Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of the registration substance was investigated in three in vitro test systems, namely bacterial reverse mutation test (Ames test), mammalian cytogenetic assay (Chromosome aberration test), and mammalian gene muatation test (HPRT). In all three tests, negative results were obtained.

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
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogenate
Test concentrations with justification for top dose:
Preliminary Toxicity Test - (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 µg/plate
Initial mutation assay - (a) 8, (b) 25, (c) 80, (d) 253 and (e) 800 µg/plate
Confirmatory mutation assay - (a) 41, (b) 86, (c) 181, (d) 380 and (e) 800 µg/plate.
Vehicle / solvent:
One hundred microliters (100 µL) of DMSO was used as the vehicle control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
- Source of the Test System:
-Salmonella typhimurium: Health Protection Agency, National Collection of Type, Cultures (NCTC), 61, Colindale Avenue, London NW9 5EQ, Great Britain
- Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

- Storage of Test System
Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8ºC. The master plates prepared on 14 March 2016 were used in the study.

- Genotypic Characterization of Test System
The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains
TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates
Evaluation criteria:
- Evaluation and Interpretation : To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of Preliminary Toxicity Test

Treatment

(mg/plate)

TA 100 revertant colonies/plate*

Presence of S9

Absence of S9

Mean

Background lawn

Precipitation

Mean

Background lawn

Precipitation

 

DMSO (100mL)

114

4+

Nil

112

4+

Nil

 

50

108

4+

Nil

107

4+

Nil

 

100

103

4+

Nil

101

4+

Nil

 

200

72

4+

Nil

70

4+

Nil

 

400

56

3+

Nil

51

3+

Nil

 

800

46

3+

Nil

43

3+

Nil

 

1600

37

3+

Nil

33

3+

Nil

 

3200

9

1+

Nil

7

1+

Nil

 

5000

4

0

Nil

3

0

Nil

*: Mean of two replicates

4+ - Normal

3+ - Slightly reduced   

1+ - Severely reduced

0  - Absent

Table 2: Viable Counts of the Overnight Culture of the Tester Strains

Tester Strains

Viable Counts ( x 109CFU/mL*)

Initial Mutation Assay

Confirmatory Mutation Assay

TA98

1.55

1.55

TA100

1.64

1.54

TA1535

1.53

1.52

TA1537

1.55

1.52

WP2uvrA (pKM101)

1.62

1.63

* Required Cell count: 1-2x109Colony Forming Units (CFU)/mL

Table 3: Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control

DMSO

26

2

NA

116

1

NA

14

2

NA

11

2

NA

134

3

NA

8

26

2

1.00

114

1

0.98

13

2

0.93

10

2

0.91

130

3

0.97

25

28

1

1.08

112

1

0.97

11

2

0.79

11

2

1.00

127

3

0.95

80

26

2

1.00

112

2

0.97

14

1

1.00

12

1

1.09

129

2

0.96

253

21

2

0.81

71

2

0.61

9

2

0.64

7

1

0.64

105

3

0.78

800

14

1

0.54

44

4

0.38

2

2

0.14

3

2

0.27

93

2

0.69

Positive controls

545c

6c

20.96c

882c

11c

7.60c

139c

9c

9.93c

126c

1c

11.45c

572d

4d

4.27d

aValues are means of three replicates calculated from Appendix 2 and are rounded off to the nearest whole number

bRatio of treated/vehicle control (mean revertants per plate)

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                         

dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate)

NA: Not applicable                             SD: Standard deviation                       

Table 4: Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

27

1

NA

115

2

NA

13

3

NA

12

2

NA

132

3

NA

8

27

2

1.00

112

2

0.97

13

2

1.00

12

1

1.00

129

4

0.98

25

27

2

1.00

112

3

0.97

14

3

1.08

11

2

0.92

129

3

0.98

80

27

1

1.00

112

1

0.97

14

2

1.08

10

2

0.83

128

2

0.97

253

21

1

0.78

75

3

0.65

9

1

0.69

8

1

0.67

109

2

0.83

800

15

1

0.56

40

1

0.35

2

1

0.15

3

2

0.25

91

2

0.69

Positive controls

241c

5c

8.93c

530d

6d

4.61d

143d

5d

11.00d

128e

2e

10.67e

572f

7f

4.33f

aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number 

bRatio of treated/vehicle control (mean revertants per plate)

cTA98: 2-Nitrofluorene (2 µg/plate),             dTA100, TA1535: Sodium azide (1 µg/plate),           

eTA1537: 9-Aminoacridine (50 µg/plate),      fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)            

NA: Not applicable                                         SD: Standard deviation                                   

Table 5: Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA (pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

