Registration Dossier
Registration Dossier
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EC number: 482-160-5 | CAS number: 130786-09-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2006
negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2006
negative, in vitro mammalian gene mutation test (with and without S-9 activation), OECD TG 490, 2018
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07-12-2005 to 03-02-2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: August 2005 ; signature: November 2005
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine or tryptophan locus
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9
- Test concentrations with justification for top dose:
- Preliminary toxicity test: All strains: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method):
TA100 & TA1535: 5, 15, 50, 150, 500, 1500 µg/plate
TA98 & TA1537: 15, 50, 150, 500, 1500, 5000 µg/plate
WP2uvrA- : 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation methodd): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle. - 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
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)
DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC 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).
Experiment 2. All of the plates were incubated at 37 ± 3 ºC 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).
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal. - Statistics:
- Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using both the Ames plate incorporation and pre incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 5 to 1500 µg/plate for Salmonella strains TA100 and TA1535, 15 to 5000 µg/plate for Salmonella strains TA98 and TA1537 and 50 to 5000 µg/plate for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (dimethyl sulphoxide) 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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on cytotoxicity. The test item caused a visible reduction in the growth of the bacterial background lawn, both in the presence and absence of S9, to Salmonella strains TA100 and TA1535 from 500 µg/plate and at 5000 µg/plate to TA1537. The test item caused no visible reduction in the growth of the bacterial background lawn to either Salmonella strain TA98 or Escherichia coli strain WP2uvrA-. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type. The test item was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02-03-2006 to 14-08-2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: August 2005 ; signature: November 2005
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable (chromosome aberration test)
- Species / strain / cell type:
- lymphocytes: Human lymphocytes
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a screened volunteer. 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 17 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fraction: PB/βNF S9/29/01l06 and S9/01l04106
- Test concentrations with justification for top dose:
- The maximum dose level was 1712 µg/mL or 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1712 μg/mL range (full results recorded in the full study report).
The purity of the test item was accounted for in the test item formulations (see 'confidential details on test material'), where required.
I. Preliminary toxicity test: 0 (control) , 6.69, 13.38, 26.75, 53.50, 107, 214, 428, 856 and 1712 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2% or 1% for PT and Exp 1 or Exp 2, respectively), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
II. Main Test:
Experiment 1:
4(20)-hour without S9: 0*, 26.75*, 53.5*, 107*, 160.5, 214, 321, MMC 0.4* μg/mL
4(20)-hour with S9: 0*, 26.75*,53.5*, 107*, 160.5, 214, 321, CP 7.5* μg/mL
Experiment 2:
4(20)-hour with S9: 0*, 13.38, 26.75*, 53.5*, 107*, 160.5, 214, CP 4* μg/mL
24-hour without S9: 0*, 13.38*, 26.75*, 53.5*, 80.25*, 107, 214, MMC 0.2* μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was soluble in acetone at 1712 µg/mL, in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 1712 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1712 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system. - Untreated negative controls:
- other: Vehicle control served as the negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- Full details on the positive controls is reported in the full study report.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Other:
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, 15% (FCS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood
DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% or 1% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment 1 or Main Experiment 2. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), UK EMS (1983). Where the analysis of the slide resulted in a large frequency of aberrant cells then the analysis was terminated after a total of 15 metaphases with aberrations (excluding gaps) were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. - Evaluation criteria:
- The test was evaluated in accordance with the OECD TG 473 guidelines.
Statistical analysis is also performed. The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
- Species / strain:
- lymphocytes: Human lymphocytes
- 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
- Effects of osmolality: here was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: a precipitate was observed in the blood-free cultures at the end of exposure, at or above 214 µg/mL in all the exposure groups.
Main test: a precipitate was observed in the blood-free cultures at the end of exposure, at or above 214 µg/mL in all the exposure groups.
- Other confounding effects: In the preliminary test: Haemolysis was observed at and above 26.75 and/or 107 µg/mL in the absence of S9 in the 4(20)-hour and 24-hour exposure groups, respectively.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1712 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.
COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).
ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%): 0, 26.75, 53.5, 107, 160.5, 214, 321 μg/mL. Secondly, the doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (1%) and 24-hour without S9: 13.38, 26.75, 53.5, 80.25, 107, 214 μg/mL. All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system. The test item was adequately tested to cytotoxic dose levels. Toxicity was demonstrated by the reduction in the mitotic index in all four exposure conditions and with no metaphases for scoring at the next dose level below which scoring was conducted. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05-09-2018 to 29-10-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Japanese MITI/MHW guidelines: `Kanpoan No. 287 - Environment Protection Agency`; `Eisei No. 127 - Ministry of Health and Welfare` and `Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry`
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell transformation assay
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 856 μg/mL exposure medium. The test item precipitated in the exposure medium at concentrations of 428 μg/mL and above after 3 and 24 hours of incubation. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 856 μg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in DMSO. Serial dilutions prepared from stock.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Liquid test item dissolved in stock.
- Final preparation of a solid: Not applicable.
FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the test item.
