Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In reliable in vitro genotoxicity tests, the substance was found to induce genemutation in bacteria and induce chromosomal aberrations in mammalian cells. Consequently, the substances was tested in in vivo-assays and was found to not induce micronuclei. Therefore, the substance is considered to be not mutagenic.

Link to relevant study records

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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:
guideline study with acceptable restrictions
Remarks:
Original report is only available in Japanese. The translation is in progress. Therefore, the report is cited by a peer-reviewed OECD SIDS.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
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: Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Without S9 mix: 156, 313, 625, 1250, 2500, 5000 µg/plate
With S9 mix: 156, 313, 625, 1250, 2500, 5000 µg/plate
Confirmative test without S9 mix: 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 µg/plate
Vehicle / solvent:
Distilled water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98, WP2 uvrA), Sodium azide (TA1535) and 9-Aminoacridine (TA1537). With S9 mix: 2-Aminoanthracene (all strains).
Details on test system and experimental conditions:
By the preliminary test to decide the highest concentration, toxicity was observed at 5000 ug/plate in the direct method without S9 mix for TA 98 and TA 1537. Then the highest concentration was set at 5000 µg/plate for all tests.

- Procedure: Pre-incubation method
- No. of replicates: 2
- No. of plates/test: 3

Two trial tests were done for all cells and a confirmation test was conducted for TA 98 and TA 1537 which showed positive results in the trial tests.
Evaluation criteria:
1) The revertant colony increase should be more than two times of the control.
2) The revertant colony increase should increase proportionally to the concentration of the test substance (concentration dependency).
3) The same revertant colony increase should be observed repeatedly by more than two tests.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 2500 and 3000 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 3500 µg/plate
Additional information on results:
In the two tests, the test substance caused a revertant colony increase of 2-times as much as that of the control to S. typhimurium TA 1537 at 2500 µg/plate without S9. However, a concentration dependency was not clear. Additionally, a revertant colony increasing tendency was observed for S. typhimurium TA98 at 2500 µg/plate and 5000 µg/plate without S9.
Consequently, to confirm the concentration dependent increase of revertant colonies at 2500 - 5000 µg/plate, the confirmation test was conducted for S. typhimurium strains TA 1537 and TA 98 by the direct method without S9. In this test, toxic effects were observed at 3500 µg/plate and more to TA 98 and TA 1537 without S9 mix. For TA 1537 a revertant colony increase was observed by more than 2-times of the control at 2500 and 3000 µg/plate. Furthermore, a concentration dependency was observed. For TA 98, although a revertant colony increase was observed at 2500 and 3000 µg/plate, it was less than 2-times as much as that of the control.
According to this study, the test substance is considered to be positive in this bacterial reverse mutation test. The number of the induced revertant colonies per mg was calculated to be 3.6/mg.

Number of revertant colonies (mean ± SD):

 

Dose/plate

(µg)

Without S9 mix

With S9 mix

Mean

SD

Mean

SD

1st Experiment

Salmonella typhimurium TA 98

0 (Water)

20

4

35

9

625

 

 

36

3

1250

20

4

42

7

2500

35

8

37

5

5000

34*

14

48

9

AF-2 0.1 µg

389

11

 

 

2-AA

 

 

275

23

 

Salmonella typhimurium TA 1537

0 (Water)

7

1

12

4

625

 

 

14

1

1250

7

1

15

2

2500

15

4

18

3

5000

4*

2

17

3

9-AA 80 µg

946

132

 

 

2-AA

 

 

89

12

 

2nd Experiment

Salmonella typhimurium TA 98

0 (Water)

26

6

37

7

625

 

 

35

5

1250

23

7

34

10

2500

39

2

40

6

5000

38*

14

43

14

AF-2 0.1 µg

406

31

 

 

2-AA

 

 

372

20

 

Salmonella typhimurium TA 1537

0 (Water)

6

1

19

2

625

 

 

19

5

1250

8

3

16

2

2500

15

3

17

2

5000

2*

2

27

3

9-AA 80 µg

964

18

 

 

2-AA

 

 

90

9

 

Confirmation Test

Salmonella typhimurium TA 98

0 (Water)

25

2

 

 

1000

23

2

 

 

1500

26

6

 

 

2000

33

3

 

 

2500

44

7

 

 

3000

42

6

 

 

3500

34*

10

 

 

4000

18*

3

 

 

4500

11*

4

 

 

5000

14*

4

 

 

AF-2 0.1 µg

365

26

 

 

 

Salmonella typhimurium TA 1537

0 (Water)

5

1

 

 

1000

7

2

 

 

1500

9

2

 

 

2000

8

1

 

 

2500

13

3

 

 

3000

15

5

 

 

3500

7*

2

 

 

4000

4*

1

 

 

4500

3*

1

 

 

5000

5*

3

 

 

9-AA 80 µg

933

29

 

 

 

* = Toxic effect was observed.

