Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
in vitro micronucleus assay in cultured peripheral human lymphocytes
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 February 2022 to 30 September 2022
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc methacrylate
EC Number:
236-144-8
EC Name:
Zinc methacrylate
Cas Number:
13189-00-9
Molecular formula:
C4H6O2.1/2Zn
IUPAC Name:
zinc methacrylate
Test material form:
solid
Details on test material:
Physical Description: White powder
Storage Conditions: At room temperature
Test item handling: No specific handling conditions required
Volatile: No, vapour pressure: 0.00000784 Pa
Expiry date: 17 January 2023
Sample Supplier Total Energies
Specific details on test material used for the study:
Physical Description: White powder
Storage Conditions: At room temperature
Test item handling: No specific handling conditions required
Volatile: No, vapour pressure: 0.00000784 Pa
Expiry date: 17 January 2023
Sample Supplier Total Energies

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
peripheral human lymphocytes
Cytokinesis block (if used):
Cytotoxicity of the test substance in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
Test concentrations with justification for top dose:
In order to select the appropriate dose levels for the in vitro micronucleus test, cytotoxicity data was obtained in a dose-range finding test. The test concentrations were 7.8, 15.6, 31,3, 63, 125, and 250 μg test material/mL. The highest tested concentration was determined by the solubility of the test material in the culture medium.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.
In the first cytogenetic assay, the following dose levels were selected: 10, 75, 100, 125, 150, 175, and 200 μg/mL culture medium (3 hours exposure time, 27 hours harvest time) with and without metabolic activation. The following dose levels were selected for scoring of micronuclei:
- Without S9-mix: 10, 100, and 150 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).
- With S9-mix: 10, 75, and 150 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).
In the second cytogenetic assay, the following dose levels were selected: 10, 40, 50, 60, 70, 80, 90, and 100 μg/mL culture medium (24 hours exposure time, 24 hours harvest time) without metabolic activation. The following dose levels were selected for the scoring of micronuclei: 60, 70, and 80 μg/mL culture medium (24 hours exposure time, 24 hours harvest time) without metabolic activation.
Vehicle / solvent:
Ethanol (Extra pure, Merck, Darmstadt, Germany).
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol (vehicle for the test material)
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
1. Test system
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2021) are presented below:
Dose-range finding study: age 27, AGT = 12.5 h
First cytogenetic assay: age 27, AGT = 12.5 h
Cytogenetic assay 1A: age 30, AGT = 12.7 h
Second cytogenetic assay: age 30, AGT = 12.9 h
2. Cell Culture
Blood samples: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
3. Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 24 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.8°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Rationale for test conditions:
A solubility test was performed based on visual assessment. The test material formed a white homogenous suspension in ethanol. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test material was tested in the absence and presence of S9-mix.
The highest tested concentration was determined by the solubility of the test material in the culture medium.
Cytotoxicity of the test material in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.
Evaluation criteria:
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control materials MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Statistics:
Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test material is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Fisher’s exact test showed that there are statistically significant differences between one
or more of the test material groups and the vehicle control group. Therefore, a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
First Cytogenetic Assay
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Second Cytogenetic Assay
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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.
Appropriate toxicity was reached at this dose level.
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. Appropriate toxicity was
reached at this dose level.
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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Dose-range finding study
After 3 and 24 hours, the test material precipitated in the solubility test in the culture medium at concentrations of 250 μg/mL and above. In the dose-range finding test blood cultures were treated with 7.8, 15.6, 31,3, 63, 125, and 250 μg test material/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. No precipitation was observed at 250 μg test material/mL culture medium after 3 hours treatment.
The pH and osmolarity of a concentration of 250 μg/mL were 7.59 and 374 mOsm/kg respectively (compared to 7.83 and 374 mOsm/kg in the solvent control). This was performed was part of the solubility test.
First cytogenetic assay
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.
Second cytogenetic assay
In the second cytogenetic assay with 24 hours continuous exposure time, the test material did not induce a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei.

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

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).