OTHER SPECIFICS: Not applicable - Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Recognised supplier (documented in full study report).
- Suitability of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line has been used successfully in in vitro experiments for many years and is guideline specified (OECD TG 490). The test system has been extensively validated.
- Cell cycle length, doubling time or proliferation index: Doubling time ca. 12 hours. Determined under normal growth conditions.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The karyotype for the cell line has been published and the modal chromosome number is 40 (ref: OECD TG 490).
- Normal (negative control) cell cycle time: Doubling time ca. 12 hours.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stocks of cells are stored in the freezer at approximately -150 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (50 U/mL), Sodium pyruvate (1 mM) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes. Before the stocks of cells were cleansed of homozygous (TK -/-) mutants by culturing in R10 medium for 24 hours. This medium contained Thymidine (1.6 x10^-5 M), Hypoxanthine (1.0 x10^-4 M) and Aminopterine (2.0 x10^-7 M). For the following 48 hours the cells were cultured in R10 medium containing Hypoxanthine (1.0 x10^-4 M) and Thymidine (1.6 x10^-5 M)only before being returned to R10 medium for a further 24 hours. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix. mixing S9, 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. Filtered (0.22 μm)-sterilized. Final S9 in exposure medium was 4% (v/v).
- Test concentrations with justification for top dose:
- The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 856 μg/mL exposure medium. The test item precipitated in the exposure medium at concentrations of 428 μg/mL and above after 3 and 24 hours of incubation. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 856 μg/mL.
I. Preliminary toxicity test: 0 (control), 27, 54, 107, 214, 428*, 856* μg/mL
Where: * = test item precipitated in the exposure medium
Within three exposure groups:
i) 3-hours exposure to the test item without S9-mix
ii) 3-hours exposure to the test item with S9-mix (4%)
iii) 24-hour exposure to the test item without S9-mix
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 3-hour exposure groups in both the absence and presence of metabolic activation, and in the 24 hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at 428 µg/mL in the 4-hour and 24-hour exposure groups.
II. Main Test:
3-hour without S9: 0 (control), 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/ml and MMS 15 μg/ml
3-hour with S9: 0 (control), 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/ml and CP 7.5 μg/ml
24-hour without S9: 0 (control), 1.3, 2.5, 5, 10, 20, 30, 40, 50, 60, 70 μg/mL and MMS 5 μg/ml
where:
MMS = Methylmethanesulfonate
CP = Cyclophosphamide
The cultures were then daily sub-cultured subject to acceptable limits of mean cell count. Giving total of 48 hours for expression period. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 856 μg/mL exposure medium. The test item precipitated in the exposure medium at concentrations of 428 μg/mL and above after 3 and 24 hours of incubation. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 856 μg/mL. The test item was formulated within one hour of it being applied to the test system.
Applicant assessment indicates: The test item had been demonstrated to be insoluble in aqueous solution in previous OECD genetic toxicity tests. DMSO is a guideline accepted vehicle with an available laboratory historic control data set. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Remarks:
- Full details of the positive control substances, is available in the full study report
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation with test item.
- Cell density at seeding (if applicable): Preliminary Test: 3-hour exposure: 1 x10^6 cells/mL ; 24-hour exposure: 1.25 x10^5 cells/mL ; Definitive Test: 3-hour exposure: 1 x10^6 cells/mL; 24-hour exposure: 1.25 x10^5 cells/mL
DURATION
- Preincubation period: None.
- Exposure duration: Treatment was for 3 hours or 24 hours, respectively at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope. In the first experiment in the absence of S9-mix, the plates for the TFT-selection were stained with MTT for 1 hour and 44 minutes. According to SOP (mutation test with mouse lymphoma L5178Y cells in micro wells) the plates should be stained for 1.5 – 2 hours. Since the staining with MTT is only performed to enhance the contrast for colony counting, and the solvent and positive controls showed acceptable responses, this deviation has no effect on the study results.
SELECTION AGENT (mutation assays): 5-Trifluorothymidine at 5 μg/mL
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.
NUMBER OF REPLICATIONS: The study conducted in single cultures but with two replicates (A and B) at in solvent and positive control groups.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope. In the first experiment in the absence of S9-mix, the plates for the TFT-selection were stained with MTT for 1 hour and 44 minutes. According to SOP (mutation test with mouse lymphoma L5178Y cells in micro wells) the plates should be stained for 1.5 – 2 hours. Since the staining with MTT is only performed to enhance the contrast for colony counting, and the solvent and positive controls showed acceptable responses, this deviation has no effect on the study results.
NUMBER OF CELLS EVALUATED: 2000 cells/well were seeded with selection agent. Cells were also diluted and plated for viability (%V) analysis. Scoring of plates daily for %RSG and %V to obtain RTG. Mutation scoring of plates was ultimately performed for the presence of mutant colonies; large and small colonies analyses was additionally conducted.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not applicable.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and cloning efficiency via viability (%)
- Any supplementary information relevant to cytotoxicity: percentage relative suspension growth (%RSG) (post exposure toxicity during the expression period); viability (%) from non-selective medium cultures.
OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable. - Rationale for test conditions:
- See tables. The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (single cultures and/or duplicate controls). In the absence of precipitation: optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
It was noted: In the range-finder: (i) in 3-hour groups: RSG was 28 and 30% at the test item concentration of 54 μg/mL compared to the RSG of the solvent control in the absence and presence of S9-mix, respectively. No cell survival was observed at test item concentrations of 107 μg/mL and above. (ii) In the 24-hour group: RSG was 17% at the test item concentration of 54 μg/mL compared to the RSG of the solvent control. No cell survival was observed at the test item concentration of 107 μg/mL and above. In the definitive test: (i) in 3-hour groups: In the absence of S9-mix, the RTG of the highest test item concentration was 6% compared to the total growth of the solvent controls, whereas RTG of one concentration below (50 µg/mL) was 31% compared to the total growth of the solvent controls. In the presence of S9-mix, the RTG of the highest test item concentration was 11% compared to the total growth of the solvent controls. (ii) In the 24-hour group: The RTG of the highest test item was 9% compared to the total growth of the solvent controls; whereas RTG of one concentration below (50 µg/mL) was 16% compared to the total growth of the solvent controls
Applicant assessment indicates: Optimum toxicity or above was achieved in the highest dose level tested in the exposure groups in the presence or absence of metabolic activation. - Evaluation criteria:
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. - Species / strain:
- mouse lymphoma L5178Y cells
- 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: Not applicable.
- Effects of osmolality: Not applicable.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable.
- Precipitation: The test item was fully soluble in DMSO. The test item formed a clear colourless solution in vehicle. The final concentration of the solvent in the exposure medium was 1%(v/v). Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 856 μg/mL exposure medium. The test item precipitated in the exposure medium at concentrations of 428 μg/mL and above after 3 and 24 hours of incubation. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 856 μg/mL.
- Definition of acceptable cells for analysis: The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (single cultures and/or duplicate controls). In the absence of precipitation: optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
- Other confounding effects: None reported.
RANGE-FINDING/SCREENING STUDIES:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Not applicable.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not applicable.
- Indication whether binucleate or mononucleate where appropriate: Not applicable.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Information provided in the full study report.
- Negative (solvent/vehicle) historical control data: Information provided in the full study report.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RSG% and RTG.
- Other observations when applicable: Not applicable. - Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 490 under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 27, 54, 107, 214, 428, 856 μg/mL concentration in DMSO vehicle (selection was based on precipitation in exposure medium at above 428 μg/mL and cytotoxicity), a definitive test was performed whereby L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 3-hour exposure with and without metabolic activation (4% S9), and a 24-hour exposure without metabolic activation. The following concentrations were used: 3-hour without S9: 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/mL, 3-hour with S9: 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/mL and 24-hour without S9: 1.3, 2.5, 5, 10, 20, 30, 40, 50, 60, 70 μg/mL. In the first experiment 3-hour exposure, the test item was tested up to concentrations of 60 and 70 µg/mL in the absence and presence S9-mix, respectively. Relative total growth (RTG) was reduced to 6 and 11% in the absence and presence of S9-mix, respectively. In the second experiment 24-hour exposure, the test item was tested up to concentrations of 60 µg/mL in the absence of S9-mix. The RTG was reduced to 9%. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Referenceopen allclose all