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation

The test substance was mutagenic in Salmonella typhimurium TA1537 without an exogenous metabolic activation system.
Executive summary:

The study was performed according to OECD TG 471 & TG 472 under GLP conditions.

The test substance induced mutations only in S. typhimurium TA1537 without metabolic activation at 2500 and 3000 µg/plate. The number of the induced revertant colonies/mg was calculated as 3.6/mg. Toxicity was observed at 5000 µg/plate (TA98, TA1537) without S9 mix. In a confirmation test, toxicity was observed at more than 3500 µg/plate (TA98, TA1537) without S9 mix.

Conclusion: The test substance was mutagenic in Salmonella typhimurium TA1537 without an exogenous metabolic activation system.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material (as cited in study report): N,N-Dimethylaminoethyl methacrylate (MADAME)
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: microsomal rat liver portion and cofactors
Test concentrations with justification for top dose:
With S9 mix: 62.5, 125, 250, 500, 1000, 1500, 2000 µg/mL
Without S9 mix: 31.25, 62.5, 125, 250, 500 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without S-9: 2.5 µg/ml MNNG. With S-9: 4 µg/ml BaP.
Details on test system and experimental conditions:
Duplicate cultures were used for each experimental point. The cells were seeded at approximately 5 x 10E+5/25 cm² and placed in an incubator at 37°C. Tw enty-four hours later, they were exposed for 3 hours to the TS, either in a medium without fetal calf serum (assay without S9 mix) or in the metabolic activation system (assay with S9 mix). After treatment, the cultures were observed under a microscope for any morphological alterations, the medium was removed and the cells were rinsed with PBS. Then the cells were used for cytotoxicity (cloning efficiency) and mutagenicity tests.
Evaluation criteria:
A test substance is considered as non-mutagenic if it does not induce a mutation frequency that is at least 3-times higher than the mutation frequency of the negative and/or solvent controls.
A test substance is considered as mutagenic if it induces a 3-fold increase in the mutation frequency when compared to the mutation frequency of the negative and/or solvent controls. In this case, a dose relationship is investigated and considered as significant if p < 0.05.
The results are considered as ambigous if a large difference is obtained between the two tests.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1000 µg/mL, round and refringent cells from 200 µg/mL on.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Although round and refringent cells were observed at 250 µg/mL, the mutation frequency in the cells from duplicate cultures treated with the TS was considered as similar to that of the negative and solvent controls, with and without S9, i.e. no significant increase (3-fold increase over the controls) was observed. The TS did not show mutagenic activity in this HPRT gene mutation assay in V79 Chinese hamster cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
By the preliminary cytotoxicity test, the cytotoxicity (decrease in the cloning efficiency and/or dead cells) was shown at the concentrations of equal or greater than 1000 µg/mL, both with or without S9 mix. At 250 µg/mL or higher, round and refringent cells were observed.
Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the TS did not show mutagenic activity in the HPRT gene mutation assay in V79 Chinese hamster cells.
Executive summary:

The study was performed according to OECD TG 476 in compliance with GLP.

The test was conducted at concentrations of 31.25 to 2000 µg/mL. With and without metabolic activation, the TS showed some cytotoxic effects at concentrations higher than 250 µg/mL, but no inerease in the mutation frequencies were observed at any concentrations tested.

Conclusion: Under the conditions of this study the TS did not show mutagenic activity in the HPRT gene mutation assay in V79 Chinese hamster cells.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Original report is only available in Japanese. The translation is in progress. Therefore, the report is cited by a peer-reviewed OECD SIDS.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Continuous treatment: 20, 39, 78, 156, 313, and 625 µg/mL
Short-term treatment: 200, 400, 600, 800, 1400, and 1600 µg/mL
Vehicle / solvent:
Distilled water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without S9 mix: N-Methyl-N'-nitro-N-nitrosoguanidine; with S9 mix: Benzo[a]pyrene.
Details on test system and experimental conditions:
- Test conditions: Continuous treatment (24 and 48 hrs) without S9; short term treatment (6 hrs) with and without S9
- No. of metaphases analysed: 200
- Dose selection rationale:
By the preliminary cytostatic test to determine the cytotoxic doses, following cytotoxicity doses were revealed:
Continuous treatment, 24 hrs: 625 µg/mL
Continuous treatment, 48 hrs: 313 µg/mL
6-hrs short-term treatment without S9 mix: 800 µg/mL
6-hrs short-term treatment with S9 mix: 1600 µg/mL
Based on these data, the doses were decided for this study.
- No. of plates per test: 2
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
clastogenic effects
Cytotoxicity / choice of top concentrations:
cytotoxicity
Additional information on results:
CONTINUOUS TREATMENT:
By the 24-hrs and 48-hrs continuous treatment without S9, structural chromosomal aberrations (including gap) were induced at 625 µg/mL with 88.5% and 76.5%, respectively. The percentage of cells with aberration except gap was 86.5% and 74.0%, respectively. Cytotoxicity was observed at 625 µg/mL and 313 µg/mL, respectively. Polyploidy was not induced under these conditions.

