimulate mixed-function oxidases in the liver. The S9 fraction was stored at -90°C to -70°C
Method of preparation of S9 mix: : S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilized before use.
Concentration of S9 in the final culture medium: 2% v/v - Test concentrations with justification for top dose:
- Preliminary toxicity test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL;
Main test in presence of S9 fraction (3 h): 5, 50, 100, 110, 120, 130, 140, 150, 160, 180 and 200 µg/mL
Main test in absence of S9 fraction (3 h): 5, 50, 60, 70, 80, 90, 100, 110 and 120 ug/mL
Main test in absence of S9 fraction (20 h): 0.2, 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 ug/mL
Additional main test in presence of S9 fraction (3 h): 5, 50, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 and 130 ug/mL
Additional main test in absence of S9 fraction (3 h): 5, 50, 100, 110, 112, 114, 116, 118, 120, 122, 124 and 126 ug/mL - Vehicle / solvent:
- Solvent used: DMSO
Justification for choice of solvent: The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures
Percentage of solvent in the final culture medium: 0.5% v/v - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls.
- Number of independent experiments : two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: Lymphocyte cultures were incubated for approximately 48 hours following stimulation with phytohaemagglutinin (PHA), before addition of the test item.
- Duration of treatment: 3 h in presence and absence of S9 mix and 20 h in absence of S9 mix
- Harvest time after the end of treatment: The cultures were incubated with Cytochalasin B for additional 17 hours until the scheduled harvest time.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis block method used for micronucleus assay: Cytochalasin B, 6 µg/mL, 17 hour incubation
- Methods of slide preparation and staining technique used including the stain used: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. Slides were stained using acridine orange.
- Criteria for scoring micronucleated cells: The incidences of mononucleate, binucleate and polynucleate cells were assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted; iInterphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible from at least 2000 binucleate cells per concentration (4000 for vehicle controls)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI), 55±5% cytostasis. - Evaluation criteria:
- Please, see 'Any other information on materials and methods incl. tables'
- Statistics:
- The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Test substance treated groups were then compared to control using Williams' tests. Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Statistical significance was declared at the 5% level for all tests.
- Key result
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected for all exposures was that which caused a reduction in CBPI equivalent to 55±5% cytotoxicity. Following a 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 53.2% cytotoxicity were obtained with the test substance at 110 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 100 and 110 µg/mL. Following 3-hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 51.3% cytotoxicity were obtained with the test substance at 124 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 112 and 124 µg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 51.4% cytotoxicity was obtained with the test substance at 17.5 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 0.2, 10 and 17.5 µg/mL.
In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20 hour treatment, the test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. None of the concentrations exceeded the upper control limits of the solvent historical control data, or were positive in a statistical trend test. Thus the outcome of the study is clearly negative. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system. - Conclusions:
- As a result of this study performed in compliance with GLP and according to OECD 487 guideline it was concluded that the test substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. Hence the test substance is concluded to be negative in this assay
- Executive summary:
This study performed in compliance with GLP and according to OECD 487 guideline was designed to assess the potential of the test substance to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro. The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture were exposed to the test substance for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 2000 µg/mL in order to test up to the maximum concentration as recommended in the current OECD Guideline 487 (2016). Vehicle (dimethyl sulphoxide [DMSO]) and positive control cultures were included in all appropriate test conditions.
Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected for all exposures was that which caused a reduction in CBPI equivalent to 55±5% cytotoxicity. Following a 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 53.2% cytotoxicity were obtained with the test substance at 110 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 100 and 110 µg/mL. Following 3-hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 51.3% cytotoxicity were obtained with the test substance at 124 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 112 and 124 µg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 51.4% cytotoxicity was obtained with the test substance at 17.5 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 0.2, 10 and 17.5 µg/mL.
In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20 hour treatment, the test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. None of the concentrations exceeded the upper control limits of the solvent historical control data, or were positive in a statistical trend test. Thus the outcome of the study is clearly negative. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
It was concluded that the test substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. Hence, the test substance is concluded to be negative in this assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Apr 2017 to 02 May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELL LINES
Absence of Mycoplasma contamination: yes
Methods for maintenance in cell culture: suitable for used cell line
Periodically checked for karyotype stability: yes
Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium, humidified incubator at 34 to 39°C in an atmosphere of 5% CO2 in air - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- S9 mix contained: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co factors were prepared, neutralized with 1N NaOH and filter sterilized before adding to S9 fraction and R0. - Test concentrations with justification for top dose:
- Preliminary test: 3.5, 7, 13.9, 27.8, 55.7, 111.4, 222.8, 445.5, 891 and 1782 μg/mL
Main test: 2.5, 25, 50, 60, 70, 80, 90, 100, 110, 110 and 120 μg/mL (-S9 and +S9) - Vehicle / solvent:
- - Vehicle used: DMSO
- Percentage of solvent in the final culture medium: 1% v/v - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: A cell suspension at 1x 10^6 cells/mL was prepared in R10p. Aliquots of the cell suspension, R0 (without metabolic activation) or S9 mix (with metabolic activation) and the test substance solution at each concentration or DMSO were combined and incubated at 37°C for three hours. Each culture contained 1.2 x 10^7 cells in 20 mL.
