4,5-dihydroxy-1,3-dimethylimidazolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key Bacterial Reverse Mutation Assay according to OECD 471, the reported data show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

In two supporting Bacterial Reverse Mutation Tests, the test substance was not mutagenic in Salmonella typhimurium TA 1535, 1537, 98 and 100.

 

In the key in vitro Mammalian Cell Gene Mutation Test according to OECD guideline 476, treatment of the cell system over a 5-hour treatment period with and without metabolic activation did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item is not mutagenic under the conditions of this test.

In a supporting in vitro Mammalian Cell Gene Mutation Test according to OECD 476 with the analogue substance Urea, reaction products with formaldehyde and glyoxal (CAS 92908-35-5), the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

In the key In Vitro Mammalian Chromosome Aberration Test according to OECD guideline 473, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the maximum recommended concentration in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

 

Overall conclusion: 4,5-Dihydroxy-1,3-dimethylimidazolidin-2-one or its analogues were not mutagenic or clastogenic in all available in vitro studies. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-06 to 2021-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2017-02-14

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster lung fibroblasts (male)
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rates (doubling time 12-14 h).
- Normal cell cycle time: 12-14 hours doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes, regularly checked
- Methods for maintenance in cell culture: The laboratory cultures were maintained in 75 cm^2 plastic flasks at 37 +/- 0.5 °C in an incubator with a humidified atmosphere, set at 5 % CO2.
- Doubling time: 12-14 hours
- Modal number of chromosomes: 22 (diploid)
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Not specified

MEDIA USED
Solvent: Dulbecco’s Modified Eagle’s medium (DMEM)
Growth medium: DMEM supplemented with L-glutamine (2 mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%).
Treatment medium: DMEM supplemented with L-glutamine (2 mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 5%).
CO2 concentration: 5%
Humidity: Humidified atmosphere
Temperature: 37 +/- 0.5°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (PB) and β-naphthoflavone (BNF) induced rats
- Method of preparation of S9 mix: 2 mL of 20 mM HEPES, 1 mL 330 mM KCl, 1 mL 50 mM MgCl2, 1 mL 40 mM NADP, 1 mL 50 mM D-Glucose-6-phosphate, 1mL DMEM and 3 mL S9 were mixed and kept in an ice bath.
- Concentration or volume of S9 mix and S9 in the final culture medium: 250 µL S9 mix containing approx 33% S9 in a final culture medium of 5 mL
- Quality controls of S9: A certificate of analysis is available from the S9 supplier

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
With and without out S9 mix: 365.3, 730.5 and 1461 (corresponding to 10 mM of the active component) μg/mL test item;

Experiment B with 20/20 h treatment/sampling time
Without S9 mix: 365.3, 730.5 and 1461 (corresponding to 10 mM of the active component) μg/mL test item;

Experiment B with 20/28 h treatment/sampling time
Without S9 mix: 365.3, 730.5 and 1461 (corresponding to 10 mM of the active component) μg/mL test item;

Experiment B with 3/28 h treatment/sampling time
With S9 mix: 365.3, 730.5 and 1461 (corresponding to 10 mM of the active component) μg/mL test item

Vehicle / solvent:

- Vehicle used: DMEM (test item and positive conrols)

- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dulbecco’s Modified Eagle’s medium (test item, EMS, CP)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Without S9
EMS: 0.4 and 1.0 μL/mL

With S9
CP: 5.0 μL/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 2 (Experiment A and B)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (experiment A, with and without metabolic activation), 20 hours (experiment B, without metabolic activation), 3 hours (experiment B, with metabolic activation)
- Harvest time: 20 hours after treatment start (experiment A, with and without metabolic activation), 20 hours after treatment start (experiment B, without metabolic activation), 28 hours after treatment start (experiment B, with and without metabolic activation)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 µg/mL), addition 2.5 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Increase in numbers of chromosome aberrations

Rationale for test conditions:

Test concentrations were based on a preliminary toxicity test and chosen up to the highest concentration of 10 mM recommended in OCD guideline 473. Sampling times were chosen to cover approx. 1.5 cell cycles (20 hours, without S9 mix only) and approx. 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance should be considered together. There is no requirement according to the respective OECD guideline for verification of a clearly positive or negative response.

