Registration Dossier
Registration Dossier
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EC number: 946-584-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: non mutagenic (OECD 471, GLP, K, rel. 1)
Micronucleus test in human lymphocytes: non clastogenic (OECD 487, GLP, K, rel. 1)
HPRT test in Chines hamster lung fibroblasts cells (V79): non mutagenic (OECD 476, GLP, K, rel. 1)
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:
- 26 July - 24 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD Guideline 471 without any deviation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- dated 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- dated August 1998
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1003395243
- Expiration date of the lot/batch: 17 May 2021
- Purity test date: 18 July 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark (closed containers)
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer and sonication for 10 minutes at 40°C. The test item was confirmed as a UVCB product and, therefore, no purity correction was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. - Target gene:
- histidine locus for Salmonella strains and tryptophan for E. coli strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in house, the test item was noted as insoluble in dimethyl sulphoxide at 50 mg/mL but fully soluble in dimethyl formamide at the same concentration and acetone at 100 mg/mL. Acetone was selected as the vehicle. Distilled water was not evaluated as a vehicle in this test system as information provided by the sponsor suggested it was unstable with the test item. - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours for both Experiment
CONTROLS:
- Vehicle/solvent control: Acetone
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
NUMBER OF REPLICATIONS: Triplicate
- OTHER: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count. - Rationale for test conditions:
- The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (i.e. maximum recommended dose level). The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
- Evaluation criteria:
- Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it will be considered to exhibit mutagenic activity in this test system (Cariello and Piegorsch, 1996).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- None
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- refer tables 7.6.1/4 and 7.6.1/5
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted in both experiments in both the presence and absence of S9 from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
MUTAGENICITY
- The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Experiment 1 (Plate Incorporation)
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
- Experiment 2 (Pre-Incubation)
Consequently, in the second mutation test (pre-incubation method), the same maximum dose level was selected as the maximum dose level. In the second experiment, the test item caused a visible reduction in all of the Salmonella bacterial lawns in the absence of S9-mix from 1500 μg/plate. In the presence of S9-mix, toxicity was noted to all of the Salmonella bacterial strains at 5000 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.
HISTORICAL CONTROL DATA [with ranges, means and standard deviation and confidence interval (e.g. 95%)]
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6
OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. - Conclusions:
- Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). In Experiment 2 (pre incubation method), the test item caused a visible reduction in all of the Salmonella bacterial lawns in the absence of S9-mix from 1500 µg/plate. In the presence of S9-mix, toxicity was noted to all of the Salmonella bacterial strains at 5000 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.
A test item precipitate (greasy and particulate in appearance) was noted in both experiments in both the presence and absence of S9 from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 January to 03 April 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD 476 Guideline without any deviation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- other: In vitro mammalian cell gene mutation test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TCH Consortium / 1002888716
- Appearance: Pale yellow paste
- Expiration date of the lot/batch: 15 December 2017
- Purity test date: 15 December 2016
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark - Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Cell cycle length, doubling time or proliferation index: The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Modal number of chromosomes: 22
MEDIA USED
- Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes; cells have a stable karyotype
- Periodically 'cleansed' against high spontaneous background: Yes; Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix contains: S9 fraction (2%) prepared from male rats, dosed with phenobarbital and β-Naphthaflavone
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation
Mutation tests:
4 h exposure with metabolic activation: 5, 10, 20, 40, 80, 120, 140 and 160 μg/mL
4 h exposure with metabolic activation: 5, 10, 20, 40, 80 and 160 μg/mL
The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of test item precipitate and test item-induced toxicity in the absence of metabolic activation, and the onset of test item precipitate in the presence of metabolic activation, as recommended by the OECD 476 guidelines. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Following solubility checks performed in-house for the Human Lymphocyte in vitro Micronucleus Test performed on the same test item (Envigo Study No. BC94DX), the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test item was a UVCB, therefore, the maximum proposed concentration level in the solubility test was set at 5000 μg/mL initially, the maximum recommended concentration level, and no correction for the purity of the test item was applied. The test item formed a suspension unsuitable for dosing at 500 mg/mL. However, a suspension suitable for dosing was formed 250 mg/mL. Therefore, the test item was formulated at 250 mg/mL and dosed at 1% to give a maximum achievable concentration level of 2500 μg/mL in the subsequent preliminary toxicity test. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9,10-dimethylbenzanthracene
- Remarks:
- with S9-mix
- Details on test system and experimental conditions:
- CELL CULTURE
The stock of cells was stored in liquid nitrogen. For use, a sample of cells were removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, Lglutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
METHOD OF APPLICATION: Eagles Minimal Essential (MEM) with 10% fetal bovine serum (FBS)
- Cell density at seeding: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 h being exposed to the test or control items.
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
All cell cultures were incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG) at a final concentration of 11 μg/mL; At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency.
NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and vehicle controls
Main test: Duplicate cultures for test item, vehicle and positive controls
NUMBER OF CELLS EVALUATED:
200 cells/flask were seeded for cloning efficiency and 2 x 10^5 cells/flask were analyzed for mutant frequencies.
DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency
- OTHER:
- Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
- The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes. - Rationale for test conditions:
- The test item was a UVCB, therefore, the maximum proposed concentration level in the solubility test was set at 5000 μg/mL initially, which was the maximum recommended concentration level in regulatory guidelines.
- Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful. - Statistics:
- When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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: There was no significant change in pH when the test item was dosed into media at 9.77 to 2500 μg/mL.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the concentration levels investigated (9.77 to 2500 μg/mL).
- Precipitation: At the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL.
PRELIMINARY CYTOTOXICITY TEST:
- A concentration range of 9.77 to 2500 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was the maximum recommended dose level.
- At the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL.
- There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. However, the greatest reductions were observed at dose levels at or beyond the onset of test item precipitate.
MAIN TEST:
- At the end of the exposure period, the onset of precipitate of the test item was observed at 80 μg/ml in the absence of metabolic activation, and at 40 μg/mL in the presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the subsequent dose levels were excluded from the analysis as they were considered to be surplus to requirements.
- There were very modest concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in any of the concentration levels, therefore indicating that residual toxicity had not occurred.
- The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.
HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mutant Frequencies per survivor (x 10^-6): 226.52 ± 77.63 (Ethyl methanesulphonate, -S9); 319.07 ± 157.07 (Dimethyl benzanthracene, +S9)
- Negative (solvent/vehicle) historical control data:
Mutant Frequencies per survivor (x 10^-6): 13.92 ± 7.04 (-S9); 20.62 ± 13.64 (+S9) - Conclusions:
- The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
- Executive summary:
In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item for 4 h, with and without metabolic activation (2% S9); S9 fraction prepared from male rats, dosed with phenobarbital and β-Naphthaflavone.
Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation
Mutation tests:
4 h exposure with metabolic activation: 5, 10, 20, 40, 80, 120, 140 and 160 μg/mL
4 h exposure with metabolic activation: 5, 10, 20, 40, 80 and 160 μg/mL
In a preliminary toxicity test, at the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL. There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. However, the greatest reductions were observed at dose levels at or beyond the onset of test item precipitate. The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of test item precipitate and test item-induced toxicity in the absence of metabolic activation, and the onset of test item precipitate in the presence of metabolic activation, as recommended by the OECD 476 guidelines.
In main test, at the end of the exposure period, the onset of precipitate of the test item was observed at 80 μg/ml in the absence of metabolic activation, and at 40 μg/mL in the presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the subsequent dose levels were excluded from the analysis as they were considered to be surplus to requirements. There were very modest concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in any of the concentration levels, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.
The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 January to 29 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD 487 Guideline without any deviation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TCH Consortium / 1002888716
- Appearance: Pale yellow paste
- Expiration date of the lot/batch: 15 December 2017
- Purity test date: 15 December 2016
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark - Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- At the end of the exposure period, the cell cultures were incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (2% final concentration of S9): S9 fraction, prepared from male rats dosed with phenobarbital and β-Naphthaflavone
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix
Main Test
4 h exposure to the test item formulations without S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
4 h exposure to the test item formulations with S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
24 h exposure to the test item without S9-mix: 10, 20, 40, 60, 80, 120 and 160 μg/mL
The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 μg/mL, however, due to formulation difficulties the maximum dose level that could be achieved was 2500 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- The test item was insoluble in Minimal Essential Medium at 50 mg/mL, and acetone and DMSO at 500 mg/mL. The test item was partially soluble/ suspendable in DMSO at 250 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: Demecolcine
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- CELLS:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are: Preliminary Toxicity Test: female, aged 21 years; Main Experiment (4 h exposures): male, aged 27 years; Main Experiment (24 h exposure): male, aged 27 years
CULTURE OF LYMPHOCYTES:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood
METHOD OF APPLICATION: in medium; Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS).
DURATION
- Exposure duration: 4 h (± S9) and 24 h exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h exposure (-S9) in main experiments
- Fixation time: 24 h incubation period in treatment-free media after exposure to the test item in all experiments
SPINDLE INHIBITOR (cytogenetic assays): At the end of the exposure period in both experiments, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.
STAIN (for cytogenetic assays): Slides were stained in 5% Giemsa for 5 minutes
NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture was prepared for test item and vehicle control
- Main tests: Duplicate cultures were prepared for test item, vehicle and positive controls
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cell harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C prior to slide making.
Preparation of microscope slides: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Then the slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
NUMBER OF CELLS EVALUATED:
- A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.
- The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = vehicle control culture
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
- OTHER:
Qualitative slide assessment: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation. - Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. - Statistics:
- The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
- 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:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media at 9.77 to 2500 μg/mL
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at 9.77 to 2500 μg/mL when compared to vehicle control.
