Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The genetic toxicity of the test item was determined in an Ames test equivalent to OECD guideline 471. No genetic toxicity was observed in this study.

Chromosome aberration test: The ability of the test item to induce chromosome aberrations was tested in a study according to OECD guideline 473. The test item is clastogenic in human lymphocyctes under the experimental conditions described in this report.

HPRT test: The test item, tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in vitro in Chinese hamster ovary cells according to OECD 476.Thus, the test item was not mutagenic under the conditions of this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
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 E.coli strain tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix Aroclor 1254 induced (rat liver)
Test concentrations with justification for top dose:
0, 0.0005, 0.001, 0.002, 0.1 µL test liquid/0.1 mL acetone/plate (1st test)
0, 0.0005, 0.001, 0.002, 0.01 µL test liquid/0.1 mL acetone/plate (2nd test)
0, 0.1, 0.25, 0.5, 1.0 (positive control) µg 2-AA/0.1 mL DMSO/plate
Vehicle / solvent:
DMSO and aceton
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All strains with and without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days at 37 °C

SELECTION AGENT (mutation assays): L-Histidine

NUMBER OF REPLICATIONS: 3


Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid

The addition of 2 -aminoanthracene showed a significant bacterial growth by the addition of S-9 mix, no significant bacterial growth was observed without the addition of S-9 mix. The amount of S-9 mix used was not optimal for the metabolic activation of 2 -AA. The positive control was not valid in this test.

Conclusions:
The genetic toxicity of the test item was determined in an Ames test equivalent to OECD guideline 471. No genetic toxicity was observed in this study.
Executive summary:

The mutagenic activity of the test item was tested in a study equivalent to OECD guideline 471. A set of 5 histidine requiring mutants of S. typhimurium (TA1535, TA1537, TA 1538, TA 98 and TA100) were used in the Ames-test with and without metabolic activation.

Doses of 0, 0.0005, 0.001, 0.002, 0.1 µL test liquid/0.1 mL acetone/plate (1st test) and 0, 0.0005, 0.001, 0.002, 0.01 µL test liquid/0.1 mL acetone/plate (2nd test) were tested. The strains were incubated over a period of 3 days at 37 °C. Incorporation of the test substance up to non-inhibitory levels did not increase the number of his+ revertants in any of the five tester strains, either in the presence or in the absence of the liver microsome activation system. It was concluded that the present results did not reveal mutagenic acitivity in the Salmonella mutagenicity test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-10 to 2004-04-02
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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix Aroclor 1254 induced (rat liver)
Test concentrations with justification for top dose:
Based on results of the dose range finding test the following dose levels were selected:

First cytogenetic assay - cytogenetic assay
Without S-9 mix: 33, 56, 100, 150, 200 and 250 µg/mL
With S-9 mix: 33, 100, 150, 175 and 200 µg/mL

To obtain more information about the possible clastogenicity of the test item a second cytogenetic assay was performed:

Second cytogenetic assay
without S-9 mix
10, 33, 66 and 100 µg/mL (24 h exposure)
33, 66, 100, 125, 150, 175 and 200 µg/mL (48 h exposure)

with S-9 Mix
33, 100, 200 and 250 µg/mL

The second cytogenetic assay was repeated to obtain more information about clastogenic properties of the test item:

Cytogenetic assay 2A
without metabolic activation: 10, 33, 42, 50, 66 and 80 µg/mL
with metabolic activation: 33, 100, 200 and 225 µg/mL

Cytogenetic assay 2B
Without S9 mix: 33, 66, 80, 100, 150, 200 and 333 µg/mL
With S9-Mix: 33, 100, 133, 166, 200, 225 and 250 µg/mL


Vehicle / solvent:
DMSO and HBSS (Hanks´Balanced Salt Solution for positive control)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h, 24h and 48 h
- Fixation time: 24 h and 48 h