SHORT-TERM TREATMENT:
By the 6-hrs short-term treatment without S9, concentration-dependent structural chromosomal aberrations (including gap) were induced at 200 µg/mL, 400 µg/mL and 600 µg/mL with 6.5%, 49.5% and 87.5%, respectively. The percentage of cells with aberrations except gap was 6.5%, 46.0% and 86.0%, respectively.
By the 6-hrs short-term treatment with S9, concentration-dependent structural chromosomal aberrations (including gap) were induced at 800 µg/mL, 1400 µg/mL and 1600 µg/mL with 13.5%, 99.5% and 100%, respectively. The percentage of cells with aberration except gap was 13.0%, 99.5% and 100.0%, respectively. Polyploidy was not induced under these conditions. At more than 800 µg/mL on 6-hrs short-term treatment without S9 mix and at more than 1600 µg/mL with S9 mix, cytotoxicity was observed and the metaphase figures were not observable.

Chromosome analysis of Chinese hamster cells (CHL) continuously treated with MADAME without S9 mix:

 

Concentration

(µg/ml)

No. of structural aberrations

No. of cells with aberrations / %

gap

ctb

cte

csb

cse

oth

total

Including gaps

Excluding gaps

Time of exposure: 24 hours

Solvent

0

0

0

0

1

0

1

1/0.5

1/0.5

20

3

0

0

0

0

0

3

0/0

3/1.5

39

0

0

1

0

1

0

2

2/1.0

2/1.0

78

0

1

1

0

0

0

2

1/0.5

1/0.5

156

0

0

0

0

1

0

1

1/0.5

1/0.5

313

0

1

0

1

2

0

4

4/2.0

4/2.0

625

22

119

131

42

0

0

314

173/86.5*

177/88.5*

MNNG 2.5

11

32

185

7

0

0

235

188/94.0*

189/94.5*

 

Time of exposure: 48 hours

Solvent

1

0

1

0

0

0

2

1/0.5

2/1.0

20

0

0

0

0

1

0

1

1/0.5

1/0.5

39

0

0

0

0

0

0

1

0/0

1/0.5

78

2

0

0

1

1

0

4

2/1.0

4/2.0

156

0

0

0

0

0

0

0

0/0

0/0

313

1

0

0

0

2

0

3

2/1.0

3/1.5

625

21

61

123

46

0

0

251

148/74.0*

153/76.5*

MNNG 2.5

11

39

136

25

17

0

228

159/79.5*

159/79.5*

 

Time of exposure: 6(-18) hours

Solvent

0

0

0

0

1

0

1

1/0.5

1/0.5

200

1

1

6

0

6

0

14

13/6.5*

13.6.5*

400

18

23

86

6

0

0

133

92/46*

99/49.5*

600

28

115

141

21

0

0

305

172/86.0*

175/87.5*

800

Toxicity

1400

Toxicity

1600

Toxicity

BP 10

1

1

2

0

0

0

4

3/1.5

4/2.0

 

* = Significantly different from solvent group data at P<0.01 by Fisher’s exact test.

gap: gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring), oth: others

_

Chromosome analysis of Chinese hamster cells (CHL) continuously treated with MADAME with S9 mix:

 

Concentration

(µg/ml)

No. of structural aberrations

No. of cells with aberrations / %

gap

ctb

cte

csb

cse

oth

total

Including gaps

Excluding gaps

Time of exposure: 6(-18) hours

Solvent

0

0

1

0

0

0

1

1/0.5

1/0.5

200

0

0

0

0

2

0

2

2/1.0

2/1.0

400

0

0

0

0

0

0

0

0/0

0/0

600

1

1

3

0

2

0

7

6/3.0

7/3.5

800

2

2

24

0

2

0

30

26/13.0*

27/13.5*

1400

13

146

194

45

0

0

398

199/99.5*

199/99.5*

1600

6

60

81

21

0

0

168

84/100*

84/100*

BP 10

9

11

112

1

2

0

138

116/58.0*

117/58.5*

 

* = Significantly different from solvent group data at P<0.01 by Fisher’s exact test.

gap: gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring), oth: others

Conclusions:
Interpretation of results (migrated information):
positive

According to this study the TS is considered to induce chromosomal aberrations with and without metabolic activation. The aberrations observed were mainly chromatid breaks and chromatid exchanges.
Executive summary:

The study was performed according to OECD TG 473 in compliance with GLP using Chinese hamster lung (CHL/IU) cells.

