Registration Dossier
Registration Dossier
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EC number: 443-870-0 | CAS number: 163520-33-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test, plate incorporation, ±S9-mix): negative for mutagenic potential in bacteria with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, HPRT, V79 cells, ±S9-mix): negative for mutagenic potential in mammalian cells in vitro with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosomal Aberration, V79 cells, ±S9-mix): equivocal results in mammalian cells in vitro (questionable positive result with metabolic activation at highly cytotoxic and precipitating concentrations)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 Feb - 03 Mar 1994
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Only 2-aminoanthracene was used to test the efficacy of the S9 mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9 mix. Historical control data was not provided.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Current version adopted in 2020
- Deviations:
- yes
- Remarks:
- Only 2-aminoanthracene was used to test the efficacy of the S9 mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9 mix. Historical control data was not provided.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1983
- Deviations:
- not applicable
- 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 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
- Version / remarks:
- Adopted in 1985
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Adopted in 1985
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon for S. typhimurium strains, trp operon for E. coli strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : liver homogenate of 5 - 6 male Sprague Dawley rats (200 - 300 g) pretreated with Aroclor 1254 (single i.p. injection of 500 mg/kg bw) 5 days prior to sacrifice
- method of preparation of S9 mix: The S9 homogenate was diluted 1:10 with a co-factor solution. The resulting S9 mix contained the following components and concentrations: 33 mM KCl, 8 mM MgCl2, 5 mM, glucose-6-phosphate, 4 mM NADP, and 100 mM phosphate buffer (pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 concentration in the S9-mix was 10%. The volume of S9 mix in the final culture medium was 0.5 mL.
- quality controls of S9: Sterility of the S9 mix was indicated by the absence of contamination on the S9 mix sterility check plates. - Test concentrations with justification for top dose:
- First experiment (= range-finding experiment), second experiment, and cytotoxicity experiment: 4, 20, 100, 500, 2500, and 5000 µg/plate, with and without metabolic activation, all strains (except for the cytotoxicity experiment, here only TA 100 was tested)
5000 µg/plate was selected as the highest test concentration based on the results of the range-finding experiment, in which no cytotoxicity was observed up to and including the highest concentration of 5000 µg/plate tested. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG, 2.5 µg/plate, -S9, WP2uvrA), 2-aminoanthracene (2-AA, 0.5 to 10 μg/plate, +S9, all strains)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 independent experiments were performed.
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Following exposure, his+ and trp+ revertant colonies were counted. - Evaluation criteria:
- A test substance is considered mutagenic if either of the following conditions under a) and b) is achieved:
a) a test substance produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test substance induces a dose-dependent increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test substance at complete bacterial background lawn.
The test results must be reproducible. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (precipitation was observed at concentrations of 2500 µg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (precipitation was observed at concentrations of 2500 µg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (precipitation was observed at concentrations of 2500 µg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (precipitation was observed at concentrations of 2500 µg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- (precipitation was observed at concentrations of 2500 µg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: visible precipitation of the test substance on the plates has been observed at 2500 µg/plate and above
- Definition of acceptable cells for analysis: Identification of the different bacterial strains was performed periodically and all criteria for a valid assay were achieved as described in B.N. Ames, J. McCann and E. Yamasaki: Methods for detecting carcinogens and mutagens with the Salmonella / mammalian-microsome mutagenicity test (Mutation Res. 31 (1975) 347 - 364) and M.H.L. Green and W.J. Muriel: Mutagen testing using trp reversion in Escherichia coli (Mutation Res. 38 (1976) 3 - 32).
RANGE-FINDING/SCREENING STUDY:
The test substance was tested at concentrations of 4 to 5000 µg/plate in TA 100 and proved to be not toxic to the bacterial strain, therefore 5000 µg/plate was selected as the highest concentration tested.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : see Table 1 and 2 under "Any other information on results incl. tables".
Ames test:
- Signs of toxicity : No signs of toxicity were noted in any strain up to and included the highest concentration tested, i.e 5000 µg/plate.
- Mean number of revertant colonies per plate and standard deviation : The mean number of revertant colonies was not significantly increased for any strain at any concentration tested. See Table 1 and 2 under "Any other information on results incl. tables". - Conclusions:
- Under the conditions of the present study, the test substance was negative for genotoxicity in bacteria with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 May - 14 Jul 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The following parameters were not reported for the cell line used: stability of the modal chromosome number/karyotype stability, typical cell cycle length, spontaneous mutant frequency in the master cell stock.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Current version adopted in 2016
- Deviations:
- yes
- Remarks:
- The following parameters were not reported for the cell line used: stability of the modal chromosome number/karyotype stability, typical cell cycle length, spontaneous mutant frequency in the master cell stock.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1984
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Adopted in 1987
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
- Version / remarks:
- Adopted in 1986
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: cell line V79 of Chinese hamster lung fibroblasts derived from the cell bank of "Genetic Toxicology", Hoechst AG, Germany
For cell lines:
- Absence of Mycoplasma contamination: stocks of V79 cells were free of mycoplasma contamination
- Methods for maintenance in cell culture: Thawed stock cultures were kept at approx. 37 °C and approx. 4% CO2 in 175 cm² plastic flasks. About 5E+05 to 1E+06 cells were seeded into each flask in 30 mL of minimal essential medium (MEM) supplement with approx. 10% (v/v) fetal calf serum (FCS) containing approx. 2 mM L-glutamine and approx. 0.1% (w/v) neomycinsulfate. The cells were subcultured twice a week.
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes. For the selection of mutants the medium was supplemented with approx. 11 µg/mL thioguanine.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Cell culture medium was composed of minimal essential medium (MEM) with Hanks-salts and 25 mM Hepes buffer. For cell culturing, 30 mL of
MEM-cell culture medium was supplement with approx. 10% (v/v) FCS containing approx. 2 mM L-glutamine and approx. 0.1% (w/v) neomycinsulfate. The CO2 concentration was approx. 4%. The temperature was approx. 37 °C. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was prepared from male Sprague Dawley rats (Harlan Winkelmann, Borchen, Germany). Animals received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) 5 days before killing. After separation of the S9 fraction from liver homogenates, small portions were rapidly frozen and stored at approx. -80 °C for not longer than 6 months.
