Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: - in vitro (bacteria): negative (OECD 471). - in vitro (chromosome aberration mammalian cells): negative (OECD 473) - in vitro (mammalian cells): negative (OECD 476). Genetic toxicity in vivo: No data available.

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:
2012
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
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0.13 0.25 0.5 1.0 2.0 3.0 mg/L (experiment 1; 4h, without S9)
0.94 1.9 3.8 7.5 15.0 30.0 mg/L (experiment 1; 4h, with S9)
46.9 93.8 187.5 375.0 750.0 1500.0 mg/L (experiment 2; 24h, without S9)
0.94 1.9 3.8 7.5 15.0 30.0 mg/L (experiment 1; 4h, with S9)
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9:150 μg/mL ethyl methanesulfonate, with S9: 1.1 μg/mL 7,12-dimethylbenz(a)anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium):3-4 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen¬trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
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:
Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 23.4 µg/mL and above in the absence of metabolic activation and at 187.5 µg/mL with metabolic activation following 4 hours treatment. After 24 hours treatment without metabolic activation cytotoxic effects occurred at 93.8 and 187.5 µg/mL and at the maximum concentration of 3000 µg/mL.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 23.4 µg/mL and above in the presence and absence of metabolic activation following 4 hours treatment and at 750.0 µg/mL and above without metabolic activation following 24 hours treatment.
There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test material is not geno-toxic under the conditions of the test.
Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material was considered to be non-genotoxic. The HPRT test was conducted in Chinese hamster lung fibroblasts cells with and without metabolic activation. Cytotoxicity was clearly observed in the 1st Experiment in the presence of metabolic activation at the two highest concentrations tested. Test concentrations were tested up to 3000 µg/l.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Directive 2000/32/EEC, L1362000, Annexe 4.D
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
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:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction of rats after treatment with phenobarbital and b-naphtoflavone on three subsequent days.
Test concentrations with justification for top dose:
Plate incorporation test: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (Experiment I)
Preincubation: 33; 100; 333; 1000; 2500; and 5000 µg/plate (Experiment II)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: With S9-mix: all strains 2-aminoanthracene; without S9-mix: sodium azide (TA 1535, TA100); 4-nitro-o-phenylene-diamine (TA1537,TA98); methyl methane sulfonate (WP2 uvrA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3


METHOD OF APPLICATION: preincubation;

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data performed
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate in the presence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar at the upper concentrations in experiment II (pre-incubation test). The undissolved particles had no influence on the data recording.

Revertants per plate (mean from three plates):without S9 mix

Dose (µg/plate)

 

TA 1535

TA 1535

TA 1537

TA 1537

TA 98

TA 98

TA 100

TA 100

WP2

WP2

 

 

 I

II

I

II

I

II

I

II

I

II

Neg. control

 

11

12

5

7

22

33

132

161

39

54

Solvent

 

11

11

7

6

21

27

132

139

37

51

Pos. control

 

608

1019

52

61

211

194

706

988

725

393

3

 

9

/

7

/

25

/

121

/

43

/

10

 

8

/

5

/

21

/

113

/

43

/

33

 

7

10

7

6

24

31

117

147

42

56

100

 

7

11

10

6

25

28

118

134

44

54

333

 

9

9

11

7

26

33

127

134

36

59

1000

 

7

13

12

8

25

28

127

133

40

56

2500

 

7

13

9

5

28

28

123

136

38

55

5000

 

6

16

4

5

31

25

124

138

42

53

 

 Revertants per plate (mean from three plates):with S9 mix

Dose (µg/plate)

 

TA 1535

TA 1535

TA 1537

TA 1537

TA 98

TA 98

TA 100

TA 100

WP2

WP2

 

 

 I

II

I

II

I

II

I

II

I

II

Neg. control

 

8

12

11

11

29

43

139

166

35

56

Solvent

 

13

12

10

10

25

39

127

142

40

45

Pos. control

 

126

309

121

110

1009

442

1104

562

177

264

3

 

13

/

5

/

31

/

134

/

47

/

10

 

15

/

5

/

26

/

119

/

39

/

33

 

12

17

6

9

26

29

110

185

34

61

100

 

8

14

2

11

24

35

139

185

40

61

333

 

16

16

6

11

25

41

125

202

47

51

1000

 

13

9

7

8

19

41

126

194

46

55

2500

 

16

10

7

6

17

36

135

183

40

47

5000

 

15

12

4

5

12

36

151

173

35

32

 

 Positive controls:

With S9-mix: all strains 2-aminoanthracene;

Without S9-mix: sodium azide (TA 1535, TA100); 4-nitro-o-phenylene-diamine (TA1537,TA98); methyl methane sulfonate (WP2 uvrA).

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:
The test substance is neither genotoxic nor cytotoxic under the conditions of the test.
Executive summary:

In this guideline (OECD 471) study conducted with GLP certification, the test material (EC 438-340-0) was determined not to be genotoxic or cytotoxic. The reverse mutagenicity study was conducted in S. typhimurium (TA98, TA 100, TA 1535 & TA 1537) and E. coli (WP2 uvr A) with and without metabolic acitvation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
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)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction of rats after treatment with phenobarbital and b-naphtoflavone on three subsequent days.
Test concentrations with justification for top dose:
-S9-mix
Test 1: 3.1, 6.3*, 12.5*, 25*, 50, 100 µg/ml; (4 h exposure )
Test 2: 0.6, 1.3, 2.5*, 5.0*, 7.5, 10* µg/ml; (18 h exposure)
Test 2: 12.5*, 25, 50, 100 µg/ml; (28 h exposure)