After 24 hrs and 48 hrs continuous treatment without S9, structural chromosomal aberrations (including gap) were induced at 625 µg/mL with 88.5% and 76.5%, respectively. The number of cells with aberrations excluding gaps were 86.5% and 74.0%, respectively. Cytotoxicity was observed at 625 µg/mL and 313 µg/mL. In the 6-hrs short-term treatment without S9, concentration-dependent structural chromosomal aberrations (including gap) were induced at 200 µg/mL, 400 µg/mL and 600 µg/mL with 6.5 %, 49.5 % and 87.5 %, respectively. The number of cells with aberrations excluding gaps were 6.5%, 46.0% and 86.0%, respectively. In the 6-hrs short-term treatment with S9, concentration-dependent structural chromosomnal aberrations (including gap) were induced at 800 µg/mL, 1400 µg/mL and 1600 µg/mL with 13.5 %, 99.5 % and 100 %, respectively. The number of cells with aberrations excluding gaps were 13.0%, 99.5% and 100.0%, respectively. Polyploidy was not induced under any of these conditions. At more than 800 µg/mL of the 6-hrs short-term treatment without S9 mix and at more than 1600 µg/mL with S9 mix, cytotoxicity was observed.

Conclusion: The TS is considered to induce chromosomal aberrations with and without metabolic activation. The aberrations were mainly chromatid breaks and chromatid exchanges.

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:
guideline study with acceptable restrictions
Remarks:
GLP guideline study (OECD TG No. 471 from 26th May 1983).
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD TG No. 471 from 26th May 1983
Deviations:
yes
Remarks:
5th strain is missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material (as cited in study report): N,N-Dimethylaminoethyl methacrylate (MADAME)
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: microsomal rat liver portion and cofactors
Test concentrations with justification for top dose:
100, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-anthramine (2AM)
Details on test system and experimental conditions:
This study was performed according to the direct plate incorporation method.
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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 1538
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:
cytotoxicity
Remarks:
slight cytotoxicity at 5000 µg/plate
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
Additional information on results:
The test substance did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains tested. The negative and solvent control results were equivalent to those usually obtained in this laboratory. The number of revertants induced by the positive control was higher than the spontaneous one, which demonstrateted the sensitivity of this test and the efficacy of the S9 mix throughout this study.
Conclusions:
Interpretation of results (migrated information):
negative

The test substance was negative in any of S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at doses of 100, 500, 1000, 2500, and 5000 µg/plate with and without S9.
Executive summary:

The study was performed according to OECD TG 471 under GLP conditions.

The test substance did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains tested (S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100) at doses of 100, 500, 1000, 2500, and 5000 µg/plate. The negative and solvent control results were equivalent to those usually obtained in this laboratory. The number of revertants induced by the positive control was higher than the spontaneous one, which demonstrateted the sensitivity of this test and the efficacy of the S9 mix throughout this study. A slight toxicity was observed at 5000 µg/plate in starin TA 100.

Conclusion: The test substance was negative in any of S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without S9.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material (as cited in study report): N,N-dimethylaminoethyl methacrylate (MADAME)
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
Without S-9: 663.2, 884.3 and 1179 µg/ml
With S-9: 884.3, 1179 and 1572 µg/ml
Vehicle / solvent:
Distilled water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without S9: 50 µg/mL MMS. With S9: 25 µg/mL CPA.
Details on test system and experimental conditions:
- TREATMENT: Blood cultures were established and incubated at 37°C for approx. 48 hours. S-9 mix or KCl were added appropriately. One set of quadruplicate cultures (A,B,C and D) for each of the 2 sampling times was then treated with the solvent and one set of duplicate cultures with the TS (0.1 ml per culture). Additional duplicate cultures for sampling at the first harvest only, were treated with 0.1 ml of the positive control chemicals.
Short treatments of 3 hours were considered preferable to continous treatments of 20 and 44 hours, in the absence of S-9, because hydrolysis of the parent compound to methacrylic acid and dimethylaminoethanol in aqueous media could be expected if treatment were prolonged. Cultures were therefore treated in the absence and presence of S-9 for 3 hours only. They were then pelleted (200 x g, 10 minutes), washed twice with sterile saline, and resuspended in fresh medium containing foetal calf serum and gentamycin. Cultures were incubated for a further 17 or 41 hours before harvesting. The delayed sample was adopted for test and negative control cultures only, not for positive controls.

- HARVESTING AND PREPARATION OF METAPHASE SPREADS: One and a half hours prior to harvest, colchicine was added to give a final concentration of approximately 1 pg/ml to arrest dividing cells in metaphase. Cells were then harvested, and metaphase spreads were prepared.

