Registration Dossier
Registration Dossier
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EC number: 605-140-1 | CAS number: 158237-07-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Mutagenicity (OECD 471, GLP), Ames: negative with and without metabolic activation
Cytogenicity/chromosome aberration in mammalian cells (OECD 473, GLP): negative with and without metablic activation
Gene mutation in mammalian cells (OECD 476, GLP): negative with and without metablic activation
Supporting data:
DNA repair in bacteria (GLP): negative with and without metabolic activation
Unscheduled DNA synthesis in vitro in rat primary hepatocytes (OECD 482, GLP): negative
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:
- 17-27 May, 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 26 May 1983
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium) and trp operon (for E. coli)
- 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- First experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation (tested up to the recommended maximum concentration)
Second experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation (tested up to the recommended maximum concentration) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2-Aminoanthracene (2-AA); 2-(2-Furyl)-3-(5-nitro- 2-furyl) acrylamide (AF-2);
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: observation of bacterial growth inhibition - Rationale for test conditions:
- based on the results of a previous study.
- Evaluation criteria:
- If more than two fold and dose-related increase in revertant colonies were observed as compared with the solvent control, it was evaluated as positive in terms of mutagenicity.
- Statistics:
- Mean numbers of the revertant colonies of each concentration of each test system was calculated and compared to that of the solvent control.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- a slight decrease in bacterial growth was detected in the 1st experiment without S9 (colony numbers reduced to up to 84% of the respective vehicle control in a dose-dependent manner)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant effect on bacterial growth was observed in any strain at any test concentration (a slight effect on bacterial growth was observed in the 1st experiment in tester strain TA 100 without metabolic activation) - Conclusions:
- Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 March - 3 May 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Adopted: 29 July 2016
- Deviations:
- yes
- Remarks:
- dose selection (highest concentration applied without S9 mix reduced the MI to 66%), number of metaphases scored (200 instead of 300 metaphases scored), no historical data included in study report
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. Utesch, Merck AG, Darmstadt
- Cell cycle length: 14 h
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's minimal essential medium (MEM, Earle) with the following supplements was used: nonessential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHC03-solution (final concentration: 0.225 %), penicillin (50 units/ml), streptomycin (50 jug/ml), heat-inactivated fetal calf serum, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Cytokinesis block (if used):
- Colcemid-solution (40 µg/mL in water)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (CCR, Roßdorf, Germany (Lot.280294; protein content: 37.4 mg/ml))
- Test concentrations with justification for top dose:
- Pre-test with and without S9: 5, 10, 25, 50, 100, 250 and 500 µg/mL
Main test: Without S9: 5, 25 and 50 µg/mL
With S9: 10, 50 and 100 µg/mL
Precipitation of the test substance was observed in the culture medium of the pre-test at 50 µg/mL and above. Decreased mitotic indeces were observed without S9 supplementation starting at 50 µg/mL (to 42.2%) and with S9 supplementation starting at 100 µg/mL (to 26.4%) in a dose-related manner. - Vehicle / solvent:
- - Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in Ham's F12 medium or Hanks'balanced salt solution, acetone was selected as the vehicle - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Fixation time: 18 h and 30 h (main test), 24 h (pre-test)
SPINDLE INHIBITOR: colcemid-solution (40 µg/mL)
NUMBER OF REPLICATIONS: dublicate cultures, two slides were generated per culture(pre-test and main test)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 5% Giemsa solution, two slides per culture
NUMBER OF CELLS EVALUATED: Mitotic index: 1000 cells /culture, 2000 cells in total
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases/slide/concentration, 200 metaphases in total
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
OTHER:
- chromosome disintegration as an indication of a cytotoxic effect was also recorded if observed and if fewer than half of the chromosomes revealed characteristic structural features within a given metaphase - Evaluation criteria:
- Evaluation criteria
- Slides were evaluated using a light microscope at a magnification of about 630. Mitotic index: 1000 cells/culture. Duplicate cultures were examined.
- Slides were evaluated using a light microscope at a magnification of about 1000.
- Chromosomes of 200 metaphases/concentration, 100 metaphases from each of two parallel cultures, were examined.
- Only metaphases containing the modal chromosome number (22) were analysed unless exchanges were detected.
- Both chromatid and chromosome-type aberrations were assessed.
- Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids.
- Chromosome-type aberrations were defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.
- Classes of aberrations characterized: gap, break, fragment, deletion, exchange, multiple aberration
- In addition: polyploid metaphases, metaphases showing chromosome disintegration
Assessment Criteria
- An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
- A test was considered positive if there was a relevant and statistically significant increase in the aberration rate.
- A test was considered negative if there was no such increase at any time interval.
- A test was considered equivocal if there was an increase which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.
Assay Acceptance Criteria
- An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from our laboratory and from published studies. - Statistics:
- Mitotic index: chi2-test.
Numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges: Fisher's exact test was
A difference was considered to be significant if the probability of error was below 5 %. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 50 µg/mL without S9 (MI was reduced to 66.8%)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: poorly soluble in aqueous solutions,
- Precipitation: precipitation was observed starting at 50 µg/mL (pre-Test), without S9 no precipitation was observed (concentrations tested 5, 25 and 50 µg/mL (main test), with S9 precipitation occured at the highest tested concentration of 100 µg/mL (concentrations tested: 10, 50, 100 µg/mL) (main test)
RANGE-FINDING/SCREENING STUDIES: yes
CYTOKINESIS BLOCK: colcemid-solution (40 µg/mL)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 March - 12 April 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Adopted: 29 Jul 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EEC Directive 88/302/EEC Mutagenicity In vitro Mammalian Cell - Gene Mutation Test
- Qualifier:
- according to guideline
- Guideline:
- other: New and Revised Health Effects Test Guidelines October 1983. (U.S.) Environmental Protection Agency Washington DC (PB 84-233295) HG - Gene Muta - Somatic Cells
- Version / remarks:
- October 1983
- 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
- Source of cells: Prof. G. Speit, University of Ulm, Germany
- Cell cycle length, doubling time or proliferation index: 10 - 14 h
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: hypoxanthine-free Eagle's Minimal Essential Medium (MEM, Earle) (Biochrom) supplemented with nonessential aminoacids, L-glutamine (2 mM), MEM-vitamins, NaHCC>3, penicillin (100 units/ml), streptomycin (100 /µg/ml) and heat-inactivated fetal calf serum (f.c.: 10%) (Gibco or Biochrom) at 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (Cytotest Cell Research, FRG)
- Test concentrations with justification for top dose:
- With and without S9 mix (preliminary cytotoxicity test): 1, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 µg/mL
Without S9 mix: 5, 10, 20, 30, 45, 60 µg/mL (Experiment 1)
Without S9 mix: 5, 10, 20, 25, 30, 45 µg/mL (Experiment 2)
With S9 mix: 1.88, 3.75, 7.5, 15, 30, 60 µg/mL (Experiment 3, 4)
Justification: Dose selection was based on limit of solubility starting at 62.5 µg/mL and cytotoxicity without S9 mix starting at 15.6 µg/mL observed in the pre-test. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO) 1% (v/v) or less
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: Dimethylbenzanthracene (DMBA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6 to 7 days
SELECTION AGENT (mutation assays): 10 µg/ml 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: six replications each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning efficiency - Evaluation criteria:
- - At least one independent repetition (+/- metabolic activation).
- The average cloning efficiency of the negative/vehicle controls should be at least 50%.
- The highest test item concentration should produce a low level of relative survival (0 - 30%) or should be the first concentration where insolubility occurs. The survival at the lowest concentration should approximate the negative control.
- The background mutant frequency in a trial should be > 25x10-6 cells.
- A mutant frequency is acceptable only if the absolute cloning efficiency is ≥ 10%.
- Mutant frequencies for at least 4 concentrations of the test substance (from a minimum of 5 dishes) should be determined
- The positive control must induce an average mutant frequency of at least 3x that of the vehicle control.
- An assay is positive if a dose-dependent, significant and in parallel cultures reproducible increase in mutant frequency is observed. It is desirable to obtain this dose-relationship for at least 3 doses. To be significant, the mutagenic response to the item should be at least approximately 2 to 3 times that of the highest negative/vehicle control. If this result can be reproduced in a second assay, the test item is considered to be mutagenic. If a reproducible increase greater than two times the minimum criterion is observed for a single dose near the highest testable concentration, the test substance is also considered mutagenic.
- A test is equivocal if there is no dose-dependency but one or more doses induce a reproducible, significant mutant frequency in all assays.
- An assay is negative if none of the doses tested (for a range of applied concentrations which extendsto toxicity causing about 30% survival or less) induces a reproducible mutant frequency which is considered significant
- In the case of a positive result, a test item is only considered mutagenic, if no significant change in osmolality compared to the vehicle control can be observed. - Statistics:
- Analysis of variance: Dunnett test, p < 0.05
Mutation frequency: Weighted ANOVA and regression result p < 0.05 - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations > 15.6 µg/mL (-S9) tested up to limit of solubility
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: The highest concentration was checked for altering osmolality. The obtained value (428.7 mOsmol/kg at 60 /µg/ml) did not differ from the measured value of the vehicle control (428.7 mOsmol/ kg).
