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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance was found negative in bacterial reverse mutation test and in gene mutation test in mammalian cells. When analysing data available on soluble zinc compounds and methacrylic acid/methyl methacrylate, many studies show ambiguous or positive responses in in vitro tests assessing clastogenicity, but overall negative response in in vivo tests.

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:
weight of evidence
Study period:
24 April - 18 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His+ for S. typhimurium; trp+ for E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day by oral route.
Test concentrations with justification for top dose:
Preliminary toxicity test (plate incorporation method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9 mix in TA 100 and WP2uvrA strains
Mutagenicity tests:
Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all 5 strains
Experiment 2 (preincubation method):
- Salmonella strains TA 1535, TA 100 and TA 1537 (without S9 mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.
- Salmonella strains TA 1535, TA 100 and TA 1537 (with S9 mix), TA 98 and Escherichia coli strain WP2uvrA (without and with S9 mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: Test item was insoluble in sterile distilled water, DMSO, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility tests. The test item formed the best doseable suspension in DMSO, therefore, this solvent was selected as the vehicle.
Formulation procedure:
- Test item was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for WP2uvrA, 3 μg/plate for TA100 and 5 μg/plate for TA1535; 9-Aminoacridine (9AA): 80 μg/plate for TA1537; 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA): 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537 and 10 μg/plate for WP2uvrA; Benzo(a)pyrene (BP): 5 μg/plate for TA98
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All five strains of S.typhimurium and E.coli were obtained from University of California, Berkeley, USA.

METHOD OF APPLICATION: In agar (direct plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes at 37 °C
- Incubation period: 48 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
- Preliminary experiment: 1 plate/dose
- Mutagenicity experiments: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of bacterial background lawn.

OTHER: After 48 h of incubation at 37 °C, revertant colonies were assessed using a Domino colony counter.
Evaluation criteria:
Criteria for positive results:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Not reported.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Remarks:
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
- Test item was toxic to TA100 at 5000 μg/plate and non-toxic to WP2uvrA.

MUTAGENICITY TESTS:
- In the first experiment, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 and 1500 μg/plate in the absence and presence of S9-mix respectively.
- In the second experiment, the test item induced a slightly stronger toxic response with weakened bacterial background lawns initially from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical vehicle and positive control data of 2010-11.

Table 7.6.1/2: Mean revertant frequencies

Strains

Doses (µg/plate)

Mean revertants per plate

Experiment 1

Experiment 2

-S9

+S9

-S9

+S9

Mean

SD

Mean

SD

Mean

SD

Mean

SD

TA 100

0

98

7.2

80

8.7

95

4.4

89

9.2

1.5

-

-

-

-

86

6.0

-

-

5

102

5.6

79

1.0

101

2.1

108*

8.2

15

98

12.2

78

15.6

96

4.9

100

9.3

50

92

8.5

71

6.4

92

6.0

102

9.8

150

101

10.4

75

8.5

99

16.3

101

6.8

500

99

3.8

74

2.1

91

16.1

101

9.2

1500

106

6.0

88

4.0

92

4.0

93

7.5

5000

94

4.2

89

1.7

N/T

 

99

3.6

PC

435

23.7

1543

74.0

433

10.2

1216

90.4

TA 1535

0

24

1.5

13

2.6

19

0.0

14

1.5

1.5

-

-

-

-

20

4.0

-

-

5

22

5.7

11

0.6

21

1.7

9

2.1

15

19

5.5

14

4.5

21

1.2

9

2.3

50

25

3.1

12

3.1

16

4.9

11

2.0

150

23

3.1

11

1.5

16

4.7

10

1.2

500

24

7.5

12

2.5

13

2.6

11

2.5

1500

29

0.6

13

2.3

14

0.6

12

2.3

5000

18

2.9

11

4.0

N/T

 

7

2.3

PC

171

31.3

267

2.6

221

22.1

218

16.6

WP2uvrA

0

36

8.1

33

6.4

34

2.1

49

3.5

1.5

-

-

-

-

N/T

-

-

-

5

40

5.0

36

5.5

37

7.0

47

4.6

15

33

1.5

33

2.3

33

4.6

47

6.0

50

31

1.5

39

4.6

35

2.1

48

6.4

150

36

1.5

36

4.0

38

3.8

42

8.5

500

34

4.0

35

6.2

29

2.5

43

4.4

1500

31

5.9

35

6.0

30

4.5

41

6.0

5000

29

0.6

37

5.0

32

2.6

47

5.1

PC

512

13.5

313

20.1

683

30.5

313

33.7

TA 98

0

37

4.0

32

4.5

32

2.6

34

1.5

1.5

-

-

-

-

N/T

-

-

-

5

33

1.0

35

11.7

31

9.0

33

3.5

15

31

3.0

35

7.0

29

4.9

27

0.6

50

36

8.7

25

3.5

31

1.5

31

0.6

150

35

2.1

33

6.6

32

5.7

31

3.8

500

33

3.2

35

1.2

27

4.0

34

4.2

1500

31

1.2

38

3.8

26

3.1

30

2.3

5000

30

1.4

30

4.2

28

4.4

33

0.6

PC

141

6.7

160

2.1

136

27.0

203

13.2

TA 1537

0

11

3.2

9

0.6

12

3.2

12

1.5

1.5

-

-

-

-

13

2.5

-

-

5

10

1.5

10

0.6

8

2.5

14

1.7

15

12

2.1

8

2.6

13

3.2

13

3.1

50

10

4.6

8

3.1

10

3.0

11

1.2

150

12

4.2

14

1.5

12

1.5

13

2.1

500

15

4.0

12

2.6

14

1.2

12

2.6

1500

10

2.1

11

6.5

10

2.6

12

1.5

5000

8

1.5

9

3.1

N/T

 