- method of preparation of S9 mix: An appropriate quantity of S9 fraction (protein concentration 62.6 g/L) was thawed and then mixed with S9 cofactor solution, which was kept on ice until usage. This preparation was termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCI2
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration of 0.3 mg/mL in the final cultures
- quality controls of S9: The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g.2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes. - Test concentrations with justification for top dose:
- Experiment I
Without S9 mix: 15, 50, 150, 375, 750, and 1500 µg/mL (4 h)
With S9 mix: 50, 150, 375, 750, 1500, and 2953.4 μg/mL (4 h)
Experiment II
Without S9 mix: 15, 50, 150, 375, 750, and 1500 µg/mL (4 h)
With S9 mix: 15, 50, 150, 375, 750, and 1500 μg/mL (4 h)
2953.4 µg/mL (= 10 mM) were tested only in the first experiment, because of high cytotoxicity. At this concentration no mutant selection could be performed. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: 9,10-dimethyl-1,2-benzanthracene, DMBA, 7.7 µg/mL in DMSO, +S9
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration : Treatment of cells with the test substance in the presence and absence of S9-mix was done in one flask per experimental point. Cells were sub-cultured in 5 flasks per dose for mutant selection 7 days post treatment.
- Number of independent experiments : 2 independent experiments, each with and without metabolic activation
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 1E+05 - 1E+06 cells/175 cm² flask 24 h before test substance application
- Test substance added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h with and without S9 mix
- Harvest time after the end of treatment (sampling/recovery times): After test substance exposure in flasks for 4 h (Day 2), the cells were subcultured and incubated for 7 days in flasks (until Day 9). On Day 9, cells were again subcultured in 5 flasks (75 cm² each) with culture medium containing 6-thioguanine for mutant selection (about 300 000 cells/flask) and incubated again for 7 days. On Day 16, cells were fixed, stained and counted.
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
- Selective agent: 6-thioguanine, 11 µg/mL, cells were exposed to the selective agent for a period of 7 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 300 000 cells/75 cm² flask; in the main experiments cytotoxicity of the test substance was assessed by the plating efficiency of cells from the culture flasks; the cultures were stained with methylene blue (10% (v/v) methylene blue in approx. 0.01% KOH solution) and survival was determined by counting the colonies after the incubation period
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth (RTG)
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Methylene blue stained colonies were counted. - Rationale for test conditions:
- At a concentration of 2953.4 µg/mL (= 10 mM) the test substance was soluble, but it precipitated after being added to the cell culture medium. The highest concentration at which no visible precipitation was observed, was 50 µg/mL without S9-mix and 150 µg/mL with S9-mix.
Precipitation of the test substance in the cell culture medium affected the measurement of the extinction in microtiterplates in the preliminary toxicity test. Therefore, in the main experiments, cytotoxicity of the test substance was assessed by the plating efficiency of cells from the culture flasks. - Evaluation criteria:
- This assay was considered valid if the following criteria were met:
- the spontaneous control data were within the laboratory's normal control range for the spontaneous mutant frequency.
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range.
The test compound is classified as mutagenic if:
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. Survival of the responding dose group must be at least 30%. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data. - Statistics:
- Mean values and standard deviation were calculated. Mann-Whitney-U-Test was performed as non-parametric significance test with positive controls only. Otherwise statistical evaluation was not necessary, because mutation frequencies for all test substance dose groups were in the range of the solvent controls.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 375 µg/mL -S9 and 1500 µg/mL +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The addition of test compound solutions did not have any obvious effect on the pH of the treatment medium.
- Effects of osmolality: The addition of test compound solutions did not have any obvious effect on the osmolality of the treatment medium.
- Precipitation: In the main experiments, the highest concentration at which no visible precipitation was observed was 50 µg/mL with S9-mix and 150 µg/mL without S9-mix.
RANGE-FINDING/SCREENING STUDIES
In preliminary toxicity experiments, cells were exposed to the test substance at concentrations up to 2953.4 µg/mL for 4 h, in the presence or absence of metabolic activation. 24 h after the treatment the cells were stained with crystal violet. Survival was determined by measurement of crystal violet extinction. However, high precipitation of the test substance in the cell culture medium affected the measurement of the extinction in the microtiter plates. Microscopic evaluation of the wells showed a low survival rate with metabolic activation and only a few surviving cells after treatment without metabolic activation in the high dose groups. Therefore, in the main experiments the method was changed and the toxicity of the test substance was assessed by determining the plating efficiency of cells in the culture flasks.
Based on plating efficiency results, 2953.4 µg/mL was selected as the highest dose level in the main mutation tests in the presence of metabolic activation. 5 lower concentrations were included in the treatment series. Without metabolic activation, a dose range from 15 µg/mL to 1500 µg/mL was chosen.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of metabolic activation in both experiments a dose-related decrease in survival (relative plating efficiency) was observed reaching 8.8% and 1.8%, respectively, of the solvent control value at the highest dose level tested (1500 µg/mL).
In the presence of metabolic activation survival (relative plating efficiency) decreased in a dose-related manner reaching 25.0% and 1.0% (at 1500 µg/mL and 2953.4 µg/mL in the first main experiment, respectively) and 27.6% (at 1500 µg/mL in the second main experiment), respectively, of the solvent control value.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not given in study report
- Negative (solvent/vehicle) historical control data: not given in study report - Conclusions:
- The test substance was evaluated as non-mutagenic with and without metabolic activation under the conditions of the present test.