Statistics:

For statistical analysis, the CHI^2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control data were calculated with C-chart. The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment concentrations using the adequate regression analysis by Microsoft Excel software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no relevant changes in pH after treatment with the test item.
- Data on osmolality: There were no relevant changes in osmolality after treatment with the test item.
- Water solubility: The test item was soluble in DMEM
- Precipitation and time of the determination: There was no precipitation in the medium at any concentration tested.
- Definition of acceptable cells for analysis: Metaphase cells containing 22 ± 2 chromosomes

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study, a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay the cells were seeded at 5 x 10^5 cells each and were incubated for 24 hours in 10 mL of DMEM containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/mL) and were incubated at 37 °C for 3 hours. Cell counts were performed after 20 hours. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. In the absence and in the presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index: See "Attached background material" and "Details on mammalian cell type"
- Genotoxicity results: See "Attached background material"
o Definition for chromosome aberrations, including gaps: According to the nomenclature and classification of chromosome aberrations given in ISCN, 1985, and Savage, 1976, 1983.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Gaps were scored but not included in the evaluation. Results are given under "Attached background material"
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): No increase in the rate of polyploid and endoreduplicated metaphases were found after treatment with the different concentrations.

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In an in vitro Mammalian Chromosome Aberration Test according to OECD guideline 473, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the maximum recommended concentration in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Executive summary:

The test item 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473 and GLP. Based on the result of the pre-test on cytotoxicity, the concentration levels for the performed Chromosome Aberration Assay were chosen according to the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight, 10 mM was chosen as the highest dose (based on the updated OECD Guideline 473 (2016).

 

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

with S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Following treatment and recovery, the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for approx. 10 minutes before being placed on slides and stained. In each experimental group, duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). In Experiment A, there were no increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation and up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to the maximum recommended concentration without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with a harvest time of 28-hour after the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In the experiment B in the presence of metabolic activation one value at the dose of 1461 μg/mL (10 aberrant cells including gaps/150 cells) was slightly above the 95% control limits of the historical control data (upper limit approximately 8.55 aberrant cells including gaps/150 cells). Furthermore, in the experiment B in the absence of metabolic activation (20/20 hours) one value at the dose of 365.3 μg/mL (9 aberrant cells including gaps/150 cells) was slightly above the 95% control limits of the historical control data (upper limit approximately 8.10 aberrant cells including gaps/150 cells). However, no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups. Therefore, the findings were considered as accidential and not being biologically relevant. No polyploid and endoreduplicated methaphases were observed in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

In conclusion, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the maximum recommended concentration in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-03 to 2021-03-29

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:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO
- Suitability of cells: Yes, CHO cells have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency, a stable karyotype, and a stable spontaneous mutant frequency.
- Normal cell cycle time (negative control): Approx. 22 hours doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes (Each batch of frozen cells was tested for mycoplasma infections)
- Methods for maintenance in cell culture: Growing cells were subcultured in an appropriate number of flasks.
- Doubling time: Approx. 22 hours
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Yes (each batch of frozen cells was purged of HPRT mutants)

MEDIA USED
Solvent: Ham's F12 medium supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine
Growth medium: Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 10 %)
Treatment medium: Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 5 %)
Selection medium: Ham's F12*-SEL (EX-CELL® CD CHO Serum-Free Medium for CHO Cells F12 medium without hypoxanthine, without thymidine) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.03 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 10 %) and 3.4 μg/mL 6-thioguanine (6-TG)
CO2 concentration: 5 %
Humidity: Humidified atmosphere (approx. 95%)
Temperature: 37 °C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (PB) and β-naphthoflavone (BNF) induced rats
- Method of preparation of S9 mix: 0.2 mg/mL of 20 mM HEPES, 0.1 mg/mL 330 mM KCl, 0.1 mg/mL 50 mM MgCl2, 0.1 mg/mL 40 mM NADP, 0.1 mg/mL 50 mM D-Glucose-6-phosphate, 0.1 mg/mL Ham’s F12 medium and 0.3 mL/mL S9 were mixed and kept in an ice bath.
- Concentration or volume of S9 mix and S9 in the final culture medium: 600 µL S9 mix containing approx 33% S9 in a final culture medium of 15 mL
- Quality controls of S9: A certificate of analysis is available from the S9 supplier