9

2.0

PC

276

28.6

254

45.2

537

184.6

276

17.7

Conclusions:
Under the test conditions, Zinc Dimethacrylate was not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains, with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli were exposed to Zinc Dimethacrylate at the following concentrations: Preliminary toxicity test (plate incorporation method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9 mix in TA 100 and WP2uvrA strains; Mutagenicity tests: Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all 5 strains; Experiment 2 (preincubation method): (a) Salmonella strains TA 1535, TA 100 and TA 1537 (without S9 mix): 1.5, 5, 15, 50, 150,500 and 1500 μg/plate (b) Salmonella strains TA 1535, TA 100 and TA 1537 (with S9 mix), TA 98 and Escherichia coli strain WP2uvrA (without and with S9 mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. Metabolic activation system used in this test was10 % (v/v) S9 mix. S9 fraction was prepared from liver homogenates of rats induced with phenobarbitone/β- Naphthoflavone at 80/100 mg/kg bw/day by oral route. Vehicle and positive control groups were also included in mutagenicity tests. Test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. In the first experiment, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 and 1500 μg/plate in the absence and presence of S9-mix respectively. In the second experiment, the test item induced a slightly stronger toxic response with weakened bacterial background lawns initially from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence S9-mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 5 μg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.  Under the test conditions, Zinc Dimethacrylate was not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains, with and without metabolic activation (Thompson, 2012).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 November 2012 - 29 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source: European Collection of Cell Cultures
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Four days prior to exposure to Zinc Dimethacrylate, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures are maintained at 37 °C in an atmosphere of 5 % CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (25% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254 (500 mg/kg bw by IP).
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL, with and without S9 mix (4 h exposure)

Experiment 1:
- Without S9 mix (4 h exposure): 5, 10, 20, 30, 40, 50 and 60 μg/mL
- With S9 mix (4 h exposure): 5, 10, 20, 25, 30, 35 and 40 μg/mL

Experiment 2:
- Without and with S9 mix (4 h exposure): 5, 10, 20, 25, 30, 35 and 40 μg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Zinc Dimethacrylate was found to form a dosable suspension at 23.5 mg/mL in water (purified by reverse osmosis). A solution of 23.5 mg/mL, when dosed at 10 % in medium, showed no precipitate in the culture medium. Therefore, water was selected as vehicle.
- Zinc Dimethacrylate was dissolved and diluted in water, shortly before dosing. The final concentration of water added to the cultures was 10 % v/v.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulfonate at 250 μg/mL
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylcholanthrene at 5 μg/mL
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Ham’s Nutrient Mixture F12 medium
- Ham’s Nutrient Mixture F12, supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium was referred to as H0.
- H0 medium supplemented with 10 % HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures/dose for test item and 2 cultures/dose for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures for test item and positive controls

NUMBER OF CELLS EVALUATED: 200 cells/plate were seeded for cloning efficiency and 10^6 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Survival and Relative Survival
Cloning efficiency: Total no of colonies for each culture / (Number of plates scored for colony formation x 200)
Survival: Cloning efficiency x Cell count Correction Factor
Relative Survival (RS): (Individual survival value x100) / Mean control survival value
Following the expression period, three plates were scored for the presence of colonies from each culture and the CE was calculated.
Relative Cloning Efficiency (RCE): (Individual CE x100) / Mean control CE

OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: (Total no. of mutant colonies x 600) / Total no of viable colonies
Evaluation criteria:
The criteria for a positive response will be:
- Demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
- Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
- Demonstration of reproducibility in any increase in mutant frequency;
- Mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10^6 survivors with a corresponding survival rate of 20 % or greater.
Statistics:
Statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the test substance in medium was tested at concentrations of 1175, 1500, 2000, 2350 μg/mL; no fluctuations in pH of the medium of more than 1 unit were observed compared with the vehicle control at 1175, 1500 and 2000 μg/mL, 2350 μg/mL caused a change of greater than 1 unit.
- Effects of osmolarity: Osmolality of the test substance in medium was tested at concentrations of 1175, 1500, 2000, 2350 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: Solution of 23.5 mg/mL, when dosed at 10 % in medium, showed no precipitate in the culture medium.

PRELIMINARY TOXICITY TEST:
- In a 4-h exposure at concentrations from 15.63 to 2000 μg/mL Zinc Dimethacrylate, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 82 to 0 % and 67 to 0 %, respectively.
- Precipitate was seen at 1000 μg/mL and above post dosing in the absence and presence of S9 mix. Precipitate was seen at 1000 μg/mL and above at the end of treatment in the absence of S9 mix and at 250 μg/mL and above at the end of treatment in the presence of S9 mix. Concentrations for the main test were based on toxicity observed from 62.5 μg/mL.

MAIN STUDY:
- See table 7.6.1/1

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (11 June 2012 - 07 January 2013)

Table 7.6.1/1: Summary results

Test Article

Dose

Level

μg/mL

Experiment 1

Experiment 2

4 h Treatment -S9-mix

4 h Treatment +S9-mix

4 h Treatment -S9-mix

4 h Treatment +S9-mix

Mean

Relative

Survival

(%)

Mean

Mutant

Frequency

(x10-6)

Mean

Relative

Survival

(%)

Mean

Mutant

Frequency

(x10-6)

Mean

Relative

Survival

(%)

Mean

Mutant

Frequency

(x10-6)

Mean

Relative

Survival

(%)

Mean

Mutant

Frequency

(x10-6)

Water

0

100

7.12

100

12.83

100

10.38

100

6.52

Zinc Dimethacrylate

5

95

10.06

101

14.15

110

5.76

100

10.02

10

72

12.14

90

10.70

104

17.02

88

7.90

20

64

10.31

68

13.62

100

9.03

89

8.77

25

-

-

46

13.29

79

9.27

81

7.51

30

23

19.15

26

15.86

51

5.53

60

8.73

35

-

-

12

6.26

49

8.98

43

7.33

40

9

19.28

-

-

24

13.13

22

0.69

Ethyl methansulphonate

250

83

373.14***

-

-

83

329.37***

-

-

3-methylcholanthrene

5

-

 

106

427.05***

-

-

85

439.72***

 

