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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial and mammalian cell gene mutation tests revealed no mutagenic potential. The chromosome aberration test in vitro resulted in a clastogenic response which could not be confirmed in the in vivo assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Feb 2010 - 30 Jun 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL) and 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
1st Experiment (without light protection)
(without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL)
(with S9 mix: 0; 312.5; 625; 1 250; 2 500; 3750; 5000 μg/mL)
2nd Experiment (without light protection)
without S9 mix: 0; (5); (10); (20); 40; 60; 80 μg/mL
with S9 mix: 0; (78.1); (156.3); 312.5; 625; 1250; (2500); (3750) μg/mL
3rd Experiment (under light protection)
without S9 mix: 0; 6.3; 12.5; 25; (50); (100), (200) μg/mL
with S9 mix: 0; 156.3; 312.5; 625; (1250); (2500); (5000) μg/mL
(test groups in brackets were not evaluated)
Vehicle / solvent:
acetone 1% (v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS: 500 μg/mL; CPP: 0.5 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h (both with and without S9)
- Expression time (cells in growth medium): 14 h (both with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (both with and without S9)

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS: All cultures were prepared in duplicate.

NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations. Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; a mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in all cytogenetic experiments.

OTHER EXAMINATIONS:
Aneuploid and polyploid cells were recorded separately.
Evaluation criteria:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 – 22 chromosomes, they were analyzed for structural chromosome aberrations.
The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the main experiments, test substance precipitation in culture medium at the end of exposure period was observed from 625 μg/mL onward only in the presence of S9 mix in all experimental parts.

RANGE-FINDING/SCREENING STUDIES:
In the pretest performed without light protection the pH was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in the vehicle acetone was observed up to the highest required concentration of 5000 μg/mL (stock solution). In culture medium test substance precipitation occurred at 625 μg/mL and above 4 hours after start of treatment in the absence and the presence of S9 mix as well as at 18 hours continuous treatment in the absence of S9 mix. At these concentrations an oily film of the test substance was observed on surface of the culture medium.
After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced cell numbers of below 50% of control was observed at 78.1 μg/mL and above. In addition, in the presence of S9 mix, clearly reduced cell numbers were observed after treatment with 5000 μg/mL. Besides, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 156.3 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
Second experiment: In the presence of metabolic activation after 4 hours treatment a statistically significant increase in the number of chromosomally damaged cells was observed at the highest scorable concentration of 1250 μg/mL (18% aberrant metaphases excl. gaps). This value clearly exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).
Third experiment: In the absence and presence of S9 mix a statistically significant increase in the number of aberrant metaphases was observed at all concentrations scored for cytogenetic damage performed under light protection conditions. In both experimental parts most values exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).

CELL MORPHOLOGY
Cell attachment was influenced from 40.0 μg/mL onward in the 1st or 2nd Experiment without S9 mix. When applying light protection in the 3rd Experiment the cell attachment was slightly reduced from 12.5 μg/mL onward in the absence of S9 mix. After the addition of S9 mix cell morphology was influenced from about 625 μg/mL onward in all experiments performed with or without light exclusion.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix 4 hours after treatment in the 2nd and 3rd Experiment, no suppression of the mitotic activity was observed at any dose analyzed for chromosomal aberrations. In the 2nd Experiment in the absence of metabolic activation after 4 hours treatment up to 80 μg/mL no reduction of mitotic activity was obtained. In the 3rd Experiment in the absence of metabolic activation the slides from 50 μg/mL onward were not scorable due to low metaphase numbers. Besides, in the presence of S9 mix in the 2nd and 3rd Experiment after 4 hours treatment the concentrations showing clearly reduced mitotic indices were not scorable for cytogenetic damage due to low metaphase numbers and/or poor metaphase quality (from 2 500 μg/mL or 1 250 μg/mL onward, respectively).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system. The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either.