25

2

NA

114

2

NA

14

2

NA

11

2

NA

133

4

NA

41

25

2

1.00

112

1

0.98

14

2

1.00

11

2

1.00

135

5

1.02

86

26

1

1.04

111

1

0.97

15

1

1.07

12

1

1.09

131

5

0.98

181

27

1

1.08

96

2

0.84

14

2

1.00

10

1

0.91

130

3

0.98

380

23

3

0.92

63

2

0.55

8

1

0.57

8

2

0.73

102

4

0.77

800

15

2

0.60

44

3

0.39

3

1

0.21

3

1

0.27

91

2

0.68

Positive control

557c

17c

22.28c

876c

10c

7.68c

134c

6c

9.57c

129c

4c

11.73c

580d

6d

4.36d

aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number         

bRatio of treated/vehicle control (mean revertants per plate)

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate)

NA: Not applicable                                                                             SD: Standard deviation                                                           

Table 6: Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

25

1

NA

114

2

NA

15

1

NA

12

1

NA

130

4

NA

41

26

1

1.04

111

2

0.97

15

2

1.00

11

2

0.92

131

5

1.01

86

25

1

1.00

111

1

0.97

14

2

0.93

12

1

1.00

128

3

0.98

181

26

1

1.04

96

3

0.84

15

1

1.00

10

1

0.83

127

2

0.98

380

21

1

0.84

64

3

0.56

9

2

0.60

6

1

0.50

99

3

0.76

800

16

3

0.64

43

2

0.38

2

2

0.13

3

1

0.25

90

2

0.69

Positive control

246c

6c

9.84c

537d

3d

4.71d

133d

6d

8.87d

133e

4e

11.08e

584f

5f

4.49f

aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number

bRatio of treated/vehicle control (mean revertants per plate)

cTA98: 2-Nitrofluorene (2 µg/plate),             dTA100, TA1535: Sodium azide (1 µg/plate)            

eTA1537: 9-Aminoacridine (50 µg/plate),      fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)            

NA: Not applicable                                         SD: Standard deviation                                   

Conclusions:
The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. No mutagenicity was found.
Executive summary:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. In the preliminary cytotoxicity test, the S.typhimurium TA 100 cultures were treated up to 5000 µg/plate with and without metabolic activation. Significantly reduced background lawn was observed at 400 µg/plate or higher. Two independent experiments for mutagenicity were performend, each with and without metabolic activation up to the concentration of 800µg/plate. No increase of number of reverstants were found in any of performed experiments.

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:
2016
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:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
Experiments 1 & 2:a) 3 b) 10 and c) 30 µg/mL (factor of 3)
Experiment 3:b) 2 b) 5 and c) 15 µg/mL (factor of 3)

Vehicle / solvent:
DMSO was used as the vehicle control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:
- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data

c.The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required to clarify such results.
Statistics:
Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
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 applicable
Positive controls validity:
valid

 

TABLE 1. Results of Preliminary Cytotoxicity Test

Treatment

(mg/mL)

Presence of metabolic activation

(3-hour exposure)

Absence of metabolic activation

(3-hour exposure)

Absence of metabolic activation

(21-hour exposure)

Final – initial cell count

(1x106/flask)

Cell growth index

RICC (%)

Cell growth inhibition (%)

Final – initial cell count

(1x106/flask)

Cell growth index

RICC (%)

Cell growth inhibition (%)

Final – initial cell count

(1x106/flask)

Cell growth index

RICC (%)

Cell growth inhibition (%)

DMSO

2.715

100

0

2.590

100

0

2.240

100

0

10

2.465

91

9

1.965

76

24

1.490

67

33

20

2.315

85

15

1.615

62

38

0.915

41

59

40

0.69

25

75

0.44

17

83

Negligible cell counts

 

80

Negligible cell counts

Negligible cell counts

160

No cell monolayer

No cell monolayer

No cell monolayer

320

640

1280

2000

Note: Baseline cell count (No. of cells at the beginning of treatment) 2.31x 105cells/mL

Relative Increase in Cell Count (RICC)

=

[Increase in number of cells in treated cultures (final – initial)]

x 100

[Increase in number of cells in control cultures (final – initial)]

TABLE 2.    Summary Results of Chromosomal Aberration Assay - Experiment 1

eatment

(µg/mL)

No. of metaphases scored

No. (%) of metaphases with aberrations@

Total No.(%) of aberrant metaphases*

Cell Growth Inhibition

(%)

Gaps

Breaks

Exchanges

Including Gaps

Excluding Gaps

Cs

Ct

Cs

Ct

Cs

Ct

RC

 

DMSO

(150 µL)

300

 

0

 

1

(0.3)

 

0

 

0

 

0

 

0

 

0

 

1

(0.3)

 

0

0

 

3

300

 

0

 

2

(0.7)

 

0

 

0

 

0

 

0

 

0

 

2

(0.7)

 

0

10

 

10

300

 

0

 

0

 

 

0

 

0

 

0

 

0

 

0

 