- Precipitation: Yes
PRELIMINARY TOXICITY TEST
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 312.5 μg/mL in the 4 h exposure groups and at and above 156.25 μg/mL in the 24 h continuous exposure group.
- Haemolysis was observed following exposure to the test item at and above 312.5 μg/mL in the 4 h exposure group in the absence of S9 and at and above 625 μg/mL in the 4 h in the presence of S9 and in the 24 h continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 312.5 μg/mL in the 4 h exposures, both in the presence and absence of metabolic activation (S9). The maximum dose with binucleate cells present in the 24 h continuous exposure was 156.25 μg/mL.
- The test item induced evidence of toxicity in all three of the exposure groups. The selection of the maximum dose level for the Main Experiment was based on toxicity with consideration also being given to the lowest precipitating dose level and was 320 μg/mL for the 4 h exposure groups and was 160 μg/mL for the 24 h exposure group.
MAIN EXPERIMENT
- The qualitative assessment of the slides determined that the toxicity in the 4 h exposure in the absence of S9 was marginally greater than that observed in the Preliminary Toxicity Test and there were binucleate cells suitable for scoring up to 240 μg/mL. The toxicity in the presence of S9 and in the 24 h exposure was similar to that seen in the Preliminary Toxicity Test and there were binucleate cells up to 240 μg/mL and 160 μg/mL, respectively.
- A precipitate of test item was noted at the end of exposure in the blood free cultures at 320 μg/mL in the 4 h exposure in the absence of S9, at and above 240 μg/mL in the presence of S9 and at 160 μg/mL in the 24 h exposure.
- Haemolysis was observed at the end of exposure at 320 μg/mL in the 4 h exposure group in the absence of S9.
- The CBPI data for the short exposure groups and for the 24 h exposure group confirmed the qualitative observations in that a dose-related inhibition of CBPI was observed. In the 4 h exposure group in the absence of S9, 25%, 43% and 62% cytostasis was achieved at 120, 160 and 240 μg/mL, respectively. In the presence of S9, 13% cytostasis was demonstrated at 240 μg/mL, the lowest precipitating dose level. The 24 h exposure group achieved optimum toxicity with 49% cytostasis at 120 μg/mL. The maximum dose level selected for evaluation of micronuclei in the binucleate cells was based on toxicity in the absence of S9 and was 240 μg/mL for the 4 h exposure and 120 μg/mL for the 24 h exposure. In the presence of S9 the maximum dose level scored was the lowest precipitating dose level, 240 μg/mL.
- The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, in the presence of S9 or in the 24 h exposure group. In the 4 h exposure in the absence of S9 there was a statistically significant increase in the frequency of micronuclei in binucleate cells at 240 μg/mL, a dose level which marginally exceeded optimum toxicity with 62% cytostasis. It was considered that the response was a typical hockey-stick response which was due to cytotoxicity. To confirm this slides from the preliminary toxicity test from the vehicle control and the 312.5 μg/mL dose level of the 4 h exposure group in the absence of S9 were scored for micronuclei in the binucleate cells. The 312.5μg/mL dose level of the preliminary toxicity test achieved the upper limit of acceptable toxicity with 59% cytostasis and there was no increase in the frequency of micronuclei in the binucleate cells.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
% binucleate cells with micronuclei: 2.25-18.85 (6.31 ± 2.90) - Mitomycin C (4 h, -S9); 1.75-5.75 (3.73 ± 1.06) - Demecolcine (24 h, -S9); 1.65-7.25 (3.48 ± 1.51) - Cyclophosphamide (4 h, +S9)
- Negative (solvent/vehicle) historical control data:
% binucleate cells with micronuclei:
4 h, -S9: 0.10-1.25 (0.38 ± 0.27); 95% CI - ± 0.09 (0.29-0.47)
24 h, -S9: 0.05-0.90 (0.43 ± 0.20); 95% CI - ± 0.07 (0.36-0.50)
4 h, +S9: 0.10-1.20 (0.41 ± 0.24); 95% CI - ± 0.08 (0.33-0.49) - Conclusions:
- Under the experimental conditions, the test item did not induce any statistically and toxicologically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system using a dose range which included a dose level which achieved acceptable toxicity or was the lowest precipitating dose level. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
- Executive summary:
In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 2% S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley derived rats induced with phenobarbital and 5,6-benzoflavone.
Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix
Main Test
4 h exposure to the test item formulations without S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
4 h exposure to the test item formulations with S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
24 h exposure to the test item without S9-mix: 10, 20, 40, 60, 80, 120 and 160 μg/mL
Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.
