Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test:
Non-mutagenic with and without metabolic activation, OECD 471, EU Method
B13/14 and EPA OPPTS 870.5100, Bowles (2008)
Mammalian chromosome aberration test:
Non-clastogenic (Chinese hamster lung fibroblasts (V79) with and without
exogenous metabolic activation), OECD 473, EU Method B.10 and EPA OPPTS
870.5375, Hargitai (2014)
Gene mutation test:
Non-mutagenic and non-clastogenic (mouse lymphoma L5178Y cells, with and
without exogenous metabolic activation) OECD 476 and EU Method B.17,
Hargitai (2014)
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:
- 05 May 2008 to 29 May 2008.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon for Salmonella triphimurium
Tryptophan operon for Escherichia coli - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone
- Test concentrations with justification for top dose:
- Preliminary toxicity test:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (Range finding test)
Salmonella strains, with and without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E. coli strain WP2uvrA-, with and without S9: 50, 150, 500, 1500, 5000 µg/plate.
Experiment 2 (Main test)
Salmonella strains, with and without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E. coli strain WP2uvrA-, with and without S9: 50, 150, 500, 1500, 5000 µg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 12.5 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N'-Nitrosoguanidine 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5µg/plate for TA1535.
- Remarks:
- Without S9
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without S9 Migrated to IUCLID6: 80 µg/plate for TA1537
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline-1-oxide 0.2 µg/plate for TA98
- Remarks:
- Without S9
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2 Aminoanthracene (2AA) at 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA-
- Remarks:
- With S9
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 Migrated to IUCLID6: 5 µg/plate for TA98
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours
NUMBER OF REPLICATIONS: triplicate plating
DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction - Evaluation criteria:
- Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method with historical control ranges from 2006 and 2007 were used.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per mL.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The positive control historical ranges were used from 2006 and 2007.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.
Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none reported
- Effects of osmolality: none reported
- Evaporation from medium: none reported
- Water solubility: The test material was fully soluble in sterile distilled water at 12.5 mg/mL in solubility checks performed in-house.
- Precipitation: A pale flakey precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:
The test material caused a reduction in the frequency of TA100 revertant colonies and/or a weakening of the background lawn, initially from 500 µg/plate. Substantial reductions in the frequency of WP2uvrA- revertant colonies were also noted at 5000 µg/plate. The test material formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
None
CONTROLS
The vehicle (sterile distilled water) 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 test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E.coli strain WP2uvrA- at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number.
A pale flakey precipitate was observed at and above 1500 µg/plate, this 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 material, either with or without metabolic activation. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
The test material was considered to be non-mutagenic under the conditions of this test. - Executive summary:
The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. 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 material formulations.
Additional dose levels (5 and 15 µg/plate) were included (where applicable) to allow for test material induced toxicity, in Salmonella typhimurium strains only, ensuring that at least four non‑toxic doses were achieved.
The vehicle (sterile distilled water) 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 test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E.coli strain WP2uvrA-at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number.
A pale flakey precipitate was observed at and above 1500 µg/plate, this 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 material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.
- 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:
- 26 February 2013 to 19 July 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Read-across study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
- Justification for type of information:
- See Read-Across Justification in Section 13.
- Reason / purpose for cross-reference:
- other: Target record
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10 000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated foetal bovine serum (DMEM-10, culture medium). This was reduced to 5 % for the assays (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- ASSAYS 1 AND 3
- Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment)
- With metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment)
ASSAY 2
- Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment)
- With metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
For the cytogenetic experiments, 1 to 3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10⁵ cells/dish concentration and incubated for approximately 24 hours at 37 °C in 10 mL of culture medium (DMEM-10). After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) for the assay without metabolic activation or with 9.4 mL treatment medium with 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test material solutions, negative (vehicle) or positive control solutions (treatment volume: 100 μL/dish in all cases) for the given period of time at 37 °C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v % antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
DURATION
- Exposure duration: Assays 1 and 3: 3 hours in the presence and absence of metabolic activation. Assay 2: 3 hours in the presence of metabolic activation and 20 hours in the absence of metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours for assays 1 and 3 and 28 hours for Assay 2
SPINDLE INHIBITOR (cytogenetic assays): 2 to 2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL).
STAIN (for cytogenetic assays): 5 % Giemsa solution. The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (methanol:acetic acid 3:1 (v:v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried (at least three slides were prepared for each culture in the main tests). The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.
NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Where there were insufficient metaphases in one replicate, the total was made up to 200 cells examined per concentration using the other replicate.
DETERMINATION OF CYTOTOXICITY
- Method: % relative survival (determined through counting with a haemocytometer)
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- The assay was considered positive if the following criteria were met:
- Increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations (only data without gaps are considered).
- The increases were reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases were statistically significant.
- The increases were not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for the testing laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test material was concluded to have given a negative response if no reproducible, statistically significant increases were observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Assay 1 produced a positive response in 1 concentration without metabolic activation and 2 concentrations with metabolic activation. No positive response was observed in assays 2 and 3 (assay 3 was performed using the same conditions)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Marked cytotoxicity was observed in the high dose concentrations in all assays with and without metabolic activation
- 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: No significant effects relating to pH were observed.
- Effects of osmolality: No significant effects relating to osmolality were observed.
- Water solubility: The test material was found to be soluble in distilled water at up to 250 mg/mL. This was selected as the vehicle for use in the study.
- Precipitation: Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.