- SCORING OF ABERRATIONS: One hundred metaphases from each culture were analysed for chromosome aberrations. Only cells with 44-46 chromosomes were considered acceptable for chromosome aberration analysis although any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Aberrations were classified according to the scheme described by Scott et al. (In vitro chromosome aberration assays. In "Report of the UKEMS sub-committee on guidelines for mutagenicity testing. Part 1. Basic Test Battery." Ed B J Dean. Published by United Kingdom Environmental Mutagen Society, Swansea. pp 41 -64.).
Evaluation criteria:
The TS was to be considered as clearly positive in this assay if:
1) statistically significant increases in the proportion of structurally aberrant cells (without gaps) occurred at one or more concentrations
2) the proportion of aberrant cells at such data points exceeded the normal range.
Increases in numbers of cells with gaps or increases in numbers of cells with structural aberrations not exceeding the normal range or occurring only at very high or very toxic concentrations were likely to be concluded as "equivocal" or "probably of no biological importance". Cells with exchange aberrations or cells with greater than one aberration were to be considered of greater biological significance.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested to its limit of toxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The cells sampled at 20 hours after the start of treatment were analysed for chromosomal aberrations. At the higher two concentrations, namely 1179 µg/mL without S9 and 1572 µg/mL with S9, this chemical induced aberrations which were significantly different from those observed in the concurrent solvent controls. No exchange-type aberrations were observed, but only the deletion-type aberratians were seen. The numbers of cells with aberrations including gap (average of two tests) at 1179 µg/mL without S9 and 1572 µg/mL with S9 were 19.5% and 12.5%, respectively.The numbers of cells with aberrations excluding gap (average of two tests) at 1179 µg/mL without S9 and 1572 µg/mL with S9 were 11.0% and 7.5%, respectively. No
marked mitotic inhibition was evident in any of the doses analysed in this study.
The mitotic index at 1179 µg/mL without S9 and 1572 µg/mL with S9 (average of two tests) were 2.3% and 6.2%, respectively.

Structural aberrations observed without metabolic activation (S9):

 

Aberration

Solvent

663.2 µg/mL

884.3 µg/mL

1179 µg/mL

MMS

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Scored cells:

100

100

200

100

100

200

100

100

200

100

100

200

25

25

50

Gaps

3

3

6

2

2

4

3

6

9

11

6

17

32

5

37

Chr..

0

1

1

0

2

2

1

0

1

1

3

4

5

2

7

Chr. exch.

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Ctd. del.

0

0

0

2

2

4

8

5

13

11

7

18

4

5

9

Ctd. exch.

0

0

0

0

0

0

0

0

0

0

0

0

3

4

7

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Total incl. gaps

3

4

7

4

6

10

12

11

23

23

16

39

15

13

28

%

3.5

5.0

11.5

19.5

14.0

Total excl. gaps

0

1

1

2

4

6

9

5

14

12

10

22

12

11

23

%

0.5

3.0

7.

11.0

11.5

 Chr. del.: chromosome deletion, Chr. exch.: chromosome exchange, Ctd. del.: chromatid deletion, Ctd. exch.: chromatid exchange

 

 

Structural aberrations observed with metabolic activation (S9):

 

Aberration

Solvent

884.3 µg/mL

1179 µg/mL

1572 µg/mL

CPA

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Scored cells:

100

100

200

100

100

200

100

100

200

100

100

200

25

25

50

Gaps

2

3

5

5

2

7

4

3

7

7

3

10

5

3

8

Chr..

0

1

1

2

3

5

2

0

2

2

2

4

1

1

2

Chr. exch.

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

Ctd. del.

1

0

1

3

6

9

1

3

4

5

6

11

14

10

24

Ctd. exch.

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Total incl. gaps

3

4

7

10

11

21

7

7

14

14

11

25

21

15

36

%

3.5

10.5

7.0

12.5

18.0

Total excl. gaps

1

1

2

5

9

14

3

4

7

7

8

15

16

12

28

%

1.0

7.0

3.5

7.5

14.0

 Chr. del.: chromosome deletion, Chr. exch.: chromosome exchange, Ctd. del.: chromatid deletion, Ctd. exch.: chromatid exchange

Conclusions:
Interpretation of results (migrated information):
positive

It is concluded that the TS was able to induce structural chromosome aberrations in cultured human lymphocytes when tested to its limit of toxicity, both in the absence and presence of S-9. No exchange-type aberration figures were observed at doses where statistically significant increases were seen; only deletion-type aberrations were seen.
Executive summary:

The study was performed according to OECD TG 473 in compliance with GLP using human peripheral blood lymphocytes.