*** Statistically significant: p < 0.001

Conclusions:
Under the test conditions, Zinc Dimethacrylate was not considered as mutagenic at the hprt locus in Chinese hamster Ovary (CHO-K1) cells, in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to Zinc Dimethacrylate for 4-h, with and without metabolic activation (25 % S9 [v/v] fraction of male Sprague Dawley rats liver induced with Aroclor 1254), at the following concentrations: (a) Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL, with and without S9 mix (4 h exposure) (b) Experiment 1: Without S9 mix (4 h exposure): 5, 10, 20, 30, 40, 50 and 60 μg/mL; With S9 mix (4 h exposure): 5, 10, 20, 25, 30, 35 and 40 μg/mL (c) Experiment 2: Without and with S9 mix (4 h exposure): 5, 10, 20, 25, 30, 35 and 40 μg/mL. In preliminary toxicity test, Day 1 relative survivals of 82 to 0 % and 67 to 0 % were observed in the absence and the presence of S9 mix, respectively from 15.63 to 2000 μg/mL. Precipitate was seen at 1000 μg/mL and above post dosing in the absence and presence of S9 mix. Precipitate was seen at 1000 μg/mL and above at the end of treatment in the absence of S9 mix and at 250 μg/mL and above at the end of treatment in the presence of S9 mix. Concentrations for the main test were based on toxicity observed from 62.5 μg/mL. In the main experiment, no precipitate was seen post-dosing or at the end of treatment. In the Experiment 1, Day 1 survival values ranged from 95 to 9% were observed at 5 to 60 μg/mL in the absence of S9 mix, whereas in the presence of S9 mix, Day 1 survival values ranged from 101 to 12% were observed at 5 to 40 μg/mL. In the Experiment 2, Day 1 survival values ranged from 110 to 24% were observed at 5 to 40 μg/mL in the absence of S9 mix, whereas in the presence of S9 mix, Day 1 survival values ranged from 100 to 22% were observed at 5 to 40 μg/mL. Zinc Dimethacrylate did not induce the statistically significant increase in the mutant frequencies in any of the experiments, in the absence or presence of S9 mix. Positive control chemicals [ethyl methanesulfonate at 250 µg/mL (without S9 mix) and methylcholanthrene at 5 µg/mL (with S9 mix)] induced significant increase in the mutant frequencies indicating the validity of the study. Under the test conditions, Zinc Dimethacrylate was not considered as mutagenic at the hprt locus in Chinese hamster Ovary (CHO-K1) cells, in the presence and absence of metabolic activation (Woods, 2013).

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Method: Induction of mitotic gene conversion or non-reciprocal recombination was studied with the diploid strain D4 of Saccharomyces cerevisiae.
GLP compliance:
not specified
Type of assay:
mitotic recombination assay with Saccharomyces cerevisiae
Target gene:
Gene conversion was studied at the following two loci: ade2 and trp5.
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
Optimal or highest concentration (ppm): 5,000/1,000
Vehicle / solvent:
0.1M potassium phosphate buffer (pH 7.5)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
0.1 M potassium phosphate buffer (pH 7.5)
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
other: 1173
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 h
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
None
Remarks on result:
other: strain/cell type: diploid strain D4
Remarks:
Migrated from field 'Test system'.

Table 1: Zinc sulfate tested for the induction of mitotic gene conversion in strain D4 ofSaccharomyces cerevisiae

Convertants per 106survivors at the loci

Survivors (%)

Optimal or highest concentration (ppm)

ade2

trp5

45.7 (28.5)

2.9 (3.3)

97

5000/1000

Doses are presented at which the highest frequencies of mitotic gene convertants per survivor were observed. Those concentrations were sometimes different for conversion at the two loci. The corresponding control values are given in parentheses. Treatments were performed in 0.1Mpotassium phosphate butter (pH 7.5) with shaking for 4 h at 25 ± 0.1 °C.

Conclusions:
Read across substance zinc sulfate was considered to be non-convertogenic under the study conditions. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A study was conducted to determine the potential of read across substance zinc sulfate to induce mitotic gene conversion using diploid strain D4 of Saccharomyces cerevisiae. Diploid strain D4 (MD 20:a,+,ade2-I, trp5-27-+)was treated with the test material for 4 h in selectivesynthetic media (WICKERHAM), supplemented with amino acids and nucleobases (ROMAN) and solidified with 1.5% Difco bactoagar, usually up to 5,000 ppm/plate. The test material showed inability to induce mitotic gene conversion. Convertants per 106 survivors at the loci ade2 & trp5 was 45.7 & 2.9, respectively. Read across substance zinc sulfate was considered to be non-convertogenic under the study conditions (Siebert, 1970). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
According to Maron D.M. and Ames B.N.; Mutation Res. 113: 173 - 215 (1983)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 104
Metabolic activation:
with and without
Metabolic activation system:
from rat liver microsomes
Test concentrations with justification for top dose:
0 - 25 mg/plate
Key result
Species / strain:
S. typhimurium TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Key result
Species / strain:
S. typhimurium, other: TA 104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Read across substance methyl methacrylate was negative for mutagenicity in a Salmonella typhimurium reverse mutation assay with the additional tester strains TA102 and TA104. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A bacterial reverse mutation assay was conducted for read across substance methyl methacrylate following Maron and Ames, 1983 procedure. S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 104 were exposed to 0 - 25 mg/plate methyl methacrylate with and without metabolic activation. Read across substance methyl methacrylate was negative for mutagenicity in a Salmonella typhimurium reverse mutation assay with and without metabolic activation in S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 104 (Schweikl, 1998). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Sister chromatid exchange in CHO cells
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
AROCLOR 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
1250 ug/ml - 5000 ug/ml
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Conclusions:
As clastogenic activity was found at probably high toxic doses, it is not possible to conlude that read across substance methyl methacrylate has a real direct clastogenic effect as it is probably secondary to cytotoxicity. Similar results can be expected for zinc dimethacrylate.
Executive summary:

In a cytogenetic test with CHO cells induction of chromosomal aberrations was bound to high doses of read across substance methyl methacrylate which are assumed to be strongly cytotoxic. With S-9 mix treatment was for 2 h followed by 8 to 10 h recovery. Doses up to 1,600 μg/ml were negative, at 5000 μg/mL the frequency of aberrant cells was 30%; only one experiment was performed. Without S-9 mix, treatment time was 8 hours with 2.0 to 2.5 h recovery. Doses up to 500 μg/mL were negative, at 1600 and 3000 μg/mL aberration frequencies ranging from 5 to 10% were found. Data on cytotoxic effects were not given, however, it can be assumed from the data presentation and the general approach of the authors that the highest doses tested led to strong cytotoxic effects. Thus, methyl methacrylate showed clastogenic activity but at highly toxic doses therefore probably due to cytotoxicity more than a direct clastogenic effect (Anderson, 1990). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In addition to the mutation test at the TK locus, analyses for chromosomal aberrations and induction of micronuclei in vitro were run.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK +- -3.7.2C heterozygote of L5187Y mouse lymphoma cells
Metabolic activation:
without
Test concentrations with justification for top dose:
1000 - 3000 µg/ml (10 - 30 mM)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2072
Details on test system and experimental conditions:
Duplicate cultures were treated for four hours with test substance and with or without metabolic activation. The cultures were split for use in a mutagenicity assay, aberration analysis and micronucleus assay. For the mutation assay, colonies were incubated for 9-11 days at 37°C, counted and colony size distribution determined.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
Methyl methacrylate produced a positive mutagenic response at the tk locus with responses variable at 2-4 times background but consistently positive at doses from 2300 to 3000 ug/ml. The highest mutant frequency occurred at 2800 ug/ml with 298 mutants/10E6 survivors. Because the background frequency was relatively high after the analysis of 100 metaphases, an additional 100 metaphases were scored. Weak positives were recorded at 2200, 2500, 2700, 2800 and 3000 ug/ml. The highest response was at 2800 ug/ml with 45 aberrations in 39 of 200 metaphases (background = 16 aberrations in 15 of 200 metaphases scored). It should be noted that the high background was due to an increase in the number of chromosome rearrangements and breaks in the control culture. No concurrent increase in background TK mutant or micronucleus frequency was observed. The MMA treated cells showed a significant increase in chromatid events with no change in the number of chromosome events. Micronucleus responses were also variable with weak positive responses recorded at 2200, 2400, 2500, 2800, 2900 and 3000 ug/ml. The greatest response was at 2900 ug/ml with micronuclei in 25 of 1000 binucleates (background = 9 /1000). The 2 lowest doses, 1000 and 1750, were negative for mutagenicity, aberrations and micronuclei. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Comparison of results: Chromosome aberrations, micronucleus induction and mutations at the TK locus, small colony mutants and survival.