Referenceopen allclose all
Table 1. Test results of first experiment (plate incorporation)
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
||||
(μg/plate) |
(average of 3 plates ± standard deviation) |
|||||
|
Base-pair substitution type |
Cross-linking type |
Frameshift type |
|||
|
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA 1537 |
|
– |
0 |
173.0 ± 16.1 |
10.0 ± 2.0 |
40.7 ± 6.8 |
36.0 ± 5.2 |
17.7 ± 2.5 |
– |
0 (DMSO) |
162.3 ± 17.2 |
14.0 ± 6.1 |
37.0 ± 6.2 |
32.0 ± 5.3 |
18.0 ± 4.6 |
– |
4 |
176.0 ± 7.5 |
14.0 ± 4.4 |
37.3 ± 9.6 |
27.3 ± 4.0 |
15.0 ± 2.6 |
– |
20 |
175.7 ± 1.5 |
13.0 ± 3.6 |
35.3 ± 3.5 |
34.0 ± 5.3 |
20.7 ± 2.5 |
– |
100 |
173.3 ± 7.5 |
15.3 ± 2.3 |
33.0 ± 0.0 |
30.0 ± 7.2 |
12.3 ± 1.5 |
– |
500 |
187.3 ± 22.1 |
18.3 ± 6.0 |
34.3 ± 2.1 |
30.3 ± 8.5 |
17.0 ± 1.7 |
– |
2500 |
190.7 ± 11.0 P |
14.0 ± 3.5 P |
36.7 ± 1.5 P |
29.7 ± 2.1 P |
12.7 ± 3.5 P |
– |
5000 |
206.7 ± 13.1 P |
12.0 ± 4.0 P |
38.7 ± 3.5 P |
30.7 ± 3.8 P |
14.0 ± 3.0 P |
Positive controls, -S9 |
Name |
SA |
SA |
MNNG |
2-NF |
9-AA |
Concentrations (μg/plate) |
1 |
1 |
2.5 |
2.5 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
762.7 ± 29.9 |
537.0 ± 32.1 |
225.3 ± 12.6 |
643.0 ± 83.0 |
114.3 ± 4.6 |
|
+ |
0 |
170.7 ± 26.6 |
13.7 ± 5.9 |
43.3 ± 6.4 |
36.3 ± 9.1 |
10.3 ± 0.6 |
+ |
0 (DMSO) |
159.0 ± 12.1 |
11.7 ± 0.6 |
42.3 ± 4.7 |
41.3 ± 6.1 |
11.7 ± 3.2 |
+ |
4 |
158.7 ± 15.6 |
12.0 ± 1.0 |
37.3 ± 7.4 |
41.0 ± 2.6 |
9.7 ± 4.0 |
+ |
20 |
166.0 ± 17.1 |
14.3 ± 1.2 |
48.0 ± 7.5 |
34.7 ± 2.3 |
9.3 ± 4.5 |
+ |
100 |
167.3 ± 18.6 |
12.0 ± 4.6 |
40.0 ± 7.2 |
43.0 ± 7.8 |
10.0 ± 1.0 |
+ |
500 |
180.3 ± 11.5 |
17.3 ± 4.5 |
42.0 ± 5.2 |
40.3 ± 11.6 |
12.0 ± 1.0 |
|
2500 |
168.7 ± 6.7 P |
16.3 ± 6.4 P |
39.7 ± 1.5 P |
37.3 ± 10.3 P |
10.7 ± 1.5 P |
+ |
5000 |
192.0 ± 12.8 P |
12.0 ± 5.3 P |
36.0 ± 2.6 P |
33.3 ± 4.5 P |
13.0 ± 5.0 P |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
0.5 |
1 |
10 |
0.5 |
1 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1194.7 ± 49.4 |
137.7 ± 10.1 |
273.7 ± 6.7 |
996.0 ± 82.3 |
244.0 ± 12.5 |
|
2-NF = 2-nitrofluorene
SA = sodium azide
MNNG= N-methyl-N-nitro-N-nitrosoguanidine
9-AA = 9-aminoacridine
2-AA = 2-aminoanthracene
P = precipitate
Table 2. Test results of second experiment (plate incorporation)
With or without S9-Mix |
Test substance concentration |
Mean number of revertant colonies per plate |
||||
(μg/plate) |
(average of 3 plates ± standard deviation) |
|||||
|
Base-pair substitution type |
Cross-linking type |
Frameshift type |
|||
|
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA 1537 |
|
– |
0 |
222.3 ± 23.4 |
15.3 ± 2.1 |
33.0 ± 7.0 |
36.0 ± 2.6 |
20.3 ± 2.1 |
– |
0 (DMSO) |
190.7 ± 16.8 |
12.3 ± 4.0 |
32.3 ± 5.5 |
37.0 ± 2.6 |
24.0 ± 6.6 |
– |
4 |
188.3 ± 11.6 |
19.3 ± 5.1 |
30.0 ± 4.4 |
35.3 ± 4.0 |
16.0 ± 9.2 |
– |
20 |
186.3 ± 14.5 |
10.7 ± 5.5 |
32.7 ± 9.0 |
32.7 ± 10.1 |
17.7 ± 4.6 |
– |
100 |
175.0 ± 18.5 |
12.0 ± 4.6 |
36.3 ± 3.5 |
37.7 ± 2.3 |
17.3 ± 4.5 |
– |
500 |
187.3 ± 1.2 |
15.7 ± 7.5 |
37.0 ± 8.5 |
32.3 ± 7.0 |
16.3 ± 4.2 |
– |
2500 |
199.7 ± 30.0 P |
18.3 ± 2.5 P |
34.3 ± 8.5 P |
27.7 ± 2.9 P |
20.3 ± 3.5 P |
– |
5000 |
233.7 ± 15.8 P |
15.7 ± 1.2 P |
34.7 ± 3.2 P |
26.7 ± 3.8 P |
19.7 ± 3.5 P |
Positive controls, -S9 |
Name |
SA |
SA |
MNNG |
2-NF |
9-AA |
Concentrations (μg/plate) |
1 |
1 |
2.5 |
2.5 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
949.7 ± 64.5 |
731.7 ± 16.8 |
242.7 ± 62.5 |
1334.7 ± 82.7 |
155.7 ± 27.0 |
|
+ |
0 |
186.0 ± 10.1 |
16.7 ± 6.0 |
40.7 ± 6.8 |
39.0 ± 8.2 |
21.0 ± 3.0 |
+ |
0 (DMSO) |
163.0 ± 6.0 |
16.7 ± 1.5 |
38.0 ± 4.6 |
40.0 ± 4.4 |
19.0 ± 2.6 |
+ |
4 |
179.3 ± 17.2 |
13.0 ± 1.0 |
36.3 ± 11.2 |
44.7 ± 3.8 |
15.7 ± 4.9 |
+ |
20 |
199.3 ± 4.2 |
14.3 ± 1.5 |
36.0 ± 5.3 |
41.0 ± 9.2 |
14.3 ± 1.2 |
+ |
100 |
199.0 ± 8.2 |
18.3 ± 2.5 |
38.7 ± 1.5 |
42.7 ± 4.9 |
14.7 ± 0.6 |
+ |
500 |
198.0 ± 7.8 |
13.3 ± 1.2 |
39.3 ± 2.3 |
46.0 ± 7.2 |
19.0 ± 2.6 |
|
2500 |
199.0 ± 24.3 P |
14.7 ± 3.5 P |
39.7 ± 9.0 P |
40.0 ± 7.8 P |
18.3 ± 3.5 P |
+ |
5000 |
207.7 ± 4.5 P |
16.3 ± 2.9 P |
34.0 ± 6.1 P |
28.0 ± 7.5 P |
10.7 ± 3.1 P |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
0.5 |
1 |
10 |
0.5 |
1 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
2041.7 ± 80.7 |
167.7 ± 7.6 |
275.3 ± 57.5 |
1712.3 ± 42.2 |
289.7 ± 0.6 |
|
2-NF = 2-nitrofluorene
SA = sodium azide
MNNG= N-methyl-N-nitro-N-nitrosoguanidine
9-AA = 9-aminoacridine
2-AA = 2-aminoanthracene
P = precipitate
Table 1: Experiment I - 4 h exposure - with metabolic activation
Concentration |
Cloning efficiency [%] |
Mutation frequency |
Mutation factor |
[µg/mL] |
|||
0 |
95.20 |
15.20 |
1.02 |
0 (DMSO) |
100.00 |
14.90 |
1.00 |
50 |
104.40 |
9.50 |
0.64 |
150 P |
97.20 |
3.60 |
0.24 |
375 P |
100.40 |
8.90 |
0.60 |
750 P |
98.60 |
16.90 |
1.13 |
1500 P |
25.00 |
1.30 |
0.09 |
2953.4 P |
1.00 |
n.d. |
n.d. |
DMBA, 7.7 |
64.40 |
168.50* |
11.31 |
DMBA = Dimethylbenzanthracene