All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Based on the toxicity, the dose levels selected for micronuclei analysis were as follows:
4 h exposure group in the absence of S9: 0, 80, 120, 160 and 240 μg/mL
4 h exposure group in the presence of S9: 0, 80, 120, 160 and 240 μg/mL
24 h continuous exposure group: 0, 40, 60, 80 and 120 μg/mL
The test item demonstrated marked toxicity but did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis in the 24 h exposure in the absence of S9 or was the lowest precipitating dose level as in the 4 h exposure in the presence of S9. The 4 h exposure group in the absence of metabolic activation did demonstrate a statistically significant increase in the frequency of binucleate cells with micronuclei but this was at a dose level which marginally exceeded acceptable toxicity. The response was considered to be a typical hockey-stick response beyond the limits of acceptable toxicity, and was not part of a dose related response and was therefore considered to be due to cytotoxicity rather than mutagenicity. To confirm the absence of a response in the 4 h exposure group in the absence of S9, slides from the preliminary toxicity test were scored where optimum toxicity was achieved.
It was concluded that the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.
Referenceopen allclose all
Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
Experiment 1 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
80 |
|
28 |
|
12 |
|
20 |
|
15 |
|
89 |
(84) |
18 |
(25) |
13 |
(16) |
22 |
(20) |
11 |
(14) |
82 |
|
28 |
|
23 |
|
18 |
|
15 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
73 |
|
31 |
|
23 |
|
16 |
|
8 |
|
80 |
(71) |
17 |
(26) |
25 |
(25) |
18 |
(19) |
16 |
(11) |
60 |
|
30 |
|
27 |
|
22 |
|
10 |
|
Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 07 August 2017 |
To: 10 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Acetone) |
80 68 68 |
(72) 6.9# |
24 38 29 |
(30) 7.1 |
18 18 24 |
(20) 3.5 |
17 23 18 |
(19) 3.2 |
19 14 14 |
(16) 2.9 |
||
1.5 µg |
71 74 74 |
(73) 1.7 |
34 27 33 |
(31) 3.8 |
21 20 25 |
(22) 2.6 |
19 20 18 |
(19) 1.0 |
16 14 9 |
(13) 3.6 |
||
5 µg |
71 74 99 |
(81) 15.4 |
29 23 22 |
(25) 3.8 |
18 18 21 |
(19) 1.7 |
15 20 25 |
(20) 5.0 |
11 12 17 |
(13) 3.2 |
||
15 µg |
102 64 74 |
(80) 19.7 |
23 31 25 |
(26) 4.2 |
26 9 17 |
(17) 8.5 |
17 13 25 |
(18) 6.1 |
17 23 20 |
(20) 3.0 |
||
50 µg |
81 74 61 |
(72) 10.1 |
24 27 26 |
(26) 1.5 |
16 19 9 |
(15) 5.1 |
33 17 10 |
(20) 11.8 |
23 19 13 |
(18) 5.0 |
||
150 µg |
82 79 87 |
(83) 4.0 |
32 22 27 |
(27) 5.0 |
16 14 10 |
(13) 3.1 |
14 22 21 |
(19) 4.4 |
13 20 15 |
(16) 3.6 |
||
500 µg |
101 74 73 |
(83) 15.9 |
30 33 36 |
(33) 3.0 |
12 16 11 |
(13) 2.6 |
23 18 25 |
(22) 3.6 |
18 21 11 |
(17) 5.1 |
||
1500 µg |
79 P 83 P 76 P |
(79) 3.5 |
25 P 28 P 24 P |
(26) 2.1 |
20 P 16 P 22 P |
(19) 3.1 |
22 P 38 P 20 P |
(27) 9.9 |
7 P 11 P 11 P |
(10) 2.3 |
||
5000 µg |
68 P 66 P 67 P |
(67) 1.0 |
31 P 34 P 33 P |
(33) 1.5 |
11 P 12 P 14 P |
(12) 1.5 |
32 P 19 P 25 P |
(25) 6.5 |
7 P 8 P 10 P |
(8) 1.5 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
346 357 357 |
(353) 6.4 |
387 507 661 |
(518) 137.4 |
554 586 539 |
(560) 24.0 |
171 169 201 |
(180) 17.9 |
113 110 113 |
(112) 1.7 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
#: Standard deviation
Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 07 August 2017 |
To: 10 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Acetone) |
65 82 81 |
(76) 9.5# |
19 22 19 |
(20) 1.7 |
24 19 17 |
(20) 3.6 |
22 16 20 |
(19) 3.1 |
14 13 12 |
(13) 1.0 |
||
1.5 µg |
95 69 86 |
(83) 13.2 |
