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EC number: 264-036-0 | CAS number: 63225-53-6
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial gene mutation (OECD 471): negative
Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative without metabolic activation, positive with metabolic activation
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2 Dec 2014 - 13 Jan 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- adopted in 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary
- Type of assay:
- other: 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):
- - Cell proliferation: doubling time 12 - 14 h
- Type and identity of media:
DME medium supplemented with:
- L-glutamine (2mM)
- 1% of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B)
- heat-inactivated bovine serum (final concentration 10 %)
During the 3 and 20 h treatments the serum content was reduced to 5%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Dose range finding study:
3 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix
3 h treatment: 5, 10, 15, 30, 60, 120 and 240 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix
Experiment 1:
3 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix
Experiment 2:
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix
4.5 µg/mL was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 20 h and 28 h; 20 h treatment: 20 h and 28 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine, 0.2 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- Validity criteria for the test:
The chromosome aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data.
- the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.
Evaluation of test results:
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed.
A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data).
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same).
- the increase is statistically significant.
Both, biological and statistical significance should be considered together. - Statistics:
- For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were as follows: number of aberration and number of cells with aberration (with and without gaps).
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 80 µg/mL and higher following the 3 h treatment
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 4 µg/mL following the 3 h and 20 h treatment
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant differences between test item treatment and control groups were observed.
- Effects of osmolality: No significant differences between test item treatment and control groups were observed.
- Precipitation: A clear solution was obtained up to a concentration of 10 mg/mL. There was no precipitation in the medium at any concentration tested.
RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection was performed. The cells were treated using increasing concentrations of test item in the absence or presence of S9 mix and were incubated at 37 °C for 3 h. Cell counts were performed after 20 h. Additional groups of cells were treated for 20 h without metabolic and for 3 h with metabolic activation, with cell counts conducted after 20 h (without S9 mix only) and 28 h (without and with S9 mix).
COMPARISON WITH HISTORICAL CONTROL DATA: In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, confirming the suitability of the cell line used. - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 - 25 Mar 2003
- 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 in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany
- 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80 mg/kg bw)
- Test concentrations with justification for top dose:
- Pre-experiment
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
Experiment 1
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
Experiment 2
33, 100, 333, 1000, 2500 and 5000 µg/plate with metabolic activation.
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
- Remarks:
- +S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535 and TA100); 4-NOPD (50 µg/plate, TA1537; 10 µg/plate, TA98); methyl methane sulfonate (4 µL/plate, WP2 uvrA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies or clearing of the background lawn - Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 2500 and 5000 µg/plate in Experiment I and II, without S9; 5000 µg/plate in Experiment II, with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1000 – 5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment I and II, with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment II, with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment II, with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated concentration.
RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as part of experiment I.
COMPARISON WITH HISTORICAL CONTROL DATA: The historical range of positive controls was exceeded without metabolic activation in strains TA1535 and TA100 (experiment I and II) and with metabolic activation in strains TA1537 and TA98 (experiment II). This effect indicates the sensitivity of the strains rather than comprising the assay.
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used, except WP2 uvrA.
Referenceopen allclose all
Table 1. Results of chromosomal aberration test.
Test item |
Concentration |
RICC |
Aberrant cells per 200 metaphase chromosome spreads |
|
|
in µg/mL |
in % |
No. of cells with aberrations |
No. of cells with aberrations |
Exposure period 3 h, sampling time 20 h, without S9 mix |
||||
DMSO |
0.45 µL |
100 |
4 |
2 |
EMS |
1.0 µL |
- |
26** |
21** |
Test substance |
0.5 |
97 |
6 |
2 |
1 |
86 |
5 |
2 |
|
2 |
77 |
5 |
2 |
|
4 |
50 |
7 |
2 |
|
Exposure period 3 h, sampling time 20 h, with S9 mix |
||||
DMSO |
9 µL |
100 |
6 |
3 |
CP |
5.0 |
- |
35** |
30** |
Test substance |
10 |
99 |
6 |
3 |
20 |
86 |
7 |
4 |
|
40 |
80 |
10 |
4 |
|
80 |
50 |
9 |
6 |
|
90 |
44 |
11 |
7 |
|
Exposure period 20 h, sampling time 20 h, without S9 mix |
||||
DMSO |
0.45 µL |
100 |
4 |
2 |
EMS |
0.4 µL |
|
32** |
26** |
Test substance |
0.5 |
94 |
5 |
3 |
1 |
82 |
4 |
2 |
|
2 |
67 |
4 |
2 |
|
4 |
49 |
6 |
3 |
|
Exposure period 20 h, sampling time 28 h, without S9 mix |
||||
DMSO |
0.45 µL |
100 |
4 |
2 |
EMS |
0.4 µL |
- |
33** |
27** |
Test substance |
0.5 |
98 |
4 |
2 |
1 |
81 |
4 |
2 |
|
2 |
74 |
4 |
2 |
|
4 |
50 |
5 |
2 |
|
Exposure period 3 h, sampling time 28 h, with S9 mix |
||||
DMSO |
9 µL |
100 |
3 |
1 |
CP |
5.0 |
- |
34** |
29** |
Test substance |
10 |
97 |
4 |
2 |
20 |
92 |
4 |
2 |
|
40 |
79 |
5 |
3 |
|
80 |
49 |
7 |
4 |
|
90 |
43 |
11* |
7* |
* (p ≤ 0.05); ** (p ≤ 0.01)
DMSO: Dimethyl sulfoxide
EMS: Ethylmethane sulphonate
CP: Cyclophosphamide
RICC: Relative Increase in Cell Counts
In Experiment 1, the test item caused a moderate increase in the number of cells with structural chromosome aberrations in the presence of metabolic activation, up to and including cytotoxic concentrations. This increase was dose associated and biologically important.
