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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 lot/batch No.of test material: BASF AG; Batch No. 0767
- Expiration date of the lot/batch: March 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at < 25°C, in die dark
- Stability of the test substance in the solvent/vehicle: The analysis of the formaldehyde content in the test substance revealed negligible loss for a period of 4 hours.

FORM AS APPLIED IN THE TEST: dissolved in distilled water; dose solution was prepared daily prior to dosing

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as a test system because it is a readily available laboratory rodent. species. lt has been historically shown to be a suitable model for repeated dose toxicity assessment and is recommended by the OECD and other regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jai Research Foundation, Valvada - 3 96 108, Dist. Valsad, Gujarat, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Fasting period before study: not specified
- Weight at start of treatment: males: 118 - 158 g; females: 95 - 130 g
- Housing: individually; during acclimation period 5/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Every new batch of feed used was analysed or microbial contaminants. Rat pellet feed and drinking water were analysed for potential contaminants once in six months.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24°C
- Humidity (%): 67% (mean)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: June 16, 2001 To: July 20 / August 03, 2001

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral administration represents a possible route of human exposure.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantities of test substance were dissolved in distilled water to get the desired dose solutions with appropriate concentrations for various treatment groups. Dose solution was prepared daily just prior to dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Purity of formaldehyde content in the test substance in dose solution was detected by the folowing procedure:

A volume of 1 ml samnple was pipetted out and diluted to 100 ml with distilled water. A volume of 100 ml diluted sample was placed in different distillation apparatus with the addition of 85% phosphoric acid (15 ml)
and distilled out. A series of standard solutions was pipetted out from 10 ml standard solutions, i. e., 0.25, 0.5, 1.0, 1.5, 2.0 ml in to 25 ml of volumetric flask with addition of10 ml of reagent solution and volume was made with distilled water (concentration of standard solutions were 1 ppm , 2 ppm, 4 ppm, 6 ppm, 8 ppm). A volume of 1.0 ml of the above distilled solution was pipetted out into a 25 ml volumetric flask with the addition of 10 ml of antona reagent and volume was adjusted with distilled water. Then standard and sample flasks were kept for 3.0 minutes in a water bath at 40°C and was cooled down at 20°C. The Blank solution was prepared with distilled water using the same procedure. Above prepared standard and sample solution was again diluted to 5 times with distilled water. The absorbance of the diluted standard and sample was measured against die blank at 412 nm.

Duration of treatment / exposure:

28 days; recovery group animals were kept under observation for further 14 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5; 3 males and females in one additonal control and high dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to find out die irritancy, if any, of the test substance at the dose level of 1000 mg/kg body weight two additional groups of animals i. e., one control and one high dose, each consisting of 3 males and 3 females, were included in die 28 day study and dosed
for a period of 14 days. On the 15th day these animals were subjected to a complete gross pathology examination which showed no treatment related irritation in the gastrointestinal tract. Hence, the 28 day repeated dose oral toxicity study was performed at the same highest dose.

- Post-exposure recovery period in satellite groups: yes, 14 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day; twice daily observation for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of initiation of treatment and at weekly intervals thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before commencement of the treatment and prior to terminal sacrifice
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and revovery period
- Anaesthetic used for blood collection: Yes; light ether anesthesia
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and revovery period
- Anaesthetic used for blood collection: Yes; light ether anesthesia
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked in table [No. 1] were examined.

NEUROBEHAVIOURAL OBSERVATIONS: yes
- Time schedule: once before commencement of the treatment and at weekly intervals thereafter
- Battery of functions tested: posture, convulsions, ease of removal from the home cage, handling reactivity of the animal, autonomic signs such as palpebral closure, lacrimation, salivation and piloerection
- Open field observations (gait, monilty score, arousal level, vocalisations, rearing, respiration, clonic or tonic movements, urination and defecation, stereotypy, bizarre behaviour)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table no. 2)

HISTOPATHOLOGY: Yes; All the preserved organs (see table no. 2) from control and high dose groups as well as liver, kidneys, heart, spleen and lungs of all the animals