11 17 29 |
(19) 9.2 |
25 24 16 |
(22) 4.9 |
17 24 22 |
(21) 3.6 |
16 7 16 |
(13) 5.2 |
||
5 µg |
71 70 81 |
(74) 6.1 |
21 16 26 |
(21) 5.0 |
23 22 17 |
(21) 3.2 |
37 24 15 |
(25) 11.1 |
10 13 15 |
(13) 2.5 |
||
15 µg |
75 72 74 |
(74) 1.5 |
10 33 19 |
(21) 11.6 |
15 29 26 |
(23) 7.4 |
22 23 21 |
(22) 1.0 |
21 19 9 |
(16) 6.4 |
||
50 µg |
75 60 78 |
(71) 9.6 |
21 22 24 |
(22) 1.5 |
28 22 24 |
(25) 3.1 |
35 23 23 |
(27) 6.9 |
15 14 13 |
(14) 1.0 |
||
150 µg |
69 83 76 |
(76) 7.0 |
31 29 23 |
(28) 4.2 |
12 26 22 |
(20) 7.2 |
26 25 26 |
(26) 0.6 |
14 23 10 |
(16) 6.7 |
||
500 µg |
74 94 86 |
(85) 10.1 |
21 14 23 |
(19) 4.7 |
19 22 15 |
(19) 3.5 |
21 20 27 |
(23) 3.8 |
10 17 15 |
(14) 3.6 |
||
1500 µg |
80 P 78 P 72 P |
(77) 4.2 |
32 P 29 P 29 P |
(30) 1.7 |
9 P 17 P 16 P |
(14) 4.4 |
19 P 17 P 28 P |
(21) 5.9 |
16 P 12 P 17 P |
(15) 2.6 |
||
5000 µg |
77 P 71 P 75 P |
(74) 3.1 |
21 P 22 P 26 P |
(23) 2.6 |
19 P 17 P 24 P |
(20) 3.6 |
22 P 26 P 16 P |
(21) 5.0 |
15 P 17 P 13 P |
(15) 2.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
849 1124 1178 |
(1050) 176.4 |
223 235 242 |
(233) 9.6 |
120 123 132 |
(125) 6.2 |
188 184 196 |
(189) 6.1 |
388 349 395 |
(377) 24.8 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Test item precipitate
#: Standard deviation
Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 21 August 2017 |
To: 24 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Acetone) |
61 79 71 |
(70) 9.0# |
25 22 30 |
(26) 4.0 |
30 19 22 |
(24) 5.7 |
23 17 15 |
(18) 4.2 |
17 9 11 |
(12) 4.2 |
||
15 µg |
70 75 77 |
(74) 3.6 |
20 25 27 |
(24) 3.6 |
18 18 27 |
(21) 5.2 |
22 18 23 |
(21) 2.6 |
16 9 8 |
(11) 4.4 |
||
50 µg |
66 66 64 |
(65) 1.2 |
22 10 24 |
(19) 7.6 |
19 23 20 |
(21) 2.1 |
19 19 22 |
(20) 1.7 |
14 18 7 |
(13) 5.6 |
||
150 µg |
71 69 69 |
(70) 1.2 |
16 13 22 |
(17) 4.6 |
22 33 21 |
(25) 6.7 |
22 16 24 |
(21) 4.2 |
12 11 9 |
(11) 1.5 |
||
500 µg |
60 79 68 |
(69) 9.5 |
27 23 17 |
(22) 5.0 |
26 21 21 |
(23) 2.9 |
12 21 15 |
(16) 4.6 |
15 11 13 |
(13) 2.0 |
||
1500 µg |
71 PS 74 PS 71 PS |
(72) 1.7 |
22 PS 26 PS 11 PS |
(20) 7.8 |
35 P 19 P 20 P |
(25) 9.0 |
21 PS 31 PS 16 PS |
(23) 7.6 |
18 PS 7 PS 12 PS |
(12) 5.5 |
||
5000 µg |
71 PS 90 PS 81 PS |
(81) 9.5 |
0 PV 0 PV 0 PV |
(0) 0.0 |
13 P 11 P 15 P |
(13) 2.0 |
19 PS 15 PS 17 PS |
(17) 2.0 |
8 PS 8 PS 7 PS |
(8) 0.6 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
1138 945 1001 |
(1028) 99.3 |
877 980 960 |
(939) 54.6 |
885 856 915 |
(885) 29.5 |
234 210 244 |
(229) 17.5 |
309 405 634 |
(449) 167.0 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
V: Very weak bacterial background lawn
S: Sparse bacterial background lawn
#: Standard deviation
Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 21 August 2017 |
To: 24 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Acetone) |
67 61 66 |
(65) 3.2# |
23 26 26 |
(25) 1.7 |
30 31 36 |
(32) 3.2 |
26 22 24 |
(24) 2.0 |
9 17 8 |
(11) 4.9 |
||
15 µg |
64 67 67 |
(66) 1.7 |
24 31 16 |
(24) 7.5 |
36 31 44 |
(37) 6.6 |
20 21 23 |
(21) 1.5 |
15 22 17 |
(18) 3.6 |
||
50 µg |
76 76 65 |
(72) 6.4 |
19 25 23 |
(22) 3.1 |
26 34 35 |
(32) 4.9 |
29 15 36 |
(27) 10.7 |
9 8 20 |
(12) 6.7 |
||
150 µg |
63 60 62 |
(62) 1.5 |
23 18 29 |
(23) 5.5 |
38 25 35 |
(33) 6.8 |
25 18 16 |
(20) 4.7 |
9 14 7 |
(10) 3.6 |
||
500 µg |
68 66 66 |
(67) 1.2 |
21 21 18 |
(20) 1.7 |
26 26 36 |
(29) 5.8 |
22 21 28 |
(24) 3.8 |
18 13 16 |
(16) 2.5 |
||
1500 µg |
61 P 67 P 61 P |
(63) 3.5 |
25 P 19 P 26 P |
(23) 3.8 |
25 P 23 P 28 P |
(25) 2.5 |
27 P 29 P 29 P |
(28) 1.2 |
15 P 16 P 21 P |
(17) 3.2 |
||
5000 µg |
62 PS 70 PS 76 PS |
(69) 7.0 |
