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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic were observed in two in vitro studies (Ames test, HPRT test) in the presence of absence of a metabolic activation system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 6, 1995 to Jun. 27, 1995
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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
CIBA-GEIGY Limited, Genetic Toxicology
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the original test:
with S9 mix: 7.41, 22.22, 66.67 and 200.00 µg/ml
without S9 mix: 0.19, 0.56, 1.67 and 5.0 µg/ml

Concentration range in the confirmatory test:
with S9 mix: 7.50, 15.0, 30.0 and 60.0 µg/ml
without S9 mix: 0.75, 1.50, 3:00 and 6.00 µg/ml

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with S9 mix
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (with S9 mix), 21 hours (without S9 mix)
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2.5-5.0 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


Evaluation criteria:
The test substance will be considered to be mutagenic if:
- The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20;
-There is a significant dose-relationship as indicated by the linear trend analysis; and
-The effects described above are reproducible.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200.0 µg/ml with metabolic activation and 5.0 µg/ml without metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of the test substance were tested. The concentrations selected ranged from 0.195 to 400.00 µg/ml and separated by 2-fold intervals. In the part with metabolic activation, at the highest concentration of 400.0 µg/ml, an acute growth inhibiting effect of 91.0% could be seen, while the next lower concentration of 200.0 µg/ml inhibited 76.0%. Without metabolic activation treatment with the test substance proved fully growth inhibiting down to the concentration of 25.0 µg/ml. The next two lower concentrations of 12.5 and 6.25 µg/ml revealed an acute inhibition of growth of 99.3 and 96.7%, respectively. Accordingly, four concentrations were selected for the original experiment ranging from 7.41 to 200.0 µg/ml and from 0.19 to 5.0 µg/ml in the presence and absence of metabolic activation, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutagenicity test with metabolic activation (Table 1):

The original experiment was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9%* and 57.4%* respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 99.7%*. The mean growth inhibitory effect after the expression period was 65.5%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation (Table 2):

The original experiment was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0%* and 31.9%* respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. The next lower concentration revealed a mean acute growth inhibitory effect of 70.5%*. The mean growth inhibition after the expression period was 11.6%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

*Mean of two independent cultures

Table 1: With metabolic activation

Treatment

Mean mutant frequency

(x 10E-6)

Mean mutant factor

Significance (P)

Original Experiment

Vehicle control

13.17

-

-

Positive control

172.14

13.07

P<0.001

TS, 200.00 µg/ml

T

T

T

TS, 66.67 µg/ml

19.60

1.49

0.001<p<0.002

TS, 22.22 µg/ml

14.35

1.09

Ns

TS, 7.41 µg/ml

12.46

0.95

Ns

Confirmatory Experiment

Vehicle control

7.26

-

-

Positive control

115.86

15.96

P<0.001

TS, 60.00 µg/ml

12.11

1.67

0.02<p<0.05

TS, 30.00 µg/ml

9.92

1.37

Ns

TS, 15.00 µg/ml

7.35

1.01

Ns

TS, 7.50 µg/ml

7.88

1.09

Ns

 

Table 2: Without metabolic activation

Treatment

Mean mutant frequency

(x 10E-6)

Mean mutant factor

Significance (P)

Original Experiment

Vehicle control

13.78

-

-

Positive control

2203.81

159.90

P<0.001

TS, 5.00 µg/ml

19.56

1.42

P<0.001

TS, 1.67 µg/ml

13.52

0.98

Ns

TS, 0.56 µg/ml

13.35

0.97

Ns

TS, 0.18 µg/ml

11.57

0.84

Ns

Confirmatory Experiment

Vehicle control

7.48

-

-

Positive control

1147.38

153.46

P<0.001

TS, 6.00 µg/ml

T

T

T

TS, 3.00 µg/ml

5.78

0.77

Ns

TS, 1.50 µg/ml

2.51

0.34

Ns

TS, 0.75 µg/ml

5.35

0.72

Ns

TS: test substance; T: toxic

Conclusions:
Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test substance and its metabolites did not show any mutagenic activity.
Executive summary:

The test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in ethanol. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment. In a preliminary cytotoxicity test the concentrations for the main test were determined at 200.0 µg/ml and 5.0 µg/ml, with and without metabolic activation, respectively. The original experiment with S9-mix was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9% and 57.4%, respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The original experiment without S9-mix was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0% and 31.9%, respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. In both experiments with and without S9-mix, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Therefore, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jun. 4, 1986 to Aug. 19, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Remarks:
but QAU statement included
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration in the preliminary toxicity test: 0.08 to 5000 µg/0.1 ml
Concentration range in the main tests (with and without S9 mix): 1.2, 4.9, 19.5, 78.1 and 312.5 µg/0.1 ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
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 hours at 37 ± 1.5 °C

NUMBER OF REPLICATIONS: 3 plates per test
-Independent repeat experiment was conducted.
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
Revertant colony numbers were calculated and arithmetic mean and standard deviation were calculated. No significance test was performed.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
78.1 µg/0.1 ml (- S9) and 312.5 µg/0.1 ml (-S9 and +S9)
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 concentration of 312.5 µg/0.1 ml the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 ml. Thereafter, the concentration of 312.5 µg/0.1 ml was used as the highest in the mutagenicity test due to a growth-inhibiting effect.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A growth-inhibiting effect of the test substance was observed in the experiments without microsomal activation at the upper concentrations. In the experiments carried out with microsomal activation, this effect was less pronounced.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Main test: Number of back-mutant colonies per plate (arithmetic mean)

Dose (µg/0.1ml)

