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Description of key information

A study was conducted following guideline OECD TG 407 (1995) and 96/54/EEC, using a 28 day study on rats (Wistar strain). The test animals (10; 5 males, 5 females) were housed individually and were given adequate time to acclimatise (10 days) before the test was conducted. The test animals then received regular daily doses (10 mL/kg bw) of the test substance at different concentrations (100, 300 and 1,000 mg/kg/bw/day) via the oral gavage route, along with a vehicle control group. The vehicle used was aqua bidest containing 0.5% carboxymethylcellulose. The observable effects of the substance were also noted for the differences in sexes.

From these no notable clinical effects were observed other than a slight increase in appetite during the second week for the females within the 50 mg/kg bw however, their appetite returned to a similar amount to the control group after this week. Within the 28 day test period and the standard recovery period after, no mortality or clinical signs were observed. After the recovery period the test subjects were sacrificed and necropsies were conducted. There were no reported abnormalities observed in the necropsy of all test animals. Under the conditions of the study, the test substance does not cause toxicity for repeated dose exposure.

 

From this it was seen that there was no potential chronic toxicity effects from the substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch number: Fixapret 140 Nr. 766.
Date of manufacturing: 24 March 2001.
Purity: 96.3% (confirmation by HPLC method).
Species:
rat
Strain:
Wistar
Remarks:
CrIGlxBrIHan:WI
Details on species / strain selection:
Wistar rats CrIGlxBrIHan:WI were supplied by Charles River, Sulzfeld, Germany. Animals free from clinical signs of disease were utilised. Wistar rats were selected since there is extensive experience available in the laboratory conducting the study with this strain of rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals
Source: Charles River, Sulzfeld, Germany.
Age at study initiation: 42±1 days.
Mean weight at study initiation: male animals 149.8g (142.3 - 160.1g); female animals 124.9g (119.7 - 133.2g).
Housing: Single.
Diet: Ground Kilba maintenance diet meal, available ad libitum.
Water: Drinking water (from water bottles), available ad libitum.
Acclimatisation period: 10 days.

Environmental conditions
Temperature (°C): 20 - 24.
Humidity (%): 30 - 70.
Photoperiod (hrs dark / hrs light): 12/12.
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered daily by gavage using 3 and 5 mL syringes (Fa.Becton & Dickinson) for about 4 weeks.
Vehicle:
other: 0.5% carboxymethylcellulose in doubly distilled water.
Details on oral exposure:
Test substance was mixed with doubly distilled water to the relevant concentrations (150, 500, 1500, 4500 mg/100 mL). These were prepared on a weekly basis. 10 mL/kg body weight was administered to each test animal.
Analytical verification of doses or concentrations:
yes
Remarks:
At the start of the administration period via HPLC.
Details on analytical verification of doses or concentrations:
Prior to the study starting, samples of each concentration were sent to an external laboratory and were assessed to confirm the concentration of the samples via HPLC.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Daily.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5.
Control animals:
yes, concurrent vehicle
Details on study design:
The control animals were administered with the vehicle only. This was performedn daily by gavage using 3 and 5 mL syringes (Fa.Becton & Dickinson) for about 4 weeks.
Positive control:
No positive controls were performed.
Observations and examinations performed and frequency:
Animlas were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) during the working days and once a day during Saturdays, Sundays and public holidays. Further clinical examinations were carried out daily just before treatment (due to technical reasons except days 0, 7, 14) and 3 to 4 hours after treatment.

Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined (findings were ranked according to the degree of severity, if applicable):

-behaviour during handling, fur (piloerection), skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exopthalmus, faeces (appearance/consistency), urine, and pupil size.

Functional observational battery (FOB) tests were performed in all animals at the end of the administration period, starting mid-morning at around 10:00AM, starting with passive observations, then more gradually involved handling of the test animals; removal from the home cage, open field observations and sensorimotor tests (including reflex tests).

Motor activity of the test animals were measured on the same day the FOB tests were carried out. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. The measurements started mid-afternoon at 14:00PM. The number of beams interrupts were counted over 12 intervals, each lasting 5 minutes. Measurements ended exactly 60 minutes thereafter. Note that during the procedure of this assessment, the animlas received no food or water.
Sacrifice and pathology:
All test animals were sacrified by decapitation under CO2 anaesthesia. The exsanguinated animals were necropsied and assessed by gros pathology. Organ weights were recorded for; anaesthetised animals, liver, kidney, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus). The following organs/tissues were fixed in 4% formaldehyde solution; all gross lesions, brain, pituitary gland, thyroid glands with parathyroid glands, thymus, trachea, lungs, heart, liver*, spleen, kidneys, adrenal glands, testes/ovaries, uterus/vagina, accessory genital organs (epididymides, prostate gland, seminal vesicles), stomach (glandular and non-glandular), duodenum/jejunum/ileum, cecum/colon/rectum, urinary bladder, lymph nodes (mandibular and mesenteric), sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar chord).

*Parts of the liver dedicated for routine investigation were fixed in Camoy's solution. After fixation, the liver was embedded in paraplast.

After fixation and histotechnical processing, slides were examined by light microscopy.
Other examinations:
Food consumption was determined weekly over a period of 7 days and calculated as a mean food consumptions in grams per animal per day.