SPINDLE INHIBITOR (cytogenetic assays): Cell cultures were treated with colchicine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose related statistically significant (Chi-square test, P<0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay
The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Second cytogenetic assay
In the absence of S9-mix, at the 24 h continuous exposure time, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
In the absence of S9-mix at the 48 h continuous exposure time test concentrations of 100 and 125 µg/ml the test item caused statistically significant, dose related increases in the number of cells with chromosome aberrations both when gaps were included and excluded.
In the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Conclusions:
Since there is a dose-related, statistically significant increase in the number of cells with chromosome aberrations (both when gaps were included and excluded) following treatment with the test item in the absence if a metabolic activation system, the test item is clastogenic in human lymphocyctes under the experimental conditions described in this report.
Executive summary:

Evaluation of the ability of the test item to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of the test item on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of the test item was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

The test substance was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, the test item was tested up to 150 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9~mix. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, the test item was tested up to 100 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 125 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction the test item was tested up to 166 µg/mL for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Second cytogenetic assay

In the absence of S9-mix, at the 24 h continuous exposure time, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the absence of S9-mix at the 48 h continuous exposure time test concentrations of 100 and 125 µg/mL the test item caused statistically significant, dose related increases in the number of cells with chromosome aberrations both when gaps were included and excluded, in the presence of S9 mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Since there is a dose-related, statistically significant increase in the number of cells with chromosome aberrations (both when gaps were included and excluded) following treatment with the test item in the absence of a metabolic activation system, the test item is clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-23 to 2017-03-02
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)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
There is a deviation from the guidelines regarding the confirmation of negative results. Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
yes
Remarks:
There is a deviation from the guidelines regarding the confirmation of negative results. Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation assay in mammalian cells
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 °C. Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections.
Metabolic activation:
with and without
Metabolic activation system:
microsomal fraction (S9) of phenobarbital and beta-naphtoflavone induced male rats
Test concentrations with justification for top dose:
5-hour treatment period:
- without S9 mix: 10, 15, 20, 25, and 30 µg/mL
- with S9 mix: 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice of vehicle: the test item was well soluble in ethanol and ethanol was compatible with the survival of the mammalian cells and the external metabolic activation system.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Study object:
The mutation assay used in this study is based on the detection of mutations in the hprt locus located on the X chromosome of mammalian cells. HPRT is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis. If a toxic base analogue 6 thioguanine (6-TG) is present in the medium, then the analogue is phosphorylated via the HPRT pathway and incorporated into the nucleic acid. Thus, the cells die unless the enzyme is rendered inactive, by mutation.

Formulation: the test item was dissolved in ethanol in a concentration of 5 mg/mL. The appropriate amount of these dosing formulations was diluted with Ham's F12 medium or Ham's F12 medium + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.

Mutation Assay:
- Dose selection: In order to determine the treatment concentrations of test item in the gene mutation test a dose selection (cytotoxicity assay) was performed.

- Main assay: The CHO KI cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL). For the 5-hour treatment, 5 x10^6 cells were placed in each of the sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix. On the day of treatment the culture medium of exponentially growing cell cultures was replaced with medium (F12-5) containing the test item. Following the exposure period of 5 hours the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Barker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survived, the cell number was adjusted to 105 cells/mL. Throughout the expression period; cells were transferred to dishes for growth or diluted to be plated for survival.

- Plating for survival: Following adjustment of the cultures to 10^5 cells/mL, samples from these cultures were diluted to 40 cells/mL. 5 mL (200 cells/dish) of the final concentration of each culture was plated in parallel into 3 dishes (diameter was approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere and at 5 % CO2 in air for 6 days for colony growing. The colonies were then fixed with methanol and counted after staining with Giemsa. Survivals were assessed by comparing the cloning efficiency of the treated groups to the solvent control.

- Expression of the mutant phenotype: During the phenotypic expression period the cultures were subcultured. Subcultures were employed on days 1, 3, and 6, and selected on day 8.

- Plating for viability: At the end of the expression period, the cell number in the samples was adjusted to 2 × 10^5 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test..