Summary table – experimental parts without S9 mix

Exp. Schedule Test groups S9 mix P Genotoxicity Cytotoxicity*
Exposure/ preparation period Aberrant cells [%] Polyploidy cells [%]
Incl. gaps# Excl. gaps# With exchanges Cell no. [%] Mitotic index [%]
2 4/18 h Vehicle control1 - - 7 3 0.5 0 100 100
5.0 mg/mL - - n.d. n.d. n.d. n.d. 98 n.d.
10.0 mg/mL - - n.d. n.d. n.d. n.d. 85 n.d.
20.0 mg/mL - - n.d. n.d. n.d. n.d. 81.8 n.d.
40.0 mg/mL - - 5 3.5 1 0 82.9 111.6
60.0 mg/mL - - 4.5 3.5 1 0 81.8 105.5
80.0 mg/mL - - 7 5.5 3.5 0 38.7 103.4
Positive control2x - - 21.0s 18.0s 10.0s 0 n.t. 103.4
     Performance under light protection
2 4/18 h Vehicle control1 - - 4 1.5 0.5 0 100 100
6.3 mg/mL - - 8.5s 5.5s 3 1 74.8 89.4
12.5 mg/mL - - 13.5s 9.5s 7.5s 0.5 47.6 105.3
25.0 mg/mL - - 14.5s 10.0s 6.5s 0 46.5 104.6
50.0 mg/mL - - n.s. n.s. n.s. n.s. 4.1 n.s.
100.0 mg/mL - - n.s. n.s. n.s. n.s. 3.2 n.s.
200.0 mg/mL - - n.s. n.s. n.s. n.s. 4.8 n.s.
Positive control2x - - 20.0s 17.0s 10.0s 0 n.t. 82.8

Summary table – experimental parts with S9 mix

Exp. Schedule Test groups S9 mix P Genotoxicity Cytotoxicity*
Exposure/ preparation period Aberrant cells [%] Polyploidy cells [%]
Incl. gaps# Excl. gaps# With exchanges Cell no. [%] Mitotic index [%]
2 4/18 h Vehicle control1 + - 5 3.5 1.5 0 100 100
78.1 mg/mL + - n.d. n.d. n.d. n.d. 88.9 n.d.
156.3 mg/mL + - n.d. n.d. n.d. n.d. 89.8 n.d.
312.5 mg/mL + - 7.5 5 3.5 0 78.5 103
625.0 mg/mL + - 6 4.5 3 0 78.5 79.2
1250.0 mg/mLx + + 18.0s 18.0s 16.0s 0 55.2 81.7
2500.0 mg/mL + + n.s. n.s. n.s. n.s. 56.7 29.2
3750.0 mg/mL + + n.s. n.s. n.s. n.s. 27.4 n.s.
Positive control3x + - 27.0s 25.0s 10.0s 0 n.t. 60.4
     Performance under light protection
2 4/18 h Vehicle control1 + - 4.5 2 0.5 0 100 100
156.3 mg/mL + - 11.5s 9.0s 4.0s 0.5 87.1 84.8
312.5 mg/mL + - 13.0s 9.0s 4.0s 0.5 83 99
625.0 mg/mLx + + 31.0s 30.0s 23.0s 0 73 85.3
1250.0 mg/mL + + n.s. n.s. n.s. n.s. 48.8 40.6
2500.0 mg/mL + + n.s. n.s. n.s. n.s. 51.2 n.s.
500.0 mg/mL + + n.s. n.s. n.s. n.s. 41.4 n.s.
Positive control3x + - 26.0s 22.0s 12.0s 0 n.t. 77.7

P Precipitation occured at the end of exposure period

* Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

s Aberration frequency statistically significant higher than corresponding control values

x Evaluation of a sample of 100 metaphase only due to strong clastogenicity

1 Acetone 1% (v/v)

2 EMS500 μg/mL

3 CPP 0.5mg/mL

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity and/or poor metaphase quality

n.t. Not tested

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that the test article is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolizing system. During the experimental phase of this study the lab was informed by the sponsor that the test substance is an UV-curing agent. Therefore, it is light sensitive and has to be stored and handled protected from light. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested and the test groups in bold type were evaluated:

1st Experiment (without light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 312.5; 625; 1250; 2500; 3750; 5000 μg/mL

The 1st Experiment with and without S9 mix had to be repeated due to severe cytotoxicity (strongly reduced mitotic rates) and partly poor metaphase quality.