- Other confounding effects: In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation, and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 2, carried out one week later with the same cell line, demonstrated that the result was not reproduced. Therefore, an additional main test (Assay 3) was carried out with identical conditions to Assay 1 for clarification. Assay 1 was considered to be invalid and was discounted.
RANGE-FINDING/SCREENING STUDIES
Two concentration selection cytotoxicity assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed to establish appropriate concentration ranges for the definitive tests.
A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
In assay A, without metabolic activation 24 % relative survival was noted at 185.2 µg/mL; above this concentration (555.6, 1666.7 and 5000 µg/mL), 0 % relative survival was recorded. With metabolic activation, % relative survival was 65 % at 185.2 µg/mL; above this concentration, % relative survival was 0 % (555.6, 1666.7 and 5000 µg/mL).
In assay B without metabolic activation, relative survival was 10 % at 61.73 µg/mL and 0 % at concentrations above this level (185.2, 555.6, 1666.7 and 5000 µg/mL). With metabolic activation, relative survival at 185.2 µg/mL was 68 %; above this concentration (555.6, 1666.7 and 5000 µg/mL) relative survival was 0 %.
MAIN STUDY
In Assay 1, marked cytotoxicity was observed at 300, 200 and 150 μg/mL c without metabolic activation (relative survival values were 0, 18 and 33 %, respectively) and 400, 300 and 250 μg/mL with metabolic activation (relative survival values were 0, 0 and 26 %, respectively).Therefore, concentrations of 150, 125, 100 and 75 μg/mL were chosen for evaluation without metabolic activation and 250, 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.
In Assay 2, cytotoxicity was also observed at 100, 80 and 60 μg/mL concentrations without metabolic activation (relative survival values were 0, 15 and 29 %, respectively) and 400, 300, 250 and 200 μg/mL with metabolic activation (relative survival values were 0, 0, 23 and 45 %, respectively). Therefore, concentrations of 60, 50 and 40 μg/mL were chosen for evaluation without metabolic activation and 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.
In Assay 3, marked cytotoxicity was observed at 300, 200 and 150 μg/mL without metabolic activation (relative survival values were 0, 18 and 37 %, respectively) and 400, 300 and 250 μg/mL with metabolic activation (relative survival values were 0, 0 and 22 % , respectively)4. Therefore, concentrations of 150, 125, 100 and 75 μg/mL were chosen for evaluation without metabolic activation and 250, 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.
Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.
Polyploid metaphases (1-6) or endoreduplicated (1-4) were found in some cases in the negative (vehicle) control, positive control or test material treated cells in Assays 1 and 3. No polyploidy or endoreduplicated metaphases were detected in Assay 2.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative (vehicle) control data were within the acceptable range for spontaneous aberration frequency.
POSITIVE CONTROL DATA: The positive control substances caused a statistically significant increase in the number of structural aberrations in the experiments with or without metabolic activation, demonstrating the sensitivity of the test system.
In the 3 hour treatment without metabolic activation for Assay 1 and 3, the aberration frequency of the EMS positive control was slightly lower than anticipated. However, the increase in the number of aberrant cells compared to the negative control value was statistically highly significant and the proper status of the cells was also proven in the same assay in the experiment with metabolic activation. Furthermore, there was a good response using this positive control substance using 20-hour treatment without metabolic activation. Therefore, the observed values were considered to be acceptable. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations. - Executive summary:
The clastogenicity of the test material was investigated in Chinese hamster lung fibroblasts (V79) in a chromosome aberration assay performed in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375 under GLP conditions.
Cells were seeded into culture dishes (5 x 10⁵ cells/dish concentration) and incubated for 24 hours prior to exposure to the test material. The cells were exposed to the test material in the presence and absence of metabolic activation (S9). The cells were exposed to the test material at the following concentrations:
- Assays 1 and 3
Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment).
- Assay 2
Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment) and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment).
Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.
Marked cytotoxicity was observed at the higher doses; however a minimum of three analysable concentrations were available for evaluation for each assay.
In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 1 was considered to be invalid and was discounted.
Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.
Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read across material
- Justification for type of information:
- See Read-Across Justification in Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Assay 1 produced a positive response in 1 concentration without metabolic activation and 2 concentrations with metabolic activation. No positive response was observed in assays 2 and 3 (assay 3 was performed using the same conditions)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Marked cytotoxicity was observed in the high dose concentrations in all assays with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- 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:
- 28 October 2013 to 16 December 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Read-across study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
- Justification for type of information:
- See Read-Across Justification in Section 13.
- Reason / purpose for cross-reference:
- other: Target record
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase, TK+/- locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI-10 medium. The RPMI-10 medium consisted of RPMI 1640 medium with the following components added (final concentration in medium): heat inactivated horse serum 10 % v/v, antibiotic-antimycotic solution 0.01 mL/mL (containing 10 000 IU/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin–B), Pluronic-F68 0.5 mg/mL and pyruvic avid 0.2 mg/mL. L-glutamine 0.3 mg/mL was also added freshly to the media. NaHCO₃ 2 mg/mL was already included in the ready to use liquid media.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- ASSAY 1
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL
ASSAY 2
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Based on a previous chromosome aberration study performed by the same laboratory, the test material was soluble in distilled water. The highest achievable concentration using this vehicle was 250 mg/mL. As this vehicle is also compatible with the test system, it was selected to be the vehicle (solvent) for this study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
0.2 mL of RPMI-5 medium, vehicle (solvent), test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 150 mM KCl (in the case of 3 h treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, at least 10⁷ cells were placed in each of a series of 75 cm² sterile flasks. For the 24-hour treatment, at least 4 x 10⁶ cells were placed in each of a series of 25 cm² sterile flasks.
