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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 1989 - 02 March 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,3-dimethyl-2-imidazolidinone (also known as 1,3-dimethylethyleneurea)
- Lot/batch No.: 58-048

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 28 +/- 1 day
- Weight at study initiation: 62 to 81 g
- Housing: All animals were initially caged, in groups of five according to sex. Each animal was identified within each cage by ear-punch and
individually by tail mark (tattoo).
- Diet (e.g. ad libitum): Biosure LAD1 Diet
- Water (e.g. ad libitum): Free access to tap water
-Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean minimum of 21.2 and mean maximum 21.9
- Humidity (%): mean minimum 55.4and mean maximum 58.1%
- Air changes (per hr):20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 05 January 1989 To: 02 March 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on analytical verification of doses or concentrations:
Aliquots (20ml) of the test substance formulations and the vehicle were retained on Days 1, 22 and 26 for concentration analysis by HRC Department of Analytical Chemistry.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, seven days per week for four weeks
Doses / concentrationsopen allclose all
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Two groups consisting of ten males and ten females and two groups consisting of five males and five females.
Control animals:
yes
Details on study design:
- Dose selection rationale: The dosage levels administered were selected on the basis of acute oral toxicity data available from the Sponsor and a 14 day preliminary dose range-finding toxicity study carried out at HRC (Report No. 89860/MTC)

Control animals received distilled water (10 ml/kg/day).

The test substance was administered by oral gavage to rats of groups 2 to 4 inclusive using a syringe and rubber catheter at a dosage volume of 10 ml/kg/day.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. At weekends a similar procedure was followed except that the final check was carried out at approximately mid-day

BODY WEIGHT: Yes
All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Daily monitoring by visual appraisal of the water bottles was maintained throughout the study.

HAEMATOLOGY: Yes

The following parameters were analysed with an Ortho ELT-1500 analyser using standard Ortho methodology:
Packed cell volume (PCV)
Haemoglobin (Hb)
Red blood cell count (RBC)
Platelet count (Plts)
Absolute indices:
Mean corpuscular haemoglobin concentrations (MCHC)
Calculated: Hb (g/dl) x 100 + PCV (%)
Mean corpuscular volume (MCV)
Calculated: PCV (%) x 10 ? RBC (x 10 e6/mm3)
Total white blood cell count (WBC)

Analysed were: differential white blood cell count (Diff) - by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)

The following parameter was also analysed:
Thrombotest (TT)

CLINICAL CHEMISTRY: Yes
The following parameters were analysed with a Roche Cobas centrifugal analyser:
Glucose
Triglycerides
Glutamic-pyruvic transaminase (GPT), also known as "alanine aminotransferase)
Glutamic-oxaloacetic transaminase (GOT), also known as "aspartate aminotransferase"
Gamma-glutamyltransferase

The following estimations were performed:
Alkaline phosphatase
Total bilirubin
Cholestrol
Urea nitrogen
Total protein
Albumin
Globulin
Sodium
Potassium
Calcium
Chloride
Inorganic phosphorus
Creatinine

URINALYSIS: Yes
Volume
pH
Specific gravity (SG) - by refractometry, compared to water with a value of 1000
Protein

Qualitative tests:
Total reducing substances
Glucose
Ketones
Bile pigments
Urobilinogen
Haem pigments
Microscopic examination





Sacrifice and pathology:
GROSS PATHOLOGY: The following organs from each animal killed after four weeks(Day 29) or six weeks ( after 14- day post- treatment observation period - Day 43):
adrenals
brain
kidneys
liver
ovaries
testes (with epididymides)

HISTOPATHOLOGY: Yes
Samples of the following tissues from all rats were preserved in 10% buffered formalin:
adrenals*,aort, brain, caecum, colon, duodenum, eyes, femur, Harderian gland, head (to preserve nasal cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes, mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatc nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen*, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, any other abnormal tissue.
* Tissues required for histopathological examination

These fixed tissues were embedded in paraffin wax (m.p. 56°C), sections cut at 4 ~m and stained with haematoxylin and eosin.
In the first instance, microscopic examinations were carried out for tissues marked with an asterisk under 'Terminal studies' from all rats of
Group 1 (Control group) and Group 4 (High dosage group) killed on Day 29. In response to changes noted in organ weights and at initial
histopathological examination, microscopic pathology was subsequently extended to include:
(a) Ovaries from female rats in the control and high dosage groups killed at termination or following the 14-day recovery period.
(b) Kidneys from male rats in the low, intermediate and recovery groups.
(c) Testes from male rats in the control, low, intermediate and recovery groups.
Statistics:
All statistical analyses were carried out separately for males and females.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pilo-erection was noted on one day only for rats in the low dosage group receiving DMI at 6 mg/kg/day.
For rats receiving DMI at 30 or 150 mg/kg/day, pilo-erection, hunched posture and abnormal gait (waddling) were noted during the treatment period.These signs were accompanied by lethary and body tremors for rats in the high dosage group.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gains for male and female rats receiving DMI at 150 mg/kg/day were lower than those of the controls during the treatment period with statistical significance being achieved in most instances. Lower, but not statistically significant, bodyweight gains were also recorded during week 4 for female rats receiving D.M.I at 30 or 6 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for male rats receiving DMI at 150mg/kg/day was lower than that of the controls throughout the treatment and can be related to the lower bodyweight gains recorded for these animals.
Food efficiency:
not examined
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):
Significantly higher (P < 0.01) neutrophil counts were recorded during Week 4 for female rats receiving D.M.I. at 150 mg/kg/day in comparison with controls.
During Week 6, red blue cell counts, packed cell volume and haemoglobin levels for male and female previously treated with DMI at 150mg/kg/day were significantly lower (P < 0.05, P < 0.01 and P < 0.001) than those of the controls.
Thrombotest times for female rats in the high dosage group were significantly higher (P < 0.01) than those of the controls during Week 6.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.05) glutamic-pyruvic transaminase (GPT) levels and higher (P < 0.05) or P < 0.01) triglyceride levels were recorded during Week 4 for both male and female rats receiving DMI at 150 mg/kg/day in comparison with controls. Cholesterol levels for female rats in this high dosage group were significantly lower (P < 0.05) than controls.
Significantly lower (P < 0.05) globulin and total protein levels were also recorded during Week 4 for female rats receiving DMI at 150 mg/kg/day.

