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Genetic toxicity in vitro

Description of key information

Ames: negative (BASF, 2015) HPRT: negative (BASF, 2015) Micronucleus: negative (BASF, 2015)

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Sep 2014 - 08 Apr 2015
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)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815) supplemented with 10% (v/v) fetal calf serum (FCS).
- 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 and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
1st Experiment (discontinued due to technical reasons)
without S9 mix (4-hour exposure period): (0); (0.16); (0.31); (0.63); (1.25); (2.50); (5.00); (10.00); (20.00) μg/mL
with S9 mix (4-hour exposure period): (0); (0.16); (0.31); (0.63); (1.25); (2.50); (5.00); (10.00); (20.00) μg/mL
2nd Experiment
without S9 mix (4-hour exposure period): 0; 0.16; 0.31; 0.63; 1.25; (2.50); (5.00); (10.00); (20.00) μg/mL
with S9 mix (4-hour exposure period): 0; (0.16); (0.31); 0.63; 1.25; 2.50; 5.00; (10.00); (20.00) μg/mL
3rd Experiment
without S9 mix (4-hour exposure period): 0; (0.06); (0.13); 0.25; 0.50; 1.00; 2.00; (4.00); (8.00) μg/mL
with S9 mix (4-hour exposure period): 0; (0.13); (0.25); (0.50); 1.00; 2.00; 4.00; 8.00; (16.00) μg/mL
numbers in parantheses were not evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used solvents (acetone, ethanol, tetrahydrofurane), dimethyl sulfoxide (DMSO) was the most suitable one.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
with S9: 1.25 µg/ml DMBA; without S9: 400 µg/ml EMS
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 5-7 days
- Selection time (if incubation with a selection agent): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:
Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
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: no precipitation in culture medium was observed up to the highest applied test substance concentration.


RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 2300 μg/mL (approx. 10 mM) test substance was used as top concentration both with and without S9 mix at 4-hour exposure time. After 4 hours treatment in the absence and presence of S9 mix cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% in all applied concentrations (9 µg/ml was the lowest dose level).

CELL MORPHOLOGY
In the absence and presence of S9 mix, after 4 hours treatment in nearly all experiments the morphology and attachment of the cells was adversely influenced (grade > 2) in at least the highest applied concentration. In detail, in the absence of S9 mix after 4 hours treatment the cell morphology and attachment of the cells was adversely influenced from 10 μg/mL onward in the 1st Experiment, from 5 μg/mL onward in the 2nd Experiment and at 8 μg/mL in the 3rd Experiment. Besides, in the 2nd and 3rd Experiment in the presence of S9 mix after 4 hours treatment the cell morphology and attachment of the cells was adversely influenced at 20 μg/mL and at 16 μg/mL, resepctively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in all evaluated experiments in the absence and presence of S9 mix, at least at the highest applied concentrations. Without S9 mix, there was a decrease in the number of colonies from 2.50 μg/mL onward after an exposure period of 4 hours in the 1st and 2nd Experiment (CE1 relative: 8.1% or 0.0%, respectively). Cytotoxic effects were observed in the 3rd Experiment from 4.00 μg/mL (CE1 relative: 0.0%) onward. The cell densities were distinctly reduced in the expression period. In contrast, with S9 mix, there was a decrease in the number of colonies from 10.00 μg/mL onward in the 2nd Experiment and at 16.00 μg/mL in the 3rd Experiment (CE1 relative: 0.0%, each). The cell densities were distinctly reduced in the expression period. Due to microbial contamination the cultures seeded for determination of survival (CE1) were not scorable in the 1st Experiment with S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results – experimental parts without metabolic activation

