Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-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 - 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus assay in bone marrow cells

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

EC Number:

226-408-0

EC Name:

Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

Cas Number:

5395-50-6

Molecular formula:

C8H14N4O6

IUPAC Name:

1,3,4,6-tetrakis(hydroxymethyl)-octahydro-[1,3]diazolo[4,5-d]imidazole-2,5-dione

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF AG and THOR GmbH and batch no.: 0767

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent: The stability of the test substance in water was determined analytically and may be requested from the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test substance was formulated in deionised water.
- Purity: 49.5 g / 100 g (aqueous solution)

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse has been used for many years as suitable experimental animal. Thus, many data are available which may be helpful in the interpretation of the results from the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllingsdorf
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: mean weight males: 39.6 +/- 3.3 g and females: 31.7 +/- 2.0 g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum five days
- number of animals: 84 (42 males and 42 females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Relative humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

deioised water
The vehicle was chosen to its relative non-toxicity for the animals.

Details on exposure:

- Single injection intraperitoneally
- Dose volume: 10 mg/kg bw

Duration of treatment / exposure:

- single expsoure
- 24 h and additionally 48 h for the vehicle control and highest dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

male animal

Doses / concentrations 2

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Remarks:

female animal

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

male animal

Doses / concentrations 4

Dose / conc.:

875 mg/kg bw/day (nominal)

Remarks:

female animal

Doses / concentrations 5

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

male animal

Doses / concentrations 6

Dose / conc.:

1 750 mg/kg bw/day (nominal)

Remarks:

female animal

No. of animals per sex per dose:

six males and six females were assigned to each test group and additionally six males and six females for control group and highest dose for the 48 h period. Animals were identified by their cage number as shown in table 1 (any other information).

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide disolved in deionised water
- Route of administration: intraperitoneally, once
- Doses: 40 mg/kg bw
- Dose volume: 10 ml/kg bw

Examinations

Tissues and cell types examined:

bone marrow
polychromatic erythrocytes (PCE)
normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in this study and reported presently are expressed in terms of this indicated content.
lt is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose leveis spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animais (including the controls) were weighed and the individual volume to be administered was adjusted to the animais body weight. The animais received the test substance, the vehicle or the positive control substance once i.p.. Six males were treated per dose group and sampling time. The animais of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test substance. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The animais were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf Serum, using a syringe. The ceII Suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supenatant was discarded. A small drop of the resuspended celI pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the sildes was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To desoribe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Five males per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.20 - 1.50 %; mean = 0.86 ± 0.32 % PCEs with micronuclei).
- the positive controls are in the range of our historical control data (10.0 - 27.1 %;mean = 16.78±4.79 % PCEs with micronuclei).
- at least 80 % of animals are evaluable

The test substance was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes (which clearly exceeds the negative control range) or a relevant positive response for at least one of the test points. Statistical methods can be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) can be used as an aid in evaluating the results. Statistical significance at the five percent level (p < 0.05) was evaluated. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In animals treated with the highest dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1,000 - 2,000 mg/kg
- Clinical signs of toxicity in test animals: clear signs of toxicity in the highest test groups. Reduction of spontaneous activity, abdominal position, eyelid closure, apathy, ruffled fur

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): affected by treatment (see tables 1 and 2 in any other information on results)
- Statistical evaluation: Mann-Whitney Test
- Appropriateness of dose: yes

Any other information on results incl. tables

In comparison to the corresponding vehicle control there was no substantial enhancement in the frequency of the detected micronuclei at the 24 h preparation interval. The value obtained for the males treated with 1500 mg/kg at the 48 h preparation interval (1 .6 %) was statistically significant. However, this increase is within the historical range for the negative control males (0.0 - 2.1%) and significance was obtained due to the low vehicle control value. Therefore, it is considered as biologically irrelevant.

Historical controls (1999 - 2000)

	Micronucleated PCE’s per thousand (‰)
	Negative controls		Positive controls
	Males	Females	Males	Females
Mean*	0.76	0.62	18.70	14.54
SD**	0.42	0.34	5.79	5.32
range	0.0 – 2.1	0.0 – 1.4	9.30 – 34.10	7.00 – 35.50

*= mean of 62 experiments

Table 1: Summary of micronucleus test results for males

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [‰]	Range	PCE/NCE	significance	P
Vehicle	0	24	0.3	0 – 1	2000/1587
Test substance	375	24	0.2	0 – 1	2000/1778	n.t.	-
“	750	24	1.4	0 – 6	2000/1612	-	0.0873
“	1500	24	1.2	0 – 4	2000/2176	-	0.0873
Positive control	40	24	15.5	12 – 46	2000/1419	+	0.0040
vehicle	0	48	0.0	0 – 0	2000/1706	-	-
Test substance	1500	48	1.6	2 – 6	2000/2564	+	0.0040

-     = not significant

+ = significant (p < 0.05)

n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

Table 2: Summary of micronucleus test results for females

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [‰]	Range	PCE/NCE	significance	P
Vehicle	0.0	24	0.8	0 – 3	2000/1288
Test substance	437.5	24	0.4	0 – 2	2000/1615	n.t.	-
“	875.0	24	0.5	0 – 3	2000/1854	n.t.	-
“	1750.0	24	0.9	1 – 2	2000/2335	-	0.5000
Positive control	40.0	24	16.5	27 – 39	2000/1894	+	0.0040
vehicle	0.0	48	0.4	0 – 2	2000/1572	-	-
Test substance	1750.0	48	0.6	1 – 2	2000/2191	-	0.3571

-   = not significant

+ = significant (p < 0.05)

n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow celIs of the mouse. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test substance was formulated in deionised water, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mI/kg b.w.. 24 h and 48 h after a single i.p. administration of the test substance the bone marrow cells were collected for micronuclei analysis. Five males or five females per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The test substance is an aqueous solution. The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in

this study are expressed in terms of this indicated content. The exact value of the test substance purity is, however, 49.5 g / 100 g as indicated later by the sponsor.

The following dose levels of the test substance were investigated:

males:

24 h preparation interval: 375, 750, and 1500 mg/kg b.w..

48 h preparation interval: 1500 mg/kg b.w..

females:

24 h preparation interval: 437.5, 875, and 1750 mg/kg b.w..

48 h preparation interval: 1750 mg/kg b.w..

The highest dose (1500 mg/kg b.w. for males and 1750 m g/kg b.w. for females) was estimated by pre-experiments to be suitable.

After treatment with the highest dose of the test substance the number of NCEs was increased as compared to the mean value of NCEs of the vehiole controls thus indicating that the test substance had cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered intraperitoneally (i.p.) was used as positive control which showed a substantial increase of induced micronucleus frequency.