Dose levels up to 1572 µg/mL were investigated (maximum solubility). Cells sampled 20 hours after start of the treatment were analysed for chromosomal aberrations. At the higher two concentrations, namely 1179 µg/mL without S9 and 1572 µg/mL with S9, the TS induced aberrations which were significantly different from those observed in the concurrent solvent controls. No exchange-type aberrations were observed. Only deletion-type aberrations were seen. The number of cells with aberrations excluding gaps (average of two tests) at 1179 µg/mL without S9 and 1572 µg/mL with S9 were 11.0% and 7.5%, respectively. No marked mitotic inhibition was evident in any of the doses analysed in this study. The mitotic index at 1179 µg/mL without S9 and 1572 µg/mL with S9 was 2.3 % and 6.2 %, respectively.

Conclusion: It is concluded that the TS may induce chromosomal aberrations in human peripheral blood lymphocytes with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material (as cited in study report): Dimethylaminoethyl methacrylate (MADAME)
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
-Strain: OF1/ICO: OF1 (IOPS Caw)
- Source: Iffa Crédo (69210 L'Arbresle, France)
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: males 21-36 g, females 24-29 g
- Housing: in polycarbonate cages (33.5 X 18.7 X 13.0 cm) and each cage contained 5 mice of the same sex and group
- Diet: A04 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France) ad libitum
- Water: filtered tap water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
Physiological solution of 0.9% NaCl
Details on exposure:
- Application volume: 10 ml/kg bw
Duration of treatment / exposure:
Sampling time 14 or 48 hours after second administration.
Frequency of treatment:
2 administrations separated by 24 hrs
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
25 mg/kg bw cyclophosphamide (2 i.p. injections)
Tissues and cell types examined:
Bone marrow smeares were prepared 24 and 48 hrs after the 2nd administration. The presence of micronuclei was analysed in 2000 polychromatic erythrocytes (PE) per mouse, and the PE/NE ratio was determined.
Details of tissue and slide preparation:
At the time of sacrifice, all the animals were sacrificed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. Two slides/animal were prepared, but only one was used for scoring. All the slides were coded for scoring.
Evaluation criteria:
The following criteria were used as an aid for deterrnining a positive response:
- a statisticaliy significant increase in the number of MPE for at least one of the sampling times when cornpared to the vehicle group,
- this increase should double the number of MPE of our historical data.
The results were considered negative if the above criteria were not fully met.
Statistics:
At each sarnpling time, the mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to the simultaneous vehicle groups. The intergroup comparison was performed using for MPE, the X² test, and for the PE/NE ratio, the Student's "t" test, in which p = 0.05 was used as the lowest level of significance.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
cytotoxicity
Additional information on results:
In all groups treated with the TS, the mean values of micronucleated polychromatic erythrocytes were similar to those of their respective vehicle control groups at each sampling time, and no statiscally significant differences were observed. The PE/NE ratio did not differ from that of the respective vehicle control group.

Summary of the test results:

 

Group

Dose

(mg/kg bw)

MPE/PE

PE/NE ratio

Mean

SD

Mean

SD

Time of sacrifice: 24 hours after the 2ndadministration

Vehicle

-

2.0

0.8

0.7

0.2

Test substance

200

1.9

1.1

0.6

0.2

CPA

25

18.2***

3.8

0.4***

0.1

 

Time of sacrifice: 48 hours after the 2ndadministration

Vehicle

-

1.9

0.8

0.9

0.4

Test substance

200

1.7

1.0

1.2

0.6

 *** = P<0.001

Vehicle: physiological solution (0.9% NaCl)

CPA: cyclophosphamide

PE: polychromatic erythrocytes

NE: normochromatic erythrocytes

MPE/PE: micronucleated polychromatic erythrocytes/2000 polychromatic erythrocytes

SD: standard deviation

Conclusions:
Interpretation of results (migrated information): negative
The TS did not induce cytogenetic damage to the bone marrow cells of mice when treated twice separated by 24 hrs by intraperioneal route at 200
mg/kg in the micronucleus test.
Executive summary:

The study was performed according to OECD TD 473 in compliance with GLP.

Swiss OF1/ICO:OF1 IFFA-CREDO mice (5 males and 5 females per group) received two administrations separated by 24 hrs of 200 mg/kg bw TS by the intraperitoneal route. The application of the vehicle (0.9% NaCl solution) served as negative control, cyclophosphamide at 25 mg/kg bw (two-times i.p. injection) served as the positive control. The test animals were killed 24 or 48 hrs after the 2nd administration and bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic erythrocytes (PE) per mouse and for the PE/NE ratio. The number of micronucleated polychromatic cells (MPE) in the dosed animals was not significantly different from that of the animals in the control groups. The PE/NE ratio did not differ from that of the respective vehicle control group.

Conclusion: The TS did not induce cytogenetic damage to the bone marrow cells of mice when treated twice separated by 24 hrs by intraperioneal route at 200 mg/kg in the micronucleus test.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of test material (as cited in study report): Dimethylaminoethylmethacrylate
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: at least 10 weeks
- Weight at study initiation: approx. 30 g
- Fasting period before study: approx. 18 hours before treatment
- Housing: single housing in Makrolon Type I cages with wire mesh tops
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, FRG)
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: Distilled water
- Justification for choice of solvent/vehicle: nontoxicity for the animals
Details on exposure:
ADMINISTRATION VOLUME: 10 ml/kg
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the TS was dissolved in distilled water.


