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Registration Dossier
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EC number: 422-120-6 | CAS number: 166432-53-7
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, 1995
negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2001
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:
- 11-01-1995 to 28-03-1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. Relevant validity criteria were met with acceptable deviations which were in accordance with the guidelines at the time of the study.
- Remarks:
- study did not include TA102 or WP2uvrA strains
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- study did not include TA102 or WP2uvrA strains ; relevant validity criteria were met with acceptable deviations which were in accordance with the guidelines at the time of the study.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- see above
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: January 1994 ; signature: March 1994
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine or tryptophan locus
- Species / strain / cell type:
- other: TA98, TA100, TA1535, TA1537 and TA1538
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased Aro. S9/24/11/94 prepared in house (dates within full study report)
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory - Test concentrations with justification for top dose:
- Preliminary toxicity test (TA100): 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method):
Without S9: All strains between dose range: 0, 0.05, 0.15, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
With S9 (10%) : All strains between dose range: 0, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation method):
Without S9: All strains between dose range: 0, 0.05, 0.15, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
With S9 (10%) : All strains between dose range: 0, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
Testing was conducted in up to eight dose levels and/or to ensure a minimum of three or four non-toxic dose levels was achieved and testing up to the maximum recommended dose level of 5000 µg/plate, depending on strain specific cytotoxicity. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was fully miscible in acetone at the required concentrations in solubility checks performed. Acetone was selected as the vehicle. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-Aminoanthracene (2AA) ; 4-nitro-o-phenylenediamine (4NOPD)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)
DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity) of the background lawn. 0.1 mL aliquots of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten. trace histidine supplemented. top agar at 45°C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or vehicle control was also added to each of the two tubes followed by either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates Cone tube per platel. This procedure was repeated. In triplicate. for each bacterial strain and for each concentration of test material. Positive controls were similarly prepared with and without S9 activation system, as applicable.
Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity) of the background lawn.
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.
2. A reproducible increase at one or more concentrations.
3. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. To be considered negative the number of induced evertants compared to spontaneous revertants should be less than two-fold at each dose level employed. the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was both toxicity and the maximum recommended dose depending on bacterial strain type and presence/absence of S9 mix.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
To be considered negative the number of induced evertants compared to spontaneous revertants should be less than two-fold at each dose level employed. the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was both toxicity and the maximum recommended dose depending on bacterial strain type and presence/absence of S9 mix. - Statistics:
- Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium 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
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD Guideline 471 and EU Method B.14 for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 range finding test was predetermined based on the results of a preliminary toxicity assay. The dose levels were 0.05 to 5000 µg/plate depending on the specific strain of S.typhimurium. The experiment was repeated on a separate day using a dose range based on Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. 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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on presence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 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:
- 16-06-2000 to 23-11-2000
- 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
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: February 2000 ; signature: April 2000
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable (chromosome aberration test)
- Species / strain / cell type:
- lymphocytes: Human lymphocytes
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a screened volunteer. Who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 17 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fraction: PB/βNF S9 08/4/00 and 14/7/00
- Test concentrations with justification for top dose:
- The maximum dose level was 2063.3 µg/mL or 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2063.3 μg/mL range (full results recorded in the full study report).
I. Preliminary toxicity test: 0 (control) , 8.06, 16.12, 32.24, 64.48, 128.96, 257.91, 515.83, 1031.65 and 2063.3 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2% or 1% for PT and Exp 1 or Exp 2, respectively), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
II. Main Test:
Experiment 1:
4(20)-hour without S9: 0*, 16.12*, 32.24*, 64.68*, 128.96, 193.44, 257.91, MMC 0.4* μg/mL
4(20)-hour with S9: 0*, 32.24*, 64.68*, 128.96*, 257.91, 386.87, 515.83, CP 25* μg/mL
Experiment 2:
4(20)-hour with S9: 0*, 8.06, 16.12, 32.24*, 64.48*, 96.72, 128.96*, CP 12.5* μg/mL
24-hour without S9: 0*, 8.06, 16.12*, 32.24*, 64.48*, 96.72, 128.96, MMC 0.2* μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble in DMSO at 2063.3 µg/mL, in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 2063.3 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2063.3 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:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- Full details on the positive controls is reported in the full study report.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 15% (FCS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood
DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 10% or 20% S9-mix (i.e. 1% or 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment 1 or Main Experiment 2, respectively. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), UK EMS (1983). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. - Evaluation criteria:
- The test was evaluated in accordance with the OECD TG 473 guidelines.