MMA [µg/ml] Cells w/Ab. Cells w/MN Total MF/106cells Small colonies MF/106 % Small colonies Survival [%]
0 15 9 68 39 57% 1OO
1000 17 6 74 60 81% 53
1750 24 10 121 88 73% 34
2102 28 22 143 115 80% 26
2400 21 20 168 144 86% 22
2499 33 18 226 180 80% 15
2601 27 17 179 140 78% 19
2700 30 12 169 147 87% 19
2799 39 20 298 260 87% 11
2901 16 25 255 227 89% 12
3000 35 22 254 222 87% 12

Increases in any type of mutations correlate strongly with a decrease in survival. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM). The increase in mutations at the TK locus is primarily due to small colony mutants with a trend to more small colonies at higher concentrations, indicating that these are primarily due to deletions (chromosome mutations) rather than gene mutations.

Conclusions:
In a mouse lymphoma assay which was only run without metabolic activation, weak effects were obtained for read across substance methyl methacrylate doses producing high toxicity. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations). Similar results can be expected for zinc dimethacrylate.
Executive summary:

In a mouse lymphoma assay which was only run without S-9 mix, weak effects were obtained for read across substance methyl methacrylate doses producing high toxicity. According to the authors, 2,000 μg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 2,499 μg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3,100 μg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations) (Moore, 1988). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In addition to the mutation test at the TK locus, analyses for chromosomal aberrations and induction of micronuclei in vitro were run.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79B
Metabolic activation:
with and without
Metabolic activation system:
rat liver
Test concentrations with justification for top dose:
0, 10, 20 mM
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1342 9,10-dimethyl 1,2-benzanthracene and ethyl methane sulfonate
Details on test system and experimental conditions:
1.5E+6 cells were plated onto cell culture plates with culture medium and incubated for 20-24 hours. Cells were exposed to varying concentrations of the test substance for 24 hrs in the absence of S9 mix or 4 hrs with or without S9 mix. The cells were subcultured after four days, cell numbers counted and expressed relative to cell counts in solvent control cultures or the plating efficiency. 3E+6 cells were subcultured for eight days and mutant isolation started on day 10 by replating the cells in selective media containing 6-thioguanidine, and the cloning efficiency determined.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
taking into account the range of control values
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>20 mM/L
Positive controls validity:
valid
Additional information on results:
A very weak mutagenic response in V79 cells was observed at the concentrations tested after a direct exposure for 24 hours. Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per million surviving cells were observed. Other control data from parallel experiments reported in the same paper range from 2 to 10 mutants per million surviving cells. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively. Data from the trial with metabolic activation were not reported in detail. The authors interpret the data as a very weakly positive finding. In the light of the range of control data, the result is interpreted as ambiguous.
Conclusions:
Under the study conditions, the result was interpreted as ambiguous for read across substance methyl methacrylate. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A very weak mutagenic response in V79 cells was observed at the concentrations tested after a direct exposure for 24 hours for read across substance methyl methacrylate. Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM methyl methacrylate were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per million surviving cells were observed. Other control data from parallel experiments reported in the same paper range from 2 to 10 mutants per million surviving cells. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively. Data from the trial with metabolic activation were not reported in detail. The authors interpret the data as a very weakly positive finding. In the light of the range of control data, the result is interpreted as ambiguous for methyl methacrylate (Schweikl, 1998). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
X-linked recessive lethal mutations were examined in Drosophila melanogaster upto F3 generations to evaluate the mutagenic potential of the test material.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Berlin K (wild-type) and Basc
Sex:
male/female
Details on test animals or test system and environmental conditions:
No data
Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: no data
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 5 % saccharose
Duration of treatment / exposure:
3 successive broods
Frequency of treatment:
No data
Post exposure period:
No data
Remarks:
Doses / Concentrations:
5 mM
Basis:
no data
No. of animals per sex per dose:
no data
Control animals:
yes
Positive control(s):
Trenimon
Tissues and cell types examined:
X-chromosomes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose close to LD50 was selected for the study
Evaluation criteria:
No data
Statistics:
Significance was calculated according to the Kastenbaum-Bowman tables.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: A significant (P < 0.01) increase of sex-linked recessive lethals was observed in the Basc strain in only one test, whereas non-significant results were observed in two repeats. It has been assumed that the single increased value was due to chance.

Table 1 : Results of the Basc test in Drosophila Melanogaster

Compound

Expt. no.