RPMI-5 medium was made in the same way as the RPMI-10, with the exception that it contained a reduced level of heat inactivated horse serum (5 % v/v).
Cultures were incubated at 37 ± 1 °C (approximately 5 % CO₂ in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Measurement of pH and osmolality was also performed after the treatment period.
Where a sufficient number of cells survived, the cell density was adjusted to a concentration of 2 x 10⁵ cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.
DURATION
- Exposure duration: Assay 1: 3 hours in the presence and absence of metabolic activation. Assay 2: 3 hours in the presence of metabolic activation, 24 hours in the absence of metabolic activation.
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): about 2 weeks
SELECTION AGENT (mutation assays): trifluorothymidine (TFT). The cultures were sub-cultured daily and the cell density adjusted to 2 x 10⁵ cells/mL.
NUMBER OF REPLICATIONS: Performed in duplicate
DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency and the corresponding relative survival (%) and relative total growth
Plating for survival - Cultures of cell density 2 x 10⁵ cells/mL were further diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture were placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.
Plating for viability - At the end of the expression period (cultures treated with TFT), the cell density in the selected cultures was determined and adjusted to 1 x 10⁴ cells/mL with RPMI-20 (20 % heat inactivated horse serum, no addition of Pluronic F-68) for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture was placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted. - Evaluation criteria:
- The test material was considered to be mutagenic in this assay if all the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency were observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There was a significant concentration-relationship as indicated by linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10⁶ viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results which only partially satisfied the acceptance and evaluation criteria were evaluated on a case-by-case basis. - Statistics:
- The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis in any assay with or without S9
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Due to excessive toxicity in the cultures, not all assays had eight analysable concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH
- Effects of osmolality: There were no large changes in osmolality
- Precipitation: In assay 1, minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the presence of metabolic activation (3 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the absence of metabolic activation (24 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate with the exception of the lowest dose tested in the series.
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed at the following concentrations: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 3 hour treatments in the presence and absence of metabolic activation (toxicity tests A and B, respectively) and for 24 hours in the absence of metabolic activation (toxicity test C).
All concentrations were performed in duplicate. Due to the solubility of the test material, the test solution administration in the preliminary toxicity test was 0.4 mL. Precipitate was observed at all concentrations in all three tests in at least one replicate.
In test A, relative survival at 78.13 µg/mL was 41 % on day 0 and 92 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).
In test B, relative survival at 78.13 µg/mL was 5 % on day 0 and 45 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival). At 36.06 µg/mL, relative survival was 61 % on day 0 and 84 % on day 3.
In test C, relative survival at 19.53 µg/mL was 21 % on day 1 and 80 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).
COMPARISON WITH HISTORICAL CONTROL DATA:
Comparison with the historical control data confirmed that the test system was functioning correctly.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Assay 1 with metabolic activation, excessive cytotoxicity of the test material was observed at 160, 140 and 120 μg/mL concentrations; cells of these samples did not survive the treatment or the expression period. Evaluation of the mutagenicity was made using data of the first surviving concentration of 100 μg/mL (relative survival value of 24 %) and the five lower concentrations (a total of six concentrations).
In Assay 1 without metabolic activation, excessive cytotoxicity of the test material was observed at the 100 μg/mL concentration; cells of this sample did not survive the treatment period. Evaluation was performed on data from the next concentration of 90 μg/mL (relative survival value of 18 %) and seven lower concentrations (a total of eight concentrations).
In Assay 2, for the 3-hour treatment with metabolic activation, cells of the 160, 140 and 120 μg/mL concentration samples did not survive the treatment or the expression period. Evaluation was performed on the data from the first surviving concentration which was 100 μg/mL (relative survival value of 46 %) and the five lower concentrations (a total of six concentrations).
In Assay 2, for the 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at the 40, 35, 32.5, 30 and 27.5 μg/mL concentrations; cells of these samples died during the treatment or in the expression period. Evaluation of the data was made using the next concentration of 25 μg/mL (relative survival of 8 %) and the following six lower concentrations (a total of seven concentrations). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations. - Executive summary:
The mutagenicity of the test material was assessed in an in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) which was performed in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions.
The test was performed using the mouse lymphoma L5178Y cell model up to cytotoxic and precipitating concentrations in the absence and presence of an exogenous metabolic activation system (S9 -mix).
The following conditions were used in the study:
ASSAY 1
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL
ASSAY 2
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL
The expression time and selection time for both assays (both in the presence and absence of metabolic activation) were 3 days and 2 weeks, respectively.
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis was observed in any of the assays either with or without metabolic activation.
Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read across material
- Justification for type of information:
- See Read-Across Justification in Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis in any assay with or without S9
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Due to excessive toxicity in the cultures, not all assays had eight analysable concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
Referenceopen allclose all
The test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E .coli strain WP2uvrA- at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number.
