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EC number: 700-956-5 | CAS number: -
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, 2013
negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 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:
- 2013
- 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: July 2012; signature: November 2012
- Type of assay:
- bacterial reverse mutation assay
- 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: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 1 range-finding test (plate incorporation method): 0 (solvent control), 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2 (pre-incubation method):
Salmonella strains and E.coli strain with and without S9-mix: 0 (solvent control), 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Up to sevent test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at 100 mg/ml in solubility checks performed. Acetone was therefore 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 (pre-incubation)
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.
Experiment 2. Measured aliquots (0.1 ml) of one of the bacterial cultures followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten amino acid supplemented, top agar. Subsequently, the procedure for incubation and duration was the same as in Experiment 1.
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).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
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).
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
Under the conditions of this study the test material 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 B13/14, US EPA OPPT 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance 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 substance using both the Ames plate incorporation and pre incubation methods at up to seven 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 the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of S9-mix), initially from 1500 μg/plate in both the presence and absence of S9-mix. In the main test (pre-incubation method) the test item induced toxicity to all of the tester strains with Salmonella strain TA1537 exhibiting weakened lawns from 500 μg/plate in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. It was concluded that, under the conditions of this assay, the test substance 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:
- 2014
- 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:
- 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
- Qualifier:
- according to guideline
- Guideline:
- other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.
- 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: March 2014; signature: May 2014
- 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 non-smoking 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. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fraction: PB/βNF S9 20/10/13 and PB/βNF S9 15/12/13
- Test concentrations with justification for top dose:
- The proposed maximum dose level following initial assessment was 5000 μg/mL, the maximum recommended dose level. The purity of the test item was was accounted for in the test item formulations. 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 5000 μg/mL range (full results recorded in the full study report).
I. Preliminary toxicity test: 0 (control) , 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 4465 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%), 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. Micronucleus Test - Experiment 1:
4(20)-hour without S9: 0, 40, 80, 160, 320, 480, 640 μg/mL
4(20)-hour with S9: 0, 40, 80, 160, 320, 640 and 800 μg/mL
III. Micronucleus Test - Experiment 2:
24-hour without S9: 0, 22.5, 45, 90, 100, 180, 360 and 640 μg/mL
4(20)-hour with S9: 0, 45, 90, 180, 360, 640, 800 and 1080 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in aqueous media at 50 mg/mL and dimethyl sulphoxide at
500 mg/mL but was miscible in acetone at 500 mg/mL in solubility checks. Due to toxicity issues with acetone, all cultures were dosed using 0.05 mL (50 μL) aliquots. Consequently, the test item was formulated at double the usual concentration to allow a maximum achievable concentration in the culture flasks. The maximum dose level achieved was 893 mg/mL (4465 μg/mL) because of the volume of the test item in the final formulation. Therefore, the maximum practical dose level was 4465 μg/mL.
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 5000 μg/mL range (full results recorded in the full study report). - 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, 10% (FBS); 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 0.05 mL (50 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% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
- In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL. 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:
- In Experiment 1, 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 0.05 mL (50 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.
- In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. 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 0.05 mL (50 μL) 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 (%) 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 culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. - Evaluation criteria:
- Positive response criteria
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of laboratory historical control data.
And
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Negative response criteria
A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Assay acceptance criteria
- Negative Control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.
- Positive Control: All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix. - 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 cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 78.13 μg/mL, in all of the exposure groups, which became greasy/oily at and above 625 μg/mL in the 4(20)-hour exposure in the absence of S9-mix but at and above 312.5 μg/mL in the other two exposure groups. Experiment 1 and 2 indicated: The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Preliminary Toxicity Test. For Experiment 1: Precipitate observations were made at the end of exposure and cloudy precipitate was noted at 80 μg/mL in both exposure groups which became greasy/oily at and above 160 μg/mL. For Experiment 2: A greasy/oily precipitate of the test item was observed at the end of exposure, at and above 90 μg/mL, in both the 4(20)-hour exposure group and the 24-hour continuous exposure group.
- Other confounding effects: In the preliminary test: Haemolysis was observed following exposure to the test item between 78.13 μg/mL and 625 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix only. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 4465 μg/mL. The maximum dose was the maximum practical dose level. The selection of the maximum dose level was based on toxicity for both Experiment 1 and Experiment 2.
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
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
Under the conditions of this study, the test substance was considered to be non-clastogenic to human lymphocytes in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 473, EC Method B.10, US EPA 870.5375 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, 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 three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. Within experiment 1, 4 hours 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 and a 4 hours exposure in the absence of metabolic activation (S9-mix) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9-mix was repeated (using a 1% final S9-mix concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test which included the following groups and final concentrations of test item: 4(20)-hour without S9-Mix: 0, 40, 80, 160, 320, 480, 640 μg/mL; 4(20)-hour with S9-Mix (2%): 0, 40, 80, 160, 320, 640, 800 μg/mL; 24-hour without S9-Mix: 0, 22.5, 45, 90, 100, 180, 360, 640 μg/mL and 4(20)-hour with S9-Mix (1%): 0, 45, 90, 180, 360, 640, 800, 1080. 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 was deemed to be toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that generally induced approximately 50% mitotic inhibition. Under the conditions of this study, the test substance was considered to be non-clastogenic to human lymphocytes in vitro.