Conc. (mM)

Sex-linked recessive lethals/chromosomes tested and percentage

Brood 1

Brood 2

Brood 3

Zinc sulphate

 -

5

13/1206** (1.08)

2/1217 (0.16)

4/1200 (0.33)

 -

5

2/1233 (0.16)

2/1175 (0.17)

8/1193 (0.67)

 -

5

3/1185 (0.25)

1/1215 (0.08)

1/1023 (0.10)

 

 

18/3624* (0.50)

5/3607 (0.14)

13/3416 (0.38)

Controls (cumulated)

6

 -#

19/7130 (0.27)

8/5525 (0.14)

19/4871 (0.39)

Positive control (trenimon)

1

0.005

35/682** (5.13)

23/598** (3.85)

14/584** (2.40)

# Different solvents were used in separate controls,  *P ≤0.05, **P ≤0.01.

Conclusions:
Under the test conditions, a single increased value observed was due to chance and did not indicate genotoxicity of read across substance zinc sulfate. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A sex-linked recessive lethal test was conducted in Drosophila melanogaster to evaluate the mutagenic potential of read across substance zinc sulfate. Berlin K (wild-type) and Base strains were used for the study. 5mM test concentration (dose close to the LD50) was applied by the adult feeding method in 5% saccharose. Controls and positive control (trenimon) were also included. Appoximately 1200 X-chromosomes were tested in each of 3 successive broods (3-3-4 days). In repeat experiments, sometimes only single broods were tested. To confirm X-linked recessive lethal mutations, F2 progeny cultures with 2 or fewer wild-type males were routinely retested in the F3 generation. Mosaics were not counted. "Clusters" of 2 were included because their occurrence was compatible with statistical expectation of independent origin. Significance was calculated according to the Kastenbaum-Bowman tables. A significant (P < 0.01) increase of sex-linked recessive lethals was observed in the Basc strain in only one test, whereas non-significant results were observed in two repeats. Under the test conditions, a single increased value observed was due to chance and did not indicate genotoxicity of read across substance zinc sulfate (Gocke, 1981). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1974
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
A dominant lethal assay was conducted in rats to evaluate the genotoxic potential of zinc sulphate.
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: gavage
Duration of treatment / exposure:
5 days
Frequency of treatment:
Daily
Post exposure period:
No data
Remarks:
Doses / Concentrations:
2.75, 27.5 or 275 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
No data
Control animals:
not specified
Tissues and cell types examined:
Not applicable
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified

None

Conclusions:
Under the study conditions, read across substance zinc sulphate was non-mutagenic in dominant lethal assay in rats. Similar results can be expected for zinc dimethacrylate.
Executive summary:
No chromosomal aberrations were induced when rats were given 2.75, 27.5 or 175 mg/kg bw zinc (as zinc sulphate) by gavage once or daily for 5 consecutive days. Under the study conditions, read across substance zinc sulphate was non-mutagenic in dominant lethal assay in rats (EU, 2008). Similar results can be expected for zinc dimethacrylate.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
A micronucleus test on mouse bone marrow was conducted to evaluate the mutagenic potential of test material.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: S. Ivanovas GmbH and Co., Kisslegg/Allgau (Germany)
- Diet: Standard chow (Altromin GmbH, Lage, Germany), ad libitum
- Water: Ad libitum
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: solution in 0.9% Sodium chloride
Details on exposure:
No data
Duration of treatment / exposure:
30 h
Frequency of treatment:
Twice, at 0 and 24 h
Post exposure period:
No data
Remarks:
Doses / Concentrations:
2 X 86.3, 2 X 57.5 and 2 x 28.8 mg/kg (treated twice, at 0 and 24 h)
Basis:
actual ingested
No. of animals per sex per dose:
2
Control animals:
yes
Positive control(s):
No data
Tissues and cell types examined:
Polychromatic erythrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Bone marrow smears were prepared at 30 h.

METHOD OF ANALYSIS: Slides were coded and 1000 polychromatic erythrocytes were scored per animal.
Evaluation criteria:
No data
Statistics:
Significance was calculated according to the Kastenbaum-Bowman tables.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data

Table 1:Results of the micronucleus test on mouse bone marrow

Surviving/treated mice

Dose

Route of application

Micronuclcated polychromatic erythrocytes (%)

mg/kg

mmole/kg

3/4

2 X 86.3

2 X 0.3

i.p.

2.9

4/4

2 X 57.5

2 X 0.2

i.p.

1.9

4/4

2 X 28.8

2 X 0.1

i.p.

1.7

4/4

0

 0

i.p.

1.9

 

Conclusions:
Under the test conditions, read across zinc sulfate was found to be non-mutagenic. Similar results can be expected for zinc dimethacrylate.
Executive summary:

A micronucleus test was conducted to evaluate the mutagenic potential of read across zinc sulfate. Male and female NMRI mice were used for the study. Feed and water were provided ad libitum. 4 animals (2 male, 2 female) were used in each treatment and control groups. 86.3, 57.5 and 28.8 mg/kg doses were administered i.p. at 0 and 24 h. Bone marrow smears were prepared at 30 h. 1000 polychromatic erythrocytes were scored per mouse. Significance was calculated according to the Kastenbaum-Bowman tables. Under the test conditions, read across zinc sulfate was found to be non-mutagenic (Gocke, 1981). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
Similar to OECD-guideline 474, all relevant study details available but article in japanese.
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Chromosome aberration rates and sister chromatid exchange frequency were examined in the peripheral lymphocytes of 38 male workers who were engaged in organic glass production and exposed to methyl methacrylate vapors.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: ddy
Sex:
male
Route of administration:
oral: gavage
Vehicle:
Olive oil
Duration of treatment / exposure:
4 doses
Frequency of treatment:
3 doses: once, 24 h before terminal sacrifice
1 dose: 4 split doses every 24 h, the last one 24 h before terminal sacrifice, total duration 5 d
Post exposure period:
24 h
Remarks:
Doses / Concentrations:
Olive oil, 25 mL/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1130, 2260, 4520 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
4 x 1130 mg/kgbw
Basis:
actual ingested
No. of animals per sex per dose:
6 (repeated treatment of 4 x 1130 mg/kgbw : 5)
Control animals:
yes
Positive control(s):
3 mg Mitomycin C, single dose by i.p. administration 24 h prior to preparation
Tissues and cell types examined:
Sampling time for bone marrow: 3 single doses - 24 h post-administration; for repeated administration: 5 days after first administration.
Statistics:
according to Kastenbaum/Bowman
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.

Conclusions:
Read across substance, methyl methacrylate was not mutagenic in vivo under study conditions. Similar results can be expected for zinc dimethacrylate.