A pale flakey precipitate was observed at and above 1500 µg/plate, this 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 material, either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Table1: Spontaneous Mutation Rates (Concurrent Negative Controls)
Range-finding Test
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
84 |
|
14 |
|
29 |
|
19 |
|
7 |
|
95 |
(94) |
18 |
(16) |
22 |
(26) |
20 |
(19) |
5 |
(6) |
102 |
|
16 |
|
26 |
|
19 |
|
7 |
|
Main Test
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
137 |
|
21 |
|
34 |
|
15 |
|
12 |
|
140 |
(136) |
18 |
(19) |
26 |
(35) |
8 |
(11) |
11 |
(12) |
131 |
|
19 |
|
45 |
|
11 |
|
13 |
|
Table2: Test Results: Range-Finding Test– Without Metabolic Activation
Test Period |
From: 19 May 2008 |
To: 22 May 2008 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
||||||||
- |
0 |
108 91 97 |
(99) 8.6# |
12 14 13 |
(13) 1.0 |
37 20 15 |
(24) 11.5 |
12 11 16 |
(13) 2.6 |
3 5 8 |
(5) 2.5 |
|
- |
5 |
86 106 92 |
(95) 10.3 |
13 15 13 |
(14) 1.2 |
N/T |
11 11 12 |
(11) 0.6 |
4 5 4 |
(4) 0.6 |
||
- |
15 |
97 90 111 |
(99) 10.7 |
11 9 18 |
(13) 4.7 |
N/T |
15 18 15 |
(16) 1.7 |
3 7 8 |
(6) 2.6 |
||
- |
50 |
118 112 88 |
(106) 15.9 |
19 10 10 |
(13) 5.2 |
14 32 19 |
(22) 9.3 |
16 15 14 |
(15) 1.0 |
5 2 3 |
(3) 1.5 |
|
- |
150 |
87 106 89 |
(94) 10.4 |
7 7 11 |
(8) 2.3 |
26 24 15 |
(22) 5.9 |
13 11 10 |
(11) 1.5 |
1 5 4 |
(3) 2.1 |
|
- |
500 |
38 41 41 |
(40) 1.7 |
2 10 2 |
(5) 4.6 |
20 22 15 |
(19) 3.6 |
8 2 2 |
(4) 3.5 |
1 1 5 |
(2) 2.3 |
|
- |
1500 |
10 P* 9 P* 14 P* |
(11) 2.6 |
1 P* 1 P* 1 P* |
(1) 0.0 |
15 P 13 P 13 P |
(14) 1.2 |
5 P 8 P 0 P |
(4) 4.0 |
0 P 0 P 1 P |
(0) 0.6 |
|
- |
5000 |
0 P* 0 P* 0 P* |
(0) 0.0 |
0 P* 0 P* 0 P* |
(0) 0.0 |
9 P 5 P 10 P |
(8) 2.6 |
0 P* 0 P* 0 P* |
(0) 0.0 |
0 P* 0 P* 0 P* |
(0) 0.0 |
|
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 |
5 |
2 |
0.2 |
80 |
||||||||
682 696 685 |
(688) 7.4 |
354 375 305 |
(345) 35.9 |
881 841 654 |
(792) 121.2 |
588 129 155 |
(291) 257.8 |
1168 1144 1107 |
(1140) 30.7 |
|||
ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO = 4-Nitroquinoline-1-oxide
9AA = 9-Aminoacridine
N/T = Not tested at this dose level
P = Precipitate
* = Partial or complete absence of bacterial background lawn
# = Standard deviation
Table3: Test Results: Range-Finding Test– With Metabolic Activation
Test Period |
From: 19 May 2008 |
To: 22 May 2008 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
+ |
0 |
92 97 77 |
(89) 10.4# |
15 15 12 |
(14) 1.7 |
22 29 18 |
(23) 5.6 |
26 30 32 |
(29) 3.1 |
7 7 7 |
(7) 0.0 |
||
+ |
5 |
71 90 90 |
(84) 11.0 |
9 7 8 |
(8) 1.0 |
N/T |
31 22 24 |
(26) 4.7 |
13 9 3 |
(8) 5.0 |
|||
+ |
15 |
75 99 96 |
(90) 13.1 |
3 9 10 |
(7) 3.8 |
N/T |
16 21 15 |
(17) 3.2 |
10 4 4 |
(6) 3.5 |
|||
+ |
50 |
80 81 77 |
(79) 2.1 |
8 4 8 |
(7) 2.3 |
31 21 22 |
(25) 5.5 |
21 23 12 |
(19) 5.9 |
7 7 5 |
(6) 1.2 |
||
+ |
150 |
80 95 80 |
(85) 8.7 |
9 7 2 |
(6) 3.6 |
21 22 14 |
(19) 4.4 |
20 14 19 |
(18) 3.2 |
9 8 3 |
(7) 3.2 |
||
+ |
500 |
97 89 75 |
(87) 11.1 |
3 2 4 |
(3) 1.0 |
18 14 18 |
(17) 2.3 |
16 27 16 |
(20) 6.4 |
4 3 4 |
(4) 0.6 |
||
+ |
1500 |
26 P 30 P 43 P |
(33) 8.9 |
4 P* 2 P* 4 P* |
(3) 1.2 |
14 P 12 P 7 P |
(11) 3.6 |
8 P 13 P 10 P |
(10) 2.5 |
8 P 4 P 4 P |
(5) 2.3 |
||
+ |
5000 |
0 P* 0 P* 0 P* |
(0) 0.0 |
0 P* 0 P* 0 P* |
(0) 0.0 |
4 P 16 P 14 P |
(11) 6.4 |
0 P* 0 P* 0 P* |
(0) 0.0 |
0 P* 0 P* 0 P* |
(0) 0.0 |
||
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
|||||||
1 |
2 |
10 |
5 |
2 |
|||||||||
2094 1811 1780 |
(1895) 173.0 |
310 198 288 |
(265) 59.3 |
508 475 435 |
(473) 36.6 |
216 222 224 |
(221) 4.2 |
275 365 197 |
(279) 84.1 |
||||
BP = Benzo(a)pyrene
2AA = 2-Aminoanthracene
N/T = Not tested at this dose level
P = Precipitate
* = Partial absence of bacterial background lawn
# = Standard deviation
Table4: Test Results: Main Test– Without Metabolic Activation
Test Period |
From: 26 May 2008 |
To: 29 May 2008 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
- |
0 |
135 172 134 |
(147) 21.7# |
20 20 22 |
(21) 1.2 |
33 31 31 |
(32) 1.2 |
12 21 13 |
(15) 4.9 |
9 22 11 |
(14) 7.0 |