In Experiment 2, the test item caused an increase in the number of cells with structural chromosome aberrations without gaps in the presence of S9 mix at concentration of 90 µg/mL following 3 h treatment. This increase was biologically and statistically significant.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.
Table 1. Test results of experiment 1 (plate incorporation).
With or without S9 mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
- |
0 |
124 ± 9.1 |
10 ± 1.7 |
35 ± 1.7 |
21 ± 1.0 |
6 ± 1.7 |
- |
0 (DMSO) |
117 ± 2.3 |
11 ± 1.5 |
28 ± 2.1 |
20 ± 2.6 |
9 ± 2.5 |
- |
33 |
120 ± 11.1 |
10 ± 4.0 |
24 ± 1.5 |
22 ± 4.7 |
5 ± 1.5 |
- |
100 |
112 ± 9.3 |
10 ± 1.5 |
33 ± 4.5 |
23 ± 5.8 |
5 ± 1.5 |
- |
333 |
121 ± 9.9 |
7 ± 4.0 |
30 ± 3.8 |
22 ± 4.6 |
4 ± 1.5 |
- |
1000 |
121 ± 6.9 |
6 ± 0.6 |
34 ± 1.5 |
18 ± 3.2 |
2 ± 1.0 |
- |
2500 |
103 ± 16.0 |
4 ± 2.9 |
35 ± 8.7 |
12 ± 5.6 |
3 ± 1.5 |
- |
5000 |
0 |
1 ± 1.2 |
32 ± 4.7 |
0 |
1 ± 1.0 |
Positive controls, |
Name |
NaN3 |
NaN3 |
MMS |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
10 |
10 |
4 μL |
10 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
953 ± 47.8 |
1038 ± 26.4 |
776 ± 117.8 |
235 ± 20.6 |
56 ± 5.5 |
|
+ |
0 |
141 ± 7.1 |
12 ± 3.5 |
31 ± 0.6 |
28 ± 3.1 |
10 ± 1.2 |
+ |
0 (DMSO) |
135 ± 20.8 |
12 ± 2.5 |
40 ± 7.2 |
25 ± 4.2 |
8 ± 3.2 |
+ |
33 |
134 ± 19.6 |
13 ± 3.1 |
39 ± 4.7 |
22 ± 3.5 |
9 ± 4.0 |
+ |
100 |
145 ± 16.7 |
15 ± 3.5 |
39 ± 4.0 |
20 ± 1.5 |
13 ± 2.6 |
+ |
333 |
155 ± 4.0 |
18 ± 4.6 |
38 ± 8.6 |
27 ± 3.1 |
14 ± 2.1 |
+ |
1000 |
137 ± 12.3 |
15 ± 4.4 |
37 ± 7.1 |
29 ± 2.5 |
8 ± 1.5 |
+ |
2500 |
148 ± 5.0 |
10 ± 2.5 |
29 ± 4.5 |
26 ± 2.5 |
7 ± 2.5 |
+ |
5000 |
142 ± 11.2 |
10 ± 1.0 |
33 ± 3.8 |
21 ± 1.2 |
3 ± 2.3 |
Positive controls, +S9 mix |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
10 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1436 ± 74.3 |
275 ± 15.7 |
196 ± 9.8 |
768 ± 82.4 |
125 ± 5.1 |
DMSO: Dimethyl sulfoxide
2-AA: 2-aminoanthracene
MMS: Methyl methane sulfonate
NaN3: Sodium azide
4-NOPD: 4-nitro-o-phenylene-diamine
Table 2. Test results of experiment 2 (preincubation).