Test concentrations with justification for top dose:

Preliminary experiment with and without S9 mix: 250, 500, 1000 and 2000 µg/mL;

Main experiment, 5-hour treatment period with and without S9 mix: 182.7, 365.3, 730.5, 1461 μg/mL;

The top dose of 1461 μg/mL corresponds to 10 mM of the active component (highest recommended dose according to OECD guideline 476).

Vehicle / solvent:

- Vehicle used: Ham's F12 medium (test item, EMS), DMSO (DMBA)

- Justification for choice of solvent/vehicle: Solubility was tested in a preliminary solubility test. The test item was soluble in Ham's F12 medium up to a concentration of 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium (test item, EMS), DMSO (DMBA)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Remarks:

Without S9:
EMS: 1.0 μL/mL
With S9:
DMBA: 20 μg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 1 (one experiment with and one without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10 x10^6 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment: 6 days (for determination of survival), 14 days for determination of viability (8 days expression period + 6 days for colony growing), 16 days for determination of mutagenicity (8 days expression period + 8 days selection period)

FOR GENE MUTATION:
- Expression time: 8 days
- Selection time: 8 days
- Fixation time: 16 days
- Selective agent: 6-thioguanine (3.4 μg/mL), 8 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10 x10^6 cells/dish for treatment, 10^5 cells/mL for expression period (during this period, cells were regularly sub-cultured to maintain them in exponential growth. 2x10^6 cells/dish), 200 cells/dish for survival, 2x10^5 cells / dish for selection, 10^5 cells/mL for determination of viability. After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination. For viability, colonies were fixed with methanol, stained with Giemsa and counted. Cloning Efficiency was assessed
(Number of colonies/Number of cells plated).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Survival / Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10^6 cells: 2 x 5 dishes at 2x10^5 cells/dish), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10^6 clonable cells.

Rationale for test conditions:

Concentration were selected based on a preliminary cytotoxicity test

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test, all results are inside the distribution of the historical negative control data (based 95% control limit.

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
• The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No biologically relevant changes in pH of the test system were noted at the different concentration levels tested.
- Data on osmolality: No biologically relevant changes in osmolality of the test system were noted at the different concentration levels tested.
- Water solubility: The test item was soluble in Ham's F12 medium up to 100 mg/mL.
- Precipitation and time of the determination: For examined test item concentrations, no precipitation in the medium was noted at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES: A pre-test on toxicity was performed to establish an appropriate concentration range for the main mutation assay, both in the absence and in the presence of metabolic activation (rodent S9 mix). Cells were treated for 5 hours and then sub-cultured for 6 days. Cytotoxicity was determined by RS (relative survival), where the cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). In the absence and in the presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration of 2000 μg/mL. Based on the result of the preliminary cytotoxicity assay and based on the low molecular weight, 10 mM was chosen as the highest dose defined in OECD guideline 476.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o Relative survival (RS) and cloning efficiency: See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: See "Details on test system and experimental conditions"
o Number of cells plated in selective and non-selective medium: See "Details on test system and experimental conditions"
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In an in vitro Mammalian Cell Gene Mutation Test according to OECD guideline 476, treatment of the cell system over a 5-hour treatment period with and without metabolic activation did not
induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item is not mutagenic under the conditions of this test.