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
99 97 100 |
(99) 1.5# |
18 21 21 |
(20) 1.7 |
29 27 20 |
(25) 4.7 |
20 23 23 |
(22) 1.7 |
4 9 7 |
(7) 2.5 |
|
5 µg |
86 98 96 |
(93) 6.4 |
20 16 9 |
(15) 5.6 |
NT |
|
NT |
|
NT |
|
|
15 µg |
97 99 112 |
(103) 8.1 |
15 14 13 |
(14) 1.0 |
NT |
|
25 24 21 |
(23) 2.1 |
7 10 5 |
(7) 2.5 |
|
50 µg |
102 102 109 |
(104) 4.0 |
24 14 15 |
(18) 5.5 |
30 19 21 |
(23) 5.9 |
20 28 22 |
(23) 4.2 |
9 9 10 |
(9) 0.6 |
|
150 µg |
100 99 80 |
(93) 11.3 |
14 26 22 |
(21) 6.1 |
19 20 15 |
(18) 2.6 |
24 18 19 |
(20) 3.2 |
13 7 13 |
(11) 3.5 |
|
500 µg |
75 S 82 S 86 S |
(81) 5.6 |
7 S 11 S 8 S |
(9) 2.1 |
24 19 18 |
(20) 3.2 |
36 21 29 |
(29) 7.5 |
7 8 5 |
(7) 1.5 |
|
1500 µg |
0 S 0 S 0 S |
(0) 0.0 |
0 S 0 S 0 S |
(0) 0.0 |
19 20 19 |
(0) 0.6 |
21 27 17 |
(22) 5.0 |
10 9 6 |
(8) 2.1 |
|
5000 µg |
NT |
NT |
|
20 20 20 |
(20) 0.0 |
9 22 8 |
(13) 7.8 |
3 S 7 S 4 S |
(5) 2.1 |
||
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 |
|||||||
543 454 488 |
(495) 44.9 |
490 302 421 |
(404) 95.1 |
957 969 1005 |
(977) 25.0 |
302 301 279 |
(294) 13.0 |
1094 1271 1561 |
(1309) 235.8 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
115 111 84 |
(103) 16.0# |
13 14 15 |
(14) 1.0 |
32 35 30 |
(32) 2.5 |
31 34 32 |
(32) 1.5 |
19 24 19 |
(21) 2.9 |
|
5 µg |
85 90 114 |
(96) 15.5 |
14 10 10 |
(11) 2.3 |
NT |
|
NT |
|
|
|
|
15 µg |
108 104 108 |
(107) 2.3 |
11 10 13 |
(11) 1.5 |
NT |
|
25 25 22 |
(24) 1.7 |
30 20 15 |
(22) 7.6 |
|
50 µg |
104 104 99 |
(102) 2.9 |
12 10 9 |
(10) 1.5 |
34 33 20 |
(29) 7.8 |
29 28 18 |
(25) 6.1 |
19 27 20 |
(22) 4.4 |
|
150 µg |
93 78 96 |
(89) 9.6 |
12 11 11 |
(11) 0.6 |
22 22 20 |
(21) 1.2 |
23 29 40 |
(31) 8.6 |
20 28 27 |
(25) 4.4 |
|
500 µg |
57 S 57 S 43 S |
(52) 8.1 |
15 S 7 S 8 S |
(10) 4.4 |
21 22 26 |
(23) 2.6 |
29 32 29 |
(30) 1.7 |
19 19 16 |
(18) 1.7 |
|
1500 µg |
0 S 0 S 0 S |
(0) 0.0 |
0 S 0 S 0 S |
(0) 0.0 |
19 24 24 |
(22) 2.9 |
26 37 31 |
(31) 5.5 |
11 11 10 |
(11) 0.6 |
|
5000 µg |
NT
|
|
NT |
|
24 19 27 |
(23) 4.0 |
37 19 18 |
(25) 10.7 |
8 S 17 S 14 S |
(13) 4.6 |
|
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 |
|||||||
955 1002 913 |
(957) 44.5 |
288 296 338 |
(307) 26.9 |
394 410 443 |
(416) 25.0 |
344 340 302 |
(329) 23.2 |
393 464 351 |
(403) 57.1 |
||
Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
120 102 126 |
(116) 12.5# |
16 16 23 |
(18) 4.0 |
26 20 26 |
(24) 3.5 |
15 18 18 |
(17) 1.7 |
8 10 11 |
(10) 1.5 |
|
5 µg |
135 133 132 |
(133) 1.5 |
21 24 19 |
(21) 2.5 |
NT |
|
NT |
|
NT |
|
|
15 µg |
130 113 123 |
(122) 8.5 |
25 19 20 |
(21) 3.2 |
NT |
|
16 18 21 |
(18) 2.5 |
9 18 7 |
(11) 5.9 |
|
50 µg |
133 124 130 |
(129) 4.6 |
19 27 30 |
(25) 5.7 |
29 29 18 |
(25) 6.4 |
10 20 26 |
(19) 8.1 |
11 11 8 |
(10) 1.7 |
|
150 µg |
85 115 134 |
(111) 24.7 |
11 20 15 |
(15) 4.5 |
18 26 14 |
(19) 6.1 |
13 13 20 |
(15) 4.0 |
12 16 11 |
(13) 2.6 |
|
500 µg |
104 S 104 S 121 S |
(110) 9.8 |
19 S 14 S 11 S |
(15) 4.0 |
21 16 16 |
(18) 2.9 |
19 15 5 |
(13) 7.2 |
13 13 11 |
(12) 1.2 |
|
1500 µg |
0 S 0 S 0 S |
(0) 0.0 |
0 S 0 S 0 S |
(0) 0.0 |
12 12 9 |
(11) 1.7 |
18 19 15 |
(17) 2.1 |
8 4 7 |
(6) 2.1 |
|
5000 µg |
NT |
|
NT |
|
16 26 24 |
(22) 5.3 |
9 15 19 |
(14) 5.0 |
0 S 0 S 0 S |
(0) 0.0 |
|
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 |
|||||||
541 566 585 |
(564) 22.1 |
691 431 475 |
(532) 139.2 |
817 789 883 |
(830) 48.3 |
223 218 216 |
(219) 3.6 |
588 999 1518 |
(1035) 466.0 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