Executive summary:

Read across substance, methyl methacrylate was administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. Read across substance, methyl methacrylate was not mutagenic in vivo under study conditions (Hachiya, 1982). Similar results can be expected for zinc dimethacrylate.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Chromosome aberration rates and sister chromatid exchange frequency were examined in the peripheral lymphocytes of 38 male workers who were engaged in organic glass production and exposed to methyl methacrylate vapors.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Species:
other: human
Sex:
male
Route of administration:
inhalation: vapour
Vehicle:
None
Details on exposure:
The time-weighted average methyl methacrylate concentrations by personal sampling, and methanol concentrations in venous blood and urine samples (collected at the end of a shift of the day) were measured.
Duration of treatment / exposure:
8 hours per day
Remarks:
Doses / Concentrations:
0.9 to 71.9 ppm
Basis:

No. of animals per sex per dose:
38 men
Control animals:
yes
Statistics:
A normal distribution was assumed for cytogenetic parameters, and the distribution was expressed in terms of arithmetic mean (AM) +/- arithmetic standard deviation (ASD) together with the number of cases (N). Exposure indicators distributed log-normally and the distribution was expressed with geometric means (GM) followed by geometric standard deviations (GSD) in parentheses. Student's t-test (unpaired) was employed for evaluation of the difference in means.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Conclusions:
Data from limited number of subjects indicated that occupational read across substance methyl methacrylate exposure under the study conditions was not associated with mutagenicity. This conclusion confirms the absence of mutagenicity of read across substance methyl methacrylate in humans, and is in general agreement with a majority of the results of studies on mutagenicity in vitro, animal carcinogenicity and occupational cancer epidemiology of read across substance methyl methacrylate. Similar results can be expected for zinc dimethacrylate.
Executive summary:

Chromosome aberration rates and sister chromatid exchange frequency were examined in the peripheral lymphocytes of 38 male workers who were engaged in organic glass production and exposed to read across substance methyl methacrylate (MMA) vapors at the concentrations of 0.9 ppm to 71.9 ppm. The results were compared with the findings in the concurrent nonexposed male subjects. Comparison of the exposed group with the nonexposed controls showed that there were no exposure-related changes in chromosome aberration rate. SCE frequency was higher in the exposed group than in the controls, but this was considered to be due to higher ages of the former group than that of the latter. In fact, selection of nonsmokers and further classification of the exposed nonsmokers into two groups of those with exposure below and above a median MMA concentration (ca. 4 ppm) failed to show any difference among the three nonsmoking groups in cytogenetic parameters, or any dose-dependency. The present results, although in a limited number of subjects, indicate that occupational methyl methacrylate exposure under the conditions studied is not associated with mutagenicity. This conclusion confirms the absence of mutagenicity of read across substance methyl methacrylate in humans, and is in general agreement with a majority of the results of studies on mutagenicity in vitro, animal carcinogenicity and occupational cancer epidemiology of read across substance methyl methacrylate (Seiji, 1994). Similar results can be expected for zinc dimethacrylate.

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

Additional information

Read across approach

The registered substance contains both the zinc (II) cation and the methacrylate anion. In a dissolution/transformation study designed according to OECD TG 29, it was shown that zinc methacrylate dissociates rapidly in water into zinc and methacrylic acid. The dissociation is almost complete at the stomach pH of ~1 and reaches approximately 79% at the lung pH of 7.35-7.45. Therefore, both oral and inhalation exposure to zinc methacrylate will lead to systemic exposure to zinc and methacrylic acid, so that it is justified to refer to the toxicological properties of both moieties in order to infer the profile of the substance as a whole. More specifically, methacrylic acid (EC No. 201-204-4/CAS No. 79-41-4), methyl methacrylate (EC No. 201-297-1/CAS No. 80-62-6), zinc chloride (EC No. 231-592-0/CAS No. 7646-85-7), zinc sulphate (EC No. 231-793-3/CAS No. 7733-02-0) and zinc lactate (EC No. 240-178-9/CAS No. 16039-53-5) were used as source substances to cover genetic toxicity as described in the proposed read across included in Section 13.

In vitro genetic toxicity

Zinc methacrylate was found to be negative in a GLP-compliant bacterial reverse mutation test according to OECD TG 471 and an ‘in vitro gene mutation test’ according to OECD TG 476 (Thompson, 2013, Woods 2013). These results are in line with those observed for zinc and methacrylic acid/methyl methacrylate individually.

The genotoxicity of soluble and slightly soluble zinc compounds has been extensively investigated in a wide range of in vitro and in vivo studies. The in vitro investigations included non-mammalian and mammalian test systems covering the endpoints of gene mutation, chromosomal aberrations, sister chromatid exchange, unscheduled DNA synthesis (UDS), as well as cell transformation. Zinc compounds did not increase the mutation frequencies in the majority of bacterial or mammalian cell culture systems. For example, zinc chloride, zinc sulphate, zinc bis(dihydrogen phosphate), zinc oxide or zinc monoglycerolate were consistently negative in the Ames test. While zinc chloride was also negative for gene mutations in the mouse lymphoma assays, there was some evidence that zinc oxide, zinc acetate or zinc monoglycerolate induced the formation of mutation colonies in the absence of metabolic activation. Several reviewers noted, however, that these mutations were observed at cytotoxic concentrations and that the analysis did not distinguish between big and small colonies which could be caused by gene mutation or chromosomal aberrations (Thompson et al., 1989; WHO, 2001; EU RAR, 2004; MAK, 2009).

Conflicting information was further found on zinc compounds regarding their potential to induce chromosomal aberrations or sister chromatid exchange in mammalian cell systems or when evaluated in the cell transformation assay. Positive as well as negative results were obtained in these cell systems. In studies where chromosomal aberrations or sister chromatid exchange were observed, these were generally considered to be weak and occurred only at high, often cytotoxic concentrations. Moreover, these positive in vitro findings were also seen in the context of the impact that changes in zinc levels can have on cell system processes controlled by a strict metal homeostasis. A change of this metal homeostasis due to increased zinc levels may lead to a binding of zinc to amino acids such as cystein and therefore to an inhibition of certain enzymes. This can lead to interactions with the energy metabolism, signal transmission and apoptotic processes which in turn lead to the observed clastogenic or aneugenic effects in in vitro systems (EU RAR, 2004; MAK, 2009)

Furthermore, methyl methacrylate has the potential to induce mutagenic effects in vitro, especially clastogenicity. However, this potential seems to be limited to high doses with strong toxic effects. Methyl methacrylate was not mutagenic in S. typhimurium strains TA97, TA98, TA100, TA1535 with and without metabolic activation (Zeiger et al., 1987). Additionally, methyl methacrylate was not mutagenic in an Ames test with S. typhimurium TA102 which is sensitive to crosslinking and oxidising agents (Schweikl et al.,2001).