||
- |
5 |
153 142 123 |
(139) 15.2 |
16 20 15 |
(17) 2.6 |
N/T |
19 10 14 |
(14) 4.5 |
9 16 13 |
(13) 3.5 |
|||
- |
15 |
129 112 140 |
(127) 14.1 |
25 20 15 |
(20) 5.0 |
N/T |
11 14 10 |
(12) 2.1 |
14 14 7 |
(12) 4.0 |
|||
- |
50 |
121 136 119 |
(125) 9.3 |
25 18 24 |
(22) 3.8 |
31 36 20 |
(29) 8.2 |
19 12 12 |
(14) 4.0 |
15 9 9 |
(11) 3.5 |
||
- |
150 |
119 111 146 |
(125) 18.3 |
20 10 18 |
(16) 5.3 |
27 29 32 |
(29) 2.5 |
9 19 15 |
(14) 5.0 |
11 11 9 |
(10) 1.2 |
||
- |
500 |
56 74 68 |
(66) 9.2 |
2 3 7 |
(4) 2.6 |
38 48 36 |
(41) 6.4 |
13 9 9 |
(10) 2.3 |
0 0 0 |
(0) 0.0 |
||
- |
1500 |
27*P 18*P 25*P |
(23) 4.7 |
0*P 0*P 0*P |
(0) 0.0 |
23 P 25 P 30 P |
(26) 3.6 |
7 P 10 P 2 P |
(6) 4.0 |
0*P 0*P 0*P |
(0) 0.0 |
||
- |
5000 |
0*P 0*P 0*P |
(0) 0.0 |
0*P 0*P 0*P |
(0) 0.0 |
25P 25P 13P |
(21) 6.9 |
0*P 0*P 0*P |
(0) 0.0 |
0*P 0*P 0*P |
(0) 0.0 |
||
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||
3 |
5 |
2 |
0.2 |
80 |
|||||||||
472 498 578 |
(516) 55.2 |
255 183 196 |
(211) 38.4 |
715 713 730 |
(719) 9.3 |
112 111 121 |
(115) 5.5 |
2294 2350 1477 |
(2040) 488.7 |
||||
ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO = 4-Nitroquinoline-1-oxide
9AA = 9-Aminoacridine
N/T = Not tested at this dose level
P = Precipitate
* = Partial or complete absence of bacterial background lawn
# = Standard deviation
Table5: Test Results: Main Test– With Metabolic Activation
Test Period |
From: 26 May 2008 |
To: 29 May 2008 |
|||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||||
Base-pair substitution type |
Frameshift type |
||||||||||||
TA100 |
TA1535 |
WP2uvrA‑ |
TA98 |
TA1537 |
|||||||||
+ |
0 |
99 126 128 |
(118) 16.2# |
8 16 16 |
(13) 4.6 |
40 42 41 |
(41) 1.0 |
34 29 23 |
(29) 5.5 |
13 21 18 |
(17) 4.0 |
||
+ |
5 |
128 117 117 |
(121) 6.4 |
9 10 14 |
(11) 2.6 |
N/T |
27 26 23 |
(25) 2.1 |
16 18 21 |
(18) 2.5 |
|||
+ |
15 |
125 114 118 |
(119) 5.6 |
10 11 18 |
(13) 4.4 |
N/T |
23 21 23 |
(22) 1.2 |
14 10 15 |
(13) 2.6 |
|||
+ |
50 |
154 115 120 |
(130) 21.2 |
13 8 11 |
(11) 2.5 |
34 37 31 |
(34) 3.0 |
22 22 16 |
(20) 3.5 |
12 12 14 |
(13) 1.2 |
||
+ |
150 |
109 109 113 |
(110) 2.3 |
19 10 9 |
(13) 5.5 |
38 40 32 |
(37) 4.2 |
27 21 24 |
(24) 3.0 |
26 13 16 |
(18) 6.8 |
||
+ |
500 |
122 123 141 |
(129) 10.7 |
8 9 11 |
(9) 1.5 |
29 43 38 |
(37) 7.1 |
C 26 33 |
(30) 4.9 |
9 9 10 |
(9) 0.6 |
||
+ |
1500 |
95 P 102 P 76 P |
(91) 13.5 |
7*P 12*P 4*P |
(8) 4.0 |
24 P 24 P 23 P |
(24) 0.6 |
22 P 24 P 30 P |
(25) 4.2 |
12*P 11*P 9*P |
(11) 1.5 |
||
+ |
5000 |
0*P 0*P 0*P |
(0) 0.0 |
0*P 0*P 0*P |
(0) 0.0 |
17P 26P 28P |
(24) 5.9 |
0*P 0*P 0*P |
(0) 0.0 |
0*P 0*P 0*P |
(0) 0.0 |
||
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
|||||||
1 |
2 |
10 |
5 |
2 |
|||||||||
2319 4009 2002 |
(2777) 1078.9 |
397 379 340 |
(372) 29.1 |
451 494 472 |
(472) 21.5 |
148 179 177 |
(168) 17.3 |
330 419 653 |
(467) 166.8 |
||||
BP = Benzo(a)pyrene
2AA = 2-Aminoanthracene
C = Contaminated
N/T = Not tested at this dose level
P = Precipitate
* = Partial absence of bacterial background lawn
# = Standard deviation
Table 1: Results for Assay 1 (discounted)
Concentration (µg/mL) |
Treatment time (hours) |
Harvesting time (hours) |
% Relative survival |
Mean % aberrant cells |
No. cells observed |
Observations |
||
Including gaps |
Excluding gaps |
Polyploid |
Endo. |
|||||
Without metabolic activation |
||||||||
Vehicle control |
3 |
20 |
100 |
2.5 |
2.5 |
3 |
1 |
normal |
300 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
200 |
3 |
20 |
18 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
150 |
3 |
20 |
33 |
2.5 |
2.5 |
6 |
1 |
ppte, disc. medium |
125 |
3 |
20 |
63 |
9.5 |
9.0** |
4 |
0 |
ppte, disc. medium |
100 |
3 |
20 |
80 |
4.5 |
4.5 |
5 |
1 |
ppte, disc. medium |
75 |
3 |
20 |
94 |
1.0 |
1.0 |
1 |
0 |
ppte |
50 |
3 |
20 |
96 |
NE |
NE |
NE |
NE |
normal |
25 |
3 |
20 |
77 |
NE |
NE |
NE |
NE |
normal |
EMS 1 µL/mL |
3 |
20 |
73 |
14.4 |
14.4*** |
5 |
0 |
normal |
With metabolic activation |
||||||||
Vehicle control |
3 |
20 |
100 |
4.0 |
4.0 |
5 |
1 |
normal |
400 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