Referenceopen allclose all
Table 1 : Test Results: Range-finding test: 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 (Acetone) |
95 (100) 114 12.7# 90 |
17 (15) 17 2.9 12 |
33 (36) 41 4.2 35 |
32 (26) 27 6.6 19 |
15 (11) 7 4.0 12 |
||||||||
5 µg |
96 (94) 92 2.1 95 |
19 (17) 18 2.1 15 |
33 (36) 39 3.1 37 |
26 (21) 17 4.7 19 |
17 (13) 12 3.2 11 |
||||||||
15 µg |
91 (101) 101 10.5 112 |
12 (11) 13 2.1 9 |
35 (37) 37 1.5 38 |
22 (24) 24 2.0 26 |
9 (9) 8 0.6 9 |
||||||||
50 µg |
81 (90) 79 17.9 111 |
19 (16) 18 4.4 11 |
38 (31) 24 7.0 31 |
25 (21) 20 3.6 18 |
11 (13) 11 2.9 16 |
||||||||
150 µg |
91 (90) 82 7.1 96 |
11 (14) 15 3.1 17 |
29 (28) 28 0.6 28 |
19 (17) 16 2.1 15 |
14 (11) 12 3.6 7 |
||||||||
500 µg |
90 (102) 108 10.7 109 |
14 (14) 13 0.6 14 |
37 (32) 31 5.0 27 |
17 (20) 21 2.3 21 |
8 (9) 11 2.1 7 |
||||||||
1500 µg |
96 TP (100) 89 TP 14.0 116 TP |
17 TP (17) 18 TP 1.5 15 TP |
29 P (30) 29 P 1.7 32 P |
35 P (24) 11 P 12.1 25 P |
7 TP (7) 7 TP 0.6 6 TP |
||||||||
5000 µg |
84 TP (56) 59 TP 30.1 24 TP |
6 TP (9) 7 TP 3.8 13 TP |
28 TP (36) 35 TP 8.0 44 TP |
16 TP (17) 19 TP 1.5 17 TP |
6 TP (6) 5 TP 0.6 6 TP |
||||||||
Positive controls S9-Mix (-) |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||
Dose |
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
Level No. of Revertants |
851 (702) 616 129.5 639 |
626 (555) 462 84.2 577 |
1155 (1031) 1068 146.6 869 |
180 (190) 184 14.6 207 |
1096 (846) 696 217.7 747 |
||||||||
|
|
||||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|
||||||||||
Base-pair substitution strains |
Frameshift strains |
|
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
||||||||
Solvent Control (Acetone) |
116 (106) 102 8.4# 101 |
16 (15) 10 5.0 20 |
38 (35) 36 3.6 31 |
37 (35) 39 5.9 28 |
11 (10) 12 2.1 8 |
|
|||||||
5 µg |
110 (105) 103 4.4 102 |
13 (13) 11 1.5 14 |
30 (30) 30 0.6 31 |
28 (27) 26 1.2 26 |
7 (8) 10 2.1 6 |
|
|||||||
15 µg |
86 (100) 111 12.8 103 |
16 (14) 8 4.9 17 |
38 (36) 34 2.0 36 |
16 (20) 16 6.4 27 |
12 (12) 12 0.6 11 |
|
|||||||
50 µg |
101 (98) 103 7.6 89 |
9 (11) 13 2.1 12 |
33 (35) 44 8.6 27 |
29 (26) 26 2.5 24 |
9 (9) 7 2.0 11 |
|
|||||||
150 µg |
103 (110) 123 11.0 105 |
13 (12) 13 1.2 11 |
35 (36) 35 1.2 37 |
22 (25) 30 4.6 22 |
12 (11) 12 1.7 9 |
|
|||||||
500 µg |
122 (101) 71 26.8 111 |
14 (13) 9 3.2 15 |
42 (38) 40 4.7 33 |
32 (32) 32 0.6 33 |
6 (9) 10 2.3 10 |
|
|||||||
1500 µg |
119 TP (110) 111 TP 9.5 100 TP |
11 TP (13) 17 TP 3.8 10 TP |
29 P (36) 39 P 5.8 39 P |
32 P (32) 29 P 3.5 36 P |
10 P (10) 10 P 0.0 10 P |
|
|||||||
5000 µg |
94 TP (91) 95 TP 6.7 83 TP |
10 TP (12) 13 TP 1.7 13 TP |
38 TP (37) 38 TP 2.3 34 TP |
30 P (31) 34 P 2.6 29 P |
18 TP (11) 8 TP 5.8 8 TP |
|
|||||||
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|
||||||
Dose |
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|
|||||||
Level No. of Revertants |
1376 (1533) 1484 186.4 1739 |
274 (259) 249 13.4 253 |
334 (377) 440 55.6 358 |
269 (283) 285 12.7 294 |
301 (243) 182 59.5 245 |
|
|||||||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-AminoacridineENNG:
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
T: Partial absence of bacterial background lawn
P: Precipitate
#: Standard deviation
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 (Acetone) |
115 (120) 123 4.6# 123 |
21 (22) 24 2.1 20 |
24 (33) 42 9.0 33 |
19 (25) 21 8.7 35 |
10 (11) 16 4.2 8 |
|||||||
5 µg |
104 (116) 105 19.9 139 |
16 (18) 18 2.5 21 |
28 (27) 25 1.7 28 |
18 (16) 16 2.5 13 |
4 (7) 9 2.5 7 |
|||||||
15 µg |
134 (107) 68 34.6 119 |
18 (20) 20 2.0 22 |
25 (31) 36 5.7 33 |
15 (21) 20 6.0 27 |
9 (12) 16 3.8 10 |
|||||||
50 µg |
100 (99) 98 1.2 100 |
17 (17) 18 0.6 17 |
25 (28) 31 3.1 29 |
19 (18) 17 1.2 19 |
10 (12) 15 2.6 11 |
|||||||
150 µg |
99 (106) 108 6.7 112 |