- Fixation and staining of colonies: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.
Evaluation criteria:
Test item is considered able to induce gene mutations in cultured mammalian cells in this test system. Providing that all acceptability criteria were fulfilled, a test chemical is considered clearly negative because:

• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are compatible the distribution of the historical negative control data (based 95% control limit).
• The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
On Day 1, there was very clear evidence of toxicity with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
There were no statistically and biologically relevant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation.
Several values of the concurrent solvent control (with and without metabolic activation) and the test item (with and without metabolic activation) slightly exceeded the 95% confidence interval of the historical control data, but these values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures. These findings are therefore considered as being biologically irrelevant.
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

Table 1 HPRT ASSAY MAIN MUTATION ASSAY/a, b, c and d (5-HOUR TREATMENT WITHOUT S9-MIX);

Solvent: ethanol; Expression period: 8 days; Selective agent: 3.4g/mL 6-thioguanine; Cells seeded for analysis: 2x105 cells/dish; For mutant selection: 200 cells/dish (for C. E.)

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control a

201.7

±

0.58

100

101

2

3

2

0

1

8

101

7.92

Solvent control a

201.0

±

3.46

100

100

2

0

0

3

3

8

100

8.00

Pos. control
(EMS 1.0 µL/mL) a

55.0

±

2.65

27

66

196

192

183

200

186

957

66

1450.00**

TEST ITEM

 

10g/mL a

194.7

±

2.08

97

100

0

1

3

2

1

7

100

7.00

15g/mL a

183.0

±

1.00

91

99

0

1

2

1

3

7

99

7.07

20g/mL a

141.7

±

1.53

70

100

3

3

1

1

0

8

100

8.00

25g/mL a

111.7

±

3.21

56

100

2

2

1

1

1

7

100

7.00

30g/mL a

41.0

±

1.00

20

98

1

2

2

0

3

8

98

8.16

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control b

200.7

±

1.15

100

100

2

3

0

1

1

7

100

7.00

Solvent control b

201.0

±

2.65

100

100

1

0

5

2

0

8

100

8.00

Pos. control
(EMS 1.0 µL/mL) b

54.7

±

0.58

27

65

198

184

184

191

181

938

65

1443.08**

TEST ITEM

 

10g/mL b

194.3

±

1.53

97

99

2

2

3

1

0

8

99

8.00

15g/mL b

182.0

±

2.65

91

100

1

4

2

0

0

7

100

7.07

20g/mL b

141.3

±

2.08

70

100

3

1

0

2

2

8

100

8.00

25g/mL b

112.3

±

2.52

56

99

3

2

1

2

0

8

99

8.08

30g/mL b

41.0

±

2.65

20

99

2

3

0

2

1

8

99

8.08

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control c

201.7

±

0.58

100

100

3

1

2

0

3

9

100

9.00

Solvent control c

201.7

±

1.53

100

100

3

1

2

0

2

8

100

8.00

Pos. control
(EMS 1.0 µL/mL) c

54.7

±

2.89

27

65

207

192

202

197

193

991

65

1524.62**

TEST ITEM

 

10g/mL c

197.0

±

1.73

98

100

2

1

3

2

1

9

100

9.00

15g/mL c

185.0

±

2.00

92

99

1

1

1

4

0

7

99

7.07

20g/mL c

143.7

±

1.53

71

100

2

2

0

0

3

7

100

7.00

25g/mL c

109.3

±

2.52

54

99

5

1

1

0

1

8

99

8.08

30g/mL c

43.0

±

2.00

21

99

1

1

2

3

1

8

99

8.08

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control d

201.3

±

1.53

100

101

1

2

2

1

3

9

101

8.91

Solvent control d

201.0

±

1.00

100

100

3

3

0

0

2

8

100

8.00

Pos. control
(EMS 1.0 µL/mL) d

54.7

±

2.08

20

65

188

194

186

197

200

965

65

1484.62**

TEST ITEM

 

10g/mL d

196.3

±

1.53

98

99

1

2

3

1

1

8

99

8.00

15g/mL d

183.3

±

2.08

91

100

2

3

1

1

2

9

100

9.09

20g/mL d

143.7

±

3.21

71

100

1

4

2

0

1

8

100

8.00

25g/mL d

110.0

±

3.00

55

99

1

4

1

2

1

9

99

9.00

30g/mL d

41.0

±

1.00

20

99

3

1

1

2

1

8

99

8.08

 

 

 

  

Table 2 CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-HOUR TREATMENT WITH S9-MIX);

Solvent: ethanol; Expression period: 8 days; Selective agent: 3.4g/mL 6-thioguanine; Cells seeded for analysis: 2x105 cells/dish; For mutant selection: 200 cells/dish for C. E.