Regarding gene mutation in mammalian cells, Litton Bionetics (1981) reported on a mouse lymphoma assay which was weakly positive in the TK locus in the presence and negative in the absence of S-9 mix. Without S-9 mix, doses of methyl methacrylate up to 100 nL/mL were tested and the higher doses led to total toxicity. With S-9 mix, the substance was positive in the range of 100 to 250 nL/mL, however clear effects were observed only at doses with high toxicity below 20% relative growth. Additionally, in a HPRT assay with and without metabolic activation, methyl methacrylate was weakly positive in V79 cells (Schweikl et al.,1998). Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per 10E+6 surviving cells were observed. The cell numbers of the low and high dose treatment were 71 and 49% of controls, respectively. Data from the trial with metabolic activation were not reported in detail. In a further lymphoma assay conducted only without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 μg/mL was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20 and 30%; in one experiment the highest dose of 499 μg/mL induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 μg/mL induced 220 mutants with 11% relative survival. The majority of induced colonies were small ones, indicating that the genetic effect was derived from clastogenicity and not from gene mutations.

Finally, in a cytogenetic test with CHO cells, induction of chromosomal aberrations was bound to high doses of methyl methacrylate which are assumed to be strongly cytotoxic (Anderson et al., 1990). With S-9 mix, treatment for 2 h was followed by 8 to 10 h recovery. Doses up to 1600 μg/mL were negative, at 5000 μg/mL the frequency of aberrant cells was 30%; only one experiment was performed. Without S-9 mix, treatment time was 8 h with 2.0 to 2.5 h recovery. Doses up to 500 μg/mL were negative; at 1600 and 3000 μg/mL aberration frequencies ranging from 5 to 6% were found. Data on cytotoxic effects were not given, however, it can be assumed from the data presentation and the general approach of the authors that the highest doses tested led to strong cytotoxic effects. Thus, methyl methacrylate seems to be a high toxicity clastogen, i.e., the induction of chromosomal aberrations is bound to highly toxic doses.

In vivo genetic toxicity

Available in vivo genotoxicity assays on soluble zinc compounds include the micronucleus test, sister chromatid exchange (SCE) and chromosomal aberration tests and the dominant lethal mutation assay in mouse or rat as well as investigations for sex-linked recessive lethal mutations in Drosophila melanogaster. Various soluble and slightly soluble zinc compounds have also been studied in a range of in vivo studies including the micronucleus test, SCE and chromosomal aberration tests or dominant lethal mutation assays in mice or rats as well as in the Drosophila melanogaster SLRL test. The zinc compounds were consistently negative in the micronucleus test and in the assay with Drosophila melanogaster. Zinc sulphate was further negative in a dominant lethal assay in rats.

Equivocal and sometimes contradicting results were found for the induction of chromosomal aberrations in bone marrow cells harvested from animals exposed to zinc chloride or zinc oxide. Negative findings for chromosome aberrations were produced after intraperitoneal injection of zinc chloride into mice (Vilkina et al., 1978) or when rats were given zinc sulphate by gavage once or daily for 5 consecutive days (Litton Bionetics, 1974). In contrast, increased aberrations were reported in rats after inhalation exposure to zinc oxide (Voroshilin et al., 1978), in rats after oral exposure to zinc chloride and in mice after multiple intraperitoneal injections of zinc chloride (Gupta et al., 1991). Moreover, increased chromosomal aberrations were found in calcium-deficient mice when fed zinc (in form of zinc chloride) via the diet (Deknudt, 1982). These equivocal finding are likely a reflection of inter-study differences in routes, levels and duration of zinc exposure, the nature of lesions scored (gaps compared to more accepted structural alterations) and great variability in the technical rigour of individual studies (WHO, 2001). The German MAK committee reviewed the existing in vivo evidence and concluded that particularly those studies indicating clastogenic effects involved a lot of methodological uncertainties which do not allow to overrule the in vivostudies which did not provide evidence for chromosomal aberrations. Moreover, the Dutch rapporteur for the EU risk assessment of zinc compounds under the EU existing substance legislation considered the positive in vitro findings for chromosomal aberration and SCE assays to be overruled by the overall weight of evidence of negative in vivo tests for this endpoint (EU RAR, 2004).

Furthermore, the mutagenicity of methacrylate compounds has been extensively tested in vivo. A dominant lethal assay in male mice was negative after inhalation exposure to doses ranging from 0.4 to 36.5 mg/L (corresponding to 100 to 9000 ppm; Zeneca/ICI, 1976) of methyl methacrylate. Specific data on toxicity were not given. However, in the 36.6 mg/L group, 6/20 males died. Treated males were mated to 2 females each for 8 periods of 1 week; females were killed 13 days after the assumed dates of fertilization. There was no significant increase in dominant lethal mutations.

Two chromosomal aberration tests were conducted by ICI (1976, 1979) investigating the effect of inhalation exposure to methyl methacrylate at doses ranging from ca. 0.4 to 36.5 mg/L (100 to 9000 ppm). In both studies, acute exposure was for 2 h (sampling 24 h after treatment) and subacute exposure for 5 h per day on 5 consecutive days (sampling 24 h after last treatment). Data on toxicity were not given. Group sizes varied from 2 to 9; as far as possible 50 metaphases were analysed per animal. The first study was negative for chromosomal aberration frequencies when - as usual - gaps were excluded. Including gaps and combining two acute experiments conducted independently some increases in aberration frequency were statistically significant. This was also due to a particularly low control rate in this experimental segment. Compared to other, almost twofold higher, control values in other segments of the report, this finding appears to be of little biological importance. In the second study frequencies of chromosomal aberrations excluding gaps were not given (ICI, 1979). Including gaps, increases were recorded at some experimental entries. Furthermore, combined data on chromosomal aberration frequencies exclusively gaps from both studies were given, then weak increases were obtained for 400 and 700 ppm in the acute study (not for 100, 1000 or 9000 ppm) and 9000 ppm in the subacute study. Both studies suffer from inadequate description; the second study in particular demonstrates severe methodological issues, e.g., analysis of 50 metaphases was not possible for 10 out of 27 animals in the acute and 10 out 26 in the subacute test. Altogether, a clear conclusion cannot be drawn from these studies.