- Water solubility: poorly soluble in aqueous solutions, therefore highest tested concentration was 60 µg/mL in DMSO
- Precipitation: precipitation of the test substance occurred at 45 µg/mL without S9 supplementation and at 60 µg/mL with S9 supplementation
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was conducted with a series of 9 concentrations of the test substance ranging from 1.0 µg/ml to 250 µg/ml both, with and without metabolic activation.Cytotoxicity was only be observed in the test without S9 mix at concentrations above 15.6 µg/ml. Precipitation of the test substance was observed starting at 62.5 µg/mL.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- S9-activation (EMS) 887.3 ± 193.9 (range 531.1 to 1291.5)
+ S9-activation (DMBA) 93.4 + 42.2 (range 21.8 to 240.2)
- Negative historical control data:
Mutation Frequency of Negative Controls
- S9-activation 6.1 ± 3.8 (range 0.5 to 15.0)
+ S9-activation 5.7 + 5.9 (range 0.5 to 28.8)
Mutation Frequency of Vehicle Controls (DMSO)
- S9-activation 5.6 ± 4.4 (range 1.0 to 22.6)
+ S9-activation 5.0 ± 5.5 (range 0.5 to 26.7)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative population growth
- Other observations when applicable: cytotoxicity was only observed in the test without S9 mix at concentrations above 15.6 µg/mL in the pre-test and at 60 µg/mL in the main test. - Conclusions:
- negative
Referenceopen allclose all
Table 1: The result of the first experiment (without S-9)
Compound |
S-9 Mix |
concentration |
TA 100 |
TA 1535 |
WP2 uvra |
TA 98 |
TA 1537 |
DMSO |
- |
0 |
94 98 84 (92) |
13 12 10 (12) |
13 16 7 (12) |
16 17 17 (17) |
7 5 5 (6) |
Test substance |
- |
5000 |
62 84 86 (77) |
5 7 8 (7) |
16 20 25 (20) |
15 15 19 (16) |
5 8 7 (7) |
|
- |
2500 |
88 81 80 (83) |
5 6 5 (5) |
15 12 13 (13) |
17 15 23 (18) |
9 7 6 (7) |
|
- |
1250 |
84 86 80 (83) |
7 4 6 (6) |
10 9 10 (10) |
18 23 19 (20) |
6 8 5 (6) |
|
- |
625 |
85 96 74 (85) |
4 6 9 (6) |
10 12 10 (11) |
19 11 25 (18) |
6 5 4 (5) |
|
- |
313 |
88 100 82 (90) |
8 5 8 ( 6) |
7 13 15 (12) |
16 18 23 (19) |
6 7 8 (7) |
Positive control |
- |
|
412a) 357 379 (383) |
175b) 172 182 (176) |
184a) 194 197 (192) |
362c) 303 333 (333) |
379d) 441 392 (404) |
( ) : mean
a) AF-2: 0.01 µg/plate; b) NaN3: 0.5 µg/plate; c) AF-2: 0.1 µg/plate; d) 9-AA: 80 µg/plate
Table 2: The result of the first experiment (with S-9)
Compound |
S-9 Mix (10%) |
concentration |
TA 100 |
TA 1535 |
WP2 uvra |
TA 98 |
TA 1537 |
DMSO |
+ |
0 |
84 90 96 (90) |
10 8 5 (8) |
19 16 25 (20) |
26 28 29 (28) |
13 12 11 (12)
|
Test substance |
+ |
5000 |
90 99 92 (94) |
6 3 5 (5) |
16 14 14 (15) |
20 19 17 (19) |
6 7 4 (6) |
|
+ |
2500 |
89 83 85 (86) |
11 8 9 (9) |
25 10 17 (17) |
28 22 30 (27) |
6 5 8 (6) |
|
+ |
1250 |
100 94 72 (89) |
3 5 6 (5) |
13 18 15 (15) |
31 26 19 (25) |
7 6 7 (7) |
|
+ |
625 |
86 90 67 (81) |
5 7 9 (7) |
18 17 13 (16) |
19 32 34 (28) |
9 5 5 (6) |
|
+ |
313 |
95 88 82 (88) |
7 5 9 (7) |
18 19 20 (19) |
31 29 36 (32) |
8 5 9 (7) |
Positive control |
+ |
|
407 e) 451 477 (445) |
176 f) 192 186 (185) |
219 g) 315 304 (303) |
188 h) 192 196 (192) |
70 f) 78 83 (77) |
( ) : mean
e) 2-AA: 1.0 µg/plate; f) 2-AA: 2.0 µg/plate; g) 2-AA: 10.0 µg/plate; h) 2-AA: 0.5 µg/plate
Table 3: The result of the second experiment (without S-9)
Compound |
S-9 Mix |
concentration |
TA 100 |
TA 1535 |
WP2 uvra |
TA 98 |
TA 1537 |
DMSO |
- |
0 |
91 84 89 (88) |
9 14 15 (13) |
13 16 14 (14) |
34 28 31 (31) |
16 9 8 (11) |
Test substance |
- |
5000 |
89 77 81 (82) |
11 7 12 (10) |
11 10 16 (12) |
27 35 30 (31) |
16 10 13 (13)
|
|
- |
2500 |
84 90 90 (88) |
13 15 10 (13) |
13 11 14 (13) |
25 30 30 (28) |
4 14 12 (10) |
|
- |
1250 |
73 86 82 (80) |
7 14 15 (12) |
9 13 10 (11) |
26 23 29 (26) |
12 9 7 (9) |
|
- |
625 |
89 94 95 (93) |
15 10 7 (11) |
10 10 12 (11) |
34 26 24 (28) |
7 10 10 (9) |
|
- |
313 |
93 87 84 (88) |
10 10 9 (10) |
14 13 8 (12) |
23 31 34 (29) |
11 14 11 (12) |
Positive control |