2nd Experiment (without light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 78.1; 156.3; 312.5; 625; 1250; 2500; 3750 μg/mL

The 2nd Experiment with and without S9 mix had to be repeated due to an update on storage conditions and handling instructions indicating protection from light.

3rd Experiment (under light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 6.3; 12.5; 25; 50; 100, 200 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 156.3; 312.5; 625; 1250; 2500; 5000 μg/mL

A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures and the concentrations 1250 μg/mL and 625 μg/mL in the 2nd and 3rd Experiment, respectively, where only 50 metaphases were scored due to clearly increased aberration rates. The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation. in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system (see Table 1, page 14 – 15). The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the test article is considered to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 30, 2010 - September 10, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin.
- 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:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
without S9: 0.6 - 160 microg/mL
with S9: 20 - 320 microg/mL
Vehicle / solvent:
tetrahydrofuran (THF, not excceding 0.5% (v/v) in the culture medium
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
EMS: 0.150 mg/mL; DMBA: 1.1 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 days

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
STAIN: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: two parallel cultures for each concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 106 cells found in the solvent con¬trols falls within the laboratory historical control data from 2006 - 2008
b) the positive control substances should produce a significant increase in mutant colony frequencies.
c) the cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH
- Effects of osmolality: There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.

RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration in the pre-test on toxicity (2600 μg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 476. Strong cytotoxic effects occurred at 162.5 μg/mL without metabolic activation and at 325 μg/mL and above with metabolic activation following 4 hours treatment. After continuous treatment (24 hours), solely performed without metabolic activation, relevant toxic effects were noted at 81.3 μg/mL and above. The test medium was checked for precipitation or turbidity at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 2600 μg/mL after 4 and 24 hours treatment in the absence of metabolic activation. In the presence of metabolic activation precipitation occurred after 4 hours treatment at 1300 μg/mL treatment at 1300 μg/mL and above. In the pre-experiment the two highest concentrations were neutralised with hydrochloric acid. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the main experiments (with and without S9 mix) the range of the solvent controls was from 11.3 up to 35.1 mutants per 106 cells; the range of the groups treated with the test item was from 3.4 up to 46.1 mutants per 106 cells. The highest solvent control slightly exceeded the historical range (35.1 versus 31.5 colonies per 106 cells). This deviation was considered irrelevant since it was very minor and the solvent control of the parallel culture remained well within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50% in at least one of the parallel cultures occurred in the first experiment at 20.0 μg/mL and above without metabolic activation and at 160 μg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 80.0 μg/mL without and 240 μg/mL with metabolic activation. In the additional experiment IIA, cytotoxicity was noted at 80.0 μg/mL and above. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I was covered with and without metabolic activation.

EXPERIMENTAL RESULT

The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Experiment I without metabolic activation had to be terminated prematurely due to irregular cell growth at all concentrations. This part was repeated with modified concentrations in the absence of metabolic activation. The experimental part of the second experiment without metabolic activation was repeated as experiment IIA since the recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 was not covered.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor did not reach the threshold of three times the mutation frequency of the corresponding solvent controls in any of the experimental parts. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation and in the first culture of experiment IIA without metabolic activation. These increases however, were judged as irrelevant since the induction factor did not reach the threshold discussed above.

Summary of results:

