Registration Dossier
Registration Dossier
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EC number: 236-144-8 | CAS number: 13189-00-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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. - 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) - 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.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- 1970
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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'.
- 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:
- supporting study
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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:
- supporting study
- 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:
- Please refer to section 13 of the dataset for read-across justification.
- 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 conclude 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:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- Please refer to section 13 of the dataset for read-across justification.
- 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'.
- 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:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- Please refer to section 13 of the dataset for read across justification.
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- 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.
Referenceopen allclose all
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 |
|
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
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.
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.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Link to relevant study records
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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. - 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:
- Please refer to section 13 of the dataset for read across justification.
- 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
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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
- 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:
- Please refer to section 13 of the dataset for read across justification.
- 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.
Referenceopen allclose all
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.
None
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 |
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.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro studies
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).
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 (Woods, 2013).
An in vitro micronucleus assay was conducted in cultured peripheral human lymphocytes to evaluate the ability of zinc (di)methacrylate to induce micronuclei according to OECD Guideline 487 and EU Method B.49, and in compliance with GLP. The assay was performed in the presence and absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test substance were tested in two independent experiments. The vehicle of the test substance was ethanol. In the first cytogenetic assay, the test material was tested up to 150 μg/mL for a 3-hour exposure time with a 27-hour harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, the test material was tested up to 80 μg/mL for a 24-hour exposure time with a 24-hour harvest time in the absence of S9-mix. The highest concentrations analyzed were selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%. The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C, colchicine and cyclophosphamide all produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. In the first cytogenetic assay, in the presence of S9-mix, the test material induced a statistically significant and biologically relevant increase in the number of binucleated cells with micronuclei. In the absence of S9-mix, the test material induced a statistically significant increase in the number of binucleated cells with micronuclei at the intermediate dose. At the highest dose, no statistically significant increase was observed. Since the number of binucleated cells with micronuclei at the highest dose was above the 95% control limits of the historical solvent data and a positive trend was observed, the increase was considered to be biologically relevant. In the second cytogenetic assay with 24 hours of continuous exposure time, the test material did not induce a dose-dependent, statistically significant increase in the number of binucleated cells with micronuclei. Under the conditions of the study, zinc (di)methacrylate induced the formation of micronuclei in human lymphocytes after a 3-hour exposure in the absence and presence of S9 metabolic activation and did not induce the formation of micronuclei after 24-hour exposure in the absence of S9 metabolic activation (Groot and Verbaan, 2022).
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.
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.
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.
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.
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.
In vivo studies:
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.
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.
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.
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.
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.
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 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).
Additional consideration on genotoxicity
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).
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.
However, 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 vivo studies 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).
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. 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. Under the conditions of the study, zinc dimethacrylate induced the formation of micronuclei in human lymphocytes after a 3-hour exposure in the absence and presence of S9 metabolic activation and did not induce the formation of micronuclei after 24-hour exposure in the absence of S9 metabolic activation (Groot and Verbaan, 2022).
Considering the new available in vitro data on zinc dimethacrylate and the overall conflicting in vitro and in vivo results obtained with soluble zinc compounds and methacrylic acid/methyl methacrylate (ambiguous or positive responses in vitro for clastogenicity but overall negative response in in vivo tests), a testing proposal for a combined in vivo comet assay (OECD Test Guideline 489) and micronucleus test (OECD TG 474) with zinc dimethacrylate was introduced as part of this dossier update.
Justification for classification or non-classification
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 warrant classification for genotoxicity under the EU CLP Regulation (EC) No. 1272/2008.
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