P = Precipitate
n.d. = not detectable, due to cytotoxicity
* = Statistical significant (p <0.05) Mann-Whitney-U-Test
Mutation factor= Mutation frequency
Table 2: Experiment I - 4 h exposure - without metabolic activation
Concentration |
Cloning efficiency [%] |
Mutation frequency |
Mutation factor |
[µg/mL] |
|||
0 |
101.40 |
22.30 |
1.55 |
0 (DMSO) |
100.00 |
14.40 |
1.00 |
15 |
83.90 |
14.10 |
0.98 |
50 |
75.90 |
16.00 |
1.11 |
150 |
62.50 |
18.90 |
1.31 |
375 P |
26.10 |
2.80 |
0.19 |
750 P |
13.90 |
2.80 |
0.19 |
1500 P |
8.80 |
3.90 |
0.27 |
EMS, 1000 |
54.90 |
474.30* |
32.94 |
EMS = Ethyl methanesulphonate
P = Precipitate
* = Statistical significant (p <0.05) Mann-Whitney-U-Test
Table 3: Experiment II - 4 h exposure - with metabolic activation
Concentration |
Cloning efficiency [%] |
Mutation frequency |
Mutation factor |
[µg/mL] |
|||
0 |
90.40 |
15.40 |
0.56 |
0 (DMSO) |
100.00 |
27.40 |
1.00 |
15 |
95.50 |
15.00 |
0.55 |
50 |
97.90 |
26.70 |
0.97 |
150 P |
93.00 |
30.00 |
1.09 |
375 P |
74.40 |
15.40 |
0.56 |
750 P |
81.60 |
13.60 |
0.50 |
1500 P |
27.60 |
2.20 |
0.08 |
DMBA, 7.7 |
88.70 |
140.70* |
5.14 |
DMBA = Dimethylbenzanthracene
P = Precipitate
* = Statistical significant (p <0.05) Mann-Whitney-U-Test
Table 4: Experiment II - 4 h exposure - without metabolic activation
Concentration |
Cloning efficiency [%] |
Mutation frequency |
Mutation factor |
[µg/mL] |
|||
0 |
100.70 |
24.00 |
0.93 |
0 (DMSO) |
100.00 |
25.70 |
1.00 |
15 |
103.90 |
19.20 |
0.75 |
50 |
84.10 |
13.70 |
0.53 |
150 |
82.00 |
1.20 |
0.05 |
375 P |
14.40 |
14.00 |
0.54 |
750 P |
6.60 |
12.10 |
0.47 |
1500 P |
1.80 |
0.00 |
0.00 |
EMS, 1000 |
68.90 |
675.70* |
26.29 |
EMS = Ethyl methanesulphonate
P = Precipitate
* = Statistical significant (p <0.05) Mann-Whitney-U-Test
Mutation frequency = Mutants per 1E+06 surviving cells
Mutation factor = Mutation frequency / Mutation frequency of vehicle control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test (OECD 474, mouse): not
clastogenic in vivo
Unscheduled DNA synthesis test in hepatocytes (OECD 486): no unscheduled DNA synthesis in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 Jul - 18 Aug 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Study was performed in accordance with an old version of the OECD TG 474; some parameters do not meet current requirements.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- Current version adopted in 2016
- Deviations:
- yes
- Remarks:
- 1000 polychromatic erythrocytes were counted for each animal. Body weight, justification for single dose treatment, and historical control data not provided. Dose spacing exceeds currently recommended factor.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- Not specified
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- Adopted in 1985
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- NMRI
- Details on species / strain selection:
- The mouse has been chosen for this study since it provides a convenient in vivo mammalian model.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH i.G., Schönwalde, Germany
- Age at study initiation: 8 weeks
- Body weight at study initiation: males: 33 - 44 g (mean: 38.1 g), females: 28 - 43 g (mean: 32.6 g)
- Assigned to test groups randomly: yes
- Housing: group housed, 5 animals per cage, Makrolon cages Type 3, on softwood granulate
- Diet: rat/mice diet ssniff R/M-H (V 1534) (ssniff GmbH, Soest, Germany), ad libitum
- Water: tap water in drinking water quality in plastic bottles, ad libitum
- Acclimation period: at least 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): housing in air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 0.2, 1 and 2% (w/v)
- Amount of vehicle (gavage): 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was suspended in sesame oil at appropriate concentrations. A magnetic stirrer was used to keep the preparations homogeneous until the completion of dosing. - Duration of treatment / exposure:
- single treatment by gavage
- Frequency of treatment:
- single treatment by gavage
- Post exposure period:
- animals were sacrificed 12, 24 or 48 h post dosing and bone marrow smears were obtained
- Dose / conc.:
- 20 mg/kg bw (total dose)
- Dose / conc.:
- 100 mg/kg bw (total dose)
- Dose / conc.:
- 200 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 5 animals per sex per dose and per sampling time point
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide (Endoxan)
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw, single oral dose - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: erythrocytes - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 400 mg/kg bw test substance resulted in mortality in male and female mice. Clinical signs of toxicity (spontaneous activity decreased, increased activity when
touched) were still observed at 200 mg/kg bw. Therefore, the highest sublethal dose of 200 mg/kg bw was selected for the main study.