0

 

0

17

 

30

300

 

0

 

1

(0.3)

 

0

 

0

 

0

 

0

 

0

 

1

(0.3)

 

0

46

 

CPA 55

300

 

7

(2.3)

 

7

(2.3)

 

16

(5.3)

 

91

(30.3)

 

35

(11.7)

 

8

(2.7)

 

0

 

137

(45.7)

+

137

(45.7)

39

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

CPA: Cyclophosphamide monohydrate            +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

 

 TABLE 3.    Summary Results of Chromosomal Aberration Assay - Experiment 2

Treatment

(µg/mL)

No. of metaphases scored

No. (%) of metaphases with aberrations@

Total No.(%) of aberrant metaphases*

Cell Growth Inhibition

(%)

Gaps

Breaks

Exchanges

Including Gaps

Excluding Gaps

Cs

Ct

Cs

Ct

Cs

Ct

RC

 

DMSO

(150 µL)

300

 

0

 

 

2

(0.7)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

2

(0.7)

 

0

 

 

0

 

3

300

 

1

(0.3)

 

1

(0.3)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

2

(0.7)

 

0

 

15

 

10

300

 

3

(1.0)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

3

(1.0)

 

0

 

24

 

30

300

 

0

 

 

5

(1.7)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

5

(1.7)

 

0

 

52

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type                  RC: Ring chromosome 

Note: There were no incidences of polyploidy and endoreduplicated cells

 

 

 

 TABLE 4.    Summary Results of Chromosomal Aberration Assay - Experiment 3

Treatment

(µg/mL)

No. of metaphases scored

No. (%) of metaphases with aberrations@

Total No.(%) of aberrant metaphases*

Cell Growth Inhibition

(%)

Gaps

Breaks

Exchanges

Including Gaps

Excluding Gaps

Cs

Ct

Cs

Ct

Cs

Ct

RC

 

DMSO

(150 µL)

300

 

0

 

 

1

(0.3)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

1

(0.3)

 

0

 

 

0

 

2

300

 

1

(0.3)

 

2

(0.7)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

3

(1.0)

 

0

 

 

18

 

5

300

 

0

 

 

3

(1.0)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

3

(1.0)

 

0

 

 

35

 

15

300

 

1

(0.3)

 

1

(0.3)

 

0

 

 

0

 

 

0

 

 

0

 

 

0

 

 

2

(0.7)

 

0

 

 

47

 

EMS600

300

 

12

(4.0)

 

23

(7.7)

 

10

(3.3)

 

93

(31.0)

 

49

(16.3)

 

47

(15.7)

 

0

 

 

145

(48.3)

+

145

(48.3)

 

40

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

EMS: Ethyl methanesulfonate   +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

Conclusions:
The genotoxicity of the registration substance was investigated using in-vitro chromosome aberration test according to the Guideline OECD 473. No significant effect was found.
Executive summary:

The genotoxicity of the registration substance was investigated using in-vitro chromosome aberration test according to the Guideline OECD 473.

The CHO cells were treated at 3, 10 and 30 µg/mL for 3 hours with (+S9) or without metabolic activation (-S9), which resulted in the cell growth inhibition of up to 46% (+S9) and 52% (-S9) when compared to the solvent control.

The CHP cells were treated at 2, 5, 15 µg/mL for 21 hours without metabolic activation, which results in the cell growth inhibition of up to 47% when compared to the solvent control.

No significant clastogenic effect was found in all performed experiments and no polyploidy and no endoreduplicated cells were found.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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
Specific details on test material used for the study:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.
Target gene:
This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster (Cricetulus grieus) ovary cell line CHO-K1 (ATCCCCL-61, Lot 4765275)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
A) 10 B) 20 C) 40 and D) 80 µg/mL (factor of 2)

Vehicle / solvent:
DMSO was used as the vehicle control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:
- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data

c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).
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
Positive controls validity:
valid

TABLE 1.         Determination of pH of Test Medium

Treatment (mg/mL)

pH at the beginning of exposure to treatment

pH at the end of exposure to treatment

With S9

Without S9

With S9

Without S9

DMSO

7.05

7.13

7.00

7.10

10

7.02

7.18

6.98

7.24

20

7.02

7.18

6.99

7.21

40

7.01

7.21

7.04

7.20

80

7.05

7.21

7.03

7.21

160

7.05

7.23

7.06

7.26

320

7.01

7.22

7.09

7.29

640

7.02

7.18

7.10

7.32

1280

7.00

7.25

7.12

7.31

2000

7.00

7.25

7.06

7.24

TABLE 2.         Determination of Osmolality of Test Medium

Treatment (mg/mL)

Osmolality at the beginning of exposure to treatment

(OSMOL/kg)

Osmolality at the end of exposure to treatment

(OSMOL/kg)

