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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: neg

HPRT: neg

MNT: neg

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

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

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

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%)

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany; large stocks stored at ICCR

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Cell cycle length, doubling time or proliferation index : 12-16h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

w/out S9: 2.94; 5.29; 9.53; 17.15; 30.86µg/mL
w/S9: 303; 60.5; 121; 242; 484µg/mL
highest dose based on cytotoxicity

Vehicle / solvent:

deionised water

- Justification for choice of solvent/vehicle: non-toxic to the cell culture, good solubility of the test substance

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.5mio cells / 25cm²
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24h
- Exposure duration/duration of treatment: 4h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 8-11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-18 days
- Selective agent: 6-thioguanine, 11µg/mL
- Number of cells seeded: 4-5x10^5

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- relative survival (RS)

Rationale for test conditions:

The highest concentration evaluated already led to significant toxicity (RS of app 15-20%). At the next higher concentration, almost no cells survived (RS < 3%)

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

Statistics:

t-test and linear regression for trend analysis

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

osmolarity and pH were not changed by treatment

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, IPGA is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 mix : from male Wistar rats livers (recived phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

1st Experiment:
all strains: 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate (with and without S9 mix) SPT
2nd Experiment:
TA 1535, TA 100 and TA 1537: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (without S9 mix) SPT
Reason: Bacteriotoxicity was observed in the standard plate test. Therefore, the standard plate test was repeated with adjusted doses.
3rd Experiment:
all strains: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA strains without S9 mix); 0; 10; 33; 100; 333; 1000 and 2650 μg/plate (E.coli with and without S9 mix and TA strains with S9 mix) PIT

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Standard plate test:
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

other: TA 1535, TA 100, TA 1537, TA98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: No precipitation of the test substance was observed with and without S9 mix.
TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 μg/plate. A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 100 μg/plate.
MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:

Under the experimental conditions chosen here, it is concluded that Laromer IPGA is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames

The test substance Laromer IPGA was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Test strains are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, in a dose range of 3.3 μg - 5300 μg/plate (SPT) and 3.3 μg - 2650 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was observed with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance Laromer IPGA is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

HPRT

The study was performed to investigate the potential of IPGA, dissolved in deionised water, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed with and without liver microsomal activation and a treatment period of 4 hours. In the main experiment in the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentration was outside the recommended range of cytotoxicity and therefore not evaluated for mutagenicity.
In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test item.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
Therefore, IPGA is considered to be non-mutagenic in this HPRT assay.

 

MNT(in vitro)

The test item IPGA, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. In Experiment I and II in the absence and presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated due to strong cytotoxicity (Table 3 and 4). In Experiment III in the absence of S9 mix after 20 h treatment, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment III in the presence of S9 mix after 4 h treatment, moderate cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated due to strong cytotoxicity.

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.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Therefore, IPGA is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).