Duration of treatment / exposure:
Sampling time 24, 48 or 74 hours after oral application by gavage.
Frequency of treatment:
once
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg/kg bw cyclophosphamide (CPA) in physiological saline (NaCl).
Tissues and cell types examined:
Bone marrow smears were prepared 24, 48 and 72 hours after application of the test substance. 1000 PCE (Polychromatic Erythrocytes) per animal were analysed.
Statistics:
Non-parametric Mann-Whitney test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
cytotoxicity
Additional information on results:
In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the TS. The mean values of micronuclei observed after treatment with the TS were in the same range compared to the negative control groups. In the positive control group a distinct increase of induced micronuclei frequency was observed.

Summary of the test results:

 

Group

Dose

(mg/kg bw)

PCEs with micronuclei

(%)

Micronuclei in 1000 PCEs

(range)

PCE/NCE

(mean)

Sampling time: 24 hours

Solvent

0

0.06

0-2

1000/554

Test substance

1000

0.03

0-2

1000/653

CPA

40

0.75

1-13

1000/742

 

Sampling time: 48 hours

Solvent

0

0.04

0-2

1000/680

Test substance

1000

0.04

0-1

1000/744

 

Sampling time: 72 hours

Solvent

0

0.06

0-4

1000/594

Test substance

1000

0.09

0-2

1000/506

 CPA: cyclophosphamide, PCE: polychromatic erythrocytes, NCE: normochromatic erythrocytes

Conclusions:
Interpretation of results (migrated information): negative
The test substance did not induce micronuclei as determined by the micronucleus test in bone marrow cells of the mouse after oral application by gavage.
Executive summary:

The study was performed according to OECD TG 474 in compliance with GLP.

The maximum tolerated dose of 1000 mg/kg bw, dissolved in water, was administered by oral gavage to 3 groups of 10 NMRI mice (5 males and 5 females). Distilled water served as negative control. As positive control, cyclophospliamide in physiological serum (NaCl) was dosed at 40 mg/kg bw. The test mice were killed at 24, 48 or 72 hrs after the administration. Bone marrow smears were examined for the presence of micronuclei in 1000 polychromatic erythocytes per mouse and for the PCE/NCE ratio. No micronuclei induction due to the TS was observed. The PCE/NCE ratio was not affected as compared to the corresponding negative controls, thus indicating no cytotoxic effects.

Conclusion: The test substance did not induce micronuclei as determined by the micronucleus test in bone marrow cells of the mouse after oral application by gavage.

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

Additional information

Seven reports were reviewed. These were two bacterial in vitro test reports, three non-bacterial in vitro test reports and two in vivo genotoxicity test reports.

Bacterial tests:

Two studies were reviewed. These two studies were conducted according to OECD guidelines 471 & 472 in compliance with GLP and were identified as key studies (MHW Japan, 1998; Atochem, 1991).

1) MHW, Japan (1998): The test was conducted twice for all strains with and without S9 and the results were positive without S9 at 2500 µg/plate and higher for TA 1537 and there was an increase in the number of revertant colonies in TA 98 at 2500 µg/plate and higher without metabolic activation. Consequently a confirmation test was conducted for S. typhimurium TA 1537 and TA 98 without S9. Cytotoxic effects were observed at 3500 µg/plate and higher concentrations to TA 98 and TA 1537 without S9 mix. The result of the confirmation test was positive only for TA 1537 at 2500 and 3000 µg/plate. The number of the induced revertant colonies per milligram test substance was calculated to be 3.6.

2) Atochem (1991): Atochem reported that the test substance was negative in any of S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at doses of 10, 100, 1000, 2500, and 5000 µg/plate with and without S9.

Non-bacterial in vitro tests:

Three studies were reviewed. Two studies were chromosomal aberration tests according to OECD guideline 473 on cultured Chinese hamster lung cells (MHW Japan, 1998) and on human lymphocytes (Atochem, 1991). Another study, a HPRT test in V79 cells, was conducted according to OECD guideline 476 (Atochem, 1992). All three tests were conducted in compliance with GLP and were identified as key studies. The three studies are summarised below.