Body weight was determined before the start of the administration period in order to randomise the animals. During the administration period the body weight was determined on day 0 (at the start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change. Food efficiency (in group means) was calculated based upon individual values for body weight and food consumption.

Statistics:
Statictics of pathology were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of animals in each test group. Analyses were perfomed using the following statistical tests; non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or <0.05, a pairwise comparison of each control group was performed using the Wilcoxon test for the hypothesis of equal medians.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No substance related clinical signs were observed. The only abnormal finding was loss of tail end in a mid-dose male animal, which was incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Some statistically significant increase in body weight was observed in the females in a low dose group between days 7 -21. Due to the lack of a dose relation this was concluded to be incidental.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food efficiency was statistically significantly increased in the low and mid dose females on day 7 and the high dose females on day 21. As these were isolated occurences and there was a lack of a dose-response relationship, these were deemed to be incidental and non-treatment related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases were observed in the haemoglobin and the haematocrit count in the high dose females (peripheral blood). The haemoglobin decrease was also observed in the mid dose females. There did not appear to be any other haematological effects.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry showed that there was a slight but statistically significant increase in glucose in the mid and high dose females and a slight decrease in inorganic phosphate in the high dose females. Other blood chemistry parameters were not affected by the test compound administered. The decrease in inorganic phosphate in the high dose females was considered to be of no toxicological significance as it was marginal and non-consistent. when compared with the other sex, furthermore there was a lack of a dose-response relationship.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Effects were observed in the high dose females; increased amounts of crystrals were detected in the urine sediments. 4/5 of the high dose females precipitated crystals of unknown origin. 3/5 high dose males also precipitated unknown crystals in the urine sediments. No treatment-related changes were observed for the rest of the animals.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean terminal body weight was significantly increased in females of the high dose group (+8.8%), the mean weight of the uterus and heart in this dose group was also significantly increased (+56.3%) and (12.5%) respectively. In the females of the low dose group (nos. 27, 37 and 39), there was also a slight but statistically significant increase in the mean weight of the heart (+14.3%). The mean weight increase for this organ, was concluded to have no dose-response relationship.

The increase in the mean absolute weight of the uterus was linked to the sex cycle of the female animals (moderate dilation of the uterus horns). The dilation of the horn(s) represents a physiologic situation not a pathologic incidence, and it has developed unrelated to treatment. This increase in body weight provides a probable correlation to the relative increase of the heart weight.

The other mean absolute weight parameters did not show significant differences when compared with the control group. The mean relative weight parameters (when related to the terminal body weight) did not show significant differences when compared with the control group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
An abcess was observed in the left epididymis of animal no. 10 (male, low dose group). Further histopathological examination confirmed this to be a spermatogenic granuloma which had developed spontaneously, as such it is unrelated to the treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Although intergroup differences were observed in the haematological, clinicochemical and urinalytical analysis, such deviations are expected within biology. Thus the results were deemed to be of no significant value.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Conclusions:
From the study and all of the different aspects that were examined, there were very few substance related changes noted in either haeamatology, blood chemistry, urinalysis and body weight/organ pathology.

For haematological changes, the test compound produced mild adverse effects on the peripheral blood - red blood cell parameters in the females which were reduced haemoglobin and haematocrit vlaues. Decreased inorganic phosphate and increased glucose levels were observed in clinical chemistry for females received the test compound. The changes were considered to represent slight biological variation and not clear effects of treatment. Since there were no other changes observed, it is difficult to determine if this change is relates to a specific disorder caused by the treatment.

For urinalysis, the sedimentation of unidentified crystals was attributed to the increased renal excretion of the test compound which is a urea derivative or its metabolites. These findings were considered to be of no pathological relevance, because renal elimination is one of the predominant excretion routes of a foreign compound. Additionally, crystal formation is most likely to be caused by the storage of the samples (in cooler temperatures), as crystals are not usually found in freshly voided urine.

Considering the information above it was therefore concluded that there were no treatment-related effects in clinical and pathological examinations, the slight changes detected in clinical pathology were regarded to be of no toxicological relevance. It was therefore, calculated that that NOAEL for glycoluril was 1000 mg/kg/bw in both males and females.
Executive summary:

A study was conducted following guideline OECD 407 and EEC 84/449, using a 28 day study on rats. The test subjects were housed individually and were given adequate time to acclimatise (10 days) before the test was conducted. The test subjects then received regular daily doses (10 mL/kg bw) of the test substance along with a vehicle control group. 5% CMC mixed with doubly distilled water was used as a vehicle. The subjects were split into several groups each testing a different concentration of the substance. Further to this it was also recorded if the test subjects were male or female to allow for difference in the genders.

The concentrations tested were, 0 (Vehicle controls), 100, 300 and 1000 mg/kg bw. From these no notable clinical effects were observed other than a slight increase in appetite during the second week for the females within the 50 mg/kg bw however, their appetite returned to a similar amount to the control group after this week. Within the 28 day test period and the standard recovery period after, no mortality or clinical signs were observed. After the recovery period the test subjects were sacrificed and necropsies were conducted. There were no reported abnormalities observed in the necropsy of all test animals.

 

From this it was seen that there was no potential chronic toxicity effect from the substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat

Additional information

Justification for classification or non-classification

The substance does not meet classification criteria under (EC) No 1272/2008 for chronic toxicity.