TREATMENT AND HARVEST SCHEDULE:
Exposure duration: 3 hours
FOR GENE MUTATION:
- Expression time: 24 and 48 h
- Selection time: 10 to 12 days
- Method used: 96-well plates
- Selective agent: Trifluorothymidine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10^3 cells/well, two plates being prepared per culture
- Criteria for small (slow growing) and large (fast growing) colonies: The criteria for sizing colonies was based on morphology and generally was less than 25% of the well’s diameter for small colonies and greater than 25% of the well’s diameter for large colonies.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency (CE), mutant frequency (MF) and colony size distribution
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Mutant frequency and the Global Evaluation Factor (GEF) - Evaluation criteria:
- The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6.
- Providing that all acceptability criteria were fulfilled, the test item was considered to be clearly positive if, in any of the experimental conditions examined the increase in MF above the concurrent background exceeded the GEF and the increase was concentration related (i.e., there is a significant positive linear trend). The test item is then considered able to induce mutation in this test system.
- Providing that all acceptability criteria are fulfilled, the test item was considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test item is then considered unable to induce mutations in this test system.
- If the maximum concentration was based on cytotoxicity, the highest concentration aimed to achieve between 20 and 10% RTG. The consensus is that care should be taken when interpreting positive results only found between 20 and 10% RTG and a result would not be considered positive if the increase in MF occurred only at or below 10% RTG (if evaluated) - Statistics:
- Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989).
Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05) - 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:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Soluble
- Osmolarity: The osmolality of the test item in medium was tested at 1782 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- pH: No fluctuations in pH of the medium were observed at 1782 µg/mL of more than 1.0 unit compared with the vehicle control.
RANGE-FINDING/SCREENING STUDIES:
Precipitate (observed by eye at the end of treatment) was observed at concentrations of 445.5 ug/mL and above in the absence and presence of S9 mix, therefore, this was the highest concentration assessed for cytotoxicity. Exposure to the test substance at concentrations from 3.5 to 445.5 ug/mL in the absence and presence of S9 mix resulted in relative suspension growth (RSG) values from 102 to 0% and from 89 to 0% respectively.
STUDY RESULTS
Concurrent vehicle negative and positive control data :
- In absence of S9 metabolic activation: The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
- In presence of S9 metabolic activation: The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Gene mutation tests in mammalian cells:
Results from cytotoxicity measurements:
- Relative total growth (RTG) or relative survival (RS) and cloning efficiency in absence of S9: Relative total growth (RTG) values from 102 to 21%
were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.
- Relative total growth (RTG) or relative survival (RS) and cloning efficiency in presence of S9: RTG values from 94 to 10% were obtained relative to
the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. - Conclusions:
- Test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay performed in compliance with GLP and following an OECD 490 guideline, and afforded a clearly negative result under the experimental conditions described.
- Executive summary:
Test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay performed in compliance with GLP and following an OECD 490 guideline. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two independent mutagenicity assays. The cells were exposed for 3 hours in the absence or presence of exogenous metabolic activation (S9 mix).
Test substance was found to be soluble at 178.2 mg/mL in dimethyl sulphoxide (DMSO). A final concentration of 1782 µg/mL, dosed at 1 % v/v, was used as the maximum concentration in the preliminary toxicity test. Precipitate was observed by eye at the end of treatment at concentrations of 445.5 µg/mL and above in the preliminary toxicity test and this was, therefore, the highest concentration assessed for cytotoxicity. Following a 3-hour exposure to the test substance at concentrations from 3.5 to 445.5 µg/mL, relative suspension growth (RSG) was reduced from 102 to 0% and from 89 to 0% in the absence and presence of S9 mix respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test concentrations which spanned the toxicity range 100 to 10-20% relative total growth (RTG). Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 90 and 110 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 21 and 10% respectively. In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, and afforded a clearly negative result under the experimental conditions described.
Referenceopen allclose all
Table 1. Results Obtained in the Absence of Metabolic Activation: Experiment 1.