81 95 82 |
(86) 7.8# |
12 10 10 |
(11) 1.2 |
35 23 15 |
(24) 10.1 |
20 33 24 |
(26) 6.7 |
23 15 30 |
(23) 7.5 |
|
5 µg |
97 84 95 |
(92) 7.0 |
8 7 9 |
(8) 1.0 |
NT |
|
NT |
|
NT |
|
|
15 µg |
100 91 97 |
(96) 4.6 |
9 9 11 |
(10) 1.2 |
NT |
|
23 19 31 |
(24) 6.1 |
16 23 21 |
(20) 3.6 |
|
50 µg |
81 81 84 |
(82) 1.7 |
7 4 10 |
(7) 3.0 |
44 24 30 |
(33) 10.3 |
22 22 29 |
(24) 4.0 |
23 21 22 |
(22) 1.0 |
|
150 µg |
62 70 62 |
(65) 4.6 |
7 11 15 |
(11) 4.0 |
30 30 25 |
(28) 2.9 |
22 30 24 |
(25) 4.2 |
21 24 22 |
(22) 1.5 |
|
500 µg |
35 S 38 S 43 S |
(39) 4.0 |
9 5 7 |
(7) 2.0 |
22 25 20 |
(22) 2.5 |
29 31 23 |
(28) 4.2 |
12 20 19 |
(17) 4.4 |
|
1500 µg |
0 S 0 S 0 S |
(0) 0.0 |
0 S 0 S 0 S |
(0) 0.0 |
25 21 18 |
(21) 3.5 |
24 33 25 |
(27) 4.9 |
11 15 16 |
(14) 2.6 |
|
5000 µg |
NT |
|
NT |
|
22 30 35 |
(26) 4.0 |
30 21 18 |
(23) 6.2 |
10 S 8 S 7 S |
(8) 1.5 |
|
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 |
|||||||
762 837 873 |
(824) 56.6 |
306 261 245 |
(271) 31.6 |
560 542 637 |
(580) 50.5 |
209 163 180 |
(184) 23.3 |
578 522 536 |
(545) 29.1 |
||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
N/T: Not tested at this dose level
S: partial absence of bacterial background lawn
#: Standard deviation
Table 1: Results of range-finding test
Dose (μg/ml) |
|
Cell count after 24 hours sub-culture (cells/mL x 10^5) |
|
Cell count after 48 hours sub-culture (cells/mL x 10^5) |
SG (1) |
RSG (%) (2) |
3-hours without metabolic activation |
||||||
SC |
|
7.7 |
|
9.4 |
36 |
100 |
27 |
|
5.7 |
|
10.5 |
30 |
83 |
54 |
|
2.0 |
|
10.1 |
10 |
28 |
107 |
|
0.1 |
(4) |
1.1 |
0 |
0 |
214 |
|
0.0 |
(4) |
0.0 |
0 |
0 |
428 |
(3) |
0.0 |
(4) |
0.0 |
0 |
0 |
856 |
(3) |
0.1 |
(4) |
0.0 |
0 |
0 |
|
|
|
|
|
|
|
3-hours with metabolic activation |
||||||
SC |
|
6.1 |
|
9.8 |
30 |
100 |
27 |
|
3.4 |
|
11.0 |
19 |
63 |
54 |
|
2.0 |
|
9.1 |
9 |
30 |
107 |
|
0.2 |
(4) |
0.2 |
0 |
0 |
214 |
|
0.2 |
(4) |
0.1 |
0 |
0 |
428 |
(3) |
0.1 |
(4) |
0.0 |
0 |
0 |
856 |
(3) |
0.1 |
(4) |
0.0 |
0 |
0 |
|
|
|
|
|
|
|
24-hours without metabolic activation |
||||||
SC |
|
12.4 |
|
10.4 |
83 |
100 |
27 |
|
7.8 |
|
8.4 |
42 |
51 |
54 |
|
4.0 |
|
5.6 |
14 |
17 |
107 |
|
0.1 |
(4) |
0.2 |
0 |
0 |
214 |
|
0.1 |
(4) |
0.1 |
0 |
0 |
428 |
(3) |
0.1 |
(4) |
0.1 |
0 |
0 |
856 |
(3) |
0.1 |
(4) |
0.1 |
0 |
0 |
|
|
|
|
|
|
|
SC = solvent control = DMSO
(1) = suspension growth
(2) = relative suspension growth
(3) = the test item precipitated in the exposure medium
(4) = since less than 1.25 x 10^5 c/ml were present, no subculture was performed
Table 2: Definitive Test - summary of results: 3-hour exposure - with and without S9
Dose (μg/ml) |
RSG (%) |
CE day2 (%) |
RCE (%) |
RTG (%) |
Mutation frequency per 10^6 survivors |
||
|
|
|
|
|
total |
small |
Large |
3-hours without metabolic activation |
|||||||
SC1 |
100 |
68 |
100 |
100 |
148 |
57 |
84 |
SC2 |
|
90 |
|
|
164 |
74 |
78 |
2.5 |
106 |
79 |
100 |
106 |
192 |
75 |
103 |
5 |
91 |
71 |
90 |
82 |
194 |
63 |
119 |
10 |
101 |
78 |
99 |
100 |
198 |
95 |
83 |
20 |
90 |
70 |
89 |
80 |
275 |
138 |
110 |
30 |
84 |
77 |
97 |
82 |
219 |
98 |
103 |
40 |
40 |
85 |
107 |
43 |
222 |
119 |
83 |
50 |
26 |
95 |
120 |
31 |
214 |
58 |
138 |
60 |
4 |
121 |
153 |
6 |
154 |
41 |
101 |
MMS |
79 |
53 |
67 |
53 |
1614 |
902 |
409 |
|
|
|
|
|
|
|
|
3-hours with metabolic activation |
|||||||
SC1 |
100 |
89 |
100 |
100 |
87 |
25 |
59 |
SC2 |
|
81 |
|
|
91 |
11 |
78 |
5 |
111 |
75 |
88 |
97 |
107 |
16 |
89 |
10 |
93 |
104 |
122 |
113 |
99 |
11 |
85 |
20 |
97 |
85 |
100 |
97 |
116 |
26 |
86 |
30 |
87 |
81 |
96 |
83 |
98 |
31 |
63 |
40 |
73 |
88 |
103 |
75 |
106 |
32 |
70 |
50 |
61 |
91 |
107 |
65 |
109 |
35 |
69 |
60 |
38 |
91 |
107 |
41 |
89 |
34 |
51 |
70 |
10 |
91 |
107 |
11 |
153 |
51 |
92 |
CP |
66 |
45 |
53 |
35 |
1224 |
653 |
410 |
|
|
|
|
|
|
|
|
Table 3: Definitive Test - summary of results: 24-hour exposure - without S9
Dose (μg/ml) |
RSG (%) |
CE day2 (%) |
RCE (%) |
RTG (%) |
Mutation frequency per 10^6 survivors |