Executive summary:

The test item, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells according to OECD guideline 476 and GLP. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β- naphthoflavone induced rat liver. Based on the result of the pre-test on cytotoxicity, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight, 10 mM was chosen as the highest dose (based on the updated OECD Guideline 476 (updated 2016). The Mutation Assay was performed at the concentrations and treatment intervals given below:

 

Mutation Assay

5-hour treatment period with and without S9-mix: 182.7, 365.3, 730.5 and 1461 μg/mL

 

Following treatment, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days. In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 1461 μg/mL. In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The mutation frequency found in the negative controls ( -S9:6 and + S9:6-7 ) were within the 95% control limits of historical laboratory control data (-S9:5.36 - 7.81 and + S9:5.19 - 8.71). The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7, 12- Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

In conclusion, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one tested up to the maximum recommended concentration (1461 μg/mL, corresponding to 10 mM) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-22 to 2021-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

2011-11

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital and β-naphthoflavone-induced rat liver S9, supplied by Trinova Biochem GmbH (Rathenau Str. 2, D-35394 Giessen, Germany) and manufactured by MOLTOX INC. (P.O. BOX 1189; BOONE, NC 28607 USA)
- Method of preparation of S9 mix: 400 mL of sterile salt solution (4 mM β-NADP Na, 5 mM D-Glucose 6-phosphate Na, 8 mM MgCl2, 33 mM KCl), 100 mL rat liver homogenate (S9) and 500 mL ice cold 0.2 M sodium phosphate-buffer, pH 7.4 were mixed and kept in an ice bath until use.
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9 mix (containing 50 µL S9) in a final volume of 2700 µL culture medium
- Quality controls of S9: The quality control and production certificate of each lot of S9 was obtained from the supplier

Test concentrations with justification for top dose:

Experiment I (plate incorporation) + experiment II (pre-incubation) +/- S9: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate (a correction factor of 2.36 based on the total organic content of 42.34% was taken into consideration)

A maximum concentration of 5000 µg/plate was selected based on preliminary solubility testing, concentration range finding testing and as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Vehicles used: Water (test item, sodium azide, methyl methanesulfonate), DMSO (4-nitro-1,2-phenylenediamine, 9-aminoacridine, 2-aminoanthracene)

- Justification for choice of vehicle: The test item showed adequate solubility in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water/DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine (NPD) 2-aminoanthracene (2AA)

Remarks:

Without S9:
NPD: 4 μg/plate (TA98)
SAZ: 2 µg/plate (TA100 and TA1535)
9-AA: 50 μg/plate (TA1537)
MMS: 2 µL/plate (E. coli WP2 uvrA)

With S9:
2-AA: 2 µg/plate (TA98, TA100, TA1535, TA1537); 50 µg/plate (E. coli WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
Experiment I: Plate incorporation method
Experiment II: Pre-incubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition, reduction of revertant colony numbers

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.

Evaluation criteria:

A test item is considered mutagenic if:
- A dose-related increase in the number of revertants occurs and/or;
- A reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.;

An increase is considered biologically relevant if:
- In strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- In strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.;

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was soluble in water, in top agar and in phosphate buffer
- Precipitation and time of the determination: No precipitation occured.

RANGE-FINDING/SCREENING STUDIES
A concentration range finding test was performed applying the plate incorporation method using TA98 and TA100 strains in the presence and absence of metabolic activation. No inhibitory effect of the test item and no precipitation of the test item were observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: No signs of toxicity were observed.
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA. In conclusion, the test has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity in a GLP compliant Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included preliminary solubility investigations, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (experiment I, plate incorporation test), and a confirmatory mutation test (experiment II, pre-incubation test). Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate. The choice of this maximum test concentration was made based on the guideline criterion (OECD 471 guideline [6]) for soluble, non-toxic test compounds. For the preparation of the test item stock solution, a correction (multiplier) factor of 2.36 (1/0.4234=2.36) based on its total organic content of 42.34% was taken into consideration. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests, no inhibitory effects of the test item on bacterial growth were observed in the examined strains. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained within the range of the biological variability of the applied test system. Furthermore, the background lawn development was not affected in any case. The revertant colony numbers of the solvent control (ultrapure water, ASTM Type I) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data ranges in all cases. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies. Moreover, the number of revertants fell in the corresponding historical control data ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment the test item  at any concentration level, either in the presence or absence of metabolic activation (±S9) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo Micronucleus Test with a product basically consisting of analogue 4,5-dihydroxy-1,3-bis(hydroxymethyl)imidazolidin-2-one (CAS 1854-26-8). Male and female NMRI mice were orally administered (gavage) concentrations of 500, 1000 and 2000 mg/kg as a 73% aqueous solution (single application). Under the conditions of this study, a negative result was obtained.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

21.09.-14.10.1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Remarks:

Read-across

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

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: Fa. Mollegaard, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 20-27g
- Assigned to test groups randomly: no
- Housing: Macrolon Typ 3
- Diet (e.g. ad libitum): Altromin Nr . N 1326, ad libitum
- Water (e.g. ad libitum): drinking water
- Acclimation period: 4 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 55+-10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 h

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
3 days before application.
0.1 ml solution was applicated to 10 g body weight.