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
DMSO |
|
22.7 |
9.3 |
|
33, 20, 15 |
|
Test subst. |
5 |
22 |
3.5 |
1 |
24, 18, 24 |
|
|
15 |
32.7 |
5.5 |
1.4 |
38, 27, 33 |
|
|
50 |
23.7 |
3.5 |
1 |
24, 27, 20 |
|
|
150 |
25.3 |
8.1 |
1.1 |
24, 18, 34 |
|
|
500 |
24.7 |
3.8 |
1.1 |
23, 29, 22 |
|
|
1500 |
22 |
7 |
1 |
29, 22, 15 |
|
|
5000 |
19 |
5.2 |
0.8 |
13 S P, 22 S P, 22 S P |
TA100 |
DMSO |
|
172 |
19.2 |
|
194, 163, 159 |
|
Test subst. |
5 |
179.7 |
17.4 |
1 |
160, 193, 186 |
|
|
15 |
191 |
5.2 |
1.1 |
188, 188, 197 |
|
|
50 |
182 |
15.6 |
1.1 |
200, 172, 174 |
|
|
150 |
163.3 |
2.5 |
0.9 |
161, 166, 163 |
|
|
500 |
164.3 |
11.4 |
1 |
177, 161, 155 |
|
|
1500 |
123.3 |
13.6 |
0.7 |
139 T, 115 T, 116 T |
|
|
5000 |
49 |
14.8 |
0.3 |
59 P V, 56 P V, 32 P V |
TA1535 |
DMSO |
|
24.3 |
3.8 |
|
27, 26, 20 |
|
Test subst. |
5 |
23.3 |
7.1 |
1 |
22, 31, 17 |
|
|
15 |
27.3 |
1.5 |
1.1 |
26, 27, 29 |
|
|
50 |
21.7 |
4.7 |
0.9 |
27, 20, 18 |
|
|
150 |
20 |
4.6 |
0.8 |
21, 24, 15 |
|
|
500 |
20 |
3.6 |
0.8 |
21, 23, 16 |
|
|
1500 |
18.7 |
8.1 |
0.8 |
26, 10, 20 |
|
|
5000 |
14.7 |
5.7 |
0.6 |
10 S P, 21 S P, 13 S P |
TA1537 |
DMSO |
|
9.7 |
3.2 |
|
6, 12, 11 |
|
Test subst. |
5 |
11.3 |
4.5 |
1.2 |
7, 11, 16 |
|
|
15 |
10.3 |
0.6 |
1.1 |
11, 10, 10 |
|
|
50 |
9 |
2.6 |
0.9 |
6, 10, 11 |
|
|
150 |
9.7 |
0.6 |
1 |
10, 9, 10 |
|
|
500 |
8 |
2.6 |
0.8 |
5, 9, 10 |
|
|
1500 |
10.7 |
3.2 |
1.1 |
7, 12, 13 |
|
|
5000 |
10.7 |
5.1 |
1.1 |
5 S P, 12 S P, 15 S P |
WP2 uvrA |
DMSO |
|
238.3 |
12.7 |
|
227, 236, 252 |
|
Test subst. |
5 |
238.3 |
15.3 |
1 |
225, 255, 235 |
|
|
15 |
220 |
6.6 |
0.9 |
221, 226, 213 |
|
|
50 |
230.7 |
18.1 |
1 |
244, 210, 238 |
|
|
150 |
243.7 |
7 |
1 |
237, 243, 251 |
|
|
500 |
233.3 |
10 |
1 |
241, 237, 222 |
|
|
1500 |
222.3 |
4.2 |
0.9 |
221, 227, 219 |
|
|
5000 |
238.7 |
6.7 |
1 |
223 S P, 24 S P 6, 237 S P |
TA98 |
2NF |
2 |
265 |
76.3 |
11.7 |
353, 218, 224 |
TA100 |
NaN3 |
2 |
775.3 |
87.6 |
4.5 |
804, 677, 845 |
TA1535 |
NaN3 |
2 |
796.3 |
66.4 |
32.7 |
769, 748, 872 |
TA1537 |
AAC |
50 |
158 |
57.7 |
16.3 |
220, 148, 106 |
WP2 uvrA |
NQO |
2 |
2775 |
596 |
11.6 |
2110, 3261, 2954 |
2NF – 2-Nitrofluorene
NaN3 – Sodium azide
AAC – 9-Aminoacridine
NQO – 4-Nitroquinoline-1-oxide
P – Precipitate
S – Slight thinning of bacterial lawn
T – Thinning of bacterial lawn
V – Severe thinning of bacterial lawn
Table 2. Results Obtained in the Presence of Metabolic Activation: Experiment 1.
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
DMSO |
|
32.7 |
5.5 |
|
38, 27, 33 |
|
Test subst. |
5 |
29 |
2.6 |
0.9 |
32, 27, 28 |
|
|
15 |
31.3 |
9.1 |
1 |
21, 38, 35 |
|
|
50 |
31.3 |
6.8 |
1 |
39, 29, 26 |
|
|
150 |
36.7 |
11.9 |
1.1 |
33, 27, 50 |
|
|
500 |
35.3 |
1.5 |
1.1 |
35, 34, 37 |
|
|
1500 |
27 |
2.6 |
0.8 |
24, 29, 28 |
|
|
5000 |
10 |
2.6 |
0.3 |
7 T, 11 T, 12 T |
TA100 |
DMSO |
|
210.7 |
10.2 |
|
215, 218, 199 |
|
Test subst. |
5 |
174 |
3 |
0.8 |
174, 177, 171 |
|
|
15 |
191 |
8 |
0.9 |
183, 199, 191 |
|
|
50 |
161.3 |
9.5 |
0.8 |
161, 171, 152 |
|
|
150 |
177.3 |
19.4 |
0.8 |
156, 182, 194 |
|
|
500 |
203.3 |
18.4 |
1 |
183, 208, 219 |
|
|
1500 |
99.7 |
28.3 |
0.5 |
115 V, 117 V, 67 V |
|
|
5000 |
0 |
0 |
0 |
0 V, 0 V, 0 V |
TA1535 |
DMSO |
|
14.3 |
2.1 |
|
16, 15, 12 |
|
Test subst. |
5 |
11 |
2 |
0.8 |
9, 11, 13 |
|
|
15 |
12.7 |
3.1 |
0.9 |
10, 16, 12 |
|
|
50 |
11 |
1 |
0.8 |
11, 12, 10 |
|
|
150 |
16.7 |
6.7 |
1.2 |
24, 11, 15 |
|
|
500 |
12.3 |
4.2 |
0.9 |
9, 11, 17 |
|
|