concentration (µg/ml) S9 Mix relative cloning efficiency I (%) relative cloning efficiency II (%) mutant colonies / 106 cells induction factor relative cloning efficiency I (%) relative cloning efficiency II (%) mutant colonies / 106 cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (THF) - 100.0 100.0 11.3 1.0 100.0 100.0 15.0 1.0
positive control (EMS) 150.0 - 69.4 90.5 90.8 8.0 71.7 98.8 95.9 6.4
test item 0.6 - 102.3 culture was not continued# 94.9 culture was not continued#
test item 1.3 - 87.0 91.2 21.3 1.9 85.9 104.2 20.0 1.3
test item 2.5 - 86.2 90.3 10.1 0.9 87.7 87.7 12.5 0.8
test item 5.0 - 66.5 95.4 3.4 0.3 83.6 99.1 9.4 0.6
test item 10.0 - 57.1 94.7 10.6 0.9 72.5 95.8 24.4 1.6
test item 20.0 - 4.5 91.0 10.3 0.9 39.1 78.9 22.0 1.5
test item 40.0 - 0.0 culture was not continued## 3.4 95.3 32.4 2.2
solvent control (THF) + 100.0 100.0 19.7 1.0 100.0 100.0 16.1 1.0
positive control (DMBA) 1.1 + 55.4 71.6 1430.0 72.6 52.1 84.7 1109.9 69.0
test item 20.0 + 105.9 85.0 30.4 1.5 103.8 94.9 18.6 1.2
test item 40.0 + 96.4 99.0 17.7 0.9 98.6 88.1 18.4 1.1
test item 80.0 + 93.8 91.5 16.2 0.8 91.1 91.4 40.0 2.5
test item 160.0 + 15.1 86.8 3.9 0.2 88.5 86.8 38.8 2.4
test item 240.0 + culture was not continued## 19.9 87.2 21.0 1.3
320.0 + culture was not continued## culture was not continued##
Experiment II / 24h treatment culture I culture II
solvent control (THF) - 100.0 100.0 17.6 1.0 100.0 100.0 21.9 1.0
positive control (EMS) 150.0 - 82.9 54.6 681.5 38.7 90.2 58.1 606.2 27.7
test item 1.3 - 82.1 culture was not continued# 110.5 culture was not continued#
test item 2.5 - 89.6 culture was not continued# 103.1 culture was not continued#
test item 5.0 - 88.8 99.7 30.9 1.8 109.7 95.4 28.9 1.3
test item 10.0 - 95.0 96.8 16.7 1.0 110.9 93.8 19.2 0.9
test item 20.0 - 92.8 96.4 13.8 0.8 110.7 93.8 15.8 0.7
test item 40.0 - 90.1 87.1 23.4 1.3 111.1 93.2 15.4 0.7
test item 80.0 - 40.2 62.4 34.7 2.0 90.1 63.1 22.3 1.0
Experiment II / 4h treatment culture I culture II
solvent control (THF) + 100.0 100.0 15.8 1.0 100.0 100.0 30.1 1.0
positive control (DMBA) 1.1 + 73.8 51.7 861.0 54.4 78.4 40.3 1067.7 35.5
test item 20.0 + 92.2 culture was not continued# 101.8 culture was not continued#
test item 40.0 + 94.7 90.0 13.5 0.9 100.5 76.4 34.2 1.1
test item 80.0 + 94.0 100.8 11.1 0.7 89.7 96.9 21.5 0.7
test item 120.0 + 84.6 93.6 21.8 1.4 87.3 92.6 19.9 0.7
test item 180.0 + 50.8 95.8 17.6 1.1 82.0 80.5 32.1 1.1
test item 240.0 + 0.9 88.0 13.3 0.8 46.8 82.4 31.2 1.0
Experiment IIA / 24h treatment culture I culture II
solvent control (THF) - 100.0 100.0 18.4 1.0 100.0 100.0 35.1 1.0
positive control (EMS) 150.0 - 93.0 87.5 374.9 20.4 95.2 109.6 313.1 8.9
test item 10.0 - 98.6 85.7 17.7 1.0 97.7 102.6 23.6 0.7
test item 20.0 - 95.7 87.4 20.6 1.1 95.6 99.5 34.6 1.0
test item 40.0 - 72.1 77.5 34.6 1.9 99.5 108.0 46.1 1.3
test item 80.0 - 8.9 82.0 44.5 2.4 56.8 88.6 44.6 1.3
test item 120.0 - 0.0 culture was not continued## 2.7 culture was not continued##
test item 160.0 - 0.0 culture was not continued## 0.0 culture was not continued##

# culture was not continued since a minimum of only four analysable concentrations is required

## culture was not continued due to exceedingly severe cytotoxic effects

cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity.

cloning efficiency II (viability): cloning efficiency determined after the expression period to measure viability of the cells without selective agent.

