Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Remarks:
according to Directive 2004/9/EC
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1-dimethylurea
EC Number:
209-957-0
EC Name:
1,1-dimethylurea
Cas Number:
598-94-7
Molecular formula:
C3H8N2O
IUPAC Name:
1,1-dimethylurea
impurity 1
Chemical structure
Reference substance name:
Urea
EC Number:
200-315-5
EC Name:
Urea
Cas Number:
57-13-6
Molecular formula:
CH4N2O
IUPAC Name:
urea
impurity 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
dihydrogen oxide
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Analytical purity: 99.9 %
- Lot/batch No.: 301601

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BeiJing Vital River Laboratory Animal Technology Co., Ltd.; License No.: SCXK (Jing)2012-0001
- Age at study initiation: 49 to 65 days
- Weight at study initiation: At the point in time of grouping, the weight variation in the same group used was not exceeding ±20% of the mean body weight for either sex.
- Fasting: Animals were fasted overnight prior to necropsy, but water was available.
- Housing: Animals were raised in suspended, stainless steel cages (L 32.0 cm × W 28.0 cm × H 20.0 cm) on cage racks (L 167.0 cm × W 70.0 cm × H 171.0 cm). There were 10 cages per layer, and 4 layers per rack. Animals were housed individually for the convenient calculation of food consumption per day for each animal during the whole test. Cages were arranged that possible effects due to cage placement were minimized.
- Diet ad libitum: Animals were provided with sterilized diet with complete nutrition supplied by Beijing keaoxieli Feed Co., LTD. (Product License No: SCXK (jing) 2009-0012, Batch No.14013213). Analysis reports of diet were supplied by the supplier. All the nutrition components and contaminants were within the permitted limits described in the national standard (GB14924.3-2010 and GB14924.2-2001).
- Water ad libitum: Water was purified by HT-R01000 purity system. Water is routinely analyzed annually, and all parameters except pH were within the permitted limits described in the national drinking water standard (GB5749-2006) but pH was within the permitted limits described in the purified water standard (GB17324-2003).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 23.2 °C
- Humidity (%): 43 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
see "any other information on material and methods incl. tables"
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the initiation of the toxicity study, the stability of the test item solutions was determined by an analytical method validation. 1 mL of each formulation sample was collected from the middle strata layer on the first dose preparation and last dose preparation for analysis . At least two parallel samples were collected each time. The homogeneity analysis were not performed for “true solutions” (solution in which the analyte is completely dissolved in the liquid phase). The actual results for the analysis of the dosing formulation were within ± 10 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
Dose / conc.:
540 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
0 mg/kg bw : 10 males and 10 females.
20, 60, 180 mg/kg bw : 5 males and 5 females per dose.
540 mg/kg bw :10 males and 10 females.
Control animals:
yes, concurrent vehicle
Details on study design:
see "any other information on material and methods incl. tables"
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the first day of dosing, general clinical observations were made at regular time intervals (0.5 h, 1 h, 1.5 h, 2 h, 4 h) after dosing. The study director
decided the time intervals according to the actual clinical signs. Thereafter
general clinical observations were made l.5 h after dosing once each day .
The observation time was based on the results of the first day observation,
preferably at the same time each day and considering the peak period of
anticipated effects after dosing. During the recovery period, the observation
time was the same as in the time of dosing. The health conditions and
toxicity signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed the day before dosing (at grouping), once a week during the treatment period and recovery period. Animals were fasted overnight (16-18h) prior to necropsy and empty stomach body weights were
collected before necropsy.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All surviving animals were anesthetized by C02 inhalation at the day after terminal dosing and end of recovery period, and blood was collected via abdominal aorta for hematology, coagulation and serum
biochemistry. All animals were fasted overnight before blood collection.
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Before necropsy at the day after terminal dosing and end of recovery period urine samples were collected from all surviving animals by
abdominal extrusion. Urinalysis was conducted to measure the following
parameters using an Uritest 500B automatic urinalyzer and test paper. In
addition, the appearance of urine was recorded but not statistically analyzed.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: In the fourth week of the exposure period, nerve function observation for recovery animals in control and top dose group were made to assess general behavior, sensory reactivity by different types of stimulation (auditory visual and proprioceptive) , grip strength and motor acitivity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see any other information on material and methods)