1500 |
15 |
6.9 |
1 |
23 S, 11 S, 11 S |
|
|
5000 |
9 |
3.6 |
0.6 |
12 T, 10 T, 5 T |
TA1537 |
DMSO |
|
10 |
3 |
|
13, 10, 7 |
|
Test subst. |
5 |
14.7 |
1.5 |
1.5 |
16, 15, 13 |
|
|
15 |
12 |
1 |
1.2 |
12, 13, 11 |
|
|
50 |
16 |
5 |
1.6 |
11, 16, 21 |
|
|
150 |
15 |
4.4 |
1.5 |
12, 20, 13 |
|
|
500 |
12.3 |
4.9 |
1.2 |
9, 10, 18 |
|
|
1500 |
5 |
1 |
0.5 |
6 S, 5 S, 4 S |
|
|
5000 |
0.3 |
0.6 |
0 |
0 V, 0 V, 1 V |
WP2 uvrA |
DMSO |
|
300.3 |
18.6 |
|
302, 281, 318 |
|
Test subst. |
5 |
338.3 |
35.5 |
1.1 |
373, 302, 340 |
|
|
15 |
332 |
14.1 |
1.1 |
319, 330, 347 |
|
|
50 |
326.7 |
21 |
1.1 |
343, 334, 303 |
|
|
150 |
292.7 |
23.1 |
1 |
290, 317, 271 |
|
|
500 |
301.7 |
16.5 |
1 |
288, 320, 297 |
|
|
1500 |
271 |
20 |
0.9 |
251 S, 271 S, 291 S |
|
|
5000 |
251 |
8.7 |
0.8 |
241 S, 257 S, 255 S |
TA98 |
B[a]P |
5 |
257 |
10.8 |
7.9 |
254, 269, 248 |
TA100 |
AAN |
5 |
4488 |
115.3 |
21.3 |
4402, 4443, 4619 |
TA1535 |
AAN |
5 |
360 |
13.5 |
25.1 |
346, 373, 361 |
TA1537 |
B[a]P |
5 |
59.3 |
20.6 |
5.9 |
50, 45, 83 |
WP2 uvrA |
AAN |
10 |
1708.3 |
106.4 |
5.7 |
1588, 1747, 1790 |
B[a]P – Benzo[a]pyrene
AAN – 2-Aminoanthracene
S – Slight thinning of bacterial lawn
T – Thinning of bacterial lawn
V – Severe thinning of bacterial lawn
Table 3. Results Obtained in the Presence of Metabolic Activation: Experiment 1 repeat
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
WP2 uvrA |
DMSO |
|
263 |
13.5 |
|
276, 264, 249 |
|
Test subst. |
5 |
231 |
42.3 |
0.9 |
192, 225, 276 |
|
|
15 |
227.3 |
8.6 |
0.9 |
235, 229, 218 |
|
|
50 |
229.3 |
16.3 |
0.9 |
218, 222, 248 |
|
|
150 |
247.3 |
7 |
0.9 |
254, 240, 248 |
|
|
500 |
245.3 |
2.9 |
0.9 |
247, 247, 242 |
|
|
1500 |
252.7 |
4.7 |
1 |
251 S, 249 S, 258 S |
|
|
5000 |
242 |
23 |
0.9 |
265 S, 242 S, 219 S |
|
AAN |
10 |
938.3 |
43.2 |
3.6 |
952, 973, 890 |
AAN – 2-Aminoanthracene
S – Slight thinning of bacterial lawn
Table 4. Results Obtained in the Absence of Metabolic Activation: Experiment 2.
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
DMSO |
|
20.3 |
2.1 |
|
22, 21, 18 |
|
Test subst. |
5 |
19.7 |
1.5 |
1 |
21, 20, 18 |
|
|
15 |
23 |
1.7 |
1.1 |
24, 24, 21 |
|
|
50 |
23 |
3 |
1.1 |
20, 23, 26 |
|
|
150 |
20.3 |
3.5 |
1 |
20, 24, 17 |
|
|
500 |
19 |
6.9 |
0.9 |
15 S, 27 S, 15 S |
|
|
1500 |
4.7 |
2.3 |
0.2 |
2 T, 6 T, 6 T |
|
|
5000 |
4 |
2.6 |
0.2 |
5 V P, 1 V P, 6 V P |
TA100 |
DMSO |
|
117.7 |
9.1 |
|
111, 128, 114 |
|
Test subst. |
5 |
139 |
17.4 |
1.2 |
127, 159, 131 |
|
|
15 |
151 |
14 |
1.3 |
167, 145, 141 |
|
|
50 |
151.7 |
8.1 |
1.3 |
143, 159, 153 |
|
|
150 |
62 |
36.4 |
0.5 |
40 V, 42 V, 104 V |
|
|
500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
|
|
1500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
|
|
5000 |
0 |
0 |
0 |
0 A P, 0 A P, 0 A P |
TA1535 |
DMSO |
|
15 |
2.6 |
|
17, 16, 12 |
|
Test subst. |
5 |
21.3 |
11.5 |
1.4 |
10, 33, 21 |
|
|
15 |
17.7 |
3.8 |
1.2 |
16, 22, 15 |
|
|
50 |
17.3 |
4 |
1.2 |
13, 21, 18 |
|
|
150 |
21.7 |
0.6 |
1.4 |
22, 22, 21 |
|
|
500 |
11 |
4.6 |
0.7 |
10 T, 16 T, 7 T |
|
|
1500 |
8 |
2.6 |
0.5 |
7 T, 6 T, 11 T |
|
|
5000 |
7 |
5.3 |
0.5 |
1 P V, 9 P V, 11 P V |
TA1537 |
DMSO |
|
9.3 |
3.5 |
|
6, 13, 9 |
|
Test subst. |
5 |
11.7 |
3.8 |
1.2 |
9, 10, 16 |
|
|
15 |
9.3 |
3.8 |
1 |
11, 12, 5 |
|
|
50 |
8.3 |
2.1 |
0.9 |
10, 9, 6 |
|
|
150 |
2.3 |
1.5 |
0.3 |
1 V, 2 V, 4 V |
|
|
500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
|
|
1500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
|
|
5000 |