19 PS 18 PS 16 PS |
(18) 1.5 |
33 P 39 P 14 P |
(29) 13.1 |
15 PS 20 PS 18 PS |
(18) 2.5 |
16 PS 24 PS 4 PS |
(15) 10.1 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1102 1076 1067 |
(1082) 18.2 |
272 252 242 |
(255) 15.3 |
246 220 247 |
(238) 15.3 |
145 134 127 |
(135) 9.1 |
322 196 386 |
(301) 96.7 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Test item precipitate
S: Sparse bacterial background lawn
#: Standard deviation
Table 7.6.1/6:History Profile of Vehicle and Positive Control Values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values† |
274 |
278 |
504 |
285 |
26 |
13 |
461 |
229 |
526 |
299 |
506 |
282 |
42 |
51 |
39 |
49 |
|||||||||||||||||
Min |
60 |
61 |
7 |
7 |
222 |
278 |
10 |
12 |
11 |
10 |
4 |
6 |
87 |
98 |
89 |
93 |
|||||||||||||||||
Max |
166 |
175 |
31 |
29 |
376 |
388 |
58 |
43 |
45 |
46 |
27 |
27 |
237 |
254 |
174 |
177 |
|||||||||||||||||
Mean |
91 |
95 |
16 |
14 |
286 |
333 |
24 |
27 |
21 |
24 |
12 |
13 |
156 |
164 |
123 |
137 |
|||||||||||||||||
SD |
19.3 |
19.1 |
4.5 |
4.0 |
48.7 |
37.6 |
5.6 |
5.9 |
6.2 |
6.1 |
3.8 |
3.4 |
42.2 |
35.6 |
23.1 |
21.2 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2015 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
276 |
280 |
252 |
264 |
13 |
13 |
231 |
227 |
262 |
276 |
253 |
261 |
20 |
35 |
20 |
35 |
|
||||||||||||||||
Min |
222 |
250 |
79 |
118 |
953 |
673 |
116 |
103 |
100 |
78 |
164 |
97 |
430 |
494 |
745 |
325 |
|
||||||||||||||||
Max |
2266 |
2402 |
2779 |
457 |
3140 |
1655 |
2769 |
550 |
502 |
705 |
2318 |
823 |
1696 |
2264 |
3662 |
1174 |
|
||||||||||||||||
Mean |
614 |
927 |
472 |
246 |
2303 |
1093 |
792 |
266 |
222 |
218 |
911 |
336 |
761 |
1461 |
2257 |
569 |
|
||||||||||||||||
SD |
260.6 |
452.5 |
434.8 |
55.7 |
815.2 |
376.5 |
342.1 |
97.7 |
70.2 |
107.6 |
412.4 |
135.7 |
350.0 |
382.0 |
790.7 |
220.3 |
|
||||||||||||||||
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values |
399 |
401 |
758 |
393 |
60 |
30 |
690 |
345 |
788 |
415 |
762 |
398 |
32 |
32 |
16 |
24 |
|||||||||||||||||
Min |
63 |
66 |
8 |
8 |
216 |
221 |
10 |
13 |
8 |
12 |
3 |
4 |
97 |
104 |
78 |
52 |
|||||||||||||||||
Max |
154 |
156 |
34 |
39 |
340 |
375 |
53 |
53 |
49 |
51 |
24 |
23 |
268 |
243 |
148 |
166 |
|||||||||||||||||
Mean |
90 |
93 |
15 |
15 |
268 |
310 |
22 |
27 |
21 |
25 |
12 |
13 |
161 |
159 |
118 |
110 |
|||||||||||||||||
SD |
14.5 |
14.3 |
4.5 |
5.2 |
26.4 |
31.1 |
5.8 |
6.3 |
4.8 |
5.7 |
3.5 |
3.5 |
39.2 |
32.3 |
17.0 |
29.3 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2016 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
409 |
406 |
381 |
386 |
30 |
28 |
341 |
335 |
388 |
385 |
379 |
381 |
14 |
24 |
8 |
16 |
|
||||||||||||||||
Min |
221 |
284 |
84 |
92 |
897 |
629 |
107 |
102 |
100 |
96 |
95 |
101 |
445 |
574 |
1674 |
372 |
|
||||||||||||||||
Max |
2222 |
2863 |
2994 |
879 |
2326 |
2140 |
1611 |
637 |
449 |
4357 |
1413 |
639 |
1117 |
1855 |
2823 |
945 |
|
||||||||||||||||
Mean |
724 |
1264 |
854 |
240 |
1633 |
950 |
718 |
240 |
186 |
188 |
406 |
290 |
743 |
1271 |
2379 |
535 |
|
||||||||||||||||
SD |
320.4 |
562.9 |
664.9 |
62.1 |
564.5 |
382.7 |
338.6 |
98.2 |
49.8 |
230.8 |
227.0 |
92.7 |
214.6 |
326.5 |
426.2 |
143.3 |
|
||||||||||||||||
None
Table 7.6.1/1: CBPI and Micronucleus Data
Exposure Time +/- S9 |
Dose Level (μg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
Mean CBPI |
% Cytostasis |
Micronuclei (MN) per 1000 Binucleate cells |
% Binucleate cells with MN |
Mean % Binucleate cells with MN |
||||
Mono |
Bi |
Multi |
1 MN |
2 MN |
>2 MN |
||||||||
4 h Exposure Without Metabolic Activation (-S9) |
|||||||||||||
4 h (-S9) |