Statistical analysis is also performed. The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
- Species / strain:
- lymphocytes: Human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There was no significant change in osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 128.96 μg/ml, in the 4(20)-hour pulse exposure groups without S9, at and above 1031.65 μg/ml in the presence of S9 and at and above 257.91 μg/ml in the continuous exposure group. Main test 1: Same as preliminary test. Main Test 2: No precipitate observed.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 2063.3 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.
COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).
ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (1%): 0, 32.24, 64.68, 128.96, 257.91, 386.87, 515.83 μg/mL and without S9-Mix: 0, 16.12, 32.24, 64.68, 128.96, 193.44, 257.91 μg/mL. The doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (2%): 0, 8.06, 16.12, 32.24, 64.48, 96.72, 128.96 μg/mL and 24-hour without S9: 0, 8.06, 16.12, 32.24, 64.48, 96.72, 128.96 μg/mL. All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system which included a dose level that induced approximately 50% mitotic inhibition. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Referenceopen allclose all
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
|||||||
Solvent Control (acetone) |
139 151 128 |
(139) 11.5# |
18 15 18 |
(18) 1.7 |
20 30 19 |
(23) 6.1 |
18 18 18 |
(18) 0.0 |
18 22 23 |
(21) 2.6 |
|
0.05 µg |
N/T
|
|
N/T
|
|
N/T
|
|
N/T
|
|
17 17 12 |
(15) 2.9 |
|
0.15 µg |
N/T
|
|
N/T
|
|
N/T
|
|
15 22 20 |
(19) 3.6 |
20 17 22 |
(20) 2.5 |
|
0.50 µg |
N/T
|
|
20 22 15 |
(19) 3.6 |
N/T
|
|
17 19 23 |
(20) 3.1 |
20 13 29 |
(20) 8.0 |
|
1.50 µg |
N/T
|
|
25 17 15 |
(19) 5.3 |
N/T
|
|
15 28 20 |
(21) 6.6 |
17 8 13 |
(13) 4.5 |
|
5 µg |
N/T
|
|
19 20 10 |
(16) 5.5 |
N/T
|
|
19 25 14 |
(19) 5.5 |
17 17 13 |
(16) 2.3 |
|
15 µg |
N/T |
|
14 13 32 |
(20) 10.7 |
38 30 22 |
(30) 8.0 |
20 20 5 |
(18) 2.9 |
28 15 8 |
(17) 10.1 |
|
50 µg |
128 121 125 |
(125) 3.5 |
18 10 15 |
(14) 4.0 |
32 33 24 |
(30) 4.9 |
18V 17V 15V |
(17) 1.5 |
10S 12S 9S |
(10) 1.5 |
|
150 µg |
132 94 122 |
(116) 19.7 |
14 10 13 |
(12) 2.1 |
27 25 32 |
(28) 3.6 |
13V 17V 15V |
(15) 2.0 |
9V 14V 9V |
(11) 2.9 |
|
500 µg |
116 95 128 |
(113) 16.7 |
12S 8S 12S |
(11) 2.3 |
13 29 24 |
(22) 8.2 |
13V 17V 14V |
(15) 2.1 |
N/T
|
|
|
1500 µg |
102 91 125 |
(106) 17.3 |
N/T |
|
23S 20S 20S |
(21) 1.7 |
N/T |
|
N/T
|
|
|
5000 µg |
49V 48V 32V |
(43) 9.5 |
N/T |
|
15V 19V 22V |
(19) 3.5 |
N/T
|
|
N/T
|
|
|
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
|||||
3 µg |
5 µg |
5 µg |
0.2 µg |
80 µg |
|||||||
466 427 430 |
(441) 21.7 |
174 169 183 |
(175) 7.1 |
384 364 343 |
(364) 3.5 |
165 160 191 |
(172) 16.6 |
470 281 344 |
(365) 96.2 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