+S9-mix
Test 1: 5, 10*, 20*, 30*, 40, 60 µg/ml; (4 h exposure)
Test 2: 3.1, 6.3, 12.5*, 25*, 50*, 100 µg/ml; (4 h exposure)

*Cultures selected for metaphase analysis.
Vehicle / solvent:
DMSO
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Ethylmethane sulfonate; With metabolic activation: Cyclophosphamide
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps). and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects indicated by reduced cell numbers and/or mitotic indices of 50 % of control and below were observed in all experimental parts.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No influence of the test item on the pH value was observed
- Effects of osmolality: no influence on the osmolarity in the main experiments was observed
- Precipitation: Precipitation of the test item in culture medium was observed 4 hrs after start of treatment with 93.8 µg/ml and above in the absence of S9 mix and with 23.4 µg/ml and above in the presence of S9 mix (pre-test data)



RANGE-FINDING/SCREENING STUDIES:
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/ml were applied. Clear toxic effects were observed after 4 hrs treatment with 23.4 to 1500 µg/ml in the absence of S9 mix and with 46.9 µg/ml in the presence of S9 mix. In addition, 24 hrs continuous treatment with 23.4 to 1500 µg/ml in the absence of S9 mix induced strong toxic effects. In the absence and the presence of S9 mix concentrations higher than the concentrations mentioned above showed slight toxicity or no toxicity only.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, exclusive gaps) were near the range of the solvent control values (0.0 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

 

However, a single significant (p < 0.05) increase was observed in experiment II at preparation interval 28 hrs after treatment with 12.5 µg/ml in the presence of S9 mix. Although this increase of 2.5 % aberrant cells, exclusive gaps, was statistically significant compared to the low response (0.0 % aberrant cells, exclusive gaps) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant.

 

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3- 4.6 %) as compared to the rates of the solvent controls (1.4 - 4.5 %).

 

In both experiments EMS (100 µg/ml) and CPA (0.7 and 1.0 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.

Conclusions:
The substance is not genotoxic and exhibits slight cytoxic effects under the conditions of the test.
Executive summary:

In this guideline (OECD 473) study conducted with GLP certification, the test material (EC 438-340-0) was determined not to be genotoxic, but did exhibit cytotoxic effects. The test was carried out over a range of concentrations (0.6 to 100 µg/l) in Chinese hamster lung fibroblasts with and without metabolic activation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

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:

In-vitro:

- Gene mutation in bacteria:

A Ames-test was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA (OECD 471; GLP, RCC, 2002). The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

 

A second Ames test was carried out in accordance with the method described by(Ciba 1989). The strains TA 98, TA 100 and TA 1537 were treated with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg/0.1 ml. The treatment with the test item did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

 

 - In vitro cytogenicity test in mammalian cells:

The test substance was tested for clastogenic effects on Chinese hamster lung fibroblasts (OECD 473, GLP, RCC, 2002). V79 cells were exposed to the test item at concentrations of:

 

Test 1: 3.1, 6.3*, 12.5*, 25*, 50, 100 µg/ml; (4 h exposure, -S9-mix)

Test 1: 5, 10*, 20*, 30*, 40, 60 µg/ml; (4 h exposure, +S9-mix)

Test 2; 3.1, 6.3, 12.5*, 25*, 50*, 100 µg/ml; (4 h exposure, +S9-mix)

Test 2: 0.6, 1.3, 2.5*, 5.0*, 7.5, 10* µg/ml; (18 h exposure, -S9-mix)

Test 2: 12.5*, 25, 50, 100 µg/ml; (28 h exposure, -S9-mix)

*: Cultures selected for metaphase analysis.

 

Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. 100 well spread metaphase plates per culture were scored for cytogenetic damage. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, exclusive gaps) were near the range of the solvent control values (0.0 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. However, a single significant (p < 0.05) increase was observed in experiment II at preparation interval 28 hrs after treatment with 12.5 µg/ml in the presence of S9 mix. Although this increase of 2.5 % aberrant cells, exclusive gaps, was statistically significant compared to the low response (0.0 % aberrant cells, exclusive gaps) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3- 4.6 %) as compared to the rates of the solvent controls (1.4 - 4.5 %).

 

 - In vitro gene mutation in mammalian cells:

The test substance was tested for mutagenic effects on Chinese hamster V79 cells in vitro (OECD 476, GLP, BASF 2012). Based on the solubility properties of the test item the range finding pre-experiment test was performed using a concentration range of 23.4 to 3000 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.

Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 23.4 µg/mL and above in the absence of metabolic activation and at 187.5 µg/mL with metabolic activation following 4 hours treatment. After 24 hours treatment without metabolic activation cytotoxic effects occurred at 93.8 and 187.5 µg/mL and at the maximum concentration of 3000 µg/mL. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 23.4 µg/mL and above in the presence and absence of metabolic activation following 4 hours treatment and at 750.0 µg/mL and above without metabolic activation following 24 hours treatment.

There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50% were observed in the first experiment at 2.0 µg/mL without metabolic activation and at 46.9 µg/mL and above in the second experiment without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered in the absence of metabolic activation

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second experiment without metabolic activation at 46.9 µg/mL (both cultures) and at 93.8 µg/mL (culture II). However, the increase was judged as irrelevant fluctuation as it was based on a rather low mutation frequency of the solvent controls of just 3.6 and 7.6 colonies per 106cells, respectively. Furthermore, there was no dose dependent increase as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.6 up to 17.1 mutants per 106cells; the range of the groups treated with the test item was from 1.6 up to 34.8 mutants per 106cells.

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Cytogenicity in vivo:

No data available

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 not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC 944/2013.