0 |
0 |
0 |
0 A P, 0 A P, 0 A P |
WP2 uvrA |
DMSO |
|
192.3 |
14.8 |
|
176, 205, 196 |
|
Test subst. |
5 |
186.7 |
13 |
1 |
174, 186, 200 |
|
|
15 |
191.3 |
12.2 |
1 |
178, 194, 202 |
|
|
50 |
201 |
36.1 |
1 |
187, 174, 242 |
|
|
150 |
210.7 |
7.6 |
1.1 |
214, 216, 202 |
|
|
500 |
201.3 |
7.5 |
1 |
197, 210, 197 |
|
|
1500 |
215 |
36 |
1.1 |
208, 254, 183 |
|
|
5000 |
206.7 |
17.9 |
1.1 |
222 S P, 187 S P, 211 S P |
TA98 |
2NF |
2 |
163.7 |
54.4 |
8 |
199, 101, 191 |
TA100 |
NaN3 |
2 |
745 |
247.7 |
6.3 |
908, 867, 460 |
TA1535 |
NaN3 |
2 |
677 |
22.5 |
45.1 |
664, 664, 703 |
TA1537 |
AAC |
50 |
117.3 |
6.7 |
12.6 |
119, 110, 123 |
WP2 uvrA |
NQO |
2 |
2909.3 |
273.5 |
15.1 |
3173, 2627, 2928 |
2NF – 2-Nitrofluorene
NaN3 – Sodium azide
AAC – 9-Aminoacridine
NQO – 4-Nitroquinoline-1-oxide
P – Precipitate
S – Slight thinning of bacterial lawn
T – Thinning of bacterial lawn
V – Severe thinning of bacterial lawn
Table 5. Results Obtained in the Presence of Metabolic Activation: Experiment 2.
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
DMSO |
|
20.7 |
3.1 |
|
18, 24, 20 |
|
Test subst. |
5 |
22.7 |
4.5 |
1.1 |
23, 18, 27 |
|
|
15 |
24 |
6.1 |
1.2 |
27, 17, 28 |
|
|
50 |
21 |
1 |
1 |
21, 20, 22 |
|
|
150 |
22 |
2 |
1.1 |
20, 22, 24 |
|
|
500 |
21.7 |
5.9 |
1 |
15 T, 24 T, 26 T |
|
|
1500 |
18 |
3.6 |
0.9 |
22 T, 17 T, 15 T |
|
|
5000 |
8 |
1.7 |
0.4 |
9 V, 6 V, 9 V |
TA100 |
DMSO |
|
134 |
6.1 |
|
127, 137, 138 |
|
Test subst. |
5 |
134 |
3.6 |
1 |
131, 138, 133 |
|
|
15 |
156 |
3.5 |
1.2 |
154, 160, 154 |
|
|
50 |
150.3 |
8.7 |
1.1 |
143, 148, 160 |
|
|
150 |
144 |
22.6 |
1.1 |
165, 120, 147 |
|
|
500 |
93.7 |
15.7 |
0.7 |
76 T, 99 T, 106 T |
|
|
1500 |
81.7 |
9.9 |
0.6 |
93 V, 77 V, 75 V |
|
|
5000 |
9.3 |
4 |
0.1 |
10 V, 5 V, 13 V |
TA1535 |
DMSO |
|
10.7 |
1.5 |
|
9, 12, 11 |
|
Test subst. |
5 |
14.7 |
4.7 |
1.4 |
20, 13, 11 |
|
|
15 |
20 |
7.2 |
1.9 |
26, 22, 12 |
|
|
50 |
7.7 |
2.1 |
0.7 |
7, 6, 10 |
|
|
150 |
13.7 |
2.1 |
1.3 |
13, 16, 12 |
|
|
500 |
14.7 |
3.5 |
1.4 |
11, 18, 15 |
|
|
1500 |
7 |
2 |
0.7 |
7 S, 5 S, 9 S |
|
|
5000 |
8.7 |
6.5 |
0.8 |
2 T, 15 T, 9 T |
TA1537 |
DMSO |
|
7.7 |
2.1 |
|
10, 6, 7 |
|
Test subst. |
5 |
10.3 |
5.1 |
1.3 |
6, 16, 9 |
|
|
15 |
8.7 |
2.3 |
1.1 |
6, 10, 10 |
|
|
50 |
11.3 |
4.7 |
1.5 |
15, 6, 13 |
|
|
150 |
9.3 |
2.1 |
1.2 |
7, 10, 11 |
|
|
500 |
6.7 |
5 |
0.9 |
2 T, 12 T, 6 T |
|
|
1500 |
3.7 |
3.2 |
0.5 |
0 V, 5 V, 6 V |
|
|
5000 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
WP2 uvrA |
DMSO |
|
268 |
19.3 |
|
260, 254, 290 |
|
Test subst. |
5 |
260 |
14.7 |
1 |
252, 277, 251 |
|
|
15 |
264.7 |
10 |
1 |
264, 255, 275 |
|
|
50 |
253.3 |
18.5 |
0.9 |
263, 232, 265 |
|
|
150 |
255.3 |
4.5 |
1 |
260, 255, 251 |
|
|
500 |
255.3 |
22.2 |
1 |
281, 243, 242 |
|
|
1500 |
249 |
11.1 |
0.9 |
259 S, 251 S, 237 S |
|
|
5000 |
231.3 |
19.4 |
0.9 |
248 S, 210 S, 236 S |
TA98 |
B[a]P |
5 |
220 |
6.6 |
10.6 |
213, 226, 221 |
TA100 |
AAN |
5 |
2970.3 |
275.6 |
22.2 |
2659, 3183, 3069 |
TA1535 |
AAN |
5 |
260.7 |
17.6 |
24.4 |
263, 277, 242 |
TA1537 |
B[a]P |
5 |
59.3 |
3.8 |
7.7 |
62, 55, 61 |
WP2 uvrA |
AAN |
10 |
1485.3 |
77 |
5.5 |
1404, 1495, 1557 |
B[a]P – Benzo[a]pyrene
AAN – 2-Aminoanthracene
S – Slight thinning of bacterial lawn
T – Thinning of bacterial lawn
V – Severe thinning of bacterial lawn
Table 6. Results Obtained in the Absence of Metabolic Activation: Experiment 2 repeat.
Strain |
Addition |
Concentration per plate (µg) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA100 |
