2-Propenoic acid, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU method B.49: In vitro Mammalian Cell Micronucleus Test

Version / remarks:

2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(2,2-dimethyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

EC Number:

814-155-4

Cas Number:

13188-82-4

Molecular formula:

C9H14O4

IUPAC Name:

(2,2-dimethyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Test material form:

liquid

Details on test material:

Analytical study no. 20L00019
Batch: 0812-HS-0030

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Manufacturer, Batch 0812-HS-0030
- Purity: 96.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: lymphocytes from healthy, non-smoking donors
- Normal cell cycle time (negative control): 16-20h

For lymphocytes:
- Sex, age and number of blood donors: Exp.1: 32 year old femald, Exp.2: 25 year old male, Exp. 3: 19 year old female
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: PHA

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

4h without S9: up to 67.7 µg/mL
20h w/out S9: up to 82.3 µg/mL
4h w/ S9: up to 1489 µg/mL
dose selection based on cytotoxicity as observed in the main experiment and the pre-test

Vehicle / solvent:

de-ionized water (final concentration in medium was 10%)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Mitomycin C (Pulse treatment ), Demecolcine (continuous treatment)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48h
- Exposure duration/duration of treatment: 4h /20h
- Recovery time: 16h (for 4h exposures only)
- Cytochalsasin B exposure: 20h
- harvest time: 40h after start of exposure
- total culture period: 88h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used: ice-cold hypotonic solution followed by Giemsa staining
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 1000 binucleated cells per culture
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Micronuclei were counted in cells showing clearly visible cytoplasm. Micronuclei have to be stained in the same way as the main nucleus. The area should not exceed one third of the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Rationale for test conditions:

The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

With regard to the molecular weight (186.21 g/mol) and the purity (96.2 %) of the test item, 1936 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.6 to 1936 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation in the absence of S9 mix, this preliminary test was designated Experiment I.
The experimental part with S9 mix was repeated in Experiment II with the same top dose but with narrower concentration spacing due to lack of evaluable concentrations in the optimal cytotoxicity range. The experimental part without S9 mix was repeated in Experiment II as confirmatory experiment with a top dose of 140 μg/mL due to a positive finding in Experiment I.
Clear cytotoxic effects were observed in Experiment I after 4 hours treatment with 118 μg/mL and above and in Experiment II with 81.3 μg/mL and above in the absence of S9 mix. Therefore, 123 μg/mL were chosen as top concentration in Experiment III without S9 mix and 20 hours treatment.
The experimental part with S9 mix was repeated in Experiment III with a top dose of 1936 μg/mL due to a positive finding in Experiment II.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realised as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in section 5.6.3 were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− The criteria for the selection of top concentration are consistent with those described above

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).
A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly ne

Statistics:

Statistical significance was confirmed by the Chi square test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

the test item did not change pH or osmolarity of the medium

cytotoxicity:
At least moderate cytotoxicity (app 30%) was observed at the highest evaluated concentration. The next higher tested concentration, however, separated by a factor smaller than requested by the guideline, could not be evaluated due to strong cytoxicity.

Any other information on results incl. tables

In Experiment I in the absence of S9 mix, the value of 1.78 % micronucleated cells at the highest evaluated concentration (67.4 μg/mL) is statistically significantly increased in comparison to the solvent control. The value exceeded the 95% control limit (0.00 – 0.99 % micronucleated cells) and the min-max range (0.15 – 1.25 % micronucleated cells) of the historical control data. Dose-dependency, tested by a trend test was not observed. In the confirmatory Experiment II in the absence of S9 mix, this finding was not confirmed. Therefore, the finding in Experiment I can be considered as biologically irrelevant.

In Experiment II in the presence of S9 mix, the values of 1.03 % and 1.80 % micronucleated cells at the two highest evaluated concentrations (678 and 881 μg/mL) are statistically significantly increased in comparison to the solvent control. The value of 1.80 % exceeded the 95 % control limit (0.02 – 1.04 % micronucleated cells) and the min-max range (0.10 – 1.18 % micronucleated cells) of the historical control data. Dose-dependency, tested by a trend test was not observed. In the confirmatory Experiment III in the presence of S9 mix, this finding was not confirmed, even at higher concentrations. Therefore, the finding in Experiment II can be considered as biologically irrelevant.

In Experiment III in the absence of S9 mix after 20 h treatment, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, IPGA is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.