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster following OECD guideline 476. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. A third experiment without metabolic activation was performed since the recommended cytotoxic range of approximately 10-20% was not covered in the second experiment without metabolic activation. The third experiment, designated experiment IIA, was performed with a treatment time of 24 hours. The cell cultures were evaluated at the following concentrations: Experiment I: without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 μg/mL; with S9 mix: 20.0; 40.0; 80.0; and 160.0 μg/mL Experiment II: without S9 mix: 5.0; 10.0; 20.0; 40.0; and 80.0 μg/mL; with S9 mix: 40.0; 80.0; 120.0; 180.0; and 240.0 μg/mL Experiment IIA: without S9 mix: 10.0; 20.0; 40.0; and 80.0 μg/mL The highest concentration of the main experiments was limited by the cytotoxicity of the test item. Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50% occurred in the first experiment at 20.0 µg/mL and above without metabolic activation and at 160 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 80.0 µg/mL without and 240 µg/mL with metabolic activation. In the additional experiment IIA, cytotoxicity was noted at 80.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor did not reach the threshold of three times the mutation frequency of the corresponding solvent controls in any of the experimental parts. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation and in the first culture of experiment IIA without metabolic activation. These increases however, were judged as irrelevant since the induction factor did not reach the threshold discussed above. In the main experiments (with and without S9 mix) the range of the solvent controls was from 11.3 up to 35.1 mutants per 106 cells; the range of the groups treated with the test item was from 3.4 up to 46.1 mutants per 106 cells. The highest solvent control slightly exceeded the historical range (35.1 versus 31.5 colonies per 106 cells). This deviation was considered irrelevant since it was very minor and the solvent control of the parallel culture remained well within the historical range. EMS (0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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:
From Jul. 16, 1980 to Sep. 8, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No independent repeat test conducted.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively.
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Toxicity test (performed on strain TA 100): 0.0001, 0.001, 0.01, 0.1, 1, 10, 50, 100, 500, 1000, and 2000 µg/0.1 mL.
Experiment (performed on all strains): 5, 10, 50, 100, 500, 1000, and 5000 µg/0.1 mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: None provided.
Method for suspension of refractory substances: Ultrasonication
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See Table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 55 hours at 37 degrees C.

NUMBER OF REPLICATIONS: 3 Petri dishes/strain/group.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated. Statistical analysis was not performed.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 500, 1,000, and 5,000 µg/0.1mL, the substance precipitated in soft agar.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

DETAILS ON EXPERIMENTAL RESULTS:

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test article revealed no marked deviations.

TA 98 TA 100 TA 1535 TA 1537 TA 1538 WP2uvrA
Dose (µg/0.1 ml) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 27 53 166 106 12 7 4 10 9 31 24 25
5 14 39 155 109 8 10 6 12 9 30 23 21
10 18 59 154 99 10 8 4 12 11 31 22 12
50 16 50 157 102 11 10 5 13 13 30 21 21
100 21 42 146 93 11 11 5 5 9 37 23 19
500 19 50 137 90 8 12 3 12 5 38 17 17
1000 15 55 171 101 10 9 2 13 10 27 16 17
5000 15 65 142 94 5 10 0 7 5 28 17 11
positive controls:
solvent control 17 45 180 146 11 12 11 17 9 32 19 15
concentration A 385 1163 712 1131 >1500 530 136 73 ~1100 374 318 617
concentration B 533 1261 1187 1932 >2000 1000 52 >1500 280 430 764

For details on positive controls see table 1 above.