|||||||
Solvent Control (acetone) |
133 102 113 |
(116) 15.7# |
24 33 20 |
(26) 6.7 |
17 17 28 |
(21) 6.4 |
24 24 25 |
(24) 0.6 |
17 13 9 |
(13) 4.0 |
|
1.5 µg
|
N/T
|
|
N/T |
|
30 23 27 |
(27) 3.5 |
N/T |
|
12 5 14 |
(10) 4.7 |
|
5 µg
|
N/T
|
|
N/T |
|
18 39 25 |
(23) 6.1 |
22 28 20 |
(23) 4.2 |
12 15 9 |
(12) 3.0 |
|
15 µg |
N/T |
N/T |
|
20 30 19 |
(27) 6.9 |
30 35 20 |
(28) 7.6 |
9 10 15 |
(11) 3.2 |
||
50 µg |
118 113 137 |
(123) 12.7 |
32 29 18 |
(26) 7.4 |
35 23 23 |
(27) 6.9 |
33 23 24 |
(27) 5.5 |
5S 10S 8S |
(8) 2.5 |
|
150 µg |
156 128 125 |
(136) 17.1 |
33 28 25 |
(29) 4.0 |
30 30 24 |
(28) 3.5 |
29 22 24 |
(25) 3.6 |
7S 8S 12S |
(9) 2.6 |
|
500 µg |
117 125 129 |
(124) 6.1 |
20 32 25 |
(26) 6.0 |
22 25 12 |
(20) 6.8 |
20 33 22 |
(25) 7.0 |
15V 9V 9V |
(11) 3.5 |
|
1500 µg |
75 77 115 |
(89) 22.5 |
5 20 13 |
(13) 7.5 |
18 14 14 |
(15) 2.3 |
15 20 27 |
(21) 6.0 |
10V 9V 10V |
(10) 0.6 |
|
5000 µg |
23S 12S 17S |
(17) 5.5 |
10S 15S 10S |
(11) 2.9 |
14 12 17 |
(14) 2.5 |
18S 28S 18S |
(21) 5.8 |
N/T |
|
|
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
2AA |
2AA |
|||||
1 µg |
2 µg |
0.5 µg |
0.5 µg |
2 µg |
|||||||
479 495 705 |
(560) 126.1 |
253 271 241 |
(255) 15.1 |
169 199 200 |
(189) 17.6 |
175 200 204 |
(193) 15.7 |
65 66 65 |
(65) 0.6 |
||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
4NOPD4-nitro-o-phenylenediamine
2AA 2-Aminoanthracene
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
T Toxic, no bacterial lawn
# Standard deviation
C Contaminated
X Plate unscorable
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 |
TA1538 |
TA98 |
TA1537 |
|||||||
Solvent Control (acetone) |
106 90 97 |
(98) 8.0# |
14 10 13 |
(12) 2.1 |
23 15 17 |
(18) 4.2 |
21 23 23 |
(22) 1.2 |
8 13 8 |
(10) 2.9 |
|
0.15 µg
|
N/T |
|
N/T |
|
N/T |
|
19 18 17 |
(18) 1.0 |
N/T |
|
|
0.50 µg
|
N/T |
|
N/T |
|
N/T |
|
15 29 18 |
(21) 7.4 |
9 8 12 |
(10) 2.1 |
|
1.50 µg
|
N/T |
|
17 17 13 |
(16) 2.3 |
N/T |
|
20 25 10 |
(18) 7.6 |
11 11 8 |
(10) 1.7 |
|
5 µg
|
N/T |
|
15 17 19 |
(17) 2.0 |
N/T |
|
17 20 20 |
(19) 1.7 |
8 8 11 |
(9) 1.7 |
|
15 µg
|
N/T |
|
24 15 13 |
(17) 5.9 |
14 9 27 |
(17) 9.3 |
17 24 19 |
(20) 3.6 |
7 11 7 |
(8) 2.3 |
|
50 µg |
107 95 72 |
(91) 17.8 |
25 19 10 |
(18) 7.5 |
15 18 15 |
(16) 1.7 |
18V 19V 17V |
(18) 1.0 |
13S 2S 3S |
(6) 6.1 |
|
150 µg |
111 86 91 |
(96) 13.2 |
14 17 14 |
(15) 1.7 |
22 18 9 |
(16) 6.7 |
N/T |
|
8V 8V 10V |
(9) 1.2 |
|
500 µg |
101 101 95 |
(99) 3.5 |
7S 14S 14S |
(12) 4.0 |
17 13 13 |
(14) 2.3 |
N/T |
|
N/T |
|
|
1500 µg |
71 65 74 |
(70) 4.6 |
N/T |
|
13S 17S 9S |
(13) 4.0 |
N/T |
|
N/T |
|
|
5000 µg |
48V 59V 63V |
(57) 7.8 |
N/T |
|
9V 14V 6V |
(10) 4.0 |
N/T |
|
N/T |
|
|
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
4NOPD |
4NQO |
9AA |
|||||
3 µg |
5 µg |
5 µg |
0.2 µg |
80 µg |
|||||||
391 368 380 |
(380) 11.5 |