With or without S9 mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
- |
0 |
111 ± 2.9 |
16 ± 1.2 |
30 ± 4.6 |
32 ± 10.0 |
6 ± 1.2 |
- |
0 (DMSO) |
92 ± 19.3 |
16 ± 6.0 |
36 ± 9.3 |
23 ± 2.0 |
6 ± 1.5 |
- |
10 |
87 ± 9.5 |
16 ± 2.0 |
26 ± 4.9 |
22 ± 2.5 |
8 ± 1.5 |
- |
33 |
92 ± 12.4 |
16 ± 2.1 |
29 ± 4.2 |
24 ± 1.5 |
6 ± 1.2 |
- |
100 |
85 ± 11.6 |
16 ± 1.5 |
33 ± 1.5 |
24 ± 4.5 |
6 ± 3.1 |
- |
333 |
67 ± 10.7 |
13 ± 3.6 |
32 ± 5.6 |
23 ± 1.7 |
6 ± 0.6 |
- |
1000 |
75 ± 26.0 |
8 ± 1.0 |
41 ± 9.8 |
18 ± 0.6 |
6 ± 2.6 |
- |
2500 |
25 ± 13.3 |
5 ± 1.2 |
41 ± 4.0 |
8 ± 2.3 |
2 ± 2.1 |
- |
5000 |
14 ± 1.5 |
0 |
29 ± 3.5 |
2 ± 0.6 |
0 |
Positive controls, |
Name |
NaN3 |
NaN3 |
MMS |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
10 |
10 |
4 μL |
10 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1182 ± 45.0 |
1034 ± 50.4 |
499 ± 36.1 |
326 ± 40.7 |
63 ± 6.1 |
|
+ |
0 |
135 ± 14.4 |
17 ± 3.1 |
39 ± 5.7 |
30 ± 6.1 |
12 ± 4.0 |
+ |
0 (DMSO) |
120 ± 2.3 |
15 ± 4.9 |
40 ± 1.2 |
27 ± 5.5 |
9 ± 1.5 |
+ |
33 |
108 ± 12.3 |
18 ± 7.0 |
38 ± 1.0 |
33 ± 4.2 |
12 ± 1.2 |
+ |
100 |
114 ± 21.5 |
19 ± 4.5 |
41 ± 6.5 |
31 ± 1.7 |
8 ± 2.6 |
+ |
333 |
122 ± 3.8 |
17 ± 3.1 |
39 ± 5.3 |
31 ± 4.0 |
8 ± 1.5 |
+ |
1000 |
132 ± 8.6 |
19 ± 2.1 |
28 ± 5.5 |
37 ± 5.2 |
8 ± 3.5 |
+ |
2500 |
150 ± 11.8 |
10 ± 2.0 |
32 ± 4.9 |
37 ± 5.7 |
9 ± 3.5 |
+ |
5000 |
27 ± 10.4 |
6 ± 1.7 |
29 ± 6.4 |
6 ± 1.7 |
3 ± 1.2 |
Positive controls, +S9 mix |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
10 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1355 ± 131.6 |
227 ± 29.3 |
230 ± 6.1 |
1338 ± 30.1 |
192 ± 12.3 |
DMSO: Dimethyl sulfoxide
2-AA: 2-aminoanthracene
MMS: Methyl methane sulfonate
NaN3: Sodium azide
4-NOPD: 4-nitro-o-phenylene-diamine
Table 3. Reduction in the number of revertants at following concentrations (µg/plate).
Strain |
Experiment I |
Experiment II |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
TA 1535 |
2500, 5000 |
/ |
2500, 5000 |
5000 |
TA 1537 |
1000 - 5000 |
5000 |
2500, 5000 |
5000 |
TA 98 |
5000 |
/ |
2500, 5000 |
5000 |
TA 100 |
5000 |
/ |
2500, 5000 |
5000 |
WP2 uvrA |
/ |
/ |
/ |
/ |
/ = no toxic effects
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
Mammalian Erythrocyte Micronucleus Test (OECD 474): Negative.