- |
|
387a) 402 424 (404) |
155b) 177 163 (165) |
147a) 160 158 (155) |
436C) 440 448 (441) |
463d) 478 442 (461) |
( ) : mean
a) AF-2: 0.01 µg/plate; b) NaN3: 0.5 µg/plate; c) AF-2: 0.1 µg/plate; d) 9-AA: 80 µg/plate
Table 4: The result of the second experiment (with S-9)
Compound |
S-9 Mix (10%) |
concentration |
TA 100 |
TA 1535 |
WP2 uvra |
TA 98 |
TA 1537 |
DMSO |
+ |
0 |
98 93 96 (96) |
11 14 11 (12) |
13 16 22 (17) |
36 39 40 (38) |
10 9 10 (10) |
Test substance |
+ |
5000 |
90 81 88 (86) |
11 16 15 (14) |
18 16 24 (19) |
37 35 27 (33) |
7 11 12 (10) |
|
+ |
2500 |
101 87 90 (93) |
11 7 13 (10) |
12 16 23 (17) |
31 37 40 (36) |
7 10 15 (11) |
|
+ |
1250 |
89 88 82 (86) |
14 15 12 (14) |
18 10 17 (15) |
34 33 40 (36) |
12 8 13 (11) |
|
+ |
625 |
79 80 83 (81) |
11 14 8 (11) |
13 17 22 (17) |
27 31 40 (33) |
8 10 12 (10) |
|
+ |
313 |
81 94 86 (87) |
11 12 12 (12) |
20 15 16 (17) |
27 29 37 (31) |
10 6 10 (9) |
Positive control |
+
|
|
455e) 398 404 (419) |
115f) 151 122 (129) |
287g) 278 265 (277) |
179h) 185 162 (175) |
79f) 72 83 (78) |
( ) : mean
e) 2-AA: 1.0 µg/plate; f) 2-AA: 2.0 µg/plate; g) 2-AA: 10.0 µg/plate; h) 2-AA: 0.5 µg/plate
Table 1: Results of experiment
Time of harvest |
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
||
in h |
|
in µg/mL |
in % |
with gaps |
without gaps |
exchanges |
|
Exposure period 4h, without S9 mix |
|
||||
18 |
Aceton |
/ |
100.0
|
2.0 3.0 2.5 |
2.0 3.0 2.5 |
0.0 2.0 1.0 |
untreated |
0 |
110.0
|
3.0 2.0 2.5 |
2.0 2.0 2.0 |
0.0 0.0 0.0 |
|
MMC |
0.1 |
107.3 |
43.0 47.0 45.0** |
43.0 44.0 43.5** |
17.0 22.0 19.5** |
|
Test substance |
5 |
109.5
|
1.0 7,0 4.0 |
1.0 7.0 4.0 |
0.0 3.0 1.5 |
|
25 |
100.9
|
4.0 1.0 2.5 |
4.0 1.0 2.5 |
0.0 0.0 0.0 |
||
50 |
66.8*
|
0.0 7.0 3.5 |
0.0 6.0 3.0 |
0.0 2.0 1.0 |
||
30 |
Aceton |
/ |
100.0
|
5.0 3.0 4.0 |
4.0 3.0 3.5 |
0.0 0.0 0.0 |
Test substance |
50 |
97.0 |
5.0 5.0 5.0 |
5.0 2.0 3.5 |
1.0 1.0 1.0 |
|
|
Exposure period 4h, with S9 mix |
|
||||
18 |
Aceton |
/ |
100.0 |
20 8.0 5.0 |
20 8.0 5.0 |
0.0 1.0 0.5 |
untreated |
|
137.9
|
6.0 3.0 4.5 |
6.0 3.0 4.5 |
2.0 1.0 1.5 |
|
CP |
2 |
111.3 |
38.0 38.0 38.0** |
36.0 34.0 35.0** |
14.0 16.0 15.0** |
|
Test substance |
10 |
124.9
|
4.0 0.0 2.0 |
3.0 0.0 1.5 |
1.0 0.0 0.5 |
|
50 |
141.8
|
3.0 2.0 2.5 |
3.0 2.0 2.5 |
0.0 0.0 0.0 |
||
100 |
97.2
|
3.0 4.0 3.5 |
3.0 4.0 3.5 |
0.0 0.0 0.0 |
||
30 |
Aceton |
/ |
100.0 |
7.0 4.0 5.5 |
7.0 4.0 5.5 |
2.0 0.0 1.0 |
Test substance |
100 |
157.9 |
3.0 3.0 3.0 |
2.0 2.0 2.0 |
0.0 0.0 0.0 |
|
Bold: Mean of two cultures
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
* p < 0.05
** p < 0.01
Table 1: Experiment 1 - Without Metabolic Activation
Concentration |
µg/mL |
Cloning efficiency ± SD [%] |
Relative population growth [% of vehicle control] |
Survival to treatment [percent vehicle control] |
Mutants per 1E+06 surviving cells |
Negative control |
|
81.0 ± 5.8 |
84.7 |
83.7 |
8.2 |
|
81.7 ± 6.5 |
110.2 |
|
13.8 |
|
Vehicle control |
|
77.8 ± 7.6 |
100.0 |
100.0 |
9.8 |
|
59.5 ± 2.0 |
100.0 |
|
14.7 |
|
Positive control |
|
68.8 ± 7.4 |
19.7 |
22.1 |
615.3 |
|
50.7 ± 5.2 |
26.9 |
|
756.9 |
|
Test item |
5 |
85.0 ± 3.5 |
103.2 |
78.1 |
5.9 |
5 |
88.5 ± 8.7 |
88.4 |
|
6.6 |
|
10 |
60.5 ± 4.8 |
127.1 |
63.0 |
3.4 |
|
10 |
81.3 ± 3.4 |
93.6 |
|
6.2 |
|
20 |
70.2 ± 3.4 |
57.2 |
90.6 |
3.0 |
|
20 |
60.5 ± 11.1 |
79.5 |
|
12.6 |
|
30 |
99.0 ± 3.1 |
13.6 |
34.3 |
1.7 |
|
30 |
62.8 ± 2.0 |
20.6 |
|
3.3 |
|
45 a |
80.3 ± 3.5 |
8.6 |
37.2 |
5.2 |
|
45 |
80.5 ± 5.2 |
13.6 |
|
3.1 |
|
60 a |
60.2 ± 2.5 |
10.7 |
18.4 |
1.4 |
|
60 |
78.5 ± 6.5 |
11.3 |
|
10.9 |
a = precipitates
SD = standard deviation
Positive control: EMS 900 µg/mL
Table 2: Experiment 2 - Without Metabolic Activation
Concentration |
µg/mL |
Cloning efficiency ± SD [%] |
Relative population growth [% of vehicle control] |
Survival to treatment [percent vehicle control] |
Mutants per 1E+06 surviving cells |
Negative control |
|
65.7 ± 5.5 |
103.8 |
118.1 |