300 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
250 |
3 |
20 |
26 |
6.0 |
6.0 |
2 |
4 |
ppte, disc. medium |
200 |
3 |
20 |
56 |
11.0 |
10.5* |
4 |
2 |
ppte, disc. medium |
150 |
3 |
20 |
75 |
12.0 |
12.0** |
2 |
2 |
ppte, disc. medium |
100 |
3 |
20 |
99 |
3.5 |
3.5 |
4 |
2 |
ppte, disc. medium |
50 |
3 |
20 |
91 |
NE |
NE |
NE |
NE |
normal |
25 |
3 |
20 |
105 |
NE |
NE |
NE |
NE |
normal |
CP 6 µg/mL |
3 |
20 |
69 |
93.8 |
93.8*** |
0 |
1 |
normal |
Endo. = Endoreduplicated
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
ppte = Precipitate
disc. medium = discoloured medium
NE = Not examined
*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control
**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control
***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control
Table 2: Results from Assay 2
Concentration (µg/mL) |
Treatment time (hours) |
Harvesting time (hours) |
% Relative survival |
Mean % aberrant cells |
No. cells observed |
Observations |
||
Including gaps |
Excluding gaps |
Polyploid |
Endo. |
|||||
Without metabolic activation |
||||||||
Vehicle control |
20 |
28 |
100 |
4.0 |
2.5 |
0 |
0 |
normal |
100 |
20 |
28 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
80 |
20 |
28 |
15 |
NE |
NE |
NE |
NE |
ppte |
60 |
20 |
28 |
29 |
4.5 |
2.0 |
0 |
0 |
normal |
50 |
20 |
28 |
58 |
6.5 |
3.5 |
0 |
0 |
normal |
40 |
20 |
28 |
97 |
2.5 |
2.0 |
0 |
0 |
normal |
30 |
20 |
28 |
95 |
NE |
NE |
NE |
NE |
normal |
20 |
20 |
28 |
102 |
NE |
NE |
NE |
NE |
normal |
10 |
20 |
28 |
108 |
NE |
NE |
NE |
NE |
normal |
5 |
20 |
28 |
120 |
NE |
NE |
NE |
NE |
normal |
EMS 0.4 µL/mL |
20 |
28 |
79 |
26.9 |
23.1*** |
0 |
0 |
normal |
With metabolic activation |
||||||||
Vehicle control |
3 |
28 |
100 |
5.0 |
2.5 |
0 |
0 |
normal |
400 |
3 |
28 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
300 |
3 |
28 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
250 |
3 |
28 |
23 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
200 |
3 |
28 |
45 |
8.5 |
3.5 |
0 |
0 |
ppte, disc. medium |
150 |
3 |
28 |
70 |
5.5 |
2.0 |
0 |
0 |
ppte, disc. medium |
100 |
3 |
28 |
93 |
3.5 |
2.0 |
0 |
0 |
ppte, disc. medium |
50 |
3 |
28 |
92 |
NE |
NE |
NE |
NE |
normal |
25 |
3 |
28 |
108 |
NE |
NE |
NE |
NE |
normal |
CP 6 µg/mL |
3 |
28 |
75 |
37.8 |
33.3*** |
0 |
0 |
normal |
Endo. = Endoreduplicated
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
ppte = Precipitate
disc. medium = discoloured medium
NE = Not examined
*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control
**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control
***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control
Table 3: Results from Assay 3
Concentration (µg/mL) |
Treatment time (hours) |
Harvesting time (hours) |
% Relative survival |
Mean % aberrant cells |
No. cells observed |
Observations |
||
Including gaps |
Excluding gaps |
Polyploid |
Endo. |
|||||
Without metabolic activation |
||||||||
Vehicle control |
3 |
20 |
100 |
2 |
1.5 |
0 |
0 |
normal |
300 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
200 |
3 |
20 |
18 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
150 |
3 |
20 |
37 |
3.5 |
2 |
1 |
4 |
ppte, disc. medium |
125 |
3 |
20 |
64 |
2 |
1.5 |
1 |
2 |
ppte, disc. medium |
100 |
3 |
20 |
69 |
5.5 |
4.5 |
0 |
1 |
ppte, disc. medium |
75 |
3 |
20 |
87 |
3.5 |
3 |
2 |
0 |
ppte |
50 |
3 |
20 |
85 |
NE |
NE |
NE |
NE |
normal |
25 |
3 |
20 |
100 |
NE |
NE |
NE |
NE |
normal |
EMS1 µL/mL |
3 |
20 |
78 |
12.5 |
11.5*** |
1 |
0 |
normal |
With metabolic activation |
||||||||
Vehicle control |
3 |
20 |
100 |
5 |
3.5 |
1 |
0 |
normal |
400 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
300 |
3 |
20 |
0 |
NE |
NE |
NE |
NE |
ppte, disc. medium |
250 |
3 |
20 |
22 |
4.5 |
3 |
0 |
2 |
ppte, disc. medium |
200 |
3 |
20 |
62 |
7 |
4 |
0 |
0 |
ppte, disc. medium |
150 |
3 |
20 |
75 |
4.5 |
3.5 |
0 |
0 |
ppte, disc. medium |
100 |
3 |
20 |
90 |
4.5 |
3.5 |
0 |
0 |
ppte, disc. medium |
50 |
3 |
20 |
90 |
NE |
NE |
NE |
NE |
normal |
25 |
3 |
20 |
100 |
NE |
NE |
NE |
NE |
normal |
CP 6 µg/mL |
3 |
20 |
66 |