DMSO |
|
165.3 |
5.9 |
|
172, 161, 163 |
|
Test subst. |
0.5 |
137 |
10.8 |
0.8 |
128, 134, 149 |
|
|
1.5 |
176.7 |
13.3 |
1.1 |
169, 169, 192 |
|
|
5 |
158 |
20.7 |
1 |
152, 141, 192 |
|
|
15 |
163.3 |
10.4 |
1 |
155, 175, 160 |
|
|
50 |
153 |
14.5 |
0.9 |
167, 154, 138 |
|
|
150 |
119.7 |
26.2 |
0.7 |
144 V, 123 V, 92 V |
|
|
500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
TA1535 |
DMSO |
|
11.7 |
2.9 |
|
10, 15, 10 |
|
Test subst. |
0.5 |
8 |
3.5 |
0.7 |
10, 4, 10 |
|
|
1.5 |
8.3 |
1.2 |
0.7 |
9, 7, 9 |
|
|
5 |
10.7 |
2.1 |
0.9 |
13, 9, 10 |
|
|
15 |
11.3 |
4 |
1 |
15, 12, 7 |
|
|
50 |
10 |
5.6 |
0.9 |
9, 5, 16 |
|
|
150 |
1.7 |
0.6 |
0.1 |
2 V, 1 V, 2 V |
|
|
500 |
0 |
0 |
0 |
0 A, 0 A, 0 A |
TA100 |
NaN3 |
2 |
773.3 |
69.8 |
4.7 |
851, 753, 716 |
TA1535 |
AAC |
50 |
125.7 |
21.7 |
10.8 |
142, 101, 134 |
NaN3 – Sodium azide
AAC – 9-Aminoacridine
V – Severe thinning of bacterial lawn
A – Lawn absent
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
No clastogenic or aneugenic effects were found in the following study:
- Mouse bone marrow micronucleus assay, OECD TG 474, Deparade, 1999
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Aug 1999 to 18 Nov 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 21 July 1997
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- August 1998
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 31 July 1992
- Qualifier:
- according to guideline
- Guideline:
- other: MITI, Ministry of International Trade and Industry, Japan, 62 Notification of Basic Industries Bureau, No. 303, (Sho-62)
- Version / remarks:
- 31 March 1987
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: Ico:CD1(CRL)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: young adults, 7 - 8 weeks old
- Weight at study initiation: 33-39 g males, 24-29 g females
- Housing: 5 animals per cage
- Diet: Certified standard diet, ad libitum
- Water: Tap water ad libitum
- Acclimation period: The animals were kept on location for at least five days prior to being used in the study. Shortly before use the health status of the animals was checked by the laboratory personnel according to veterinary/scientific standards.
ENVIRONMENTAL CONDITIONS
- Temperature: 20.5-22.0°C
- Humidity: 50-79%
- Air changes (per hr): Not reported
- Photoperiod: 12 hours dark/12 hours light
IN-LIFE DATES: 16 Aug 1999 to 18 Nov 1999 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: arachis oil
- Concentration of test material in vehicle: 16, 32, 64 mg/mL
- Amount of vehicle: 5 mL/kg bw - Frequency of treatment:
- Single dose
- Post exposure period:
- 24 hours (all groups) and 48 hours (high dose group and negative control group only)
- Dose / conc.:
- 80 mg/kg bw (total dose)
- Remarks:
- Low dose group
- Dose / conc.:
- 160 mg/kg bw (total dose)
- Remarks:
- Mid dose group
- Dose / conc.:
- 320 mg/kg bw (total dose)
- Remarks:
- High dose group
- No. of animals per sex per dose:
- 5 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Substance: cyclophosphamide (CPA)
Route of administration: Oral gavage
Doses: 64 mg/kg bw
Dosing volume: 10 mL/kg bw - Tissues and cell types examined:
- Bone marrow, Polychromatic erythrocytes (PCEs)
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Preliminary toxicity testing
DETAILS OF SLIDE PREPARATION: The animals were sacrificed by CO2 asphyxiation. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grunwald/Giemsa solution and mounted.