Statistics:

Raw data were processed by the Department of Biostatistics and Systems Information, Jai Research Foundation to give group means and standard deviations with significance between the control and treated groups, using in-house developed statistical software. All the parameters characterised by continuous data such as rearing count, urination count, defecation, body weight, feed consumption, organ weight, relative organ weight, haematological and clinical chemnistry data were subjected to Bartlett's test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett' s t-test. Where the data did not meet die homogeneity of variance, Student's t-test was performed to calculate significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In high and mid dose males, the monocyte count was significantly enhanced. This effect was not observed at the end of the recovery phase.
Females of all dose groups showed an increase in mean corpuscular volume and an according decrease in measn corpuscular haemoglobin concentration. In the absence of any other alterations in the red blood corpuscle, these changes are considered biologically insignificant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males: decreased sodium and potassion values mainly in the low dose animals were considered incidental
Females: Marginally, though statistically significantly increased phophorus values in mid and high dose animals without deviations in calcium levels and/or histopathological alterations in the kidney were considered biologically irrelevant. A statistical increase in total protein, though marginal and hardly dose related, in all dose groups was not considered adverse. All other changes occured with no relation to dose and were considered incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute prostate weights were increased in high dose recovery males, but not at the end ot the treatment period.
In females of the recovery group, the absolute spleen weight was decreased, and the relative liver weight was increased.
As for males, in the absence of histopathological correlates and since the changes were not observed at the end of the treatment period, they were considered as irrelevant and incidental.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Degenerative or inflammatory changes were observed at similar incidence and severity in control and treated animals.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) of the test substance in Wistar rats exposed over a period of 28 days is 1000 mg/kg body weight.

Executive summary:

This study was conducted to assess the toxic effects of the test substance to rats, when administered orally by gavage for 28 consecutive days according to OECD 407. Three groups of Wistar rats comprising 5 males and 5 females per group with the test substance at 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg b.wt. A concurrent control group comprising die same number of animals was also maintained in die study. In addition, a high dose recovery (1000 mg/kg b. wt.) and a control recovery group were included in the study to investigate the persistence, recovery or delayed effect of the test substance for an other period of 14 days.

Each rat was observed for visible signs of reactions to the treatment once a day and for rnortality and morbidity twice a day. Individual body weight and feed consumption were monitored weekly. Detailed clinical examination/neurobehavioural observations were conducted for each animal once prior to initiation of treatment and at weely intervals thereafter. Ophthalmological exanmination was conducted on all animals prior to treatment and prior to sacrifice. Haematological observations and biochemical analysis of blood samples were performed on all animals at the end of treatment and recovery periods. All the rats were sacrificed and subjected to a gross pathological examination at the end of treatment and recovery period. Absolute organ weights were recorded and relative organ weights were calculated for die organs viz., adrenal, testes/ovaries, epididymis/uterus, posate, kidneys, brain, heart, spleen, liver and thymus in all rats. Histopathological examination was made on all preserved organs from control and high dose group animals as well as liver, kidneys, spleen, heart and lungs of all animals.

No treatment related mortality or visible clinical symptoms were observed in any of the treatment group aimals. Body weight, feed consumption and opthalmological examination did not reveal treatmnent related alterations.

The test substance did not alter the neurobehavioural pattern of animals.

Haematology parameters studied in treated males were comparable to control group except in monocyte count; significantly increased monocyte count was observed in mid and high dose groups.

The clinical chemistry parameters did not reveal any major alterations in treated males. In female increased total protein in all the dose groups (low, mid and high dose) and phosphorus in mid and high dose groups were observed. All other parameters studied were comparable to control group.

No treatment related changes were observed in the absolute organ weight and relative organ weight of treated groups as compared to control groups.

Gross pathology examination did not reveal any treatment related lesions.

The histopathological examination of various organs /tissues from control and high dose (1000 mg/kg b. wt.) group animals did not reveal any treatment related effect.

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) of the test substance in Wistar rats exposed for a period of 28 days is 1000 mg/kg b. wt.