||
|
|
|
|
|
total |
small |
Large |
24-hours without metabolic activation |
|||||||
SC1 |
100 |
105 |
100 |
100 |
77 |
20 |
54 |
SC2 |
|
99 |
|
|
87 |
28 |
55 |
2.5 |
105 |
86 |
84 |
88 |
72 |
18 |
52 |
5 |
107 |
86 |
84 |
90 |
109 |
25 |
80 |
10 |
111 |
88 |
86 |
95 |
72 |
23 |
47 |
20 |
82 |
81 |
80 |
65 |
72 |
26 |
44 |
30 |
62 |
99 |
97 |
60 |
61 |
22 |
37 |
40 |
38 |
95 |
93 |
35 |
84 |
30 |
50 |
50 |
20 |
83 |
81 |
16 |
124 |
46 |
72 |
60 |
9 |
93 |
91 |
9 |
94 |
37 |
53 |
MMS |
99 |
68 |
67 |
66 |
706 |
413 |
215 |
|
|
|
|
|
|
|
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
negative up to 1000 mg/kg bw/day, in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus, OECD TG 474, 2006
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:
- 11-07-2006 to 06-11-2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Guideline study performed under GLP. All relevant validity criteria were met. The test was consistent with the guideline methodology applicable at the time.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- (1997), noting that the requirement to evaluate 4000 erythrocytes for micronuclei, was not implemented until a later version (2014) of the guideline. This would not be considered a deviation as the test was conducted to the methodology applicable at the time.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: August 2005 ; signature: November 2005
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: Crl:CD-l (ICR)BR
- Details on species / strain selection:
- The species and strain was selected in accordance with the OECD TG 474 guideline.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Age at study initiation: approximately 5 to 8 weeks
- Weight at study initiation: definitive test: males 22 - 30 g (males only were used in the definitive test following a preliminary toxicity test using males/females which indicated no marked intersex differences in toxicity)
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Group housed (up to 7 per group)
- Diet (e.g. ad libitum): Certified Rat and Mouse diet 5LF2, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: > 7 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark
IN-LIFE DATES: From: 2006-07-11 To: 2006-08-30 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. Formulations were freshly prepared prior to dosing.
- Concentration of test material in vehicle: Range-finding test: 2000 mg/kg and 1000 mg/kg (or 200 mg/mL and 100 mg/mL respectively); Definitive test: 1000, 500, 250 mg/kg (or 100, 50, 25 mg/mL respectively)
- Amount of vehicle (if gavage or dermal): Treatment volume was 10 mL/kg for control (negative, untreated groups) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Type and concentration of dispersant aid (if powder): Not applicable. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Arachis oil BP was considered as appropriate based on test item solubility. Formulations were freshly prepared prior to dosing.
DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable. - Duration of treatment / exposure:
- Dosed once, then termination at 24 hours (vehicle, positive control and 1000 mg/kg bw groups), and/or 48 hours (vehicle and 1000 mg/kg bw duplicate groups, plus 500 mg/kg bw, 250 mg/kg bw dose levels)
- Frequency of treatment:
- Once by oral gavage
- Post exposure period:
- Termination at 24 hours (vehicle, positive control and 1000 mg/kg bw groups), and/or 48 hours (vehicle and 1000 mg/kg bw duplicate groups, plus 500 mg/kg bw, 250 mg/kg bw dose levels)
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Vehicle control - Group
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- Low - Group
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Intermediate - Group
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- High - Group
- No. of animals per sex per dose:
- 7 male mice per group (14 males for vehicle and 1000 mg/kg dose levels, in two groups for 24 and 48 hour termination and analysis).
