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Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
RCC, Research & Consulting Company, Ltd.
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: liquid
Details on test material:
Name of the test substance used in the study report: N,N'-Dimethylpropylene urea
Purity: 99.7%

Test animals

Details on test animals or test system and environmental conditions:
The animals were assigned to identification numbers.
Air-conditioning with 10-15 air changes per hour and continuously monitored environment with a target range for room temperature of 22 ± 3°C, and for relative humidity between 40-70 % (values above 70 % during cleaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycle. Music was played during the light period.
Groups of five rats were accomodated in Makrolon type-4 cages with standard softwood bedding.
Pelleted standard Kliba 3433, batch no. 90/97 rat maintenance diet and community tap water were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Frequency of preparation: Daily, prior to each application.
Chemical analysis of dose preparations: Concentration, homogeneity and stability of the test article / vehicle mixtures were determined in samples taken during acclimatization and during week 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG according to a method supplied by the Sponsor.
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
28 days
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 10, 50, and 250 mg/kg bw/d

No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
Based on data from a 14-day dose-range fmding study (RCC selection Projeet 666786) in which the test substance was administered by gavage to 3 rats per group and sex. Treatment with the test article at 300 and 1000 mg/kg was associated with effects on food consumption and body weight in both test groups and mortality, clinical symptoms and macroscopic findings at 1000 mg/kg.


Observations and examinations performed and frequency:
Clinical signs, outside cage observations, food consumption and body weights were recorded periodically during the acclinatization, treatment and recovery periods. The animals were examined for signs of toxicity or mortality at least once a day.
Sacrifice and pathology:
At the end of the dosing period and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses and urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem. Samples of major organs from all animals of group 1 (0 mg/kg) and group 4 (250 mg/kg), as well as kidneys, mandibular lymph nodes, spleen, testes, thymus and thyroid glands from the animals of the intermediate dose groups 2 (10 mg/kg) and 3 (50 mg/kg) and gross lesions from all animals were processed as hematoxylin and eosin stained slides and examined by light microscopy.
Other examinations:
At pretest and at weeks 4 and 6 a modified Irwin screen test was performed on all rats per group and sex.
The following statistical methods were used to analyze the body weights, organ weights and all ratios as well as clinical laboratory data:
If the variables could be assumed to foilow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Details on results:
All animals survived the scheduled study period.
The only treatment-related clinical signs, observed during the daily clinical observations and 1 or the weekly outside cage observations, were slight sedation and ruffled fur, which were observed in all group 4 (250 mg/kg) animals, predominantly during weeks 2 and 3 of the dosing period. No clinical signs were noted during the recovery period. Incidental clinical observations included soft feces, increased salivation and wounds / scars / fissures on tail / ears in occasional animals.
For the group 4 (250 mg/kg) animals, food consumption was reduced throughout the 4-week dosing period. On cessation of dosing, food consumption increased and was slightly higher than that of the controls in weeks 1 (females only) and 2 (both sexes) of the recovery period. Relative food consumption was lower than that of the control group in weeks 1 and 2 of the dosing period and was higher than that of the control group in weeks 1 and 2 of the recovery period. Both food consumption and relative food consumption of the group 2 (10 mg/kg) and 3 (50 mg/kg) animals were similar to that of the control group throughout the 4-week dosing period.
For the group 4 (250 mg/kg) animals, body weight gain was retarded throughout the 4-week dosing period. For both sexes, mean body weights were significantly lower than controls from Day 8 of the dosing period. During the 2-week recovery period, there was an increase in weight gain for both sexes so that mean terminal body weight of the females, but not the males, was the same as the control value. For the group 3 (50 mg/kg) animals, there was a slight retardation in body weight gain during the 4-week dosing period, but none of the lower mean body weight values attained statistical
significance. Body weights and body weight gain of the group 2 (10 mg/kg) animals were similar to those of the control group throughout the study.
At pretest and at weeks 4 and 6, a modified Irwin screen test was performed on all rats. This involved the observation of individual animals in the home cage, in the arena, in the hand and on return to the home cage, and the measurement of hind- and forelimb grip strength and locomotor activity. During the assessments performed at both 4 and 6 weeks, all observations recorded (decreased / increased pupil diameter, vocalization, decreased exploratory activity) were considered to be incidental and not related to treatment with the test article. In all groups, values for grip strength increased over the duration of the study, as the animals increased in weight and developed from ca. 7 weeks to 13 weeks of age. The significantly lower values for fore- and hindlimb grip strength at 6 weeks (recovery period) in group 4 (250 mg/kg) were considered to be incidental as the values at the end of the 4-week dosing period were similar to those of the control group and the value for forelimb grip strength was also significantly lower than controls at pretest. The significantly increased value for forelimb grip strength after 4 weeks in group 2 (10 mg/kg) was similarly considered to be incidental. There was a large inter-individual variability in the values obtained for the animals from all groups.
Quantitative assessment of locomotor activity over a period of 60 minutes was measured using an Activity Monitor in week 4 of the study. For the group 4 (250 mg/kg) females, overall activity was significantly lower than controls and for the group 2 (10 mg/kg) females, activity was significantly increased at 50 minutes. Both of these findings were considered to be incidental and to have occurred because of the large inter-individual variability in this type of measurement.
After 6 weeks, there was evidence of slight anemia in both males and females at 250 mg/kg (RBC, HB and WBC reduced). At the end of the 4-week dosing period, there were indications of this effect in the slightly lower, but not statistically significant (p>0.05) values for total leukocyte count, but the erythrocyte values were not affected. Increased hemosiderin deposits in the spleen at histological examination correlate with these hematological findings. The slight increase in reticulocytes (botih sexes) and in nucleated erythrocytes and platelets (males) at 250 mg/kg at 6 weeks indicate a compensatory effect by the hemopoietic organs, showing that the repair mechanism had started. The decreased Iymphocyte count at both 4 and 6 weeks correlates with the lymphoid atrophy in the spleen and thymus observed at histological examination and is considered to have contributed to the decrease in the leukocyte values mentioned above. There were no changes in the hematological parameters in groups 2 (10 mg/kg) or 3 (50 mg/kg), which were considered to be related to treatment with the test substance.

Effect levels

Dose descriptor:
Effect level:
10 mg/kg bw/day (nominal)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Oral administration of the test substance to Wistar rats at doses of 10, 50 and 250 mg/kg body weight/day for 28 days was associated with clinical signs, effects on food consumption, body weight, clinical laboratory parameters, organ weights and macroscopic and microscopic findings at 250 mg/kg/day and, to a lesser extent, at 50 mg/kg/day. Target organs were identified as spleen, thymus (both sexes), kidneys, testes and thyroids (males). There was evidence of recovery in some, but not all parameters at the end of the recovery period.