5,5-Dimethylimidazolidine-2,4-dione, reaction products with oxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of three different reliable in vitro genotoxicity studies (bacterial mutation, chromosome aberration, mammalian celll mutation), conducted in both in the presence and absence of a metabolic activation system, demonstrate that the substance is non-mutagenic and non-genotoxic. The substance was also negative for mutagenicity in an reliable (with restrictions given that it did not follow any guidelines and not in accordance with GLP, but the study was conducted to nationally acceptable standards) in vitro bacterial mutation assay both in the presence and absence of a metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2007 to 23 April 2007

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:

not specified

GLP compliance:

yes

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:

S9 fraction was derived from rats induced with phenobarbitone/beta-napthoflavone mixture.

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Evaluation criteria:

Dose-related increase in revertant frequency over the dose range tested and/or reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. A test material will be considered non-mutagenic (negative) if the above criteria aare not met.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not-mutagenic

Conclusions:

The substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The substance underwent a test to evaluate the mutagenic potential in the Ames test according to the OECD Guideline 471 employing Salmonella typhimurium strains TA1535, TA1537, TA100, TA98, and Eschericia coli WP2, with and without S9 (metabolic activation). Under the conditions of these tests the substance is considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

D4

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0.001µl to 10 µl based on quantitative or qualitative evidence of some chemically induced physiological effect at the highest dose level. The low dose was below a concentration that demonstrated a toxic effect.

Vehicle / solvent:

Deionised water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

ethylmethanesulphonate

other: QM for TA-1537; anthracene; ,4-dihydroxy-2-napthoic acid for D4.

Evaluation criteria:

Strains TA1535, TA1537 and TA1538: if the solvent control value is within the normal range, a chemical that produced a positive response over three concentrations with the lowest equal to twice the solvent control is considered mutagenic.

Strains TA98, TA100 and D4: If the solvent control value is within the normalk range, a chemical that produces a positive response over three concentrations with the highest increase equal to twice the solvent control value for TA100 and 2-3 times the solvent control value for strains TA98 and D4 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Saccharomyces cerevisiae

Remarks:

D4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The substance showed no evidence of mutagenic activity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, and in Saccharomyces cerevisiae strain D4 in the presence and in the absence of a metabolic activation system.

Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, and in Saccharomyces cerevisiae strain D4 in the presence and in the absence of a metabolic activation system. Concentrations of up to 10 μl/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in either bacterial strain. It was concluded that the substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2007 to 18 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: Chromosome aberration test in human lymphocytes in vitro

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Beta naphthoflavone and phenobarbitone mixture induced rat liver homogenate metabolising system (S9).

Vehicle / solvent:

Minimum Essential Medium (MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Experiment 1 - 4-hour exposure to test material with/without S9 followed by 20-hour culture treatment-free media prior to cell harvest.
Experiment 2 - 24-hour continuous exposure to the test material with/without S9 followed by 20-hour culture treatment-free media prior to cell harvest.

Evaluation criteria:

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: not clastogenic

Conclusions:

The substance is considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The substance underwent genotoxicity testing according to OECD Guideline 473 employing human lymphocytes, with and without metabolic activation (S9 fraction). The substance was considered to non-genotoxic under the conditions of the study.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2016 to 16 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction from rats induced with Phenobarbital/beta naphthaflavone.

Test concentrations with justification for top dose:

62.5, 125, 250, 500, 1000, 2000 μg/mL.
The molecular weight of the test item was 216.23 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test (Relative Suspension Growth). This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).

Vehicle / solvent:

R0 medium.

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Evaluation criteria:

A substance is considered to be clearly positive if the increase in mutation frequency above the the concurrent background exceeds the Global Evaluation Factor of 126x10-6 and the increase is concentration-related.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

In an in vitro cell transformation assay to evaluate the mutagenic and clastogenic potential L5178Y mouse lymphoma cells were exposed to the substance at 62.5, 125, 250, 500, 1000, 2000 μg/mL. the substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo genotoxicity assay was conducted on the substance according to OECD Guideline 474. The results of the test indicates that the substance is non-genotoxic in vivo under the conditions of the study.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2013 to 16 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

On occassions the relative humidity was outside the target range of 30-70%. The animals in the main test, with the exception of the positive control group were housed in groups of seven.

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Species:

mouse

Strain:

ICR

Details on species / strain selection:

Sufficient albino Hsd: ICR (CD-1)

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

Phoshate Buffered Saline (PBS)

Duration of treatment / exposure:

Once.

Frequency of treatment:

Once.

Post exposure period:

24 hours (500, 1000 or 2000 mg/kg bw) and 48 hours (2000 mg/kg bw group) post-treatment.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (50 mg/kg bw)

Tissues and cell types examined:

Bone marrow smears prepared and polychromatic erythrocytes per animal was scored. The number of normochromatic erythrocytes were also counted and scored for incidence of micronuclei.

Evaluation criteria:

Micronuclei are cicular, but occassionally may be oval or half-moon shaped and have a sharp contour with even staining.

Statistics:

The ratio of polychromatic to normochromtic erythrocytes was calculated together with appropriate group of mean values and standard deviations.
A positive mutagenic response was demoinstrated when a statistically significant, dose-responsive, toixicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for iether the 24 or 48-hour kill times compared to their corresponding control group.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No premature deaths or clinical signs of toxicity observed in any of the animals dosed.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

An in vivo gentoxicity assay was conducted on the substance according to OECD Guideline 474. The results of the test indicates that the substance is non-genotoxic in vivo under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the findings of several reliable genetic toxicity studies (in vitro and in vivo) conducted on the substance, classification is not justified.