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:
- From: 26 Apr 2021 to 03 Aug 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- According to Chem VwV-GLP Nr. 5.3/OECD Guidance
- Type of assay:
- mammalian erythrocyte micronucleus test
- Strain:
- Wistar
- Remarks:
- Species: Han
- Details on species / strain selection:
- The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 164-201 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed in Makrolon Type IV, with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum; Supplier: Envigo Teklad Diets, Madison, Wisconsin, United States of America
- Water: tap water
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 45-65%
- Air changes (per hr): at least 8
- Photoperiod (hrs dark / hrs light): 12 /12
IN-LIFE DATES: From: 26 April 2021To: 03 August 2021 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was freshly formulated in corn oil. The formulations were prepared at room temperature, and applied to the animals within 15 minutes
after preparation - Duration of treatment / exposure:
- Not applicable
- Frequency of treatment:
- Single dose
- Post exposure period:
- Pre-Experiment on Toxicity: The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of approx. 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration of the test item.
Main study: The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after
administration of the test item. Sampling of the bone marrow was conducted at low, mid and high doses at 24 h and then at the high dose only at 48 h - Dose / conc.:
- 500 mg/kg bw (total dose)
- Dose / conc.:
- 1 000 mg/kg bw (total dose)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Pre experiment on toxicity: 2 males/2 females
Main study: 6 males per dose - Control animals:
- yes, concurrent vehicle
- other:
- Positive control(s):
- Cyclophosphamide dissolved in sterile water
- Justification for choice of positive control(s): Identified as a suitable positive control in the guidelines
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw - Tissues and cell types examined:
- The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna et. al.
- Details of tissue and slide preparation:
- Tissue preparation: The cell suspensions were passed through a column consisting of α-cellulose and cellulose. The columns were then washed with Hank’s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.
Slide preparation: A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.
Bioanalysis was performed (report number S21-02411-L1) by Eurofins Agroscience Services EAG Laboratories GmbH.
Six additional male rats were assigned to 2 groups in order to be treated as satellite animals for plasma sampling. These animals were treated once with the test item at the maximum tolerated dose level, as determined within the range finding experiment. All animals were sampled twice by retroorbital puncture (eyes alternating). A maximum blood volume of 1.5 mL was withdrawn during the first sampling time point. The first sampling was performed under light isoflurane anaesthesia. The second, terminal sampling was performed under deep CO2 anaesthesia, and the animals were humanely euthanized after sampling by cervical dislocation whilst they were are in deep anaesthesia.
Blood sampling scheme:
Group 1: 1st sampling before first treatment '2nd sampling and termination 1 hour after application
Group 2: 1st sampling 0.5 hours after application, 2nd sampling and termination 4 hours after application.
The blood of the animals was collected in tubes containing K3-EDTA. The blood samples were centrifuged at 10’000 rpm for about 5 minutes to obtain plasma samples. The obtained plasma was divided in duplicates of about 0.2 mL each. - Evaluation criteria:
- The test substance is classified as positive in the assay if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent
negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an
appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data
(e.g., Poisson-based 95% control limits)
The study was considered valid if the following criteria were met:
• the concurrent negative control is considered acceptable for addition to the laboratory historical control database (should ideally be within the 95% control limits of the distribution of the historical negative control database)
• at least 5 animals per group can be evaluated.
• the appropriate number of doses and cells will be analysed.
• PCE to erythrocyte ratio is not less than 20% of the negative control.
• The positive control shows a statistically significant increase of micronucleated PCEs compared to the negative control and is compatible to those in the historical positive control database. - Statistics:
- Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the Familywise error rate of multiple comparisons.
- 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: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Decreased activity. No substantial differences between sexes in toxicity were observed, so that only male animals were used in the main experiment.
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg bw as the upper limit for nontoxic test items.
- Harvest times: Three adequately spaced dose levels spaced by a factor of 2 were administered and samples of bone marrow were collected at the central sampling interval 24 h after treatment.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 4000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Ratio of PCE/NCE (for Micronucleus assay): The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Appropriateness of dose levels and route: As estimated by a pre-experiment at 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable as highest treatment dose. The animals treated with the test item and the vehicle control did not exhibit any clinical symptoms.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group as shown in Table 1, 2, 3, 4, 5 and 6.
A linear regression (least squares, calculated using the validated statistical program RScript
LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.1964 was obtained, demonstrating that there was no dose dependent increase of mean micronuclei values.
A bioanalysis of the test item in plasma (phase number S21-02411-L1) was performed. The method had been successfully validated by procedural recovery samples for determination of the test substance with an LOQ of 0.10 mg/L and up to 10 mg/L in rat plasma according to the guidance document SANTE/2020/12830 rev. 1 of the European Commission. With regard to selectivity, accuracy and precision, the analytical method was applied successfully for the analytical set when analysing the samples of the study. In all plasma samples of animals treated with the test substance at a concentration of 2000 mg/kg bw, however, the residues of the test substance were below the Limit of Detection (i.e., <0.03 mg/L). Rapid hydrolysis of the test substance was suspected as the most probable underlying reason for the missing proof of exposure.
