Perhydroimidazo[4,5-d]imidazole-2,5-dione

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Commision Directive 2000/32/EC, Annex 4C, dated May 19, 2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus Assay

Test material

Test material information

1

Reference substance name:

Perhydroimidazo[4,5-d]imidazole-2,5-dione

EC Number:

207-821-5

EC Name:

Perhydroimidazo[4,5-d]imidazole-2,5-dione

Cas Number:

496-46-8

Molecular formula:

C4H6N4O2

IUPAC Name:

perhydroimidazo[4,5-d]imidazole-2,5-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch number: Fixapret 140 Nr. 766.
Date of manufacturing: March 24, 2001.
Purity: 96.3g/100g.

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal model for which suitable cytogenic investigations have been made, historic data from these investigations aid to elucidate interpretation of results from the micronucleus test. Additionally, the mouse is an experimental animal model that has been traditionally utilised for toxicological studies.

Sex:

male

Details on test animals or test system and environmental conditions:

Test Animals
Strain: NMRI.
Source: RCC Ltd. Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf.
Number: 42.
Initial age at the start of acclimatisation: 8-10 weeks.
Acclimatisation period: 5 days (minimum).
Body weight at beginning of study: mean value 40.9g (SD± 3.8g).

The animals were distributed into the test groups at random and identified by cage number.

Environmental Conditions
Housing: Single.
Bedding: Granulated soft wood bedding.
Feed: Pelleted standart diet, ad libitum (ALIROMIN 1324, D-32791 Lage/Lippe).
Water: Tap water, ad libitum (Gemeindewerke, D-64380 Roßdorf).
Temperature: 21C (SD± 4C).
Humidity: 30-70%.
Lighting: 12 hours Light / 12 Hours dark.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

0.5% carboxymethyl cellulose (CMC).

Details on exposure:

Prior to treatment, all animals (including controls) were weighed and the individual volume to be administered was 10mL/kg body weight. The animals were then administered with the test substance, the vehicle or the positive control substance once intraperitoneally.

Duration of treatment / exposure:

The animals were sacrified by cervical dislocation 48 hours post test substance administration.

Frequency of treatment:

Once.

Post exposure period:

48 hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6.

Control animals:

yes, concurrent vehicle

Positive control(s):

Historical positive control(s) were reported using a known clastenogestic and aneugenic substance; cyclophosphamide; 40 mg/kg b.w (oral administration).
Micronucleated polychromatic erythrocytes per thousand;
Range: 10.00 to 27.10.
Mean: 16.53*±4.09 (*= mean of 35 experiments (caiculated from 350 animals; 2000 polychromatic erythrocytes scored per animal).

Examinations

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-64293Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

The test substance is classified as mutagenic if;
- it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, that is statistically significant when compared with the negative control range or a relevant positive response for at least one of the test points.

The test substance is considered non-mutagenic if;
- neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points.

Statistics:

Evaluation via statistical method analysis was conducted using a non-parametric Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Summary of Micronucleus Test Results

 

 

 

Test group	dose mg/kgb.w.	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/ NCE ratio
vehicle	0	24	0.50	0-2	2000/1819
test substance	500	24	1.00	0-5	2000/1953
test substance	1000	24	0.30	0-2	2000/2597
test substance	2000	24	0.40	0-2	2000/2377
positive control	40	24	19.20	25-52	2000/1866
vehicle	0	48	0.60	0-2	2000/1699
test substance	2000	48	0.70	0-4	2000/2429

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, it is concluded that the test substance did not cause any clastenogenic or aneugenic effects on the test animals.

Executive summary:

The study aimed to investigate the potential of the test substance glycoluril to introduce micronucleus abnormalities in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to the OECD Guideline No 475 (1997) and the Commission Directive 2000/32/EC, Annex 4C (2000). 

The test substance was dissolved in 0.5% CMC. 10mLkg body weight was administered intraperitoneally 24 hours, and 48 hours after a single administration of the test substance. The bone marrow cells were collected for micronuclei analysis. 5 male mice per test group were evaluated for the occurrence of micronuclei, and at least 2000 PCEs per animal were scored for the presence of micronuclei. The mice were treated with 1000 and 2000 mg/kg b.w. at the 24 hour preparation interval and 2000 mg/kg b.w. at the 48 hour preparation interval. 40 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as a positive control. This showed a substantial increase of induced micronucleus frequency.

At the 48 hour preparation interval, there were slightly increased mean normochromatic erythrocytes (NCE) numbers, as compared to the mean values of NCEs of the corresponding vehicle control. This indicated that glycoluril had cytotoxic effectiveness in the bone marrow. However, when comparing with the corresponding vehicle controls, there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used.

Therefore, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Thus, glycoluril is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.