TREATMENT AND SAMPLING TIMES: In conformity with the test procedure, the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 h after administration of the single, oral dose.
DETAILS OF SLIDE PREPARATION: Femoral bone marrow was flushed into the centrifuge tubes containing 3 mL of fetal bovine serum. A suspension was formed. The mixture was then centrifuged for 5 min at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h. Slides were fixed with methanol and stained according to May-Grünwald-Giemsa staining.
METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in the 1000 normocytes, were evaluated statistically. - Evaluation criteria:
- Both biological and statistical significances were considered together for evaluation purposes.
A test substance was considered positive if there was a significant dose-related increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. Individual and/or group mean values should exceed the laboratory historical control range. A test substance producing no significant dose related increase in the number of micronucleated polychromatic erythrocytes, and the values within the historical control range, is considered not mutagenic in this system.
For the assay to be valid, individual and/or group mean values for the positive control should exceed the laboratory's historical control range. - Statistics:
- One-sided Wilcoxon-Test was performed for each treatment interval and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5%. Tests on lower dose groups are only performed if the higher dose group is significantly different from the control. If there is a difference between the positive and negative control (24 h) (two-sided Wilcoxon-Test with a 5%-level of significance) two-sided Wilcoxon-Tests are also performed sequentially for the ratio of polychromatic erythrocytes for each treatment interval (12 h, 24 h, 48 h) at a multiple level of significance of 5%. Actual data were also compared with historical controls. Lower dose groups are only tested if the higher dose group is significantly different from the control.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- 200 mg/kg bw: 24 h sampling time point: 1 male with spotted/patterned appearing hepatic tissue
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 - 1200 mg/kg bw in 3 animals per sex and dose
- Clinical signs of toxicity in test animals:
1200 mg/kg bw: spontaneous activity decreased, squatting posture, ataxic gait, prone position, forward crawling, staggering gait, palpebral fissure narrow, irregular respiration, panting, flanks pinched in, clonic convulsions, no macroscopic findings were observed; mortality: 2/3 males, 3/3 females
1000 mg/kg bw: spontaneous activity decreased, squatting posture, unsteady gait, staggering gait, prone position, flanks pinched in, coat bristling, panting, clonic convulsions, palpebral fissure narrow, palpebral fissure very narrow, no macroscopic findings were observed; mortality: 1/3 males, 3/3 females
800 mg/kg bw: spontaneous activity decreased, squatting posture, prone position, staggering gait, stilted gait, ataxic gait, panting, clonic convulsions, no macroscopic findings were observed; mortality: 3/3 males, 2/3 females
600 mg/kg bw: spontaneous activity decreased, increased activity when touched, squatting posture, unsteady gait, forward crawling, stilted gait, ataxic gait, prone position, flanks pinched in, marked flank respiration, tonic convulsions when touched, palpebral fissure narrow, lacrimation clear, colourless; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 2/3 females
400 mg/kg bw: spontaneous activity decreased, increased activity when touched, stilted gait, forward crawling, unsteady gait, squatting posture, prone position, coat bristling, marked flank respiration, panting, palpebral fissure narrow, palpebral fissure very narrow, twitching; macroscopic findings: hepatic tissue lightly coloured, distinctly patterned appearence of hepatic tissue; mortality: 2/3 males, 1/3 females
200 mg/kg bw: spontaneous activity decreased, increased activity when touched; no macroscopic findings were observed; mortality: 0/6 animals
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups was within the normal range of the negative control groups.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test substance.
- Appropriateness of dose levels and route: In a preliminary dose range finding study, oral administration of 400 mg/kg bw test substance resulted in mortality in male and female mice and 200 mg/kg bw clinical signs of toxicity. Therefore, the highest sublethal dose of 200 mg/kg bw was selected for the main study.
- Statistical evaluation: One-sided Wilcoxon-Test was performed for each treatment interval.
For details see results tables under "Any other information on results incl. tables". - Conclusions:
- The test substance was not mutagenic (clastogenic) in the present mouse bone marrow micronucleus test.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Mar - 16 May 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Proposal for a new guideline. Unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo.
- Version / remarks:
- Drafted in 1996
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Version / remarks:
- Guideline was adopted in 1997, shortly after the study was conducted.
- Deviations:
- no
- Remarks:
- The study was conducted in accordance with the draft version of the OECD TG 486. No deviations to the final version of the OECD TG 486, which was adopted shortly after study finalisation, were determined.