Reference
Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 24 hours after treatment
Table 1: Vehicle Control
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Corn Oil |
0 |
1 |
4000 |
11 |
0.28 |
771 |
271 |
0.649 |
|
2 |
4000 |
2 |
0.05 |
934 |
434 |
0.535 |
|||
3 |
4000 |
8 |
0.20 |
707 |
207 |
0.707 |
|||
4 |
4000 |
12 |
0.30 |
812 |
312 |
0.616 |
|||
5 |
4000 |
7 |
0.18 |
823 |
323 |
0.608 |
|||
6 |
4000 |
4 |
0.10 |
845 |
345 |
0.592 |
|||
|
|
|
|
|
|
|
|||
Mean |
7.3 |
0.19 |
815.3 |
315.3 |
0.618 |
||||
SD |
3.9 |
0.10 |
75.8 |
75.8 |
0.058 |
Table 2: Test Item - Low Dose Group
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Dose 1 |
500 |
7 |
4000 |
5 |
0.13 |
801 |
301 |
0.624 |
|
8 |
4000 |
6 |
0.15 |
691 |
191 |
0.724 |
|||
9 |
4000 |
9 |
0.23 |
686 |
186 |
0.729 |
|||
10 |
4000 |
9 |
0.23 |
725 |
225 |
0.690 |
|||
11 |
4000 |
10 |
0.25 |
737 |
237 |
0.678 |
|||
12 |
4000 |
6 |
0.15 |
718 |
218 |
0.696 |
|||
|
|
|
|
|
|
|
|||
Mean |
7.5 |
0.19 |
726.3 |
226.3 |
0.690 |
||||
SD |
2.1 |
0.05 |
41.6 |
41.6 |
0.038 |
Table 3: Test Item - Medium Dose Group
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Dose 2 |
1000 |
13 |
4000 |
7 |
0.18 |
682 |
182 |
0.733 |
|
14 |
4000 |
9 |
0.23 |
782 |
282 |
0.639 |
|||
15 |
4000 |
5 |
0.13 |
730 |
230 |
0.685 |
|||
16 |
4000 |
12 |
0.30 |
751 |
251 |
0.666 |
|||
17 |
4000 |
15 |
0.38 |
790 |
290 |
0.633 |
|||
18 |
4000 |
9 |
0.23 |
719 |
219 |
0.695 |
|||
|
|
|
|
|
|
|
|||
Mean |
9.5 |
0.24 |
742.3 |
242.3 |
0.675 |
||||
SD |
3.6 |
0.09 |
40.6 |
40.6 |
0.037 |
Table 4: Test Item - High Dose Group
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Dose 3 |
2000 |
19 |
4000 |
9 |
0.23 |
739 |
239 |
0.677 |
|
20 |
4000 |
9 |
0.23 |
769 |
269 |
0.650 |
|||
21 |
4000 |
6 |
0.15 |
1129 |
629 |
0.443 |
|||
22 |
4000 |
4 |
0.10 |
784 |
284 |
0.638 |
|||
23 |
4000 |
14 |
0.35 |
910 |
410 |
0.549 |
|||
24 |
4000 |
13 |
0.33 |
1184 |
684 |
0.422 |
|||
|
|
|
|
|
|
|
|||
Mean |
9.2 |
0.23 |
919.2 |
419.2 |
0.563 |
||||
SD |
3.9 |
0.10 |
193.7 |
193.7 |
0.110 |
Table 5: Positive Control
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Positive |
20 |
25 |
4000 |
55 |
1.38 |
767 |
267 |
0.652 |
|
26 |
4000 |
105 |
2.63 |
948 |
448 |
0.527 |
|||
27 |
4000 |
76 |
1.90 |
1104 |
604 |
0.453 |
|||
28 |
4000 |
63 |
1.58 |
1115 |
615 |
0.448 |
|||
29 |
4000 |
63 |
1.58 |
1080 |
580 |
0.463 |
|||
30 |
4000 |
42 |
1.05 |
1281 |
781 |
0.390 |
|||
|
|
|
|
|
|
|
|||
Mean |
67.3 |
1.69 |
1049.2 |
549.2 |
0.489 |
||||
SD |
21.6 |
0.54 |
174.3 |
174.3 |
0.091 |
Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 48 hours after treatment
Table 6: Test Item - High Dose Group
Test Group |
Dose mg/kg b.w. |
Animal No. |
Micronuclei in Polychromatic Erythrocytes (PCE) |
Evaluation 500 PCE in |
|||||
No. PCE |
No. MN/4000 PCE |
% |
Total No |
NCE per total Ery |
Ratio PCE/Total Ery |
||||
Dose 3 |
2000 |
31 |
4000 |
15 |
0.38 |
625 |
125 |
0.800 |
|
32 |
4000 |
10 |
0.25 |
820 |
320 |
0.610 |
|||
33 |
4000 |
9 |
0.23 |
766 |
266 |
0.653 |
|||
34 |
4000 |
10 |
0.25 |
963 |
463 |
0.519 |
|||
35 |
4000 |
7 |
0.18 |
969 |
469 |
0.516 |
|||
36 |