266 216 275 |
(252) 31.2 |
343 344 324 |
(337) 11.3 |
144 129 133 |
(135) 7.8 |
687 810 709 |
(735) 65.6 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
TA1538 |
TA98 |
TA1537 |
|||||||
Solvent Control (acetone) |
80 76 75 |
(77) 2.6# |
18 23 19 |
(20) 2.5 |
30 27 34 |
(30) 3.5 |
25 17 34 |
(25) 8.5 |
14 13 18 |
(15) 2.6 |
|
0.50 µg
|
N/T |
|
N/T |
|
N/T |
|
N/T |
|
15 11 12 |
(13) 2.1 |
|
1.50 µg
|
N/T |
|
N/T |
|
N/T |
|
N/T |
|
13 9 13 |
(12) 2.3 |
|
5 µg
|
N/T |
|
N/T |
|
N/T |
|
N/T |
|
15 17 12 |
(15) 2.5 |
|
15 µg
|
N/T |
|
N/T |
|
N/T |
|
N/T |
|
12 12 7 |
(10) 2.9 |
|
50 µg |
75 125 107 |
(102) 25.3 |
15 19 24 |
(19) 4.5 |
30 34 23 |
(29) 5.6 |
34 22 32 |
(29) 6.4 |
13S 9S 10S |
(11) 2.1 |
|
150 µg |
111 86 89 |
(96) 13.7 |
14 23 22 |
(20) 4.9 |
32 24 24 |
(27) 4.6 |
33 35 27 |
(32) 4.2 |
6S 6S 11S |
(8) 2.9 |
|
500 µg |
82 101 90 |
(91) 9.5 |
22 17 28 |
(22) 5.5 |
18 19 29 |
(22) 6.1 |
35 17 24 |
(25) 9.1 |
N/T |
|
|
1500 µg |
90 91 61 |
(81) 17.0 |
9 12 7 |
(9) 2.5 |
25 17 22 |
(21) 4.0 |
28 20 17 |
(22) 5.7 |
N/T |
|
|
5000 µg |
53V 59V 10V |
(41) 26.7 |
4S 3S 13S |
(7) 5.5 |
20 24 24 |
(23) 2.3 |
17S 10S 12S |
(13) 3.6 |
N/T |
|
|
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
2AA |
2AA |
|||||
1 µg |
2 µg |
0.5 µg |
0.5 µg |
2 µg |
|||||||
635 663 725 |
(675) 46.6 |
160 172 165 |
(165) 6.0 |
325 328 399 |
(351) 41.9 |
302 335 299 |
(312) 20.0 |
151 185 193 |
(178) 18.9 |
||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key study: OECD TG 471, 1995 : The study was performed to the requirements of OECD Guideline 471 and EU Method B.14 for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 range finding test was predetermined based on the results of a preliminary toxicity assay. The dose levels were 0.05 to 5000 µg/plate depending on the specific strain of S.typhimurium. The experiment was repeated on a separate day using a dose range based on Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. 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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on presence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S-9 mix.
Key study: OECD TG 473, 2001 : The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (1%): 0, 32.24, 64.68, 128.96, 257.91, 386.87, 515.83 μg/mL and without S9-Mix: 0, 16.12, 32.24, 64.68, 128.96, 193.44, 257.91 μg/mL. The doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (2%): 0, 8.06, 16.12, 32.24, 64.48, 96.72, 128.96 μg/mL and 24-hour without S9: 0, 8.06, 16.12, 32.24, 64.48, 96.72, 128.96 μg/mL. All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system which included a dose level that induced approximately 50% mitotic inhibition. Under the conditions of this study, the test item 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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