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control a

200.3

±

0.58

101

100

3

1

2

2

1

9

100

9.00

Solvent control a

198.3

±

2.08

100

100

3

0

3

1

2

9

100

9.00

Pos. control
(DMBA 20 µg/mL) a

119.7

±

2.52

60

68

102

109

116

104

111

542

68

797.06**

TEST ITEM

 

0.31g/mL a

194.7

±

1.53

98

98

2

4

1

1

0

8

98

8.16

0.63g/mL a

172.3

±

2.08

87

99

2

2

2

2

1

9

99

9.09

1.25g/mL a

157.7

±

2.31

79

98

0

2

4

1

2

9

98

9.18

2.5g/mL a

138.0

±

2.65

70

99

2

3

0

1

2

8

99

8.08

5g/mL a

114.3

±

2.52

58

98

2

3

1

0

2

8

98

8.16

10g/mL a

40.0

±

1.00

20

99

3

2

3

0

1

9

99

9.09

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control b

199.0

±

1.00

100

100

1

1

3

1

3

9

100

9.00

Solvent control b

199.0

±

2.00

100

100

2

2

1

3

1

9

100

9.00

Pos. control
(DMBB 20 µg/mL) b

119.0

±

1.00

60

68

114

108

105

109

115

551

68

810.29**

TEST ITEM

 

0.31g/mL b

193.7

±

1.53

97

99

2

3

3

0

0

8

99

8.08

0.63g/mL b

172.7

±

1.53

87

99

0

3

3

2

1

9

99

9.09

1.25g/mL b

156.3

±

2.08

79

99

2

3

1

0

3

9

99

9.09

2.5g/mL b

138.3

±

1.53

70

99

2

1

3

3

0

9

99

9.09

5g/mL b

114.0

±

1.73

57

99

4

2

2

0

0

8

99

8.08

10g/mL b

40.3

±

0.58

20

99

3

0

1

3

2

9

99

9.09

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control c

201.0

±

2.00

101

100

2

2

3

0

1

8

101

7.92

Solvent control c

198.7

±

1.53

100

100

3

1

3

0

1

8

101

7.92

Pos. control
(DMBC 20 µg/mL) c

122.0

±

2.65

61

69

107

104

115

110

117

553

70

790.00**

TEST ITEM

 

0.31g/mL c

196.0

±

2.00

99

98

4

2

0

2

1

9

99

9.09

0.63g/mL c

173.7

±

2.31

87

100

3

2

3

0

0

8

101

7.92

1.25g/mL c

156.7

±

3.06

79

98

2

0

1

3

3

9

99

9.09

2.5g/mL c

137.0

±

1.73

69

99

2

2

2

2

0

8

100

8.00

5g/mL c

111.7

±

1.53

56

98

2

3

0

0

3

8

99

8.08

10g/mL c

42.7

±

2.31

21

98

4

1

0

2

2

9

99

9.09

 

NON
ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABSOLUTE
C.E.
%

MUTANT
FREQ.
X 10-6

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

Untreated control d

200.7

±

1.15

100

100

2

2

2

3

0

9

100

9.00

Solvent control d

200.3

±

1.53

100

100

2

3

2

1

2

10

100

10.00

Pos. control
(DMBD 20 µg/mL) d

121.3

±

1.53

61

70

103

106

118

110

109

546

70

780.00**

TEST ITEM

 