88.2 |
88.2*** |
1 |
0 |
normal |
Endo. = Endoreduplicated
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
ppte = Precipitate
disc. medium = discoloured medium
NE = Not examined
*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control
**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control
***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control
Table 1: Summary of Results for Assay 1
Concentration (µg/mL) |
Survival Data |
Viability Data |
Mutation Frequency |
||
PE |
RS (%) |
RTG (%) |
PE |
||
Metabolic activation |
With (+ S9-mix); 3 hour exposure |
||||
160 |
ND |
ND |
ND |
ND |
ND |
140 |
0.007 |
0 |
ND |
ND |
ND |
120 |
0.015 |
1 |
ND |
ND |
ND |
100 |
0.374 |
24 |
1 |
0.860 |
120.7 |
80 |
0.575 |
51 |
37 |
0.810 |
116.4 |
60 |
0.748 |
79 |
59 |
0.706 |
139.1 |
40 |
0.805 |
89 |
85 |
0.687 |
109.3 |
20 |
0.787 |
95 |
108 |
0.727 |
102.2 |
10 |
0.776 |
97 |
117 |
0.701 |
123.3 |
Vehicle control |
0.810 |
100 |
100 |
0.759 |
109.9 |
Vehicle control for positive control (DMSO) |
0.712 |
94 |
113 |
0.743 |
99 |
Untreated control |
0.737 |
98 |
91 |
0.732 |
111.8 |
Positive control CP (4 μg/mL) |
0.405 |
45 |
16 |
0.398 |
1733.3* |
Metabolic activation |
Without (- S9-mix); 3 hour exposure |
||||
100 |
ND |
ND |
ND |
ND |
ND |
90 |
0.765 |
18 |
3 |
0.732 |
100.4 |
80 |
0.732 |
31 |
13 |
0.754 |
101.6 |
70 |
0.776 |
41 |
25 |
0.781 |
104.8 |
60 |
0.712 |
65 |
54 |
0.712 |
122.6 |
50 |
0.810 |
89 |
59 |
0.759 |
108.9 |
40 |
0.707 |
84 |
78 |
0.754 |
108.6 |
20 |
0.776 |
98 |
78 |
0.712 |
106.5 |
10 |
0.816 |
115 |
108 |
0.816 |
96.5 |
5 |
0.799 |
105 |
100 |
0.787 |
110.9 |
Vehicle control |
0.781 |
100 |
100 |
0.722 |
99.8 |
Vehicle control for positive control (DMSO) |
0.816 |
108 |
94 |
0.687 |
103.8 |
Untreated control |
0.754 |
103 |
99 |
0.793 |
101.3 |
Positive control NQO (0.15 μg/mL) |
0.453 |
60 |
29 |
0.440 |
1087.9* |
CP = Cyclophosphamide
RS= Relative survival values (%) corrected with the post treatment cell concentrations
ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)
NQO = 4-Nitroquinoline-N-oxide
RTG = Relative total growth
PE = Plating efficiency
* = Statistically significant
Table 2: Summary of Results for Assay 2
Concentration (µg/mL) |
Survival Data |
Viability Data |
Mutation Frequency |
||
PE |
RS (%) |
RTG (%) |
PE |
||
Metabolic activation |
With (+ S9-mix); 3 hour exposure |
||||
160 |
ND |
ND |
ND |
ND |
ND |
140 |
0.008 |
0 |
ND |
ND |
ND |
120 |
0.008 |
1 |
ND |
ND |
ND |
100 |
0.424 |
46 |
35 |
0.787 |
141.1 |
80 |
0.644 |
83 |
47 |
0.640 |
147.4 |
60 |
0.743 |
86 |
79 |
0.835 |
99.0 |
40 |
0.841 |
105 |
89 |
0.737 |
132.1 |
20 |
0.706 |
97 |
110 |
0.701 |
120.0 |
10 |
0.770 |
100 |
99 |
0.663 |
118.9 |
Vehicle control |
0.765 |
100 |
100 |
0.653 |
94.3 |
Vehicle control for positive control (DMSO) |
0.770 |
107 |
116 |
0.776 |
90.9 |
Untreated control |
0.805 |
106 |
114 |
0.810 |
89.8 |
Positive control CP (4 μg/mL) |
0.380 |
41 |
12 |
0.357 |
1935.9* |
Metabolic activation |
Without (- S9-mix); 24 hour exposure |
||||
40 |
ND |
ND |
ND |
ND |
ND |
35 |
ND |
ND |
ND |
ND |
ND |
32.5 |
ND |
ND |
ND |
ND |
ND |
30 |
ND |
ND |
ND |
ND |
ND |
27.5 |
ND |
ND |
ND |
ND |
ND |
25 |
0.398 |
8 |
18 |
0.706 |
92.4 |
22.5 |
0.408 |
9 |
23 |
0.822 |
80.3 |
20 |
0.539 |
14 |
29 |
0.805 |
83.0 |
15 |
0.532 |
25 |
41 |
0.748 |
84.3 |
10 |
0.805 |
92 |
121 |
0.860 |
70.8 |
5 |
0.743 |
99 |
119 |
0.722 |
91.5 |
2.5 |
0.727 |
83 |
103 |
0.776 |
89.0 |
Vehicle control |
0.754 |
100 |
100 |
0.717 |
95.2 |
Vehicle control for positive control (DMSO) |
0.754 |
74 |
91 |
0.793 |
85.2 |
Untreated control |
0.727 |
99 |
122 |
0.810 |
86.1 |
Positive control NQO (0.1 μg/mL) |
0.360 |
17 |
6 |
0.386 |
1473.9* |
CP = Cyclophosphamide
RS= Relative survival values (%) corrected with the post treatment cell concentrations
ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)
NQO = 4-Nitroquinoline-N-oxide
RTG = Relative total growth
PE = Plating efficiency
* = Statistically significant
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In Vitro Bacterial Gene Mutation