Frequency of treatment:

single application

Post exposure period:

24 h (additionally 48 h in the 2000 mg/kg treatment)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

As 73 % solution in water

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

As 73 % solution in water

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

As 73 % solution in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 20/80 mg/kg b.w. (24 hrs post exposure period)

Tissues and cell types examined:

bone marrow of the femora; 2000 polychromatic erythrocytes per animal were studied for the presence of micronucleus.

Evaluation criteria:

Micronucleus
- are round, oval or crescent-shaped
- have a precise contour
Application of high doses of clastogenic substances can cause the appearence of several almond- or ring-shaped micronuclei.
- are uniformly coloured
- have a diameter of approximately 1/20 to 1/5 of an erythrocyt
- Es ist meistens nur ein Mikrokem vorhanden .
- Nach Behandlung mit hohen Dosen von Substanzen, die Chromosomenbrüche erzeugen, können
bei einigen Erythrozyten auch mehrere Mikrokeme auftreten, die auch mandelförmig oder ringförmig
sein können

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Substance	Dose (mg/kg)	Sex	post exposure period (h)	PCE with micronuclei	total number of PCE	Ratio PCE/NCE
vehicle	solvent	male	24	0.30	6.0	0.97
		female	24	0.34	6.8	0.96
test substance	500	male	24	0.31	6.2	0.99
		female	24	0.27	5.4	0.98
test substance	1000	male	24	0.36	7.2	1.02*
		female	24	0.32	6.4	1.00
test substance	2000	male	24	0.24	4.8	0.95
		female	24	0.26	5.2	0.97
test substance	2000	male	48	0.37	7.4	0.99
		female	48	0.33	6.6	0.99
positive ctrl	80	male	24	2.01	40.2	0.69
		female	24	1.90	38.2	0.69
positive ctrl	20	male	24	0.86	17.4	0.76
		female	24	0.82	16.4	0.86
	PCE = polychromatic erythrocyts (2000 were scored for micronuclei)
	NCE = normochromatic erythrocyts
	The NCE/PCE ratio (as an indicator of cytotoxicity) is based on the scoring of 1000 erythrocyts
	* = significantly different from the control, but within historical control data (0.6 to 1.2)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key, Bacterial Reverse Mutation Assay, RL1

The test item was tested with regard to a potential mutagenic activity in a GLP compliant Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included preliminary solubility investigations, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (experiment I, plate incorporation test), and a confirmatory mutation test (experiment II, pre-incubation test). Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate. The choice of this maximum test concentration was made based on the guideline criterion (OECD 471 guideline [6]) for soluble, non-toxic test compounds. For the preparation of the test item stock solution, a correction (multiplier) factor of 2.36 (1/0.4234=2.36) based on its total organic content of 42.34% was taken into consideration. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests, no inhibitory effects of the test item on bacterial growth were observed in the examined strains. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained within the range of the biological variability of the applied test system. Furthermore, the background lawn development was not affected in any case. The revertant colony numbers of the solvent control (ultrapure water, ASTM Type I) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data ranges in all cases. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies. Moreover, the number of revertants fell in the corresponding historical control data ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment the test item  at any concentration level, either in the presence or absence of metabolic activation (±S9) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

 

Supporting studies, Bacterial Reverse Mutation Assay, RL2

BASF (1995) reported a bacterial reverse mutation assay (Ames test). In this experiment, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were used. The test concentrations ranged between 20 - 5000 ug/plate. A negative result was reported with and without metabolic activation.