With S9

Without S9

With S9

Without S9

DMSO

0.493

0.477

0.479

0.460

2000

0.454

0.449

0.449

0.433

TABLE 3.         Results of Preliminary Cytotoxicity Test

 

Treatment

(mg/mL)

3-hour exposure with metabolic activation

3-hour exposure without metabolic activation

Cloning Efficiency

(CE)

Cells at end of treatment

(1x105/mL)

Adjusted Cloning Efficiency

Relative Survival (%RS)*

Cloning Efficiency

(CE)

Cells at end of treatment

(1x105/mL)

Adjusted Cloning Efficiency

Relative Survival (%RS)*

DMSO

0.96

11.90

1.31

100

0.97

11.50

1.27

100

10

0.90

11.50

1.18

90

0.92

10.30

1.08

85

20

0.77

10.05

0.88

67

0.87

9.30

0.92

72

40

0.58

8.05

0.53

40

0.63

8.50

0.61

48

80

0.32

5.00

0.18

14

0.44

4.00

0.20

16

160

0.16

1.50

0.03

2

0.20

1.00

0.02

2

320

-

Absence of cells

-

-

-

Absence of cells

-

-

640

-

-

-

-

-

-

1280

-

-

-

-

-

-

2000

-

-

-

-

-

-

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment

µg/mL

No. of Colonies /Flask

CE*

ACE

RS

%

1

2

3

DMSO

191

190

192

0.96

 

1.39

100

192

194

195

10

179

182

188

0.91

1.28

92

180

184

181

20

152

163

158

0.78

0.97

70

150

156

154

40

124

130

123

0.63

0.65

47

126

122

127

80

69

63

62

0.32

0.21

15

62

62

65

3-MCA

104

110

113

0.54

0.41

29

108

117

101

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment

µg/mL

No. of Colonies /Flask

CE*

ACE

RS

%

1

2

3

DMSO

198

192

193

0.97

1.37

100

196

190

191

10

180

187

183

0.92

1.17

85

186

183

185

20

173

179

164

0.86

1.00

73

170

176

173

40

131

133

136

0.67

0.69

50

135

139

127

80

88

93

96

0.44

0.24

18

81

89

84

TABLE 6.         Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

2

2

3

3

3

25

0.0000125

192

189

194

0.97

12.89

2

2

2

3

3

193

195

197

10

2

1

3

1

2

17

0.0000085

184

188

179

0.90

9.44

3

0

2

1

2

176

181

175

20

2

2

2

1

0

16

0.000008

168

157

159

0.81

9.88

2

1

2

3

1

154

170

163

40

1

2

2

2

2

19

0.0000095

152

152

155

0.76

12.50

3

2

1

2

2

153

149

148

80

1

1

1

1

2

15

0.0000075

109

108

117

0.58

12.93

2

1

3

1

2

129

111

116

3-MCA

36

32

33

34

32

337

0.0001685

144

158

149

0.75

224.67

38

34

30

35

33

157

143

148

TABLE 7.         Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment

µg/mL

Mutation Assay Flasks

Cloning Efficiency of Mutant Colonies

Cloning Efficiency Flasks

6-TG Mutants

per 106Clonable Cells

(MF)

TG Colonies/Flask

No. of Colonies/Flask

1

2

3

4

5

Total

1

2

3

CE*

DMSO

2

3

2

3

3

25

0.0000125

190

187

192

0.95

13.16

2

2

2

4

2

194

192

190

10

2

2

2

1

2

20

0.00001

178

177

168

0.87

11.49

3

2

3

1

2

174

176

170

20

1

1

2

2

1

18

0.000009

158

164

149

0.77

11.69

2

2

2

3

2

148

149

154

40

1

2

2

2

2

16

0.000008

135

139

136

0.67

11.94

1

0

0

4

2

133

135

129

80

2

2

1

2

2

16

0.000008

120

128

130

0.64

12.50

0

2

1

2

2

127

130

128

Conclusions:
The genotoxicity of the registration substance was investigated using in-vitro mammalian cell gene mutation tes (HPRT) according to the Guideline OECD 476. No significant effect was found.
Executive summary:

The genotoxicity of the registration substance was investigated in in-vitro mammalian gene mutation test (HPRT) according to the Guideline OECD 476.

The CHO cells were treated at 10, 20, 40 and 80 µg/mL for 3 hours with (+S9) or without metabolic activation (-S9), which resulted in reduction of the relative survival down to 15% (+S9) and 18% (-S9) when compared to the solvent control.

No significant mutagenic effect was found.

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

Additional information

Justification for classification or non-classification

The genotoxicity of the registration substance was investigated in three in vitro test systems, namely bacterial reverse mutation test (Ames test), mammalian cytogenetic assay (Chromosome aberration test), and mammalian gene muatation test (HPRT). In all three tests, negative results were obtained. No classification is justified.