HISTOPATHOLOGY: Yes (see any other information on material and methods)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals showed defect teeth (1/10), eye hemorrhage and scab (1/10), alopecia and scab (2/10) in the control group. Only one animal showed blood in the urine in the 540mg/kg dose group on Day 24 and 25 after dosing and recovered on Day 26. The symptom of blood urine may be related to the test item. No testing animals died during the test.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the 28-days dosing period and the recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences compared with the control group during the whole test. The results of the body weight measurement indicated that the body weight gain was not influenced by the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Description (incidence and severity):
There were no statistically significant differences compared with the control group during the whole test. The results of the food consumption statistical analysis indicated that the amounts of food consumed of all animals in treated groups were not influenced
by the test item.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period:
The males in all treatment groups showed a significant decrease ( p<0.05, p<0.01 ) in monocytes (MONO) compared with the control group. It was considered to be of no toxicological significance since it was within the range of the historical data of the test facility and with no dose response relationship. The males in the 540 mg/kg dose group showed a significant increase ( p<0.05 ) in Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) and the females in the 540 mg/kg dose group showed a significant increase (p<0.01) in MCHC, but the extent of these changes was small, hence it was considered to be within the normal biological variance. The small and statistically insignificant differences in RDW-CV, RDW-SD and PCT between control and dosing groups are of no toxicological relevance. The females in the 540 mg/kg dose group showed a significant decrease (p<0.05) in Platelet Count (PLT) , but it was considered to be of no toxicological significance since it was within the range of the historical data of the test facility.

At the end of the recovery period:
No abnormal findings were observed in the hematological parameters in all treated animals. There were no treatment-related findings during the study.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the dosing period:
The males at the dose of 180 mg/kg showed a significant decrease (p<0.01) in Total Bile Acids (TBA), but with no dose response relationship and no toxicological relevance.

At the end of the recovery period:
No abnormal findings were observed in the biochemical parameters in all treated animals. There were no treatment-related findings during the study.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No differences between control and dose groups of toxicological meaning (importance) were found during the whole test.
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):
At the end of the dosing period:
High relative values of heart weights were observed in female animals in the 180 mg/kg bw/d group (p<0.05), but with no dose response relationship and no toxicological relevance.

At the end of the recovery period:
No abnormal findings were observed in absolute and relative values in all treated animals. The results indicated that there were no toxicologically relevant alterations in organ weights due to the test item in any dose group during the whole test
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing and recovery period: One case of joint enlargement was observed in the control group. One case of scab on the skin in the back of the animal was observed in the control group. The results indicated that there were no signs of toxicity related to the gavage of the test item in gross necropsy and macroscopic observation during the whole test.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Low incidence of urinary bladder edema, urinary bladder mucous plug, liver focal necrosis, joint suppurative inflammation, joint edema, parathyroid fibrosis
ectopic thymus in thyroid, skin ulcer, cardiac muscle inflammation, pancreas atrophy, pituitary aberrant craniopharyngeal tissue, kidney tubule basophilic
change and inflammatory cell infiltration, urinary bladder mucous plug, stomach edema, pituitary cyst, liver lymphocyte infiltration, kidney mineralization and kidney tubule protein cast were observed in the control group and 540 mg/kg group at the end of the treatment period and the recovery period. There were no statistically significant differences and no dose-related responses by comparison of the ratios of the above abnormalities with the control in either group for both sexes. So no test item-related changes were observed in histopathology examinations.
Histopathological findings: neoplastic:
not specified
Details on results:
Clinical observation and mortality:
There were no deaths during the 28-days dosing period and the recovery period. Animals showed defect teeth (1/10), eye hemorrhage and scab (1/10), alopecia and scab (2/10) in the control group. Only one animal showed blood in the urine in the 540mg/kg dose group on Day 24 and 25 after dosing and recovered on Day 26. The symptom of blood urine may be related to the test item. No testing animals died during the test.

Body weight:
There were no statistically significant differences compared with the control group during the whole test. The results of the body weight measurement indicated that the body weight gain was not influenced by the test item.