Hill Top Research (1979) also reported a bacterial mutation assay (Ames test). In this study, Salmonella typimurium strains TA 1535, TA 1537, TA 98 and TA 98 were used. Five concentrations were used (dilutions 1:5, 1:50, 1:100, 1:500, 1:1000). A negative result was reported with and without metabolic activation.

 

Key, Mammalian Cell Gene Mutation Test, RL1

The test item, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells according to OECD guideline 476 and GLP. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β- naphthoflavone induced rat liver. Based on the result of the pre-test on cytotoxicity, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight, 10 mM was chosen as the highest dose (based on the updated OECD Guideline 476 (updated 2016). The Mutation Assay was performed at the concentrations and treatment intervals given below:

 

Mutation Assay

5-hour treatment period with and without S9-mix: 182.7, 365.3, 730.5 and 1461 μg/mL

 

Following treatment, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days. In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 1461 μg/mL. In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The mutation frequency found in the negative controls ( -S9:6 and + S9:6-7 ) were within the 95% control limits of historical laboratory control data (-S9:5.36 - 7.81 and + S9:5.19 - 8.71). The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7, 12- Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one tested up to the maximum recommended concentration (1461 μg/mL, corresponding to 10 mM) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

 

Supporting, read-across, Mammalian Cell Gene Mutation Test, RL2

An in vitro mammalian cell HGPRT-gene mutation assay in V 79 cells is available with Fixapret CP konz. (CAS: 92908-35-5; BASF, 2010), basically 4.5 -Dihydroxy-1.3-dihydroxyimidazolid-2-one, a material closely related and a possible metabolic sequel product of [3923 -79 -3]. The test concentrations ranged from 2.5 - 1400 µg/ml. A negative result was reported with and without metabolic activation.

 

Key, Mammalian Chromosome Aberration Test, RL1

The test item 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473 and GLP. Based on the result of the pre-test on cytotoxicity, the concentration levels for the performed Chromosome Aberration Assay were chosen according to the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight, 10 mM was chosen as the highest dose (based on the updated OECD Guideline 473 (2016).

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

with S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 365.3, 730.5 and 1461 μg/mL test item

 

Following treatment and recovery, the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for approx. 10 minutes before being placed on slides and stained. In each experimental group, duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). In Experiment A, there were no increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation and up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to the maximum recommended concentration without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with a harvest time of 28-hour after the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In the experiment B in the presence of metabolic activation one value at the dose of 1461 μg/mL (10 aberrant cells including gaps/150 cells) was slightly above the 95% control limits of the historical control data (upper limit approximately 8.55 aberrant cells including gaps/150 cells). Furthermore, in the experiment B in the absence of metabolic activation (20/20 hours) one value at the dose of 365.3 μg/mL (9 aberrant cells including gaps/150 cells) was slightly above the 95% control limits of the historical control data (upper limit approximately 8.10 aberrant cells including gaps/150 cells). However, no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups. Therefore, the findings were considered as accidential and not being biologically relevant. No polyploid and endoreduplicated methaphases were observed in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the maximum recommended concentration in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

 

Supporting, read-across, In Vivo Micronucleus Assay, RL2

No in vivo data are available for 4,5-Dihydroxy-1,3-dimethylimidazolidin-2-one.

Biopharm (1995) reported a micronucleus test with Fixapret CP konz. which basically consists of 4,5-dihydroxy-1,3-bis(hydroxymethyl)imidazolidin-2-one (CAS 1854-26-8). Male and female NMRI mice were orally administered (gavage) concentrations of 500, 1000 and 2000 mg/kg as a 73% aqueous solution (single application). Under the conditions of this study, a negative result was obtained.

 

Overall conclusion: 

4,5-Dihydroxy-1,3-dimethylimidazolidin-2-one or its analogues were not mutagenic or clastogenic in all key and supporting Bacterial Reverse Mutations Assays, in vitro Mammalian Cell Gene Mutation Tests and in an in vitro Mammalian Chromosome Aberration Test. Furthermore, analogue 4,5-dihydroxy-1,3-bis(hydroxymethyl)imidazolidin-2-one was not clastogenic in an in vivo Micronucleus Test.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified and labelled for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.