Following the two-week recovery period, the above changes in triglyceride, cholesterol, globulin and total protein levels were not observed and GPT levels for female rats in the high dosage group were significantly higher (P < 0.01) than those of the controls.

During Week 4 changes in sodium, potassium and phosphorus levels were recorded amongst male rats receiving DMI. However, these changes were low in magnitude and considered unlikely to be of toxicological importance.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.01) urine volumes were collected during Week 4 from female rats receiving DMI at 150 mg/kg/day in comparison with controls. Similar but not statistically significant shifts to lower urine volumes were recorded for the corresponding male rats during Week 4 and for male and female treated rats during Week 6.

For animals in the high dosage group, pH values for male rats during Week 4 and for female rats during Week 6 were significantly lower (P < 0.05 or P < 0.01) than those of the controls.

During Week 4, a slight increase in the incidence of epithelial cells and polymorphonuclear leucocytes was noted for male rats in the high dosage group.

During Week 6, no sperm cells were seen in the urine of male rats previously treated with DMI at 150mg/kg/day. This finding may be related to the histopathological changes noted in the testes of treated male rats.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower (P < 0.01 or P < 0.001) testes weights were recorded for male rats in the high dosage group following the four-week treatment period and also following the two week recovery period in comparison with controls. These changes can be related to the macroscopic and microscopic findings.

Ovary weights for female rats in the high dosage group were higher than those of the controls at the terminal and recovery kills with statistical significance being achieved (P < 0.05) for animals at the recovery kill.

At terminal sacrifice significantly lower (P < 0.05) brain weights were recorded for female rats in the high dosage group.

At recovery sacrifice, adjusted kidney weights for male rats in the treatment group were significantly higher (P < 0.01) than those of the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Small testes and pallor of the kidneys were noted at termination for all male rats in the high dose group.
Following the two-week recovery period, testes from male rats in the treatment group were similarly small and blue discoloured.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys:
At terminal sacrifice, eosinophilic droplets were seen in the cortical tubular epithelium of all male rats receiving DMI., 30 or 150 mg/kg/day.
Focal tubular basophilia was also noted in all male rats receiving DMI at 150mg/kg/day and was associated with a focus of cortical scarring in one rat.
Following the recovery period, eosinophilic droplets in the cortical tubular epithelium were seen in three males previously treated with DMI at 150mg/kg/day. Focal tubular basophilia was observed in four male rats in the treatment group.

Testes and epididymides:
For all male rats in the high dosage group killed at terminal or recovery sacrifice, testicular atrophy and numbers of multinucleate spermatogenic cells were noted. In two rats killed at recovery sacrifice, there was evidence of early spermatogenesis in a proportion of a tubules and this may represent an early stage in the recovery of testicular function. Focal mineralisation was associated with the testicular atrophy in one rat at recovery sacrifice.
The epididymides of most male rats in the high dosage group showed a reduced number of spermatozoa (terminal sacrifice) or an absence of spermatozoa (recovery sacrifice), with or without numbers of abnormal spermatogenic cells.

Ovaries:
In view of an apparent increase in ovary weights from rats receiving DMI at 150 mg/kg/day, these tissues were included at microscopic examination. There was no change noted to be of toxicological significance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOEL of 1,3 -Dimethyl-2 -Imidazolidinone is considered to be 6 mg/kg/day under the conditions of the subjected study after taking account of the clinical signs noted in male and female rats receiving 30mg/kg/day for periods of several days.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathology: Testicular atrophy and reduced epididymus sperm at 150 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The observed changes were considered unlikely to be of toxicological importance

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of above results, the No Observed Adverse Effect Level (NOAEL) of the test substance for the males was judged to be 30 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of the 150 mg/kg group.

For treated male rats, the presence of eosinophilic droplets in the cortical tubular epithelium of the kidneys also extended to rats in the intermediate dosage group receiving DMI at 30 mg/kg/day. However, this change is commonly found following treatment with low molecular weight hydrocarbons and is species and sex specific to the male rat. The finding is not therefore considered to be a significant finding in respect to the test substance toxicity.

Changes in general health and in bodyweight gains were also noted for rats receiving DMI at 6 or 30 mg/kg/day. These changes were not severe in nature and in the absence of any other significant morphological or physiological changes, were not considered to be of major toxicological importance.

Executive summary:

The repeated dose toxicity of the test material was investigated in a GLP study which was conducted accoding to a methodology similar to the standardised guideline OECD 407.

During the study, groups of rats received DMI, formulated as a solution, at dose levels of 6, 30 and 150 mg/kg bw/day, daily, for a period of 28 consecutive days, by oral gavage. Clinical findings, bodyweight changes, food consumption, haematology parameters, biochemistry parameters and urinalayses were conducted throughout the study. Following treatment on day 28, the animals were sacrificed. Organs were weighed and macroscopic and microscopic pathology investigation ensued.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) of DMI for the males was judged to be 30 mg/kg/day each in the conditions of this study because of the effects on the testis and epididymis of the150 mg/kg group.