Conclusions:
No evidence for a mutagenic effect was obtained in Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E.coli strain WP2 uvr A treated with up to 5000 µg/plate in experiments with and without microsomal activa¬tion.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 15, 2013 - February 15, 2013
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals 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: pre-experiment: 8 – 9 weeks, main experiment: 9 - 10 weeks
- Weight at study initiation: mean value 36.7 g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages with wire mesh top(EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: pelleted standard diet, ad libitum, (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 45 - 65 %
- Photoperiod (hrs dark / hrs light): 12/12
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.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in corn oil.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
24 h preparation interval: 500, 1000, and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Seven males were assigned to each test group (except the vehicle and positive control groups with five animals each).
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Route of administration: orally, once
- Volume administered: 10 mL/kg bw
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 0-1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were administered (500, 1000 and 2000 mg/kg), and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Since no gender specific differences were observed in the pre-experiment, the main study was performed using males only in accordance with the test guidelines.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. 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 (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

OTHER:
The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 0-1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
nonparametric Mann-Whitney test
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, Eyelid closure, Ruffled fur, Tumbling

RESULTS OF DEFINITIVE STUDY
In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw.
After treatment with the test item at the 24 h and 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test article did not induce cytotoxic effects in the bone marrow.
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 were below to the value of the vehicle control group and well within the historical control data range. The positive control showed a statistically significant increase of induced micronucleus frequency.

Summary of Micronucleus Test Results

Test group Dose(mg/kg bw) Sampling time (h) PCEs with micronuclei (%) Range PCE per 2000 erythrocytes
Vehicle control 0 24 0.24 2 - 9 1212
Test item 500 24 0.143 1 - 5 1286
Test item 1000 24 0.121 0 - 8 1243
Test item 2000 24 0.214 1 - 9 1210
Positive control 40 24 2.7 33 - 86 1200
Vehicle control 0 48 0.15 2 - 4 1212
Test item 2000 48 0.114 0 - 5 1202

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group Significance p
500  mg/kg bw; 24 h n.t. -
1000 mg/kg bw; 24 h n.t. -
2000 mg/kg bw; 24 h n.t. -
40 mg CPA/kg bw; 24 h + 0.004
2000 mg/kg bw; 48 h n.t. -

+ = significant;

n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse and is therefore considered to be non-mutagenic in this micronucleus assay.
Executive summary:

A GLP-compliant study following OECD guideline 474 was performed to investigate the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) 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 2000 erythrocytes. The following dose levels of the test item were investigated, based on results of a pre-experiment:

24 h preparation interval: 500, 1000, and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw. After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. The mean values of micronuclei observed after treatment were below to the value of the vehicle control group and well within the historical control data range. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test article is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames test

The test article was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophan-auxotrophic strain of E. coli (Ciba-Geigy, 1980). The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml. In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of test material revealed no marked deviations. No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.

Similar results are reported in a supporting study, which showed no marked deviations in the comparison of the number of back-mutant colonies in the controls and the treated cultures either with or without microsomal activation (Gakushi University, 1982).

HPRT test

A GLP-compliant study following OECD guideline 476 was performed with a structural analogue (see attached read across justification) to investigate its potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan, 2010). The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. A third experiment without metabolic activation was performed since the recommended cytotoxic range of approximately 10-20% was not covered in the second experiment without metabolic activation. The third experiment, designated experiment IIA, was performed with a treatment time of 24 hours. The used concentrations ranged from 0.6 - 160 µg/mL (without S9) and from 20 - 320 µg/mL (with S9). No substantial and reproducible dose dependent increase of the mutation frequency was observed in any of the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

in vitro chromosome aberration test

According to the results of the included in vitro cytogenetic study (BASF, 2010), the test substance led to a statistically significant and biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps both either without S9 mix or after the addition of a metabolizing system in a single experiment performed under light protection conditions with 4 hours treatment interval and 18 hours sampling time. In detail, in the 2nd Experiment after 4 hours treatment in the presence of metabolic activation a statistically significant increased chromosome aberration rate clearly exceeding the historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps) was obtained at 1250 μg/mL (18% aberrant metaphases excl. gaps) when handling the test substance without light exclusion. This finding was assumed as artificial observation due to missing light protection. However, in the 3rd Experiment, performed following the sponsors recent instructions on handling under light exclusion, the chromosome damaging effect was corroborated. The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

In vivo micronucleus test

A GLP-compliant study following OECD guideline 474 was performed to investigate the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Harlan, 2013). The test item was dissolved in corn oil and administered at the following dose levels: 500 and 1000 mg/kg (24h preparation interval) and 2000 mg/kg bw (24 and 48 h preparation interval). Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. The positive control showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test article is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.