4.4 |
|
62.5 ± 9.0 |
105.0 |
|
6.7 |
|
Vehicle control |
|
67.2 ± 1.3 |
100.0 |
100.0 |
6.2 |
|
61.3 ± 11.3 |
100.0 |
|
6.1 |
|
Positive control |
900 |
50.8 ± 3.0 |
36.7 |
74.3 |
691.4 |
900 |
62.7 ± 1.6 |
35.0 |
|
641.8 |
|
Test item |
5 |
67.5 ± 2.5 |
117.4 |
131.0 |
3.1 |
5 |
60.7 ± 10.5 |
87.0 |
|
4.1 |
|
10 |
69.3 ± 1.8 |
96.0 |
119.7 |
3.6 |
|
10 |
71.7 ± 4.9 |
102.3 |
|
2.3 |
|
20 |
68.7 ± 3.3 |
123.7 |
117.1 |
0.6 |
|
20 |
75.5 ± 4.5 |
88.2 |
|
1.7 |
|
25 |
65.8 ± 9.4 |
66.0 |
126.1 |
2.5 |
|
25 |
56.7 ± 16.8 |
52.2 |
|
6.7 |
|
30 |
71.7 ± 10.3 |
35.1 |
47.2 |
0.6 |
|
30 |
81.3 ± 1.1 |
11.6 |
|
2.6 |
|
45 a |
71.0 ± 2.8 |
6.9 |
62.5 |
0.6 |
|
45 |
79.3 ± 8.9 |
29.5 |
|
4.2 |
a = precipitates
SD = standard deviation
Positive control: EMS 900 µg/mL
Table 3: Experiment 3 - With Metabolic Activation
Concentration |
µg/mL |
Cloning efficiency ± SD [%] |
Relative population growth [% of vehicle control] |
Survival to treatment [percent vehicle control] |
Mutants per 1E+06 surviving cells |
Negative control |
|
63.2 ± 5.0 |
79.8 |
137.7 |
5.3 |
|
68.7 ± 8.6 |
87.9 |
|
3.0 |
|
Vehicle control |
|
58.5 ± 8.3 |
100.0 |
100.0 |
1.4 |
|
69.2 ± 4.5 |
100.0 |
|
3.0 |
|
Positive control |
20 |
62.0 ± 6.6 |
58.4 |
114.2 |
55.1 |
20 |
64.0 ± 4.8 |
54.3 |
|
57.9 |
|
Test item |
1.88 |
58.5 ± 4.8 |
67.2 |
91.1 |
1.4 |
1.88 |
69.8 ± 5.9 |
87.0 |
|
4.2 |
|
3.75 |
66.0 ± 6.8 |
92.5 |
95.0 |
0.6 |
|
3.75 |
93.3 ± 7.0 |
66.1 |
|
0.4 |
|
7.5 |
78.2 ± 2.8 |
69.8 |
98.4 |
4.3 |
|
7.5 |
72.2 ± 10.1 |
72.7 |
|
7.5 |
|
15 |
65.8 ± 7.3 |
81.2 |
112.8 |
6.3 |
|
15 |
73.7 ± 8.9 |
50.5 |
|
2.8 |
|
30 |
78.2 ± 11.6 |
74.4 |
116.3 |
1.1 |
|
30 |
83.5 ± 10.4 |
79.9 |
|
1.5 |
|
60 a |
57.3 ± 10.8 |
64.5 |
155.5 |
2.9 |
|
60 |
60.7 ± 4.3 |
88.9 |
|
4.8 |
a = precipitates
SD = standard deviation
Positive Control: DMBA 20 µg/mL
Table 4: Experiment 4 - With Metabolic Activation
Concentration |
µg/mL |
Cloning efficiency ± SD [%] |
Relative population growth [% of vehicle control] |
Survival to treatment [percent vehicle control] |
Mutants per 1E+06 surviving cells |
Negative control |
|
69.2 ± 8.6 |
74.9 |
83.8 |
8.4 |
|
78.0 ± 7.4 |
88.8 |
|
15.0 |
|
Vehicle control |
|
95.0 ± 5.9 |
100.0 |
100.0 |
7.0 |
|
67.8 ± 1.9 |
100.0 |
|
9.2 |
|
Positive control |
20 |
82.5 ± 11.7 |
57.7 |
68.4 |
67.7 |
20 |
88.7 ± 4.5 |
49.5 |
|
70.9 |
|
Test item |
1.88 |
94.7 ± 4.8 |
53.3 |
103.8 |
8.4 |
1.88 |
75.2 ± 1.2 |
77.6 |
|
5.0 |
|
3.75 |
79.0 ± 3.5 |
93.9 |
113.3 |
11.6 |
|
3.75 |
63.2 ± 4.3 |
85.9 |
|
11.9 |
|
7.5 |
78.0 ± 7.8 |
61.5 |
89.9 |
6.9 |
|
7.5 |
72.7 ± 3.7 |
76.9 |
|
4.0 |
|
15 |
91.2 ± 8.3 |
74.0 |
112.4 |
7.8 |
|
15 |
71.7 ± 2.0 |
74.8 |
|
7.0 |
|
30 |
62.8 ± 9.1 |
75.9 |
89.4 |
12.6 |
|
30 |
70.5 ± 11.0 |
53.3 |
|
8.9 |
|
60 a |
70.5 ± 7.4 |
48.3 |
73.2 |
6.5 |
|
60 |
69.5 ± 7.0 |
34.5 |
|
13.2 |
a = precipitates
SD = standard deviation
Positive Control: DMBA 20 µg/mL
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Supporting data:
Erythrocyte micronucleus test in mice (OECD 474, GLP): negative
³²P-postlabelling in rat urinary bladder epithelium for determination of DNA adducts in vivo (GLP): negative
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Various genotoxicity tests were conducted with the test substance. These include in vitro and in vivo techniques. In detail, the following studies have been performed with the test substance:
In vitro studies:
- Genetic toxicity in bacteria (Ames)
The in-vitro genetic toxicity of the test substance was assessed in a bacterial reverse mutation assay (Ames test) according to OECD TG 471 and GLP criteria (1995e). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E.coli WP2uvra at concentrations up to 5000 µg/plate in 2 independent experiments (pre-incubation method). The test substance did not induce an increase in reversions in any of the tested strains with or without metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls proved validity of the experiment.