1) MHW, Japan (1998): Chromosomal aberrations assay on cultured Chinese hamster lung cells

After 24 hours and 48 hours continuous treatment without S9, structural chromosomal aberrations (including gaps) were induced at 625 µg/mL in two replicates with 88.5% and 76.5%, respectively. The number of cells with aberrations excluding gaps were 86.5% and 74.0%, respectively. Cytotoxicity was observed at 625 µg/mL and 313 µg/mL. By the 6-hours short-term treatment without S9, concentration-dependent structural chromosomal aberrations (including gaps) were induced at 200 µg/mL, 400 µg/mL and 600 µg/mL with 6.5%, 49.5% and 87.5%, respectively. The number of cells with aberrations excluding gaps were 6.5%, 46.0% and 86.0%, respectively. By the 6-hours short-term treatment with S9, concentration-dependent structural chromosomal aberrations (including gaps) were induced at 800 µg/mL, 1400 µg/mL and 1600 µg/mL with 13.5%, 99.5% and 100%, respectively. The number of cells with aberrations excluding gaps were 13.0%, 99.5% and 100%, respectively. Polyploidy was not induced under any of the test conditions. At more than 800 µg/mL of the 6-hours short-term treatment without S9 mix and at more than 1600 µg/mL with S9 mix, cytotoxicity was observed. As a result, the test substance is considered to induce chromosomal aberrations with and without metabolic activation. The aberrations were mainly chromatid breaks and chromatid exchanges.

2) Atochem (1992): Chromosomal aberrations assay on human lymphocytes

Dose levels up to 1572 µg/mL (maximum solubility) were investigated. The cells sampled at 20 hours after the start of the treatment were analysed for chromosomal aberrations. At the higher two concentrations, namely 1179 µg/mL without S9 and 1572 µg/mL with S9, this chemical induced aberrations which were significantly different from those observed in the concurrent solvent controls. No exchange-type aberrations were observed, only deletion-type aberrations were seen. The number of cells with aberrations excluding gaps (average of two tests) at 1179 µg/mL without S9 and 1572 µg/mL with S9 were 11.0% and 7.5%, respectively. No marked mitotic inbibition was evident at any of the doses analysed in this study. The mitotic index at 1179 µg/mL without S9 and 1572 µg/mL with S9 was 2.3 % and 6.2 %, respectively. It is concluded that the test substance may induce chromosomal aberrations in human peripheral blood lymphocytes with and without metabolic activation.

3) Atochem (1992): HPRT/V79 Chinese hamster cell test

The test was conducted at concentrations from 31.25 to 2000 µg/mL. With and without metabolic activation, the test substance showed some cytotoxic effects at concentrations higher than 250 µg/mL, but no increase in the mutation frequencies was observed at any concentration tested. Under these experimental conditions, the test substance was not genotoxic.

In vivo genotoxicity tests:

Two in vivo micronucleus assays in mice were reviewed. These two studies were conducted according to OECD guideline 474 in compliance with GLP and were identified as key studies (Atochem, 1993; Roehm, 1989). The summaries of the studies are shown below.

1) Atochem (1993): Study on the clastogenic potential of the test substance in OF1 mice

The animals (5 males and 5 females per group) received two administrations separated by 24 hours of 200 mg/kg by the intraperitoneal route. Cyclophosphamide at a dose level of 25 mg/kg (two-times i.p. injection) served as the positive control. The test animals were killed at 24 or 48 hours after the second administration and the bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse and for the polychromatic-to-normochromatic erythrocytes (PCE/NCE) ratio. The number of micronucleated polychromatic cells in the dosed animals was not significantly different from that of the animals in the control groups. The test substance did not induce cytogenetic damage to the bone marrow cells of mice in this test.

2) Roehm (1989): Study on the clastogenic potential of the test substance in NMRI mice

The maximum tolerated dose of 1000 mg/kg bw, dissolved in water, was administrated by oral gavage to 3 groups of 10 NMRI mice (5 males and 5 females per group). Distilled water served as negative control. Cyclophosphamide in physiological serum (NaCl) was dosed at 40 mg/kg as positive control. The test mice were killed at 24, 48 or 72 hours after the administration, and bone marrow smears were examined for the presence of micronuclei in 1000 polychromatic erythrocytes per mouse as well as for the polychromatic-to-normochromatic erythrocytes (PCE/NCE) ratio. No significant increase of micronuclei was observed compared to the negative control group. No induction of micronuclei due to the test substance was observed.


Short description of key information:
Two independent gene mutation tests in bacteria resulted in negative results except for one positive result in S. typhimurium TA1537 at 2500 µg/plate without metabolic activation in one study. A HPRT study with Chinese hamster cultured cells was negative. Two chromosomal aberration tests in mammalian cells in vitro were positive for clastogenicity with and without metabolic activation. Two in vivo micronucleus assays in mice by i.p. or gavage, respectively, were negative for clastogenicity. Based on the weight of evidence, it is concluded that this chemical is not genotoxic in vivo.

Justification for classification or non-classification

The test substance was mutagenic in various in vitro test systems; however these results could not be confirmed in vivo in tests with mammals.

GHS classification according to Annex I EC/1272/2008 CLP (EU GHS):

- No classification required.