4000 |
8 |
0.20 |
970 |
470 |
0.515 |
|||
|
|
|
|
|
|
|
|||
Mean |
9.8 |
0.25 |
852.2 |
352.2 |
0.602 |
||||
SD |
2.8 |
0.07 |
141.3 |
141.3 |
0.113 |
Animal Weights
Dose Group |
Animal No. |
Animal weights before treatment |
Animal weights before sacrifice |
||||||||||
Initial Weight [g] |
Mean [g] |
SD [g] |
Range |
Initial Weight [g] |
Mean [g] |
SD [g] |
Range |
||||||
Vehicle Control |
1 |
179.0 |
179.0 |
5.4 |
169.3 |
- |
185.2 |
185.3 |
186.2 |
6.3 |
174.8 |
- |
193.7 |
2 |
185.2 |
193.7 |
|||||||||||
3 |
169.3 |
174.8 |
|||||||||||
4 |
182.6 |
189.8 |
|||||||||||
5 |
179.4 |
186.3 |
|||||||||||
6 |
178.7 |
187.2 |
|||||||||||
500 mg/kg b.w. |
7 |
191.4 |
180.6 |
10.1 |
165.7 |
- |
191.9 |
199.8 |
187.1 |
9.7 |
171.5 |
- |
199.8 |
8 |
181.0 |
186.6 |
|||||||||||
9 |
165.7 |
171.5 |
|||||||||||
10 |
191.9 |
194.6 |
|||||||||||
11 |
178.8 |
186.0 |
|||||||||||
12 |
174.5 |
184.0 |
|||||||||||
1000 mg/kg b.w. |
13 |
187.0 |
187.8 |
9.3 |
175.6 |
- |
200.6 |
194.9 |
193.0 |
10.8 |
177.9 |
- |
205.7 |
14 |
196.7 |
203.9 |
|||||||||||
15 |
200.6 |
205.7 |
|||||||||||
16 |
175.6 |
177.9 |
|||||||||||
17 |
183.4 |
184.8 |
|||||||||||
18 |
183.8 |
190.8 |
|||||||||||
2000 mg/kg b.w. |
19 |
169.8 |
186.6 |
11.6 |
169.8 |
- |
196.6 |
160.8 |
184.6 |
16.6 |
160.8 |
- |
201.9 |
20 |
194.6 |
201.9 |
|||||||||||
21 |
192.3 |
187.0 |
|||||||||||
22 |
192.1 |
185.8 |
|||||||||||
23 |
196.6 |
201.8 |
|||||||||||
24 |
173.9 |
170.0 |
|||||||||||
Positive Control |
25 |
182.6 |
178.7 |
4.6 |
170.4 |
- |
183.5 |
188.7 |
184.1 |
6.8 |
171.2 |
- |
188.7 |
26 |
170.4 |
171.2 |
|||||||||||
27 |
178.7 |
188.2 |
|||||||||||
28 |
178.4 |
185.7 |
|||||||||||
29 |
178.8 |
182.1 |
|||||||||||
30 |
183.5 |
188.5 |
|||||||||||
2000 mg/kg b.w. |
31 |
193.1 |
184.8 |
12.3 |
164.3 |
- |
198.7 |
198.5 |
188.0 |
11.3 |
174.6 |
- |
199.4 |
32 |
198.7 |
199.4 |
|||||||||||
33 |
181.9 |
174.9 |
|||||||||||
34 |
179.7 |
186.4 |
|||||||||||
35 |
164.3 |
174.6 |
|||||||||||
36 |
190.8 |
194.4 |
Animal Weights
Dose Group |
Animal No. |
Animal weights before treatment |
Animal weights before sacrifice |
||||||||||
Initial Weight [g] |
Mean [g] |
SD [g] |
Range |
Initial Weight [g] |
Mean [g] |
SD [g] |
Range |
||||||
Vehicle Control |
1 |
179.0 |
179.0 |
5.4 |
169.3 |
- |
185.2 |
185.3 |
186.2 |
6.3 |
174.8 |
- |
193.7 |
2 |
185.2 |
193.7 |
|||||||||||
3 |
169.3 |
174.8 |
|||||||||||
4 |
182.6 |
189.8 |
|||||||||||
5 |
179.4 |
186.3 |
|||||||||||
6 |
178.7 |
187.2 |
|||||||||||
500 mg/kg b.w. |
7 |
191.4 |
180.6 |
10.1 |
165.7 |
- |
191.9 |
199.8 |
187.1 |
9.7 |
171.5 |
- |
199.8 |
8 |
181.0 |
186.6 |
|||||||||||
9 |
165.7 |
171.5 |
|||||||||||
10 |
191.9 |
194.6 |
|||||||||||
11 |
178.8 |
186.0 |
|||||||||||
12 |
174.5 |
184.0 |
|||||||||||
1000 mg/kg b.w. |
13 |
187.0 |
187.8 |
9.3 |
175.6 |
- |
200.6 |
194.9 |
193.0 |
10.8 |
177.9 |
- |
205.7 |
14 |
196.7 |
203.9 |
|||||||||||
15 |
200.6 |
205.7 |
|||||||||||
16 |
175.6 |
177.9 |
|||||||||||
17 |
183.4 |
184.8 |
|||||||||||
18 |
183.8 |
190.8 |
|||||||||||
2000 mg/kg b.w. |
19 |
169.8 |
186.6 |
11.6 |
169.8 |
- |
196.6 |
160.8 |
184.6 |
16.6 |
160.8 |
- |
201.9 |
20 |