Further, the in-vitro genetic toxicity of the test substance was assessed in an additional bacterial reverse mutation assay (Ames test) similar to OECD TG 471 and GLP criteria with the deviation that only 4 of 5 recommended bacterial were used (1995f). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535 and 1537 at concentrations up to 5000 µg/plate in 2 independent experiments (plate incorporation and pre-incubation method). The test substance did not induce an increase in reversions in any of the tested strains with or without metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls were valid and within the range of historical control data.
- Cytogenicity/chromosome aberration in mammalian cells
A GLP conform chromosome aberration study was performed according to OECD guideline 473 (1996e). Clastogenicity of the test material was investigated in Chinese hamster V79 cells. Cells were treated with the test material both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254. The test material was suspended in acetone, due to the limited solubility of the test substance in Ham's F12 medium or Hanks' balanced salt solution. A pre-test was performed at concentrations of 5, 10, 25, 50, 100, 250 and 500 µg/mL with and without metabolic activation. Precipitation of the test substance was observed in the culture medium of the pre-test at 50 µg/mL and above. Based on the results of the pre-test, the following concentrations were tested in the main study: 5, 25 and 50 µg/mL without metabolic activation and 10, 50 and 100 µg/mL with metabolic activation. Cultures of all concentrations were exposed to the test substance for 4 h followed by harvesting after 18 h. In addition, cells treated with the highest concentrations were harvested 30 h after the beginning of the treatment. Colcemid-solution was added to each culture 2 h prior to the end of the incubation period. The positive controls used were mitomycin C in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every test and positive, solvent and negative controls were set up and handled in parallel. Cytotoxicity was determined by calculation of the mitotic index for which 2000 cells were evaluated. From each culture 200 metaphases were examined for chromosome aberrations. Without metabolic activation, the mitotic index was reduced in cultures treated with 50 µg/mL to 66.8%. Cultures treated with mitomycin C showed no reduced mitotic index compared to the negative control. Precipitates were not observed in cultures without metabolic activation. With metabolic activation, no reduction of the mitotic index was observed in cultures treated with the test substance or cyclophosphamide, but substance precipitation occurred at the highest concentration tested. There were no biologically relevant and statistically significant increases in the incidence of metaphases with aberrations in any culture examined, neither in the presence nor in the absence of S9. Positive controls with and without metabolic activation showed a clear and statistically significant increase of metaphases with aberrations. The test material was evaluated as not clastogenic under the conditions of this test.
- Gene mutation in mammalian cells:
A GLP guideline study on gene mutation in mammalian cells was performed according to OECD Guideline 476 (1996f). In this study the ability of the test material to induce reverse mutations at the HPRT locus was investigated in V79 Chinese hamster lung cells. Cells were treated with the test material solved in dimethylsulfoxide both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254.
Preliminary tests for evaluation of solubility and cytotoxicity were performed. The test substance was soluble in dimethyl sulfoxide (DMSO) up to concentrations of 200 mg/mL. When administered to the culture medium precipitates were observed at a concentration of 62.5 µg/mL and above. Cytotoxicity determined by comparison of colonies in treated cultures versus vehicle control cultures (relative cloning efficiency) was tested at concentrations between 1 and 250 µg/mL. Cytotoxicity was observed starting at 31.3 µg/mL without metabolic activation. Based on these findings concentrations used in the main test were 5, 10, 20, 25, 30, 45 and 60 µg/mL without metabolic activation and 1.88, 3.75, 7.5, 15, 30 and 60 µg/mL with metabolic activation. Osmolality was not altered at the highest concentration tested. Cells were treated with the test substance for 5 h, replated and incubated for 7 days for cytotoxicity determination. For mutagenicity testing cells were additionally incubated with selective medium containing 6-thioguanidine (6-TG) for 6 to 7 days before 6-TG resistant colonies were counted. The corresponding negative, vehicle and positive controls ethylmethanesulfonate (EMS) (without metabolic activation) and dimethylbenzanthracene (DMBA) (with metabolic activation) were treated and cultured under the same conditions. Without metabolic activation, a dose-related decrease in relative survival and relative population growth was observed. Furthermore precipitates were observed at 45 µg/mL and above. With metabolic activation no cytotoxic effects were observed, but the test substance was tested up to its limits of solubility. No biologically and no statistically significant increase in mutation frequency was observed at any tested concentration with or without metabolic activation. Both positive controls, EMS and DMBA showed a clear mutagenic effect. Therefore, the test system was considered sensitive to detect mutagens and the test material was evaluated as not mutagenic under the conditions of this test.