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Justification for choice of positive control(s): The choice of positive control was made in accordance with the OECD TG 474 guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 1 dose; 50 mg/kg bw
- Other: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 50 mg/kg bw. - Tissues and cell types examined:
- Bone marrow was extracted and smear preparations were made and stained Polychromatic (PCE) and Normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for (genetic) toxicity.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Following a range-finding test at: 2000 mg/kg and 1000 mg/kg, the test item dose levels used in the Definitive test were 1000, 500, 250 mg/kg. This was based on significant clinical signs being seen in the range finding test at and above 1000 mg/kg: Hunched posture, lethargy, ataxia, ptosis, decreased respiratory rate, laboured respiration, splayed gait, prostration and hypothermia. With the clinical signs of hunched posture, ptosis, lethargy, ataxia and decreased respiratory rate: 1000 mg/kg was selected as the maximum tolerated dose (MTD) of the test item for use in the main test, with 500 and 250 mg/kg as the lower dose
levels.
Applicant assessment indicates: the maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study based on the observed toxicity that limited the maximum dose level to the MTD dose level of 1000 mg/kg bw.
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Treatment and Control groups were treated once. 24-hours and/or 48-hours following the treatment the respective groups were terminated and then bone marrow was extracted from the femurs as part of sampling. Smear preparations were subsequently made and stained.
DETAILS OF SLIDE PREPARATION:
24-hours and/or 48-hours following treatment (as applicable), femurs were extracted, aspirated with foetal bovine serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald / Giemsa. Then allowed to air-dry and a cover slip applied using mounting medium.
METHOD OF ANALYSIS:
Stained smears were coded and examined blind using microscopy (x1000) magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with the appropriate group mean values and standard deviations.
OTHER: Not applicable. - Evaluation criteria:
- 1. Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes observed at the 24-hour or 48-hour termination time compared with the corresponding vehicle control group
- A negative response would be fulfilled if the positive response criteria were not fulfil (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.
2. A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically lower than the vehicle control group.
Data is subject to statistical analysis, where appropriate. References: UKEMS sub-committee on Guidelines of Mutagenicity Testing Reports, Part III (1989). - Statistics:
- Wherea appropriate, data is subject to statistical analysis using appropriate methods as recommended in UKEMS Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed). Any significant results would be examined by one way analysis of variance.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks:
- No statistically significant PCE/NCE decreases at up to 1000 mg/kg bw observed
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: In the range finder: There was no marked difference in toxicity of the test material between the sexes; therefore the definitive test was performed using only male mice.
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable.
- Induction of micronuclei (for Micronucleus assay):
Vehicle Control (24-h), 0 mg/kg bw: Group mean (SD) = 1.4 (2.9) PCE with micronuclei per 2000 PCE
Vehicle Control (48-h), 0 mg/kg bw: Group mean (SD) = 1.0 (1.0) PCE with micronuclei per 2000 PCE
Positive Control (24-h), 50 mg/kg bw/day: Group mean (SD) = 55.2 *** (12.9) PCE with micronuclei per 2000 PCE
Low dose (24-h), 250 mg/kg bw: Group mean (SD) = 3.1 (3.0) PCE with micronuclei per 2000 PCE
Intermediate dose (24-h), 500 mg/kg bw: Group mean (SD) = 1.6 (1.1) PCE with micronuclei per 2000 PCE
High dose (24-h), 1000 mg/kg bw: Group mean (SD) = 2.4 (1.3) PCE with micronuclei per 2000 PCE
High dose (48-h), 1000 mg/kg bw: Group mean (SD) = 1.4 (1.9) PCE with micronuclei per 2000 PCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; *** P < 0.001
- Ratio of PCE/NCE (for Micronucleus assay):
Vehicle Control (24-h), 0 mg/kg bw: Group mean (SD) = 0.99 (0.53) PCE/NCE
Vehicle Control (48-h), 0 mg/kg bw: Group mean (SD) = 2.00 (1.65) PCE/NCE
Positive Control (24-h), 50 mg/kg bw/day: Group mean (SD) = 1.33 (0.49) PCE/NCE
Low dose (24-h), 250 mg/kg bw: Group mean (SD) = 1.56 (0.82) PCE/NCE
Intermediate dose (24-h), 500 mg/kg bw: Group mean (SD) = 1.13 (0.18) PCE/NCE
High dose (24-h), 1000 mg/kg bw: Group mean (SD) = 1.65 (0.56) PCE/NCE
High dose (48-h), 1000 mg/kg bw: Group mean (SD) = 1.50 (0.50) PCE/NCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes
- Appropriateness of dose levels and route: The maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study based on the observed toxicity that limited the maximum dose level to the MTD dose level of 1000 mg/kg bw. The appropriateness of the dose and route was confirmed during the study (recorded in the full study report). With adequate toxicity being observed in the range-finding test via the oral route it was considered to be unnecessary to investigate the intraperitoneal route of administration. Furthermore, in the definitive test: clinical signs were observed when dosed with the test item at and above 250 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ptosis, ataxia, lethargy and splayed gait. The observation of clinical signs was taken to indicate that systemic absorption had occurred.