0 |
A |
201 |
295 |
4 |
1.61 |
1.53 |
0 |
12 |
0 |
0 |
1.20 |
0.70 |
B |
282 |
209 |
9 |
1.45 |
1 |
1 |
0 |
0.20 |
|||||
80 |
A |
278 |
221 |
1 |
1.45 |
1.50 |
7 |
7 |
4 |
1 |
1.20 |
0.90 |
|
B |
232 |
267 |
1 |
1.54 |
6 |
0 |
0 |
0.60 |
|||||
120 |
A |
306 |
194 |
0 |
1.39 |
1.40 |
25 |
2 |
0 |
1 |
0.30 |
0.25 |
|
B |
308 |
186 |
6 |
1.40 |
2 |
0 |
0 |
0.20 |
|||||
160 |
A |
380 |
120 |
0 |
1.24 |
1.30 |
43 |
7 |
1 |
0 |
0.80 |
0.50 |
|
B |
319 |
180 |
1 |
1.36 |
2 |
0 |
0 |
0.20 |
|||||
240 |
A |
413 |
87 |
0 |
1.17 |
1.20 |
62 |
27 |
2 |
1 |
3.00 |
4.25*** |
|
B |
384 |
116 |
0 |
1.23 |
54 |
1 |
0 |
5.50 |
|||||
Mitomycin C 0.2 |
A |
329 |
169 |
2 |
1.35 |
1.32 |
40 |
20 |
2 |
0 |
2.20 |
3.85*** |
|
B |
354 |
146 |
0 |
1.29 |
53 |
2 |
0 |
5.50 |
|||||
4 h Exposure With Metabolic Activation (+S9) |
|||||||||||||
4 h (+S9) |
0 |
A |
254 |
240 |
6 |
1.50 |
1.46 |
0 |
1 |
0 |
0 |
0.10 |
0.15 |
B |
301 |
195 |
4 |
1.41 |
2 |
0 |
0 |
0.20 |
|||||
80 |
A |
315 |
185 |
0 |
1.37 |
1.38 |
18 |
10 |
0 |
0 |
1.00 |
0.70 |
|
B |
313 |
183 |
4 |
1.38 |
4 |
0 |
0 |
0.40 |
|||||
120 |
A |
304 |
195 |
1 |
1.39 |
1.42 |
8 |
9 |
1 |
0 |
1.00 |
0.70 |
|
B |
285 |
204 |
11 |
1.45 |
3 |
1 |
0 |
0.40 |
|||||
160 |
A |
313 |
187 |
0 |
1.37 |
1.36 |
22 |
8 |
0 |
2 |
1.00 |
0.70 |
|
B |
332 |
168 |
0 |
1.34 |
3 |
1 |
0 |
0.40 |
|||||
240 P |
A |
322 |
174 |
4 |
1.36 |
1.40 |
13 |
5 |
0 |
0 |
0.50 |
0.50 |
|
B |
287 |
212 |
1 |
1.43 |
5 |
0 |
0 |
0.50 |
|||||
Cyclophosphamide 5 |
A |
390 |
110 |
0 |
1.22 |
1.18 |
62 |
25 |
4 |
0 |
2.90 |
3.40*** |
|
B |
436 |
64 |
0 |
1.13 |
33 |
4 |
2 |
3.90 |
|||||
24 h Exposure Without Metabolic Activation (-S9) |
|||||||||||||
24 h (-S9) |
0 |
A |
224 |
262 |
14 |
1.58 |
1.61 |
0 |
3 |
0 |
1 |
0.40 |
0.35 |
B |
199 |
283 |
18 |
1.64 |
2 |
1 |
0 |
0.30 |
|||||
40 |
A |
227 |
263 |
10 |
1.57 |
1.57 |
7 |
5 |
0 |
0 |
0.50 |
0.45 |
|
B |
221 |
273 |
6 |
1.57 |
4 |
0 |
0 |
0.40 |
|||||
60 |
A |
209 |
280 |
11 |
1.60 |
1.61 |
0 |
6 |
0 |
0 |
0.60 |
0.40 |
|
B |
200 |
290 |
10 |
1.62 |
2 |
0 |
0 |
0.20 |
|||||
80 |
A |
274 |
222 |
4 |
1.46 |
1.50 |
19 |
4 |
1 |
0 |
0.50 |
0.80 |
|
B |
238 |
260 |
2 |
1.53 |
9 |
2 |
0 |
1.10 |
|||||
120 |
A |
352 |
148 |
0 |
1.30 |
1.31 |
49 |
1 |
0 |
0 |
0.10 |
0.20 |
|
B |
343 |
156 |
1 |
1.32 |
3 |
0 |
0 |
0.30 |
|||||
Demecolcine 0.075 |
A |
275 |
216 |
9 |
1.47 |
1.48 |
22 |
14 |
3 |
1 |
1.80 |
1.80*** |
|
B |
272 |
216 |
12 |
1.48 |
14 |
2 |
2 |
1.80 |
|||||
Additional Score Data from the Preliminary Toxicity Test 4 h (-S9) |
|||||||||||||
4 h (-S9) |
0 |
- |
256 |
217 |
27 |
1.54 |
- |
0 |
7 |
0 |
0 |
- |
0.7 |
312.5 |
- |
396 |
99 |
5 |
1.22 |
- |
59 |
7 |
0 |
1 |
- |
0.8 |
|
*** = P<0.001
P = Precipitate observed at the end of exposure
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). In Experiment 2 (pre incubation method), the test item caused a visible reduction in all of the Salmonella bacterial lawns in the absence of S9-mix from 1500 µg/plate. In the presence of S9-mix, toxicity was noted to all of the Salmonella bacterial strains at 5000 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.
A test item precipitate (greasy and particulate in appearance) was noted in both experiments in both the presence and absence of S9 from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
Micronucleus test in human lymphocytes:
In anin vitromicronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 2% S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley derived rats induced with phenobarbital and 5,6-benzoflavone.
Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix
Main Test