194.6 |
201.9 |
|||||||||||
21 |
192.3 |
187.0 |
|||||||||||
22 |
192.1 |
185.8 |
|||||||||||
23 |
196.6 |
201.8 |
|||||||||||
24 |
173.9 |
170.0 |
|||||||||||
Positive Control |
25 |
182.6 |
178.7 |
4.6 |
170.4 |
- |
183.5 |
188.7 |
184.1 |
6.8 |
171.2 |
- |
188.7 |
26 |
170.4 |
171.2 |
|||||||||||
27 |
178.7 |
188.2 |
|||||||||||
28 |
178.4 |
185.7 |
|||||||||||
29 |
178.8 |
182.1 |
|||||||||||
30 |
183.5 |
188.5 |
|||||||||||
2000 mg/kg b.w. |
31 |
193.1 |
184.8 |
12.3 |
164.3 |
- |
198.7 |
198.5 |
188.0 |
11.3 |
174.6 |
- |
199.4 |
32 |
198.7 |
199.4 |
|||||||||||
33 |
181.9 |
174.9 |
|||||||||||
34 |
179.7 |
186.4 |
|||||||||||
35 |
164.3 |
174.6 |
|||||||||||
36 |
190.8 |
194.4 |
Historical Control Data (Oct 2014 - Dec 2020)
Vehicle Controls (%) |
Male animals |
|
min |
0.025 |
|
max |
0.750 |
|
mean |
0.255 |
|
95% Ctr. Limit |
0.001 |
0.509 |
SD |
0.127 |
|
2x SD |
0.254 |
|
Range of individual animal micronuclei values* |
1 - 30 |
|
No° indiv. values |
240 |
Positive Controls (%) |
Male animals |
|
min |
0.450 |
|
max |
4.525 |
|
mean |
1.718 |
|
95% Ctr. Limit |
-0.086 |
3.522 |
SD |
0.902 |
|
2x SD |
1.804 |
|
Range of individual animal micronuclei values* |
18 - 181 |
|
No° indiv. values |
161 |
*per 4000 Polychromatic Erythrocytes
Historical Control Data (Oct 2014 - Dec 2020)
Vehicle Controls (%) |
Male animals |
|
min |
0.025 |
|
max |
0.750 |
|
mean |
0.255 |
|
95% Ctr. Limit |
0.001 |
0.509 |
SD |
0.127 |
|
2x SD |
0.254 |
|
Range of individual animal micronuclei values* |
1 - 30 |
|
No° indiv. values |
240 |
Positive Controls (%) |
Male animals |
|
min |
0.450 |
|
max |
4.525 |
|
mean |
1.718 |
|
95% Ctr. Limit |
-0.086 |
3.522 |
SD |
0.902 |
|
2x SD |
1.804 |
|
Range of individual animal micronuclei values* |
18 - 181 |
|
No° indiv. values |
161 |
*per 4000 Polychromatic Erythrocytes
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Gene mutation in bacteria
A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (Poth, 2003). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance dissolved in DMSO using either the preincubation or the plate incorporation method. To evaluate the cytotoxicity of the test substance a pre-experiment with eight concentrations in the range of 3 - 5000 µg/plate was performed in the strains TA 98 and TA 100. In the first experiment (plate incorporation), test concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate were used with and without metabolic activation. In the second experiment (preincubation method), test concentrations of 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were used without and 33, 100, 333, 1000, 2500 and 5000 µg/plate were used with metabolic activation. No visible reduction of the background growth was observed in Experiment 1. In Experiment 2, irregular background growth was observed at the highest concentration in strain TA 98 and WP2 uvrA (with S9 mix) and in strain TA 100 (with and without S9 mix). Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used, except WP2 uvrA. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and E. coli strain in the presence and absence of metabolic activation.