17 (20) 19 3.6 24 |
25 (31) 37 6.0 31 |
13 (19) 27 7.4 16 |
10 (11) 15 4.0 7 |
|||||||
500 µg |
123 (121) 132 11.6 109 |
16 (17) 20 2.3 16 |
19 (25) 29 5.5 28 |
25 (25) 26 1.0 24 |
13 T (10) 10 T 3.5 6 T |
|||||||
1500 µg |
124 TP (110) 108 TP 13.6 97 TP |
22 TP (15) 9 TP 6.5 15 TP |
46 P (33) 27 P 11.3 26 P |
15 TP (15) 17 TP 2.0 13 TP |
10 TP (10) 10 TP 0.6 11 TP |
|||||||
5000 µg |
118 TP (100) 98 TP 16.6 85 TP |
16 TP (12) 9 TP 3.5 12 TP |
30 TP (26) 28 TP 4.7 21 TP |
18 TP (16) 16 TP 2.5 13 TP |
3 TP (4) 5 TP 1.2 3 TP |
|||||||
Positive controls S9-Mix (-) |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
Dose |
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|||||||
Level No. of Revertants |
589 (607) 627 19.1 605 |
581 (544) 526 32.0 525 |
593 (649) 695 51.8 660 |
129 (137) 147 9.3 134 |
344 (507) 646 152.5 532 |
|||||||
|
|
|||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|
|||||||||
Base-pair substitution strains |
Frameshift strains |
|
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
|||||||
Solvent Control (Acetone) |
77 (85) 72 17.8# 105 |
20 (14) 13 6.0 8 |
30 (32) 32 2.0 34 |
22 (26) 31 4.6 25 |
8 (12) 14 3.2 13 |
|
||||||
5 µg |
92 (86) 90 8.7 76 |
16 (12) 9 3.8 10 |
36 (38) 44 4.9 35 |
25 (27) 30 2.9 25 |
13 (12) 7 4.2 15 |
|
||||||
15 µg |
86 (87) 82 5.6 93 |
11 (9) 9 1.5 8 |
31 (31) 31 0.6 30 |
26 (23) 26 4.6 18 |
18 (13) 12 4.2 10 |
|
||||||
50 µg |
95 (96) 96 1.5 98 |
10 (9) 9 1.0 8 |
32 (31) 25 5.1 35 |
24 (30) 31 6.0 36 |
10 (13) 16 3.1 12 |
|
||||||
150 µg |
101 (87) 89 14.6 72 |
11 (14) 15 2.3 15 |
26 (32) 32 5.5 37 |
26 (26) 21 5.0 31 |
13 (12) 10 2.1 14 |
|
||||||
500 µg |
74 (72) 63 8.6 80 |
11 (12) 11 2.3 15 |
34 (39) 40 4.2 42 |
21 (22) 32 9.1 14 |
16 T (11) 10 T 4.6 7 T |
|
||||||
1500 µg |
81 TP (78) 75 TP 3.1 77 TP |
13 TP (11) 8 TP 2.5 11 TP |
29 P (32) 34 P 2.9 34 P |
25 P (23) 16 P 6.2 28 P |
12 TP (10) 8 TP 2.0 10 TP |
|
||||||
5000 µg |
61 TP (68) 69 TP 7.0 75 TP |
9 TP (9) 9 TP 0.6 8 TP |
31 TP (35) 33 TP 5.9 42 TP |
24 TP (19) 17 TP 4.0 17 TP |
8 TP (10) 14 TP 3.5 8 TP |
|
||||||
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|
|||||
Dose |
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|
||||||
Level No. of Revertants |
849 (736) 653 101.2 707 |
201 (193) 179 12.2 199 |
346 (325) 334 26.1 296 |
227 (252) 255 24.1 275 |
167 (174) 185 9.6 170 |
|
||||||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-AminoacridineENNG:
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
T: Partial absence of bacterial background lawn
P: Precipitate
#: Standard deviation
Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls)
Range-finding test: EXP1 |
||||
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
83 94 (91) 97 |
17 13 (19) 27 |
28 29 (32) 38 |
15 25 (22) 26 |
7 4 (7) 10 |
Main test: EXP2 |
||||
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
125 109 (114) 108 |
25 20 (21) 17 |
33 40 (36) 35 |
26 22 (24) 25 |
9 8 (9) 10 |
1. Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given below:
4(20)-hour without S9-Mix:
0*, 40, 80*, 160*, 320, 480*, 640, MMC 0.4* (μg/mL)
4(20)-hour with S9-Mix (2%):
0*, 40, 80, 160, 320*, 640*, 800*, CP 5*(μg/mL)
Where: * = Dose levels selected for metaphase analysis; MMC = Mitomycin C and CP = Cyclophosphamide
Qualitative observations in that a modest inhibition of mitotic index was observed, and that 37% mitotic inhibition was achieved at 480 μg/mL in the absence of S9-mix. In addition, there was evidence of a plateau of toxicity between 320 and 640 μg/mL in this exposure group. In the presence of S9-mix, 44% mitotic inhibition was achieved at 800 μg/mL which was considered to be satisfactory to assess for metaphases because it approached the 50% Mitotic Inhibition limit. The maximum dose levels selected for metaphase analysis were, therefore, based on toxicity and limited to these dose levels.