4 h exposure to the test item formulations without S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
4 h exposure to the test item formulations with S9-mix: 20, 40, 60, 80, 120, 160, 240 and 320 μg/mL
24 h exposure to the test item without S9-mix: 10, 20, 40, 60, 80, 120 and 160 μg/mL
Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slidesprepared, so that binucleate cells could be examined for micronucleus induction.
All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Based on the toxicity, the dose levels selected for micronuclei analysis were as follows:
4 h exposure group in the absence of S9: 0, 80, 120, 160 and 240 μg/mL
4 h exposure group in the presence of S9: 0, 80, 120, 160 and 240 μg/mL
24 h continuous exposure group: 0, 40, 60, 80 and 120 μg/mL
The test item demonstrated marked toxicity but did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis in the 24 h exposure in the absence of S9 or was the lowest precipitating dose level as in the 4 h exposure in the presence of S9. The 4 h exposure group in the absence of metabolic activation did demonstrate a statistically significant increase in the frequency of binucleate cells with micronuclei but this was at a dose level which marginally exceeded acceptable toxicity. The response was considered to be a typical hockey-stick response beyond the limits of acceptable toxicity, and was not part of a dose related response and was therefore considered to be due to cytotoxicity rather than mutagenicity. To confirm the absence of a response inthe 4 h exposure group in the absence of S9, slides from the preliminary toxicity test were scored where optimum toxicity was achieved.
It was concluded that the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.
HPRT test in V79 cells:
In anin vitromammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item for 4 h, with andwithout metabolic activation (2% S9); S9 fraction prepared from male rats, dosed with phenobarbital and β-Naphthaflavone.
Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation
Mutation tests:
4 h exposure with metabolic activation: 5, 10, 20, 40, 80, 120, 140 and 160 μg/mL
4 h exposure with metabolic activation: 5, 10, 20, 40, 80 and 160 μg/mL
In a preliminary toxicity test, at the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL. There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. However, the greatest reductions were observed at dose levels at or beyond the onset of test item precipitate. The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of test item precipitate and test item-induced toxicity in the absence of metabolic activation, and the onset of test item precipitate in the presence of metabolic activation, as recommended by the OECD 476 guidelines.
In main test, at the end of the exposure period, the onset of precipitate of the test item was observed at 80 μg/ml in the absence of metabolic activation, and at 40 μg/mL in the presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the subsequent dose levels were excluded from the analysis as they were considered to be surplus to requirements. There were very modest concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in any of the concentration levels, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.
The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
Justification for classification or non-classification
Harmonized classification:
The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.
Self-classification:
Based on the available information, no classification is proposed.
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.