0.31g/mL d

195.3

±

2.08

98

99

3

0

3

2

1

9

99

9.09

0.63g/mL d

173.0

±

2.65

86

99

1

0

3

3

2

9

99

9.09

1.25g/mL d

155.3

±

1.53

78

99

1

0

2

3

2

8

99

8.08

2.5g/mL d

135.3

±

1.15

68

99

2

1

0

2

4

9

100

9.00

5g/mL d

111.3

±

0.58

56

99

2

1

0

2

4

9

99

9.09

10g/mL d

40.7

±

1.53

20

99

3

3

0

0

3

9

99

9.09

a = parallel of first culture.

b = parallel of first culture.

c = parallel of second culture.

d = parallel of second culture.

 abs.C.E. = Absolute Cloning Efficiency

DMBA= 7,12-Dimethyl benzanthracene

** = p < 0.01 to the concurrent negative control and to the historical control

Conclusions:
The test item, tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in vitro in Chinese hamster ovary cells.
Thus, the test item was not mutagenic under the conditions of this study.
Executive summary:

The test item, dissolved in Ethanol, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 10, 15, 20, 25, and 30 μg/mL

5-hour treatment period with S9-mix: 0.31, 0.63, 1.25, 2.5, 5 and 10 μg/mL

In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.

The test item tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.

Thus, the test item was not mutagenic under the conditions of this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item, tested in the Micronucleus test in mice, was found to be not clastogenic in the micronucleus test under the experimental conditions.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-01-09 to 2007-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
other: micronucleus assay, chromosome aberration
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation: within 20 % of the mean
- Fasting period before study: 3-4 h prior to dosing
- Housing: The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type Mil height: 14 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as nest material (Enviro-dri, TecniLab-BMI BV, Someren, The Netherlands).
- Diet: Free access to standard pelleted laboratory animal diet (SM R/M-Z from SSNIFF® SpezialdiSten GmbH, Soest, Germany)
- Water: Free access to tap water.
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
Details on exposure:
Dosing volume: 10 mL/kg bw
Duration of treatment / exposure:
Single oral intubation
Frequency of treatment:
Animals were dosed once
Post exposure period:
24 and 48 h
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes: Cyclophosphamide
- Route of administration: oral intubation
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
bone marrow, eryhtrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study.

TREATMENT AND SAMPLING TIMES: Single treatment, Sampling time: 24 and 48 h

DETAILS OF SLIDE PREPARATION: The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

METHOD OF ANALYSIS: All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, onesided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time)
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500, 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In the dose range finding test, one male and one female animal dosed with 2000 mg/kg b.w. died within 20 and 44 hours after dosing, respectively. One male and one female animal dosed with 1000 mg/kg b.w. showed no reaction to treatment. Three male and three female mice dosed with 1500 mg/kg b.w. showed the following clinical signs within 1.5 hours after dosing: ataxia (3 male and 2 female animals), lethargy and hunched posture (2 male and 3 female animals). Within 3 hours after dosing all animals were still lethargic, had a hunched posture and two male and one female animal had a rough coat. Within 25 hours after dosing two female animals recovered from the treatment and all males and one female still had a hunched posture. Within 49 hours after dosing all animals had recovered from the treatment, except for one male animal that still had a hunched posture.

The animals of the groups treated with 750 and 375 mg/kg b.w. and the animals of the negative and positive control groups showed no abnormalities.

The following clinical observations were made in the groups treated with 1500 mg test item/kg b.w.

During the first 1.5 hours after dosing all animals of the groups treated with 1500 mg/kg b.w. were lethargic and had a hunched posture. Four animals also showed ataxia.

Within 4 hours after dosing all animals were still lethargic and had a hunched posture. Five animals also had a rough coat and one of.these animals also had showed the following toxic signs: eyes closed, ataxia and ventral recumbency.

Within 21 hours after dosing two animals died. Two animals had a hunched posture and a rough coat. One of these animals also showed the following toxic signs: lethargy, closed eyes, tremors and slow breathing. Six animals recovered from the treatment.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test item treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

The animals of the groups, which were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Within 45 hours after dosing all surviving animals had recovered from the treatment.