Exp. Exposure period [h] Test group [µg/ml] S9 mix Prec.* Genotoxicity**
Mfcorr [per 106 cells]
Cytotoxicity***
CE1 [%]
Cytotoxicity***
CE2 [%]
1 4 vehicle control1 - n.d. n.c.(3) 100 n.c.(3)
0.16 - - n.c.(3) 95.1 n.c.(3)
0.31 - - n.c.(3) 102.7 n.c.(3)
0.63 - - n.c.(3) 91.9 n.c.(3)
1.25 - - n.c.(3) 55 n.c.(3)
2.5 - - n.c.(3) 8.1 n.c.(3)
5 - - n.c.(3) 0 n.c.(3)
10 - - n.c.(3) 0 n.c.(3)
20 - - n.c.(3) 0 n.c.(3)
positive control2 - n.d. n.c.(3) 100.1 n.c.(3)
2 4 vehicle control1 - n.d. 1.56 100 100
0.16 - - 0.62 107.6 105.3
0.31 - - 0.65 104.2 96.9
0.63 - - 0 104.2 105.3
1.25 - - 2.17 81.2 100.3
2.5 - - n.c. (2) 0 n.c. (2)
5 - - n.c. (2) 0 n.c. (2)
10 - - n.c. (2) 0 n.c. (2)
20 - - n.c. (2) 0 n.c. (2)
positive control2 - n.d. 82.17 93.6 84.2
3 4 vehicle control1 - n.d. 1.78 100 100
0.06 - - n.c. (1) 106.5 n.c. (1)
0.13 - - n.c. (1) 99.2 n.c. (1)
0.25 - - 0 97.8 107.3
0.5 - - 4.14 104.2 115.8
1 - - 0.27 97.8 119.1
2 - - 1.63 53.2 116.3
4 - - n.c. (2) 0 n.c. (2)
8 - - n.c. (2) 0 n.c. (2)
positive control2 - n.d. 114.05 94.5 103.7

Summary of results – experimental parts with metabolic activation

Exp. Exposure period [h] Test group [µg/ml] S9 mix Prec.* Genotoxicity**
Mfcorr [per 106 cells]
Cytotoxicity***
CE1 [%]
Cytotoxicity***
CE2 [%]
1 4 vehicle control1 + n.d. n.c.(3) n.c.(3) n.c.(3)
0.16 + - n.c.(3) n.c.(3) n.c.(3)
0.31 + - n.c.(3) n.c.(3) n.c.(3)
0.63 + - n.c.(3) n.c.(3) n.c.(3)
1.25 + - n.c.(3) n.c.(3) n.c.(3)
2.5 + - n.c.(3) n.c.(3) n.c.(3)
5 + - n.c.(3) n.c.(3) n.c.(3)
10 + - n.c.(3) n.c.(3) n.c.(3)
20 + - n.c.(3) n.c.(3) n.c.(3)
positive control3 + n.d. n.c.(3) n.c.(3) n.c.(3)
2 4 vehicle control1 + n.d. 1.4 100 100
0.16 + - n.c. (1) 95.2 n.c. (1)
0.31 + - n.c. (1) 110.2 n.c. (1)
0.63 + - 3.52 110.4 99.1
1.25 + - 1.23 109.2 84.8
2.5 + - 1.78 108.4 92.1
5 + - 2.17 98.2 96.7
10 + - n.c. (2) 0 n.c. (2)
20 + - n.c. (2) 0 n.c. (2)
positive control3 + n.d. 130.29 105 77.3
3 4 vehicle control1 + n.d. 4.84 100 100
0.13 + - n.c. (1) 105.1 n.c. (1)
0.25 + - n.c. (1) 100.5 n.c. (1)
0.5 + - n.c. (1) 110.5 n.c. (1)
1 + - 0.37 105.4 95.2
2 + - 1.46 110.6 109.9
4 + - 0 105.2 113.5
8 + - 0 63.5 119.9
16 + - n.c. (2) 0 n.c. (2)
positive control3 + n.d. 140.91 106.2 114.9