In the key study (Bowles 2008), the potential for the test material to case in vitro bacterial gene mutation was determined in an Ames test. The study was performed using bacterial strains S. typhimurium TA98, TA100 TA1535 and TA1537, and E. coli WP2 uvrA. The study was conducted in accordance with the OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100 guidelines.
Bacterial strains were treated with the test material using the plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. 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 material formulations. Additional dose levels (5 and 15 µg/plate) were included (where applicable) to allow for test material induced toxicity, in Salmonella typhimurium strains only, ensuring that at least four non‑toxic doses were achieved.
The vehicle (sterile distilled water) 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 test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E.coli strain WP2uvrA-at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number.
A pale flakey precipitate was observed at and above 1500 µg/plate, this 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 material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.
Mammalian Chromosome Aberration Test
In the key study (Hargitai 2014), the clastogenicity of a structural analogue of the registered material was investigated in Chinese hamster lung fibroblasts (V79) in a chromosome aberration assay performed in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375 under GLP conditions. The study was therefore assigned a reliability score of 2 in accordance with the criteria for assessing data quality as described in Klimisch et al (1997).
Cells were seeded into culture dishes (5 x 10⁵ cells/dish concentration) and incubated for 24 hours prior to exposure to the test material. The cells were exposed to the test material in the presence and absence of metabolic activation (S9). The cells were exposed to the test material at the following concentrations:
- Assays 1 and 3
Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment).
- Assay 2
Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment) and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment).
Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.
Marked cytotoxicity was observed at the higher doses; however a minimum of three analysable concentrations were available for evaluation for each assay.
In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 1 was considered to be invalid and was discounted.
Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.
Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations.
Mammalian Cell Gene Mutation Test
In the key study (Hargitai 2014), the mutagenicity of a structural analogue of the registered material was assessed in an in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) which was performed in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al (1997).
The test was performed using the mouse lymphoma L5178Y cell model up to cytotoxic and precipitating concentrations in the absence and presence of an exogenous metabolic activation system (S9 -mix).
The following conditions were used in the study:
ASSAY 1
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL
ASSAY 2
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL
The expression time and selection time for both assays (both in the presence and absence of metabolic activation) were 3 days and 2 weeks, respectively.
No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis was observed in any of the assays either with or without metabolic activation.
Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.
Justification for selection of genetic toxicity endpoint
No single key study was selected on the basis that the available
studies all address different aspects of genetic toxicity and the data
should be considered as a whole. All three studies were conducted in
accordance with standardised guidelines under GLP conditions.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to germ cell mutagenicity.
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.