METHOD OF ANALYSIS: The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 2000 polychromatic erythrocytes (PCE) and the ratio of PCE to normochromatic erythrocytes (NCE) among a total of at least 1000 erythrocytes were determined for each slide. - Evaluation criteria:
- Please, see "Any other information on results incl. tables" section
- Statistics:
- The significance of differences between control and treated groups was assessed by the Chi-Squared-Contingency-Test (F=1, p<0.05).
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- the high dose group
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
In the first step of the tolerability test, the dose of 2000 mg/kg of the test substance, was administered to one female and one male animal. One to two hours after dosing, both mice exhibited recumbence and died within two to three hours after administration. In the second step, a pair of mice was treated with 800 mg/kg of the test substance. Both animals exhibited recumbence and decreased muscle tone. They were found dead three hours after dosing. In the third step, one female and one male were dosed with 320 mg/kg of the test substance. No symptoms of toxicity occurred during the three days observation period . In the confirmatory step, again one male and one female received 320 mg/kg of the test substance. No symptoms of toxicity were observed. Based on these results, the dose of 320 mg/kg for females and males was chosen as the highest one to be administered in the micronucleus test.
RESULTS OF DEFINITIVE STUDY
- In the high dose group (24 hours sampling time), two female and four male animals exhibited creeping movements, recumbence and decreased muscle tone on day of sacrifice. No symptoms of toxicity were noted in the intermediate and low dose groups.
- In the group of 48 hours sampling time dosed with 320 mg/kg test substance, one female and one male showed creeping movements, recumbence and decreased muscle tone on day of sacrifice. One male was found dead on day of sacrifice and was replaced by a reserve animal. No symptoms of toxicity occurred in the other animals.
- At both sampling times (24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of the test substance, as compared with the negative control animals.
- At the 24 hours sampling time after treatment with the test substance the mean percentage of micronucleated PCEs was 0.07 (80 mg/kg), 0.10 (160 mg/kg) and 0.08 (320 mg/kg ) respectively compared to a negative control value of 0.08.
- At the 48 hours sampling time after treatment with 320 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.12 compared to a negative control value of 0.10
- In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was significantly increased. The mean percentage of micronucleated PCEs evaluated was 1.80. In comparison with the negative control of 0.08% this value is highly significant (p<0.05). - Conclusions:
- In a mouse bone marrow micronucleus assay performed in compliance with GLP and following an OECD 474 guideline, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. There was no evidence for clastogenic or aneugenic effects in mice treated with the test substance.
- Executive summary:
In a mouse bone marrow micronucleus assay performed in compliance with GLP and following an OECD 474 guideline, groups of 5 male and 5 female young adult, Ico:CD1 [CRL] mice were dosed orally, by gavage, with the test substance at doses of 0, 0, 80, 160, 320 and 320 mg/kg/day. An additional group was dosed with the positive control, cyclophosphamide at 64 mg/kg/day. One group of mice at each test substance dose level, plus one negative control group and the positive control group were killed after 24 hours. The other negative control group and 320 mg/kg/day group were killed after 48 hours. Femoral bone marrow cells were harvested and polychromatic erythrocytes scored for micronuclei.
In the 320 mg/kg dosage group (24 hours sampling time), four males and two females exhibited creeping movement, recumbence and decreased muscle tone the day after administration. No symptoms of toxicity were noted in the 160 and 80 mg/kg groups. In the 320 mg/kg group (48 hours sampling time) one male was found dead two days after administration and was replaced by a reserve animal. Creeping movement, recumbence and decreased muscle tone were occasionally observed shortly before sacrifice. In the reserve group one female was found dead two days after administration. No statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed in any group when compared with the respective negative control group. The positive control induced the appropriate response.
There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. There was no evidence for clastogenic or aneugenic effects in mice treated with the test substance.
Reference
Table 1. Overall mean percentage of micronucleated PCEs
Treatment time |
320 mg/kg |
160 mg/kg |
80 mg/kg |
Positive control |
Negative control |
24 hours |
0.08 |
0.10 |
0.07 |
1.80 |
0.08 |
48 hours |
0.12 |
|
|
|
0.10 |
Analytical results
The test material in suspension was analysed by HPLC with UV detection to confirm the intended concentrations to be used in the micronucleus test and the stability of the test substance in the vehicle used.
Nominal concentrations |
Actual concentrations |
||
Experiment |
pg/mL |
pg/mL |
% of nominal value* |
24 + 48 h |
64000 |
62250/62899 |
97.3/98.3 |
24 h |
16000 |
18700/18804 |
117/118 |
* acceptable range: 100 ± 20 %
The values found by analysis of all samples were in agreement with the intended concentrations, demonstrating sufficient stability of the test substance in the vehicle.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The key study is considered to be worst-case and was selected for the CSA.