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c. (1) Culture was not continued since a minimum of only four analysable concentrations are required

n.c. (2) Culture was not continued due to strong cytotoxicity

n.c. (3) Culture was not continued due to technical reasons (microbial contamination)

n.d. Not determined

1 DMSO 1% (v/v)

2 EMS 400 μg/mL

3 DMBA 1.25 μg/mL

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the

addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test the concentrations ranged from 0.16 to 8.0 µg/ml. Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguaninecontaining medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study, in the 2nd and 3rd Experiment, at least the highest concentrations tested for gene mutations were clearly cytotoxic in the absence and presence of metabolic activation. The 1st Experiment was discontinued due to technical reasons. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep 2014 - 15 Apr 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 487, 22 Jul 2010, “In vitro Mammalian Cell Micronucleus Test”
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012; B.49
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus 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), 1% (v/v) amphotericine B (250 μg/mL). During exposure to the test substance in the presence of S9 mix MEM medium was used without FCS supplementation.
- 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
4 hours exposure, 24 hours harvest time, without S9 mix: 0; (0.39); 0.78; 1.56; 3.13; (6.25); (12.50); (25.00) μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 0; (0.78); (1.56); 3.13; 6.25; 12.50; (25.00); (50.00) μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix: 0; (0.16); (0.31); 0.63; 1.25; 2.50; (5.00); (10.00) μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix: 0; (0.63); (1.25); 2.50; 5.00; 10.00; (20.00); (40.00) μg/mL
The test groups in parentheses were not evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used solvents (acetone, ethanol, tetrahydrofurane), dimethyl sulfoxide (DMSO) was the most suitable one.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
with and without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hrs
- Expression time (cells in growth medium): 20 and 40 hrs
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 44 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide

NUMBER OF REPLICATIONS:
Two independent experiments were carried out and at least 2 cultures were prepared per test group.

NUMBER OF CELLS EVALUATED:
At least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, proliferation index
Evaluation criteria:
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the test groups is not distinctly increased above the concurrent vehicle control and is within our laboratory’s historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system. The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 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 only in the presence of metabolic activation from about 10 μg/mL onward either at 24 or 44 hours preparation interval.

RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration of 50 mg/mL (Test group: 500 μg/mL) was a homogeneous suspension in the most appropriate solvent DMSO. Test substance solutions in DMSO were obtained from 10 mg/mL (Test group: 100 μg/mL) downward. In culture medium test substance precipitation occurred 4 hours after start of treatment at 100 μg/mL and above in the absence of S9 mix and at 10 μg/mL and above in the presence of S9 mix. At 24 hours continuous treatment in the absence of S9 mix precipitation of the test substance in culture medium occurred at 500 μg/mL at the end of exposure period. After 4 and 24 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced cell numbers of about or below 40 - 50% was observed at 5 μg/mL and above. In addition, in the presence of S9 mix, clearly reduced cell counts were observed after treatment with 10 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of micronucleated cells in the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix from about 5 μg/mL onward in both experiments and in the presence of S9 mix at 25 μg/mL in the 1st Experiment and at 20 μg/mL in the 2nd Experiment the slides were not scorable for the occurrence of micronucleated cells due to low proliferation rates.

Summary table - experimental parts without S9 mix

 

         

Cytotoxicity

Exp Exposure / Preparation interval Test groups S9 Mix Prec.* Genotoxicity
Micronucleated cells**
[%]
Proliferation
index
cytostasis
(CBPI)
[%]
Cell count
[%]
1 4/24 hrs Vehicle control1 - n.d. 0.7 0.0 100.0
0.39 µg/mL - - n.d. n.d. 74.7
0.78 µg/mL - - 0.4 5.2 76.7
1.56 µg/mL - - 0.5 4.3 73.5
3.13 µg/mL - - 0.9 10.5 50.3
6.25 µg/mL - - n.s. n.s. 48.1
12.50 µg/mL - - n.s. n.s. 47.2
25.00 µg/mL - - n.s. n.s. 49.6
Positive control2 - n.d. 2.5S 4.3 101.4
2 24/24 hrs Vehicle control1 - n.d. 0.4 0.0 100.0
0.16 µg/mL - - n.d. n.d. 91.8
0.31 µg/mL - - n.d. n.d. 88.4
0.63 µg/mL - - 0.3 1.4 90.7
1.25 µg/mL - - 0.4 3.5 77.3
2.50 µg/mL - - 0.5 5.7 94.7
5.00 µg/mL - - n.s. n.s. 92.5
10.00 µg/mL - - n.s. n.s. 152.0
Positive control2 - n.d. 2.6S 16.1 148.0