Precipitate observations were made at the end of exposure and cloudy precipitate was noted at 80 μg/mL in both exposure groups which became greasy/oily at and above 160 μg/mL.
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- 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 significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
2. Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given below:
24-hour without S9-Mix:
0*, 22.5*, 45*, 90*, 100, 180, 360, 640, MMC 0.2*(μg/mL)
4(20)-hour with S9-Mix:
0*, 45, 90, 180, 360*, 640*, 800*, 1080, CP 5*(μg/mL)
Where: * = Dose levels selected for metaphase analysis; MMC = Mitomycin C and CP = Cyclophosphamide
Qualitative observations in that moderate dose-related inhibition of mitotic index was observed, and that, in the absence of S9-mix, 35% and 52% mitotic inhibition was achieved at 45 μg/mL and 90 μg/mL, respectively. In the presence of S9-mix, a dose-related inhibition of mitotic index was observed at 800 μg/mL (48%). The Mitotic Inhibition values noted at the maximum concentrations assessed for the presence of aberrations were considered to be near optimum toxicity.
Greasy/oily precipitate of the test item was observed at the end of exposure, at and above 90 μg/mL, in both the 4(20)-hour exposure group and the 24-hour continuous exposure group.
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- 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 significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
OECD TG 471, 2013 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OPPT 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance 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 substance using both the Ames plate incorporation and pre incubation methods at up to seven 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 the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of S9-mix), initially from 1500 μg/plate in both the presence and absence of S9-mix. In the main test (pre-incubation method) the test item induced toxicity to all of the tester strains with Salmonella strain TA1537 exhibiting weakened lawns from 500 μg/plate in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. It was concluded that, under the conditions of this assay, the test substance 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.
OECD TG 473, 2014 - The study was performed to the requirements of OECD TG 473, EC Method B.10, US EPA 870.5375 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, 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 three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. Within experiment 1, 4 hours 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 and a 4 hours exposure in the absence of metabolic activation (S9-mix) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9-mix was repeated (using a 1% final S9-mix concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test which included the following groups and final concentrations of test item: 4(20)-hour without S9-Mix: 0, 40, 80, 160, 320, 480, 640 μg/mL; 4(20)-hour with S9-Mix (2%): 0, 40, 80, 160, 320, 640, 800 μg/mL; 24-hour without S9-Mix: 0, 22.5, 45, 90, 100, 180, 360, 640 μg/mL and 4(20)-hour with S9-Mix (1%): 0, 45, 90, 180, 360, 640, 800, 1080. 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 was deemed to be toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that generally induced approximately 50% mitotic inhibition. Under the conditions of this study, the test substance was considered to be non-clastogenic to human lymphocytes in vitro.
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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