Further, the following supporting information on in vitro genetic toxicity is available:
- DNA repair in bacteria:
The DNA damaging effect of the test substance was determined in a GLP compliant DNA repair test in bacteria (1997l). The possibility of DNA-damaging effects was investigated in spores of Bacillus subtilis H17 strain (with recombinant repair ability) and M45 strain (without repair ability). After incubation with the test substance, the diameter of inhibition zones were measured as read-out parameter for bacterial growth. Based on the different repair abilities, the test is indicative for DNA damaging effects. If the test substance is genotoxic, growth inhibition is different in the bacterial strains with and without recombinant repair ability. The bacteria were plated in agar with and without S9 mix for metabolic activation. Subsequently the plated bacteria were treated with paper disks impregnated with 20 µl of the test or control substances and incubated for 24 h. After the incubation period the paper disks were removed and the growth inhibition zone of both strains and their difference was determined. If there were more than 5 mm difference of the diameter of growth inhibition zones between the two strains of bacteria, it was evaluated as positive for DNA damaging effects. A preliminary test revealed remarkable inhibition of bacterial growth at test substance concentrations of 12.5 ug/disk. Thus, concentrations of 0.75, 1.5, 3, 6 and 12 µg/disk were selected as for the first experiment with and without metabolic activation. Mitomycin c (MMC) and 2-Aminoantrhacene (2-AA) were used as positive controls. Kanamycin (KN) served as negative control substance and DMSO as solvent control. In the first experiment slight growth inhibition in both strains was observed for testsubstance concentrations at 6 and 12 µg/disk without metabolic activation. However, thedifferences of the diameter between both strains were within 5 mm and thus, the test substance was not considered as DNA damaging under the respective test conditions. With metabolic activation no growth inhibition was observed in both strains up to the highest dose tested. Positive controls were valid and induced growth inhibition differences between the two bacterial strains of more than 5 mm diameters and negative controls of less than 5 diameters. In solvent controls no growth inhibition was observed. Since growth inhibition was only observed in bacteria without metabolic activation a further dose range finding test was carried out at 50, 200 and 800 µg test substance/disk for test conditions with metabolic activation. Since remarkable inhibition of bacterial growth was observed at 50 µg/disk, 48 µg/disk was selected as the highest dose for a second experiment. In this experiment test substance concentrations of 0. 3, 6, 12, 24 and 48 µg/disk were used in the presence of S9. Slight growth inhibition in both strains was observed at 24 and 48 µg/disk. However, the differences of the diameter between both strains were within 5 mm. Thus, the test substance is not considered to exhibit DNA damaging properties in this test system. MMC (S-9(-)) and 2-AA (S-9(+)) as positive control substances, did not inhibit growth in H 17, but in M 45 bacterial strains, indicating that both positive control substances have a DNA-damaging activity. KM (S-9(-)) as the negative control substance, inhibited growth in both strains, but the difference of the diameter of growth inhibition zone was less than 5 mm. Thus, the respective controls verified the suitability of the test system. Based on the observed results the test substance has no DNA-damaging effect with or without metabolic activation.
- Unscheduled DNA synthesis in vitro in rat primary hepatocytes:
A GLP study to test unscheduled DNA synthesis (UDS) induced by the test material was performed according to OECD Guideline 482 in vitro in rat primary hepatocytes (1996g). DNA repair was quantified by measuring the amount of labeled thymidine incorporated into the nuclear DNA of cells that are not in S-phase. In detail, UDS was measured by counting nuclear grains and subtracting the average number of grains in 3 cytoplasmatic areas of the same size as the corresponding nucleus.
In the main study, viable rat primary hepatocytes were treated with test substance dissolved in dimethyl sulfoxide at concentrations of 1, 5, 10, 25, 50, 100 and 150 µg/mL for 20 h. The concentrations were based on preliminary solubility and cytotoxicity experiments showing a distinct decrease in relative survival at concentrations of 125 µg/mL and above. A vehicle control and a positive control with 1µg/mL 2-Aminoanthracene (2-AAF) was run in parallel. No concentration of the test substance caused nuclear grain counts which were relevantly different from the vehicle control. The percentage of cells in repair was not statistically significant increased at any concentration tested. The grain counts per nucleus and the average number of cells in repair of the vehicle control were within the normal range. The positive control 2-AAF induced strong increases in nuclear labelling indicating unscheduled DNA synthesis and thus demonstrating the sensitivity of the test system. Based on the results of this study the test substance caused no biologically relevant increase in percentage of cells in repair over the concurrent vehicle control and was therefore considered negative in the in vitro rat primary hepatocyte UDS Assay.