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity

1 DMSO 1% (v/v)

2 EMS 400 μg/mL

Summary table - experimental parts with S9 mix

           

Cytotoxicity

Exp Exposure / Preparation interval Test groups S9 Mix Prec.* Genotoxicity
Micronucleated cells**
[%]
Proliferation
index
cytostasis
(CBPI)
[%]
Cell count
[%]
1 4/24 hrs Vehicle control1 + n.d. 0.5 0.0 100.0
0.78 µg/mL + - n.d. n.d. 93.2
1.56 µg/mL + - n.d. n.d. 80.6
3.13 µg/mL + - 0.6 11.5 76.8
6.25 µg/mL + - 0.6 10.2 77.6
12.50 µg/mL + + 0.6 20.4 59.1
25.00 µg/mL + + n.s. n.s. 61.1
50.00 µg/mL + + n.d. n.d. 66.9
Positive control2 + n.d. 3.1S 26.6 83.8
2 4/44 hrs Vehicle control1 + n.d. 0.4 0.0 100.0
0.63 µg/mL + - n.d. n.d. 89.2
1.25 µg/mL + - n.d. n.d. 83.0
2.50 µg/mL + - 0.2 -1.4 90.4
5.00 µg/mL + - 1.1S 3.3 78.8
10.00 µg/mL + + 1.6S 4.2 54.0
20.00 µg/mL + + n.s. n.s. 50.6
40.00 µg/mL + + n.d. n.d. 50.6
Positive control2 + n.d. 4.6S -7.1 83.9

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

SFrequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity

1 DMSO 1% (v/v)

2 CPP 0.5 μg/mL

Conclusions:
Under the experimental conditions described, the test article is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic

activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix: 0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.16; 0.31; 0.63; 1.25; 2.50; 5.00; 10.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced cell count and/or cell proliferation was observed in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. A dose-dependancy and statistical significance was observed in Experiment 2 after 4 hours of treatment in the presence of S9 mix. However, all values were close to or within the respective negative control range and clearly within our historical negative control data range. In addition, this finding was not reproduced in Experiment 1 in the presence of S9 mix. Therefore, these findings have to be regarded as biologically irrelevant. Thus, under the experimental conditions described, the test article is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Aug 2014 - 12 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital and β-naphthoflavone induced rats
Test concentrations with justification for top dose:
1st Experiment (SPT with and without S9): 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd Experiment (SPT with and without S9): 0; 0.1; 0.3; 1.0; 3.3; 10 and 33 μg/plate; due to technical reason and strong bacteriotoxicity observed in the 1st experiment, the experimental part was repeated with lower dose groups.
3rd Experiment (SPT without S9): 0; 0.01; 0.03; 0.1; 0.3; 1.0 and 3.3 μg/plate; due to strong bacteriotoxicity observed in the 2nd experiment, the experimental part was repeated with lower dose groups using S. typhimurium TA 1535, TA 1537, TA 98, TA 100.
4th Experiment (PIT): 0; 0.01; 0.03; 0.1; 0.3; 1.0 and 3.3 μg/plate (without S9 mix); 0; 0.1; 0.3; 1.0; 3.3; 10 and 33 μg/plate (with S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
see below for details
Positive control substance:
other: see below
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: at 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn

POSITIVE CONTROLS
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO, with strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, with strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO, with strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO, with strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO, with strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO, with strain: E. coli WP2 uvrA
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strong bacteriotoxicity was observed from about 3.3 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strong bacteriotoxic effects (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) were observed in the standard plate test depending on the strain and test conditions from about 3.3 μg/plate onward. In the preincubation assay strong bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 3.3 μg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