In vitro genetic toxicity in bacteria
In the key study (Woods 2017), the mutagenic potential of the test substance performed in compliance with GLP and following the OECD TG 471, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.
Toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance in both tests. Precipitate was observed on all plates containing the test substance at 5000 µg/plate in the absence of S9 mix in the first and second test. No evidence of mutagenic activity was seen at any concentration of the test substance in either experiment. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.
It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Three supporting studies (Sokolowski 2014, Deparade 1983, Arni 1979) for this endpoint are available which support the results of the key study. The test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used in the studies. Therefore, the test substance is considered to be non-mutagenic in the tested strains.
In vitro cytogenicity/ chromosome aberration
The key study (Gilby 2017), performed in compliance with GLP and according to OECD TG 487 was designed to assess the potential of the test substance to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro. The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture were exposed to the test substance for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 2000 µg/mL in order to test up to the maximum concentration as recommended in the current OECD TG 487 (2016). Vehicle (dimethyl sulphoxide [DMSO]) and positive control cultures were included in all appropriate test conditions.
Three test substance concentrations were assessed for determination of induction of micronuclei. The highest concentration selected for all exposures was that which caused a reduction in CBPI equivalent to 55±5% cytotoxicity. Following a 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 53.2% cytotoxicity were obtained with the test substance at 110 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 100 and 110 µg/mL. Following 3-hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 51.3% cytotoxicity were obtained with the test substance at 124 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 5, 112 and 124 µg/mL. In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 51.4% cytotoxicity was obtained with the test substance at 17.5 µg/mL. Concentrations of the test substance selected for micronucleus analysis were 0.2, 10 and 17.5 µg/mL.
In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20 hour treatment, the test substance did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. None of the concentrations exceeded the upper control limits of the solvent historical control data, or were positive in a statistical trend test. Thus the outcome of the study is clearly negative. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
It was concluded that the test substance did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. Hence, the test substance is concluded to be negative in this assay.
One supporting study (Strasser 1984) for this endpoint is available which support the results of the key study. This study was conducted according to EPA 84-2 and followed GLP. No evidence of mutagenic effects was obtained in human lymphocytes in vitro treated with the test substance.
In vitro genetic toxicity in mammalian cells
In the key study (Gilby 2017), the test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay performed in compliance with GLP and following an OECD TG 490. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two independent mutagenicity assays. The cells were exposed for 3 hours in the absence or presence of exogenous metabolic activation (S9 mix).
Test substance was found to be soluble at 178.2 mg/mL in dimethyl sulphoxide (DMSO). A final concentration of 1782 µg/mL, dosed at 1 % v/v, was used as the maximum concentration in the preliminary toxicity test. Precipitate was observed by eye at the end of treatment at concentrations of 445.5 µg/mL and above in the preliminary toxicity test and this was, therefore, the highest concentration assessed for cytotoxicity. Following a 3-hour exposure to the test substance at concentrations from 3.5 to 445.5 µg/mL, relative suspension growth (RSG) was reduced from 102 to 0% and from 89 to 0% in the absence and presence of S9 mix respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test concentrations which spanned the toxicity range 100 to 10-20% relative total growth (RTG). Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 90 and 110 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 21 and 10% respectively. In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, and afforded a clearly negative result under the experimental conditions described.
Five supporting studies (Strasser 1982, Puri 1982a, Puri 1982b, Wollny 2014 and Strasser 1982) for this endpoint are available which support the results of the key study. One study is a transformation assay, two studies are DNA damage and repair assays, and two studies are gene mutation studies in mouse lymphoma cells. The test substance was negative in all of the mentioned assays.
Genetic toxicity in vivo
The key study (Deparade 1999), in a mouse bone marrow micronucleus assay, was performed in compliance with GLP and followed OECD TG 474. Groups of 5 male and 5 female young adult, Ico:CD1 [CRL] mice were dosed orally, by gavage, with the test substance at doses of 0, 0, 80, 160, 320 and 320 mg/kg/day. An additional group was dosed with the positive control, cyclophosphamide at 64 mg/kg/day. One group of mice at each test substance dose level, plus one negative control group and the positive control group were killed after 24 hours. The other negative control group and 320 mg/kg/day group were killed after 48 hours. Femoral bone marrow cells were harvested and polychromatic erythrocytes scored for micronuclei.
In the 320 mg/kg dosage group (24 hours sampling time), four males and two females exhibited creeping movement, recumbence and decreased muscle tone the day after administration. No symptoms of toxicity were noted in the 160 and 80 mg/kg groups. In the 320 mg/kg group (48 hours sampling time) one male was found dead two days after administration and was replaced by a reserve animal. Creeping movement, recumbence and decreased muscle tone were occasionally observed shortly before sacrifice. In the reserve group one female was found dead two days after administration. No statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed in any group when compared with the respective negative control group. The positive control induced the appropriate response.
There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. There was no evidence for clastogenic or aneugenic effects in mice treated with the test substance.
Five supporting studies (Hool 1982, Strasser 1982, Hool 1979, Strasser 1987, and Hool 1982) for this endpoint are available which support the results of the key study. Two studies are chromosome aberration test, one study is an MNT, another study is a point mutation assay and the final study is a dominant lethal assay. The test substance was negative in all of the mentioned studies.
Justification for classification or non-classification
Based on the available data, classification of the test substance for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.
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