- GLP compliance:
- yes
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Specific Pathogen Free outbred albino Hsd/Ola
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Olac UK Ltd, Bicester, UK
- Age at receipt: approx. 5 weeks
- Body weight at receipt: 140 - 149 g
- Assigned to test groups randomly: yes
- Housing: group housed, in plastic disposable cage with a stainless steel grid top
- Diet: Special Diet Services rat and mouse maintenance diet No. 1 (RM1(E) SQC expanded pellet), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 50 ± 20 within experimental phase (in acclimisation phase the minimum relative humity was 24%, but this did not affect integrity of the study)
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: aqueous 1% (w/v) methyl cellulose
- Concentration of test material in vehicle: 60 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were freshly prepared on the day of use (using identical methods for each phase of the test) and were diluted to the required concentration. - Duration of treatment / exposure:
- single treatment by gavage
- Frequency of treatment:
- single treatment by gavage
- Post exposure period:
- animals were sacrificed 2 and 14 h post dosing
- Dose / conc.:
- 600 mg/kg bw (total dose)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Test substance-treated and control group: 5 males (only cells from 4 animals were analysed) per dose and sampling time point.
Positive control groups: 3 males (only cells from 2 animals were analysed) per sampling time point. - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 1. Dimethylnitrosamine for 2 h expression time
- Route of administration: oral gavage
- Doses / concentrations: 4 mg/kg bw / 0.4 mg/mL in water
2. 2-Acetylaminofluorene for 14 h expression time
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw / 5 mg/mL in aequous 1% methyl cellulose - Tissues and cell types examined:
- Tissue: liver
Cell type: hepatocytes - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: During a preliminary toxicity test 4 male rats per group were dosed with 686, 980, 1400 and 2000 mg/kg bw. No mortality or clinical signs of toxicity were observed. Thus, 2000 mg/kg bw, the maximum recommended dose, was chosen for use in the DNA repair test. Two expression times were utilised to allow for variations in the rate of absorption, metabolism and accumulation of DNA damage.
TREATMENT AND SAMPLING TIMES: The animals were killed by carbon dioxide asphyxiation 2 or 14 h after treatment.
DETAILS OF SLIDE PREPARATION: Hepatocytes for primary cell culture were obtained by collagenase perfusion of the rat liver via the portal vein applying standard procedures. After an initial attachment period the medium was replaced with incomplete Willams E containing high specific activity (methyl-³H) thymidine (Amersham International TRK 686, batch number 168; specific activity 85.0 Ci/mmol) at a final activity of 10 µCi/mL. The cultures were incubated in this medium for a period of 4 h. After this labelling period, the supernatant medium was removed and replaced by incomplete Williams E containing 250 µM cold (unlabelled) thymidine. The cultures were then incubated for a 'chase' period of 24 h with 'cold' thymidine. This additional culture period helps to wash out excess radiolabel and improves cell morphology thus facilitating subsequent grain count analysis of autoradiographs. After the 24 h cold chase with thymidine, coverslips with attached cells were removed from the culture medium, given three 5 min washes in Hanks' balanced salts solution then fixed in 2.5% (v/v) acetic acid in ethanol and allowed to dry. They were mounted on glass microscope slides, with the cell layer uppermost, using DPX mountant. The mountant was allowed to harden at approximately 37 °C. Ilford K2 emulsion was applied to six slides per animal. The slides were dried and exposed in a light tight box for 14 days and subsequently developed.
METHOD OF ANALYSIS: Prior to grain count analysis the autoradiographs were examined for signs of test substance-induced toxicity (e.g. pyknosis, reduced levels of radiolabelling).The slides were randomised, encoded and grain count analysis was performed using a Zeiss Photomicroscope II connected to an AMS 40-10 image analyser via a high resolution camera fiited with a 1" chalnicon tube. Three slides per animal were used. 50 hepatocytes over several widely separated, randomly chosen fields of view, from each of 3 cultures per animal were analysed (resulting in 150 analysed cells per animal). Only results from hepatocytes not in S-phase with a normal morphology (i.e. not pyknotic or lysed) without staining artifacts or debris were recorded. For each cell the number of silver grains overlying the nucleus was estimated using the image analysis system, then the number of silver grains in an equivalent and most heavily-grained, adjacent area of cytoplasm was estimated. The cytoplasmic grain count was subtracted from the gross nuclear grain count to give the net nuclear grain count. Mean grain counts were calculated for each slide examined. For slides showing a strong response, ie where the mean net grain count was in excess of 10, only 25 cells were examined. The number of cells with a net grain count of greater than or equal to 5 was recorded in the raw data. - Evaluation criteria:
- A positive response is indicated by a substantial dose-associated statistically significant increase in the net nuclear grain count which is accompanied by a substantial increase in the gross nuclear grain count over concurrent control values.
A negative response is indicated by a mean net nuclear grain count which is not significantly greater than the concurrent control. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response. - Statistics:
- Both gross and net nuclear grain counts for treated animals were compared with vehicle control counts using classical one-way analysis of variance followed by a Student's t test with an appropriate transformation of values if indicated by excessive variance.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 686, 980, 1400 and 2000 mg/kg bw
- Solubility: given in aqueous 1% (w/v) methylcellulose
- Clinical signs of toxicity in test animals: none
- Rationale for exposure: maximum dose level recommended by the OECD guideline - Conclusions:
- Negative for induction of unscheduled DNA synthesis
Referenceopen allclose all
Table 1: Results of the in vivo micronucleus assay in male animals.
|
Mean PCEs / 1000 NCEs |
Total micronuclei per 1000 PCEs at sampling time |
Total micronuclei per 1000 NCEs at sampling time |
||||||||
Exp group |
Number of animals |
Dose [mg/kg bw] |
12 h |
24 h |
48 h |
12 h |
24 h |
48 h |
12 h |
24 h |
48 h |
Vehicle control (corn oil) |
5 |
0 |
1.1±0.08 |
0.8±0.07 |
0.8±0.10 |
1±0.0 |
1.2±0.11 |
1.4±0.09 |
1.8±0.08 |
0.8±0.08 |
1.6±0.09 |
Positive control (Cyclophosphamide) |
5 |
50 |
n.d |
0.8±0.19 |
n.d. |
n.d. |
40±0.73 |
n.d. |
n.d. |
1.60±0.05 |
n.d. |
Test substance |
5 |
20 |
1±0.22 |
1.0±0.09 |
0.8±0.10 |
1±0.1 |
2±0.12 |
0.8±0.04 |
0.6±0.05 |
0.80±0.04 |
0.8±0.11 |
Test substance |
5 |
100 |
0.8±0.09 |
0.9±0.07 |
0.8±0.07 |
1.2±0.11 |
1±0.07 |
0.8±0.08 |
1.4±0.09 |
0.8±0.08 |
1±0.10 |
Test substance |
5 |
200 |
1.1±0.09 |
1.1±0.09 |
0.9±0.19 |
2.4±0.11 |
1.8±0.11 |
0.8±0.04 |
0.8±0.13 |
0.6±0.09 |
0.8±0.13 |
n.d. = not determined
Table 2: Results of the in vivo micronucleus assay in female animals.