Food consumption:
There were no statistically significant differences compared with the control group during the whole test. The results of the food consumption statistical analysis indicated that the amounts of food consumed of all animals in treated groups were not influenced by the test item.

Nerve Function Observation:
The results of nerve function observation during the fourth week of the exposure period indicated no neurotoxicity was found related to the test item administered by gavage.

Hematological examination:
At the end of the treatment period: The males in all treatment groups showed a significant decrease (p<0.05, p<0.01) in monocytes (MONO) compared with the control group. It was considered to be of no toxicological significance since it was within the range of the historical data of the test facility and with no dose response relationship. The males in the 540 mg/kg dose group showed a significant increase (p<0.059 in Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) and the females in the 540 mg/kg dose group showed a significant increase (p<0.01) in MCHC, but the extent of these changes was small, hence it was considered to be within the normal biological variance. The small and statistically insignificant differences in RDW-CV, RDW-SD and PCT between control and dosing groups are of no toxicological relevance. The females in the 540 mg/kg dose group showed a significant decrease (p<0.05) in Platelet Count (PLT), but it was considered to be of no toxicological significance since it was within the range of the historical data of the test facility.
At the end of the recovery period: No abnormal findings were observed in the hematological parameters in all treated animals.
There were no treatment-related findings during the study.

Serum biochemical examination:
At the end of the dosing period: The males at the dose of 180 mg/kg showed a significant decrease (p<0.01) in Total Bile Acids (TBA), but with no dose response relationship and no toxicological relevance
At the end of the recovery period: No abnormal findings were observed in the biochemical parameters in all treated animals.
There were no treatment-related findings during the study.

Coagulation examination:
At the end of the dosing period: The males at the 540 mg/kg dose level showed a significant decrease (p<0.059 in Activated Partial Thromboplastin Time (APTT) (12 % lower than control). The males at 60 and 180 mg/kg dose level showed a significant decrease (p<0.05) in Fibrinogen (FIB). But the value of Fibrinogen (FIB) in the animal number 1003 of the control group was 11.86 g/L. This value is not plausible and was therefore eliminated. . Withdrawal of this abnormal value of FIB, leads to statistical insignificance, so there was no toxicological meaningful finding for FIB.
At the end of the recovery period: No abnormal differences were observed for the coagulation parameters in all treated animals.
The results of the coagulation parameter’s statistical analysis indicated that the test item at the dose of 540 mg/kg had decreased APTT in the males, but the animals recovered after stop of dosing within the two weeks recovery period.

Urinalysis:
No differences between control and dose groups of toxicological meaning (importance) were found during the whole test.

Organ weight:
At the end of the dosing period: High relative values of heart weights were observed in female animals in the 180 mg/kg bw/d group (p<0.05), but with no dose response relationship and no toxicological relevance.
At the end of the recovery period: No abnormal findings were observed in absolute and relative values in all treated animals.
The results indicated that there were no toxicologically relevant alterations in organ weights due to the test item in any dose group during the whole test.

Gross necropsy and Macroscopic observation:
At the end of the dosing period: One case of joint enlargement was observed in the control group. One case of scab on the skin in the back of the animal was observed in the control group.
The results indicated that there were no signs of toxicity related to the gavage of the test item in gross necropsy and macroscopic observation during the whole test.

Histopathology:
Low incidence of urinary bladder edema, urinary bladder mucous plug, liver focal necrosis, joint suppurative inflammation, joint edema, parathyroid fibrosis, ectopic thymus in thyroid, skin ulcer, cardiac muscle inflammation, pancreas atrophy, pituitary aberrant craniopharyngeal tissue, kidney tubule basophilic change and inflammatory cell infiltration, urinary bladder mucous plug, stomach edema, pituitary cyst, liver lymphocyte infiltration, kidney mineralization and kidney tubule protein cast were observed in the control group and 540 mg/kg group at the end of the treatment period and the recovery period.
There were no statistically significant differences and no dose-related responses by comparison of the ratios of the above abnormalities with the control in either group for both sexes. So no test item-related changes were observed in histopathology examinations.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

Discussion of the results:

After 28-days of dosing, the body weight gain and food consumption was not influenced by the gavage of the test item. The results of nerve function observation during the fourth week of the exposure indicated that no neurotoxicity was found related to the gavage of the test item. Only one animal showed blood in urine (haematuria) in the 540 mg/kg dose group on Day 24 and 25 of dosing but recovered until Day 26. And at the end of the recovery period, the animal showed no abnormalities in hematological examination, the serum biochemical, urinalysis examination and histopathology examination. The symptom of blood in urine (haematuria) may be related to the test item. There were no treatment-related findings during the studyinhematological examination, the serum biochemical examination and urinalysis.The results of the coagulation parameters statistic analysis indicated that the test item at the dose of 540 mg/kg could decrease Activated Partial Thromboplastin Time(APTT) in the males, and recovered after stop dosing two weeks later. No test item–related toxicity changes were found in organ weights, gross autopsy and macroscopic observation. Under the condition of this study there were no toxicity pathology changes in SD rats with 1,1 -Dimethylurea.

Applicant's summary and conclusion

Conclusions:
According to the results of the test, the no-observed-effect-level (NOEL) for 1,1-Dimethylurea in repeated dose 28-day oral toxicity study in SD rats was considered to be 180 mg/kg body weight/d for the males and 540 mg/kg body weight/d for females. The no-adverse-observed-effect-level (NOAEL) for 1,1-Dimethylurea in repeated dose 28-day oral toxicity study in SD rats was considered to be more than 540 mg/kg body weight for the males and females. There was no significantly delayed occurrence of toxic effects during 14-day recovery period.
Executive summary:

A study was performed to assess the toxicity of 1,1-Dimethylurea in Sprague Dawley rats. The method was designed to meet the requirements of OECD Guideline for Testing of Chemicals: Repeated Dose 28 -day Oral Toxicity Study in Rodents” (No. 407,adopted 2008).

There were five groups at different dose levels, including the control group (0 mg/kg bw/d) and the test item groups (20 mg/kg, 60 mg/kg, 180 mg/kg and 540 mg/kg bw/d). Animals in the four treatment groups were administered the concentrations of 2 mg/ml, 6 mg/ml, 18 mg/ml and 54 mg/ml of test item solution by gavage once daily for 28 days. Animals in the control group were administered the vehicle once daily for 28 days. Concentration analysis of the formulated test item were carried out on the first dose preparation and last dose preparation day.Clinical symptom observation, body weight measurement, food consumption measurement,nerve function observation and clinical pathology including urinalysis, hematological, coagulation,blood biochemistry, necropsy and anatomic pathology and histopathology were carried out after the end of the dosing period and recovery period.

ResultsOn the first dose preparation and last dose preparation day, analysis of samples showed that the actual results for the analysis of the dosing formulation were within ± 10 % of the nominal concentration and here within the limits of validity. After 28-days of dosing, the body weight gain and food consumption was not statistically significantly altered by the administered test item. The results of nerve function observation in the fourth week of the exposure period indicated that no neurotoxicity was found related to the gavage administration of the test item. Only one animal showed blood in urine (haematuria) in the 540 mg/kg dose group on Day 24 and 25. But the animal recovered, and on Day 26 and at the end of the recovery period, the animal showed no abnormalities in hematological examination, the serum biochemical values, urinalysis and histopathology. The symptom of blood in urine (haematuria) may be related to the test item. There were no treatment-related findings during the study in hematological examination, the serum biochemical examination and urinalysis.The statistic analysis of the coagulation parameters indicated that the test item at the dose of 540 mg/kg could decrease the Activated Partial Thromboplastin Time(APTT) value in the males. The APTT value normalized until the end of the recovery period. No test item related changes were found in organ weights, gross autopsy and macroscopic observation. In terms of this study, there were no pathological changes detectable in SD rats with 1,1-dimethylurea.

Conclusion: According to the above results, the no-observed-effect-level (NOEL) for 1,1 -Dimethylurea in repeated dose 28 -day oral toxicity study in SD rats was considered to be 180mg/kg body weight/d for the males and 540 mg/kg body weight/d for females. Theno-adverse-observed-effect-level(NOAEL)for 1,1 -Dimethylurea in repeated dose 28 -day oral toxicity study in SD rats was considered to be more than 540 mg/kg body weight for the males and females. There was no significantly delayed occurrence of toxic effects during 14-day recovery period.