Hachiya et al. (1982) reported on a negative bone marrow micronucleus assay with mice. In an acute test, methyl methacrylate was given by gavage at doses ranging from 1130 to 4520 mg/kg, in a subacute assay daily doses of 1130 mg/kg were given on 4 consecutive days. All groups consisted of 6 animals; sampling was done 24 h after (last) administration. There was no increase in the frequency of micronucleated polychromatic erythrocytes. The percentage of reticulocytes from all bone marrow cells was not affected data on general toxicity were not given.

Human information

No relevant human information was generated either with the registered substance or with zinc compounds. However, chromosome aberration rates and sister chromatid exchange frequency were examined in the peripheral lymphocytes of 38 male workers engaged in organic glass production and exposed to methyl methacrylate vapors at concentrations of 0.9 to 71.9 ppm (Seiji, 1994). The results were compared with the findings in concurrent non-exposed male subjects. Comparison of the exposed group with the non-exposed controls showed that there were no exposure-related changes in chromosome aberration rate. SCE frequency was higher in the exposed group than in the controls, but this was considered to be due to the higher ages of the subjects in the exposed group. Selection of non-smokers and further classification of the exposed non-smokers into two groups (exposure below and above a median methyl methacrylate concentration of ca. 4 ppm) failed to show any difference among the three non-smoking groups in cytogenetic parameters, or any dose-dependency. The present results, although in a limited number of subjects, indicate that occupational methyl methacrylate exposure under the conditions studied is not associated with mutagenicity. This conclusion confirms the absence of mutagenicity of methyl methacrylate in humans and is in general agreement with a majority of the results of studies on mutagenicity in vitro, animal carcinogenicity and occupational cancer epidemiology of methyl methacrylate. Despite its limitations, this study is considered to be critical as it is directly in humans and conducted under occupational exposure conditions.

Overall conclusion on genotoxicity

Based on a wide body of available evidence, zinc methacrylate does not appear to be directly mutagenic or to cause indirect DNA damage, including chromosomal aberrations, micronucleus formation and sister chromatid exchanges in vitro and in vivo. The registered substance was found to be negative in a bacterial reverse mutation assay and a gene mutation test in mammalian cells. When analysing data available on soluble zinc compounds and methacrylic acid/methyl methacrylate, many studies show ambiguous or positive responses in vitro for clastogenicity, but overall negative response in in vivo tests.

In addition, in response to a Notification of an adopted Decision on a Compliance Check (Decision number CCH-D-2114558120-60-01/F) dated 9th June 2021, the registrants have conducted an in vitro micronucleus study according to OECD 487, in compliance with GLP. Although a first unaudited draft report was made available by mid-June 2022, the final report is not yet available. This section will be updated to include the new findings once the final report is available.

References

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Deknudt G (1982) Étude des eets clastogéniques du zinc chez les mammifères (Clastogenic eects of zinc in mammals) (French).C R Seances Soc Biol Fil 176: 563–567.

EU RAR (2002). Methyl methacrylate.Risk assessment report, 1st priority list, Volume 22, European Commission, Institute for Health and Consumer Protection, European Chemicals Bureau.

EU RAR (2004). Zinc sulphate. Risk assessment report, 2nd priority list, Volume 46, European Commission, Institute for Health and Consumer Protection, European Chemicals Bureau.

Gocke E, King MT, Eckhardt K, Wild D (1981). Mutagenicity of cosmetics ingredients licensed by the European Communities. Mutation Research, 90: 91-109.

Gupta T, Talukder G, Sharma A (1991) Cytotoxicity of zinc chloride in mice in vitro.Biol Trace Elem Res 30: 95–101.

Hachiya N, Taketani A, Takizawa Y (1982). Research relating to the mutagenicity of substances in the living environment, report 3. Ames tests and mouse bone marrow micronucleus test for acrylic resin monomers and their main additives.Nippon Koshu Eisei Zasshi 29, 236-239.

ICI (1979) as available on ECHA dissemination portal:https://www.echa.europa.eu/web/guest/registration-dossier/-/registered-dossier/15528/7/7/3/?documentUUID=928376e5-119b-45f7-84f4-2d48b4847a3a

Litton Bionetics Inc. (1974). Mutagenic evaluation of compound FDA 71-49. Report no.: PB-245 451.

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MAK Value Documentation, (2009) Zinc and its inorganic compounds.

Moore MM, Amanda A, Doerr CL, Brock KH, Dearfield KL (1988) Genotoxicity of acrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and ethyl methacrylate in L5178Y mouse lymphoma cells. Environmental and molecular mutagenesis, 11:49-63.

Schweikl H, Schmalz G, Rackebrandt K (1998) The mutagenic activity of unpolymerized resin monomers in Salmonella typhimurium and V79 cells. Mutation Research 415: 119 – 130.

Schweikl H, Schmalz G, Spruss T (2001) The induction of micronuclei in vitro by unpolymerized resin monomers. J. of Dent. Res. 80(7): 1615 – 1620.

Seiji K, Inoue O, Kawai T, Mizunuma K, Yasugi T, Moon C-S, Takeda S and Ikeda M (1994). Absence of Mutagenicity in Peripheral Lymphocytes of Workers Occupationally Exposed to Methyl Methacrylate. Industrial Health 32: 97-105. Testing laboratory: Tohoku Rosai Hospital, Sendai 980, Japan.

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Thompson PW (2013) Reverse Mutation Assay “Ames Test” using Salmonella typhimurium and Escherichia coli. Harlan Laboratories Ltd, Derby, UK.

Vilkina GA, Pomerantzeva MD, Ramaya LK (1978) Lack of mutagenic effect of cadmium and zink salts in somatic and germ mouse cells (Russian). Genetika 14: 2212–2214.

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Justification for classification or non-classification

The basic assumption to deduce the toxicological properties of the registered substance (based on its behaviour in water) is that after intake of the substance, it is mainly transformed into the ionic species and zinc compounds and the methacrylic part of the substance are the determining factors of the biological activities of the registered substance. This assumption is confirmed by the genotoxicity studies performed on the registered substance where negative reponse was found in Ames test and in an in vitro gene mutation in mammalian cells test. These results are in line with the numerous studies performed on both zinc compounds and methacrylate compounds. Therefore the genotoxic potential of the registered substance was deduced from all the data available on these compounds. The overall weight of evidence from the existing in vitro and in vivo genotoxicity assays suggest that zinc compounds and methacrylate compounds do not have biologically relevant genotoxic activity, which was confirmed by other regulatory reviews of the genotoxicity of zinc compounds and methacrylate compounds. Thus the registered substance does not need to be classified for genotoxicity under the CLP Regulation (EC) No 1272/2008.