in vivo studies
- Erythrocyte micronucleus test in mice:
A GLP study to test the clastogenic effect of the test material in mouse (Hsd/Win: NMRI) bone marrow erythrocytes was performed similar to OECD Guideline 474 (1995g). The test material dissolved in 0.5 % aqueous Cremophor emulsion was administered intraperitoneally to 5 male and 5 female mice at a dose of 1500 mg/kg bw. Positive control animals received a single injection of cyclophosphamide (20 mg/kg bw) and negative controls received the respective vehicle. Bone marrow of test substance treated animals was sampled 16, 24 and 48 h after administration and 1000 polychromatic erythrocytes were analyzed for occurrence of micronuclei. Negative and positive control animals were sacrificed after 24 h. The animals treated with the test substance showed symptoms of toxicity including apathy, roughened fur, staggering gait, difficulty in breathing, eyelids stuck together, slitted eyes and diarrhoea after administration. All animals survived until the end of the test. An altered ratio between polychromatic and normochromatic erythrocytes was determined (1000: 798 in the negative control, 1000: 1130 in the 16 h group, 1000: 1982 in the 24 h group and 1000: 2483 in the 48 h group), thus indicating that the test substances reached the bone marrow. No blood samples were taken to permit investigation of plasma levels of the test substance. The incidence of the micronucleated cells was comparable among the negative and treated animals (1.8/1000 in the negative control, and 2.2/1000, 1.6/1000 and 1.8/1000 in the treated groups). The positive control caused a clear increase in the number of polychromatic erythrocytes with micronuclei indicated by an incidence of micronucleated cells of 12.2/1000, which represents a biologically relevant increase in comparison to the negative control. Based on the observed results, the test material was evaluated as not clastogenic under the conditions of this test.
- DNA adduct formation in rats:
Further, DNA-adduct formation after test substance exposure was investigated under in vivo conditions (1997m). Therefore, a GLP conform 32P-postlabelling assay was performed in female Wistar rats in the urinary bladder epithelium.6 female rats per dose level received a single oral administration of 2500 or 5000 mg test substance/kg bw. 2-Acetylaminofluorene (2-AAF), known to form stable DNA adducts, served as positive control at a dose of 100 mg/kg bw and vehicle controls received 0.5% Cremophor solution. 17 h after test substance administration, rats were sacrificed, target tissues were prepared and DNA was isolatedfor the determination of DNA adduct formation. Briefly the applied method involved the incorporation of 32P into non-radioactive nucleic acid constituents by an enzyme-catalysed derivatization, subsequent enrichment of adducted nucleotides employing nuclease P1 (performed with tissues from test substance treated animals and control animals) or butanol extraction (performed with control animals), followed by a chromatographic separation of radioactive products. For the test substance the P1 nuclease enrichment and for 2-AAF the butanol extraction method was considered optimal as 2-AAF-specific DNA adducts in urinary bladder epithelium DNA could only be detected by butanol enrichment in this tissue. Liver served as control tissue and demonstrated the adequate performance of the nuclease P1 enrichment, since 2-AAFspecific adducts could be detected in liver DNA after nuclease P1 enrichment, and the sensitivity of the test system for the detection of DNA adducts in urinary bladder epithelium. Analysis and comparison of the adduct pattern between treated and vehicle control animals was used as criterion for determination of chemically induced DNA adducts. Rats treated with the test substance showed no signs of toxicity. No DNA adduct formation was found in the urinary bladder epithelium up to the highest dose tested. In addition at the 5000 mg/kg bw dose the whole urinary bladder was analyzed and no DNA adducts were observed. Treatment with 2-AAF clearly induced DNA adducts and demonstrated the sensitivity of the test system. Based on this result, the test substance was considered inactive in the 32P-postlabelling assay in vivo in the rat urinary bladder epithelium of female.
In addition, an Ames test with bile from mice treated with the test substance at 7200 ppm was negative. Further, genotoxic studies were also conducted with test substance metabolites. Briefly, the rat metabolite CRT (o-chlorphenyltetrazolinone) was tested in an Ames test and in a micronucleus test, the rat metabolites 4-OH CEA (4-(ethylamino)cyclohexanol) and CEA (N-ethylcyclohexylamine) were tested for in vitro point mutation (SMT&EMT) and DNS damage in bacteria (DNA/B) and the rat metabolite CA (cyclohexylamine) was was tested for in vitro point mutation (CHO HPRT) and in vitro DNS damage in mammals (UDS) and for in vivo chromosome aberration in insects (Drosophila SLRL). All these tests were negative.
Conclusion on genetic toxicity
Overall, the available in vitro and in vivo studies on genetic toxicity do not indicate that the test substance exhibits genotoxic properties. The performed mutagenicity tests in bacteria and mammalian cells were negative. Further, chromosome aberrations were not observed in mammalian cells after treatment with the test substance. In addition, DNA repair in bacteria and unscheduled DNA synthesis in vitro in rat liver primary hepatocytes was not altered by treatment with the test substance. Analysis of micronuclei formation in mouse bone marrow erythrocytes in vivo after test substance administration was negative. Furthermore, the test substance did not form DNA adducts in rat urinary bladder epithelium in vivo, measured by ³²P-postlabelling. Thus, taking all these data in consideration, a genotoxic potential of the test substance can be excluded.
Justification for classification or non-classification
The available genotoxicity data obtained for the test substance are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP).
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