|
Mean PCEs / 1000 NCEs |
Total micronuclei per 1000 PCEs at sampling time |
Total micronuclei per 1000 NCEs at sampling time |
||||||||
Exp group |
Number of animals |
Dose [mg/kg bw] |
12 h |
24 h |
48 h |
12 h |
24 h |
48 h |
12 h |
24 h |
48 h |
Vehicle control (corn oil) |
5 |
0 |
1.1±0.13 |
1±0.10 |
1±0.16 |
2±0.17 |
1.2±0.11 |
0.8±0.08 |
0.4±0.05 |
1.2±0.13 |
0.4±0.09 |
Positive control (Cyclophosphamide) |
5 |
50 |
n.d. |
0.7±0.18 |
n.d. |
n.d. |
29.8±0.73 |
n.d. |
n.d. |
0.8±0.08 |
n.d. |
Test substance |
5 |
20 |
1.0±0.21 |
1.1±0.14 |
0.9±0.13 |
1.2±0.08 |
2.4±0.11 |
1±0.1 |
1±0.07 |
0.2±0.04 |
1±0.07 |
Test substance |
5 |
100 |
0.8±0.10 |
0.8±0.20 |
0.9±0.17 |
1.8±0.08 |
1.2±0.11 |
1±0.17 |
0.6±0.09 |
0.6±0.05 |
0.6±0.09 |
Test substance |
5 |
200 |
1.0±0.09 |
1.0±0.13 |
0.8±0.14 |
1.2±0.13 |
2.2±0.18 |
1.2±0.11 |
1±0.07 |
0.8±0.08 |
0.6±0.05 |
n.d. = not determined
Table 3: Results of the in vivo micronucleus assay.
Treatment group |
Dose [mg/kg] |
Sampling time [h] |
PCE with MN ± SD |
Mean frequency of PCE with MN |
PCE/NCE ratio |
Vehicle control
|
0 |
12 |
1.5±0.13 |
0.2 |
1.1±0.11 |
0 |
24 |
1.2±0.10 |
0.1 |
0.9±0.15 |
|
0 |
48 |
1.1±0.09 |
0.1 |
0.9±0.15 |
|
Test substance
|
200 |
12 |
1.8±0.13 |
0.2 |
1.1±0.13 |
200 |
24 |
2.0±0.14 |
0.2 |
1±0.12 |
|
200 |
48 |
1.0±0.08 |
0.1 |
0.9±0.16 |
|
Positive control (Cyclophosphamide) |
40 |
24 |
34.9±0.87* |
3.5 |
0.8±0.18 |
* statistically significant (p <0.05)
Table 1. Unscheduled DNA synthesis in ³H- thymidine labelled rat hepatocytes (2 h expression time)
|
Mean gross nuclear grain count |
Mean cytoplasmic grain count |
Mean net nuclear grain count |
|||
Exp group |
Number of animals |
Dose [mg/kg bw] |
2 h |
2 h |
2 h |
|
Vehicle control |
4 |
0 |
11.6 |
15.4 |
-3.8 |
|
Positive control (Dimethylnitrosamine) |
2 |
4 |
46.5* |
14.9 |
31.6* |
|
Positive control (2-Acetylaminofluorene) |
2 |
50 |
- |
- |
- |
|
Test substance |
4 |
600 |
14.1 |
18.2 |
-4.1 |
|
Test substance |
4 |
2000 |
12.9 |
16.7 |
-3.8 |
|
*statistically significant (on-way ANOVA followed by one-sided Students t-test, p <0.001)
Table 2. Unscheduled DNA synthesis in ³H- thymidine labelled rat hepatocytes (14 h expression time)
|
Mean gross nuclear grain count |
Mean cytoplasmic grain count |
Mean net nuclear grain count |
|||
Exp group |
Number of animals |
Dose [mg/kg bw] |
14 h |
14 h |
14 h |
|
Vehicle control |
4 |
0 |
12.2 |
16.5 |
-4.4 |
|
Positive control (Dimethylnitrosamine) |
2 |
4 |
- |
- |
- |
|
Positive control (2-Acetylaminofluorene) |
2 |
50 |
44.7* |
17 |
27.8* |
|
Test substance |
4 |
600 |
14.6 |
19.8 |
-5.2 |
|
Test substance |
4 |
2000 |
15.8 |
20.9 |
-5.1 |
|
*statistically significant (on-way ANOVA followed by one-sided Students t-test, p <0.001)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro gene mutation in bacteria
The potential for the test substance to cause mutagenicity in bacteria was assessed in a test performed according to OECD test guideline (TG) 471 and under GLP conditions (M-133133-01-1, 1994). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvr A were exposed to concentrations of 4, 20, 100, 500, 2500 and 5000 µg/plate in the plate incorporation assay, in the absence and presence of metabolic activation by rat liver S9-mix. No strain-specific bacteriotoxic effect was observed in the tester strains. Precipitation was observed in the test plates of all strains at concentrations of 2500 µg/plate and above. For all tester strains the maximum number of revertants observed in the test plates was comparable to those of the solvent and negative controls with and without metabolic activation, whereas treatment with the positive control substances caused the expected statistically significant increase in revertants per test plate, demonstrating the validity of the test system. Under the conditions of this test the test substance has no mutagenic potential in bacteria.