- Statistical evaluation: No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw relative to the concurrent vehicle control.
- Other: A statistically significant (P < 0.001) response was observed in PCE with micronuclei per 2000 PCE therefore confirming the sensitivity of the test system under the conditions of the test. - Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-genotoxic. - Executive summary:
The study was performed according the requirements of OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 and Japan MHLW/METI guidelines under GLP conditions. Within the study a preliminary range-finding test was performed to find suitable dose levels of the test item, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes at 2000 mg/kg and 1000 mg/kg dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1000 mg/kg and with 500 and 250 mg/kg. Exposure was via oral gavage and then terminations were completed at 24 or 48 hours, as applicable. The femoral bone marrow was extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Within the vehicle controls further groups of seven males were dosed with the vehicle (arachis oil) and then terminations were completed at 24 or 48 hours, as applicable. A positive control group utilising cyclophosphamide at 50 mg/kg was also employed and terminated at 24 hours. There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at and above 250 mg/kg in both the 24 and 48 hour groups where applicable, these were as follows: Hunched posture, ptosis, ataxia, lethargy and splayed gait. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test item dose groups when compared to their concurrent control groups. However, the observation of clinical signs was considered to indicate that systemic absorption had occurred. No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw relative to the concurrent vehicle control. The positive control group showed .a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system. Under the conditions of this study, the test item would not be considered as genotoxic.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Key study : OECD TG 471, 2006 : The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using both the Ames plate incorporation and pre incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 5 to 1500 µg/plate for Salmonella strains TA100 and TA1535, 15 to 5000 µg/plate for Salmonella strains TA98 and TA1537 and 50 to 5000 µg/plate for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (dimethyl sulphoxide) 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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on cytotoxicity. The test item caused a visible reduction in the growth of the bacterial background lawn, both in the presence and absence of S9, to Salmonella strains TA100 and TA1535 from 500 µg/plate and at 5000 µg/plate to TA1537. The test item caused no visible reduction in the growth of the bacterial background lawn to either Salmonella strain TA98 or Escherichia coli strain WP2uvrA-. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type. The test item was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.
Key study : OECD TG 473, 2006 : The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%): 0, 26.75, 53.5, 107, 160.5, 214, 321 μg/mL. Secondly, the doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (1%) and 24-hour without S9: 13.38, 26.75, 53.5, 80.25, 107, 214 μg/mL. All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system. The test item was adequately tested to cytotoxic dose levels. Toxicity was demonstrated by the reduction in the mitotic index in all four exposure conditions and with no metaphases available for scoring at the next dose level below which scoring was conducted. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Key study : OECD TG 490, 2018 : The study was performed to the requirements of OECD TG 490 under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 27, 54, 107, 214, 428, 856 μg/mL concentration in DMSO vehicle (selection was based on precipitation in exposure medium at above 428 μg/mL and cytotoxicity), a definitive test was performed whereby L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 3-hour exposure with and without metabolic activation (4% S9), and a 24-hour exposure without metabolic activation. The following concentrations were used: 3-hour without S9: 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/mL, 3-hour with S9: 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100 μg/mL and 24-hour without S9: 1.3, 2.5, 5, 10, 20, 30, 40, 50, 60, 70 μg/mL. In the first experiment 3-hour exposure, the test item was tested up to concentrations of 60 and 70 µg/mL in the absence and presence S9-mix, respectively. Relative total growth (RTG) was reduced to 6 and 11% in the absence and presence of S9-mix, respectively. In the second experiment 24-hour exposure, the test item was tested up to concentrations of 60 µg/mL in the absence of S9-mix. The RTG was reduced to 9%. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF(controls) + 126. Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Key study : OECD TG 474, 2006 : The study was performed according the requirements of OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 and Japan MHLW/METI guidelines under GLP conditions. Within the study a preliminary range-finding test was performed to find suitable dose levels of the test item, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes at 2000 mg/kg and 1000 mg/kg dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1000 mg/kg and with 500 and 250 mg/kg. Exposure was via oral gavage and then terminations were completed at 24 or 48 hours, as applicable. The femoral bone marrow was extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Within the vehicle controls further groups of seven males were dosed with the vehicle (arachis oil) and then terminations were completed at 24 or 48 hours, as applicable. A positive control group utilising cyclophosphamide at 50 mg/kg was also employed and terminated at 24 hours. There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at and above 250 mg/kg in both the 24 and 48 hour groups where applicable, these were as follows: Hunched posture, ptosis, ataxia, lethargy and splayed gait. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test item dose groups when compared to their concurrent control groups. However, the observation of clinical signs was considered to indicate that systemic absorption had occurred. No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw relative to the concurrent vehicle control. The positive control group showed .a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system. Under the conditions of this study, the test item would not be considered as genotoxic.
Justification for classification or non-classification
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity
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