Chromosome aberrations
The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in Chinese Hamster lung fibroblasts (V79) performed according to OECD Guideline 473 and GLP (Béres, 2015). The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments, the test substance concentrations 0.5, 1, 2, 4 and 4.5 µg/mL were used for a 3 h-short-term and for a 20 h-continuous exposure period with a 20 and 28 h sampling time without metabolic activation. In addition, the concentrations 10, 20, 40, 80 and 90 µg/mL were used for a 3 h-exposure period with a 20 and 28 h sampling time with metabolic activation (S9 mix). In Experiment 1, no biologically significant increases in the number of cells showing structural chromosome aberrations without gap were observed in the absence of metabolic activation, up to and including cytotoxic concentrations. However, the test substance caused a moderate increase in the number of cells with structural chromosome aberrations in the presence of metabolic activation, up to and including cytotoxic concentrations. This increase was dose associated and biologically important. In Experiment 2, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when the test substance was examined up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 h with a 20 and 28 h sampling time. Following the 3 h-exposure period with metabolic activation and a 28 h sampling time, the test substance caused an increase in the number of cells with structural chromosome aberrations without gaps at 90 µg/mL. This increase was biologically and statistically significant. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9 mix. Positive control chemicals, ethyl methanesulphonate and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. In conclusion, the test substance did not induce structural chromosome aberrations in Chinese Hamster lung fibroblasts (V79) in the absence of metabolic activation. However, the test substance induced structural chromosome aberrations in the presence of metabolic activation. Therefore, the test substance is considered clastogenic under the conditions of this test system.
In conclusion, the test substance did not show mutagenicity in bacteria both in the presence and absence of metabolic activation while clastogenic effects were observed in an in vitro mammalian chromosome aberration test in the presence of metabolic activation. Thus, an in vivo somatic cell study according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) was conducted to assess the genetic toxicity potential of the test substance.
In vivo Micronucleus Assay in Bone Marrow Cells of the Rat
The potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat was assessed. The test substance was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally was 10 mL/kg bw. The administered volume of the positive control was 10 mL/kg. 24 h and 48 h after a single administration of the test item to six males per test group, the bone marrow cells were collected for micronuclei analysis for the occurrence of micronuclei. 4000 polychromatic erythrocytes per animal were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw 48 h preparation interval: 2000 mg/kg bw The highest dose (maximum guideline-recommended dose) was estimated by a pre experiment to be suitable for the main experiment. The animals treated with the test substance and the vehicle control did not exhibit any clinical symptoms. A bioanalysis of the test substance in plasma (report number S21-02411-L1) was performed. The method had been successfully validated by procedural recovery samples for determination of the test substance with an LOQ of 0.10 mg/L and up to 10 mg/L in rat plasma according to the guidance document SANTE/2020/12830 rev. 1 of the European Commission. With regard to selectivity, accuracy and precision, the analytical method was applied successfully for the analytical set when analysing the samples of the study. In all plasma samples of animals treated with the test substance at a concentration of 2000 mg/kg bw., however, the residues of the test substance were below the Limit of Detection (i.e., <0.03 mg/L). Rapid hydrolysis of the test substance was suspected as the most probable underlying reason for the missing proof of exposure. After treatment with the test item the number of PCEs was slightly decreased in the 24 h high dose group (group mean decrease of approximately 9%) as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance may exert a slight, transient cytotoxic effect in the bone marrow at the maximum guideline-recommended dose level of 2000 mg/kg bw. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei at any preparation interval after administration of the test item and with any dose level used. 20 mg/kg bw cyclosphosphamide administered orally was used as positive control which induced a substantial increase in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce an increased frequency of micronuclei in the bone marrow cells of the rat after single oral administration up to a dose level of 2000 mg/kg bw. Therefore, the test substance was considered to be non-genotoxic in the in vivo micronucleus assay.
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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