In vitro genetic toxicity in mammalian cells
The mutagenic potential of the test substance in mammalian cells in vitro was assessed by a HPRT-assay performed similar to OECD TG 476 under GLP-conditions (M-143543-01-1, 1997). In the pre-test, Chinese hamster lung fibroblasts (V79) were exposed for 4 h to test substance concentrations of 100, 250, 500, 1000, 1500, 2000, 2500 and 2953.4 µg/mL (10 mM) in the absence and presence of metabolic activation by rat liver S9-mix. Dose-dependent cytotoxic effects with and without metabolic activation were observed, but a high precipitation of the test substance at all doses tested without S9-mix and≥250 µg/mL with S9-mix prevented the evaluation of cytotoxicity in the preliminary experiment. Therefore in the main assay the cytotoxicity of the test substance was assessed by determining the plating efficiency of cells in the culture plates rather than by determining survival by measurement of crystal violet extinction. Based on the results of the preliminary experiment V79 cells were exposed to the test substance at concentrations of 15, 50, 150, 375, 750 and 1500 µg/mL without S9-mix and 15 (test 2 only), 50, 150, 375, 750, 1500 and 2953,4 µg/mL (10 mM, test 1 only) with S9-mix for 4 h in 2 independent experiments. The relative plating efficiency decreased with dose, indicating cytotoxicity. Precipitation was observed at 150 µg/mL (without S9-mix) and 375 µg/mL (with S9-mix) and above. No increases in mutant colony numbers were observed in 2 independent experiments with the test substance in either the presence or absence of S9-mix. Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, indicating the sensitivity of the assay. Therefore, the test substance is regarded as not mutagenic in mammalian cells, in vitro.
The clastogenic potential of the test substance in vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD TG 473 and GLP (M-182288-01-1, 998). Following preliminary toxicity testing, Chinese hamster lung fibroblasts (V79) were exposed to 10, 25 and 40 µg/mL for 20 h without S9-mix and to 100, 250 and 750 µg/mL for 3 h with S9-mix. Duplicate cell cultures per dose level were used and cells sampled 20 or 28 h (40 µg/mL without S9-mix only) after the start of treatment were examined for chromosome aberrations. Positive and vehicle (dimethylsulfoxide, DMSO) control cultures were included in each assay. Genotoxicity, indicated by increased numbers of chromosome aberrations, was only observed at the highest concentration tested (750 µg/mL) in the presence of metabolic activation (no increased values were seen in the absence of metabolic activation). At this concentration, microscopical visible precipitation of the test substance was observed and relative survival was reduced to 13.8% of the solvent control (mitotic index was approximately 54%). Hence, this observation is considered not reliable. In the absence of metabolic activation, relative survival was reduced to approximately 63% of the solvent control value at the highest concentration tested (40 µg/mL), thus, minimally deviating from the current guideline criteria. Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, indicating the sensitivity of the assay and the efficacy of the S9-mix. In summary, under the conditions of the present study, the results are regarded as equivocal and do not allow for a definitive assessment.
In vivo genetic toxicity
The genetic toxicity of the test substance in vivo was addressed in a Mammalian Erythrocyte Micronucleus test in the mouse, performed similar to OECD TG 474 under GLP conditions (M-140262-01-1, 1996). Groups of 5 male and 5 female NMRI mice were used at each time point. Based on the results of a preliminary toxicity study, each animal was given a single dose by gavage of either 20, 100 or 200 mg/kg bw, suspended in sesame oil. Bone marrow smears were obtained from 5 males and 5 females from each of the test groups and negative control groups at the three sampling times 12, 24, and 48 h after dosing. Smears from the positive control group were obtained 24 h post dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes and 1000 normochromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examining at least 1000 erythrocytes from each animal. Clinical signs of toxicity were observed in the preliminary toxicity study at 200 mg/kg bw and above, and in one male receiving 200 mg/kg bw in the main study, demonstrating relevant systemic exposure of the animals to the test substance in the absence of mortality. Treatment with the test substance did not increase the number of polychromatic and normochromatic erythrocytes containing micronuclei. The ratio of polychromatic to normochromatic erythrocytes in both male and female animals also remained unaffected by treatment. Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes was not significantly affected. These results gave no indication of a clastogenic effect of the test substance in NMRI mice under the conditions of this test.
In addition, the potential of the test substance to induce unscheduled DNA synthesis (indicative of DNA damage and subsequent repair) in vivo was assessed in a study performed according to the draft version of OECD TG 486 under GLP conditions (M-142947-01-1, 1997). Five male rats per sampling time and dose were administered 600 and 2000 mg/kg bw (recommended limit dose) as a single gavage dose. A concurrent negative control group was treated with the vehicle (aqueous 1% (w/v) methyl cellulose). The sampling times for treatment and vehicle control group was 2 and 14 h post dosing. The positive control groups were treated with dimethylnitrosamine at 4 mg/kg bw (2 h expression) or 2-acetylaminofluorene at 50 mg/kg bw (14 h expression). The animals were sacrificed and hepatocytes were isolated by enzymatic dissociation 2 or 14 h after treatment with the positive substances, respectively. Samples from 4 treatment and 4 vehicle control group animals per dose and sampling time were analysed. Samples from 2 animals from each of the positive control groups were assessed. The isolated hepatocytes were allowed to attach to glass coverslips and were cultured in vitro with (methyl-³H) thymidine at 10 µCi/mL for 4 h to 'radiolabel’ replicating DNA. The hepatocytes were 'chased' for 24 h with unlabelled thymidine then they were fixed and processed for autoradiography. DNA repair was assessed by comparing the labelling levels of hepatocyte nuclei from treated animals with control values and with the accompanying cytoplasmic labelling levels (a total of 150 cells from 3 different slides per animal were examined). The test substance did not cause any significant increases in either the gross nuclear grain count or the net nuclear grain count at any dose level and sampling time. Positive control group animals showed a large and highly significant increase in the net nuclear grain count, which was accompanied by a large increase in the gross nuclear grain count.
In conclusion, the test substance did not induce gene mutations in bacteria and mammalian cells in vitro. Equivocal results were obtained in an in vitro chromosome aberration test with metabolic activation in Chinese hamster V79 cells. The increase in chromosome aberrations was only observed at cytotoxic dose levels. In an in vivo mouse erythrocyte micronucleus test, no indication of a clastogenic effect was observed. In addition, no unscheduled DNA synthesis, as indicator of DNA damage, was observed in cultured rat hepatocytes after in vivo treatment of rats with the test substance. There is no indication that the test substance is genotoxic in vivo.
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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