TA 98

TA 100

TA 102

TA 1535

TA 1537

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

0

41

24

114

151

246

274

12

12

20

7

1.2

45

24

103

133

243

277

12

13

16

6

4.9

48

27

122

155

285

226

12

15

16

8

19.5

34

30

122

78

110

250

10

15

12

6

78.1

43

3

152

6

3

212

8

1

11

1

312.5

26

1

10

0

9

4

6

1

4

1

PC*

1105

757

657

1035

822

1234

363

1018

126

929

 

Repeat test: Number of back-mutant colonies per plate (arithmetic mean)

Dose (µg/0.1ml)

TA 98

TA 100

TA 102

TA 1535

TA 1537

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

0

41

25

119

122

254

306

14

13

9

6

1.2

40

25

113

117

252

299

16

18

11

5

4.9

38

20

97

130

235

262

10

15

10

8

19.5

48

20

108

99

216

123

15

11

6

4

78.1

40

6

131

5

179

2

16

2

11

1

312.5

16

2

27

5

2

0

1

0

1

0

PC

653

597

889

1045

1439

1146

375

904

75

1176

* Positive control (high dose)

Conclusions:
Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.
Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES following OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 1.2, 4.9, 19.5, 78.1 and 312.5 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect of the test substance was observed in the experiments without microsomal activation at the upper concentrations. In the experiments carried out with microsomal activation, this effect was less pronounced. At the concentration of 312.5 µg/0.1 ml the substance precipitated in soft agar. In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.

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

Genetic toxicity in vivo

Description of key information

No clastogenic effects were observed in an in-vivo micronucleus assay.

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:
From Jun. 2, 1986 to Nov. 3, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
other: random outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 21-32 g (females) and 23-35 g (males)
- Housing: individual caging
- Diet: NAFAG No. 924, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3-4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23°C
- Humidity (%): 52-78%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 ml/kg
Details on exposure:
The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.
Duration of treatment / exposure:
Single dose administered orally.
Frequency of treatment:
Once
Post exposure period:
16, 24, 48 hours after treatment application.
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8/sex/sampling time
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 64 mg/kg in 20 ml/kg 0.5% CMC
Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. The highest dose causing no deaths is used as the highest in the mutagenicity test. No deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen as no death occurred at this concentration.

TREATMENT AND SAMPLING TIMES:
16, 24 and 48h after application, 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
Statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at any sampling time.
Statistics:
The significance of difference was assessed by Chi square-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the preliminary tolerability test, no deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen for the micronucleus test as no death occurred at this concentration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.66. In comparison with the negative control (0.11) this value is highly significant (p<0.05).

Table: The effect of the test substance on bone marrow cells of Chinese hamster (arithmetic mean per sex and group).

Sacrifice

Dose

Sex

PCE

NCE

Ratio of PCE/NCE

No. of PCE with MN

% of PCE with MN

16 h

Control

Female

583

417

1.40

2.0

0.20

Male

514

486

1.06

0.8

0.08

TS

Female

650

350

1.86

0.6

0.06

Male

511

489

1.04

1.2

0.12

24 h

Control

Female

595

405

1.47

1.2

0.12

Male

562

438

1.28

1.0

0.10

TS

Female

515

485

1.06

2.0

0.20

Male

508

492

1.03

0.8

0.08

48 h

Control

Female

564

436

1.29

1.8

0.18

Male

561

439

1.28

1.6

0.16

TS

Female

520

480

1.08

0.6

0.06

Male

456

544

0.83

1.0

0.10

Positive control

24 h

Control

Female

595

405

1.47

1.2

0.12

Male

562

438

1.28

1.0

0.10

CPA

Female

514

484

1.06

23.6

2.36

Male

459

541

0.85

29.0

2.96

TS: test substance; PCE: polychromatic erythrocytes; NCE: normochromatic erythrocytes; MN: micronuclei; CPA: cyclophosphamide

Conclusions:
There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of the test substance as compared with the negative control animals. Under the conditions of this experiment, no evidence of mutagenic effects was obtained in chinese hamster treated with the test substance.
Executive summary:

A micronucleus test in hamster was performed to evaluate any mutagenic effect of the test substance on polychromatic erythrocytes in bone marrow cells in vivo. To that end, the animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. Mutagenic effects present themselves in form of micronuclei in polychromatic erythrocytes in the bone marrow. These micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes which lag behind at anaphase stage during the mitotic process. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. The increase in micronucleated polychromatic erythrocytes shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. In this experiment, the bone marrow smears from animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.66% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.11%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.

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

Additional information

Ames test

The mutagenic potential of the test substance was investigated in an Ames following OECD guideline 471, performed on Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations (1.2 to 312.5 µg/plate) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control either with or without metabolic activation. Owing to a growth-inhibiting effect of the substance a reduction in the colony count was observed in the experiments without microsomal activation at the upper concentrations and to a lesser extent with microsomal activation. This result was confirmed in a second independent experiment. Therefore, based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

HPRT-Test

In an HPRT-Test according to OECD guideline 474 and in compliance with GLP, the test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. Two independent experiments were performed. Concentrations range was from 7.41 to 200 µg/ml in the experiments with S9-mix; without S9-mix concentrations from 0.19 to 6.0 µg/ml were used. The test substance was dissolved in ethanol. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. Growth inhibiting effects were observed at high doses. In both experiments with and without S9-mix, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Therefore, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.

In vivo micronucleus test

A micronucleus test in hamster was performed to evaluate any mutagenic effect of the test substance on polychromatic erythrocytes in bone marrow cells in vivo. To that end, the animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals were observed at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.66% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.11%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.

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.