1st Expermient (SPT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 26.0 28.7 - - - - - - 62.7 45.7
33 8.7 12.7 - - - - - - 0.0 49.3
100 0.0 0.0 - - - - - - 0.0 0
333 0.0 0.0 - - - - - - 0.0 0
1000 0.0 0.0 - - - - - - 0.0 0
2500 0.0 0.0 - - - - - - 0.0 0
5000 0.0 0.0 - - - - - - 0.0 0
positive control 358.3 1339.7 - - - - - - 854.3 248

Fields with "-": Technical fault

2nd Experiment (SPT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 19.0 23.0 45.3 55 13 12 6.3 8 58.3 65
0.1 21.7 23.7 54 49.3 12.3 12.7 5.7 9 50.7 59.7
0.3 16.0 24.7 63.3 50.7 10 11.3 5.3 9.3 58.3 66.7
1 21.7 19.7 55.3 48.7 9 12 5 7.3 51.7 68.3
3.3 10.3 23.3 30.3 54.3 9.3 10.3 5 6.7 49.0 61.3
10 0.0 21.0 0 58.7 0 11.7 0 7 12.3 61.7
33 0.0 12.3 0 65.7 0 5.3 0 5.7 0.0 50.7
positive control 456.3 1374.7 4979.7 1635.3 5717 194.7 2641.7 156 911.0 238

3rd Experiment (SPT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 26.3   59.7   11.3   8.7      
0.01 23.0   54   12.7   9      
0.03 24.3   59   15   7      
0.1 22.0   62.3   11.3   9.7      
0.3 22.3   55.3   12.3   6.7      
1 17.3   58.7   13.7   7      
3.3 16.3   32   8.3   5.3      
positive control 557.7   1597.3   4945.7   1091      

4th Experiment (PIT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 33.0 34.7 62.7 46 22.7 20 6 15.3 57.0 75.3
0.01 32.3 39.7 67 58.7 22.7 13.3 6.7 19 54.0 70
0.03 27.7 41.7 70.3 56 28.3 25 8.3 17 44.3 75
0.1 28.3 33.7 64.7 62.7 25.3 14 9 14.7 44.3 74.3
0.3 29.7 41.3 69.7 65.7 19 11.3 7 15.3 51.0 77.3
1 28.3 33.7 70.7 64 22.7 9.7 6.3 16.7 59.0 76.7
3.3 8.0 25.3 23 64 10.3 16.7 2.3 13 16.0 71
positive control 429.7 1328.3 3722 1624 3265.3 168.7 2169 146.7 576.7 159.7
Conclusions:
Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. Dose range was 0.01 μg - 5000 μg/plate in the standard plate test and 0.01 μg - 33 μg/plate in the preincubation test. Both test were performed with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 3.3 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

In a GLP-compliant Ames Test following OEDCD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range used was 0.01 μg - 5000 μg/plate in the standard plate test and 0.01 μg - 33 μg/plate in the preincubation test. Both tests were performed with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 3.3 μg/plate onward. The test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay). The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT-Test

In a GLP-compliant study following OECD guideline 476, the test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. According to the results of the present in vitro study, in two valid experiments performed independently of each other the test substance did not relevantly increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to or within the range of the concurrent vehicle control values and within the range of our historical negative control data. The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method 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, in the absence and the presence of metabolic activation, the test article is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

In vitro micronucleus test

In a GLP-compliant study following OECD guideline 487, the test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). According to the results of the present assay, the test substance did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were close to the range of the concurrent vehicle control values at both exposure times and clearly within the range of our historical negative control data. The statistical significance observed in the 2nd Experiment with metabolic activation has to be regarded as biologically irrelevant under the circumstance that all these values were clearly within the historical negative control data range and not reproducible in Experiment 1 with the same treatment duration in the presence of S9 mix. In addition, the dose-related increase in the number of micronucleated cells in the 1st Experiment without S9 mix and in the 2nd Experiment with and without S9 mix has to be considered as biologically irrelevant, because all micronucleus rates were clearly within the historical negative control data range. The number of micronucleated cells in 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 micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and/or 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 item has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be not classified for genetic toxicity under Regulation (EC) No. 1272/2008.