Conclusions:
The test item was tested in the Micronucleus test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. It is concluded that the test item is not clastogenic in the micronucleus test under the experimental conditions described.
Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. The test substance was dissolved in corn oil. Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight (b.w.) of cyclophosphamide (CP), respectively. Animals were dosed with the test item at 1500 (two groups), 750 (one group), and 375 (one group) mg/kg b.w.. Two animals dosed with 1500 mg/kg b.w. died within 21 hours after dosing. All animals dosed with 1500 mg/kg b.w. showed the following toxic signs after dosing: lethargy and hunched posture. Four animals also showed ataxia and five animals had a rough coat. Two animals also had closed eyes. One of these animals had ventral recumbency and the other animal also had tremors and was breathing slow. The animals dosed with 750 and 375 mg/kg b.w. and the control animals showed no abnormalities after dosing. Bone marrow of the groups treated with the test item was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that the test item is not clastogenic in the micronucleus test under the experimental conditions described in this report.

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

Additional information

Ames test

The mutagenic activity of the test item was tested in a study equivalent to OECD guideline 471. A set of 5 histidine requiring mutants of S. typhimurium (TA1535, TA1537, TA 1538, TA 98 and TA100) were used in the Ames-test with and without metabolic activation.

Doses of 0, 0.0005, 0.001, 0.002, 0.1 µL test liquid/0.1 mL acetone/plate (1st test) and 0, 0.0005, 0.001, 0.002, 0.01 µL test liquid/0.1 mL acetone/plate (2nd test) were tested. The strains were incubated over a period of 3 days at 37 °C. Incorporation of the test substance up to non-inhibitory levels did not increase the number of his+ revertants in any of the five tester strains, either in the presence or in the absence of the liver microsome activation system. It was concluded that the present results did not reveal mutagenic acitivity in the Salmonella mutagenicity test.

Chromosome aberration test

Evaluation of the ability of the test item to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of the test item on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of the test item was tested in two independent experiments.

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

The test substance was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, the test item was tested up to 150 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9~mix. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, the test item was tested up to 100 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 125 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction the test item was tested up to 166 µg/mL for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix.

Second cytogenetic assay

In the absence of S9-mix, at the 24 h continuous exposure time, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the absence of S9-mix at the 48 h continuous exposure time test concentrations of 100 and 125 µg/mL the test item caused statistically significant, dose related increases in the number of cells with chromosome aberrations both when gaps were included and excluded, in the presence of S9 mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Since there is a dose-related, statistically significant increase in the number of cells with chromosome aberrations (both when gaps were included and excluded) following treatment with the test item in the absence of a metabolic activation system, the test item is clastogenic in human lymphocytes under the experimental conditions described in this report.

HPRT test

The test item, dissolved in Ethanol, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 10, 15, 20, 25, and 30 μg/mL

5-hour treatment period with S9-mix: 0.31, 0.63, 1.25, 2.5, 5 and 10 μg/mL

In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.

The test item tested up to cytotoxic concentrations both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.

Thus, the test item was not mutagenic under the conditions of this study.

In vivo

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. The test substance was dissolved in corn oil. Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight (b.w.) of cyclophosphamide (CP), respectively. Animals were dosed with the test item at 1500 (two groups), 750 (one group), and 375 (one group) mg/kg b.w.. Two animals dosed with 1500 mg/kg b.w. died within 21 hours after dosing. All animals dosed with 1500 mg/kg b.w. showed the following toxic signs after dosing: lethargy and hunched posture. Four animals also showed ataxia and five animals had a rough coat. Two animals also had closed eyes. One of these animals had ventral recumbency and the other animal also had tremors and was breathing slow. The animals dosed with 750 and 375 mg/kg b.w. and the control animals showed no abnormalities after dosing. Bone marrow of the groups treated with the test item was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that the test item is not clastogenic in the micronucleus test under the experimental conditions described in this report.

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 (EC) No 1272/2008. As a result based on the studies the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008.