1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Unspecified to February 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

None, pre-dates OECD 408 but broadly comparable.

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

The study broadly complies with OECD 408. Deviations from the guideline included:
no ophthalmoscopy was performed
no functional observational battery was observed
inaccurate reporting of the method and results
For histopathology tissues from 10 animals/sex were examined.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

EC Number:

266-096-3

EC Name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Cas Number:

66063-05-6

Molecular formula:

C19H21ClN2O

IUPAC Name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Test animals

Species:

mouse

Strain:

ICL-ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

4-week-old JCL-ICR strain mice were used (average body weight: males; 25 g, females; 20 g). Prior to employment in the study, these animals were acclimated for one week in a cage maintained at the temperature of 25±3°C and humidity of 55±7% They were lit for 12 hours daily. One week after healthy animals were selected for this study. The average body weights of males and females were 29 g and 24 g respectively, at the beginning of the experiment. During the experiment five mice were housed each in a plastic cage. Test animals received powder feed and tap water ad libitum.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Dose levels of test compound were determined at the concentrations of 0 (control), 80, 400, 2,000 and 10,000 ppm was mixed with powder feed CE-2 at the above concentrations.

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prepared feed was analyzed to investigate mixing condition and stability of the compound in the diet, and as the result, each dosed feed was weekly prepared and kept in a refrigerator.

Duration of treatment / exposure:

The diet containing Pencycuron technical was orally administered to mice during a period of J months.

Frequency of treatment:

Continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

80 ppm

Doses / concentrations 3

Dose / conc.:

400 ppm

Doses / concentrations 4

Dose / conc.:

2 000 ppm

Doses / concentrations 5

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based on the LD50 > 1000 mg/kg food from an unspecified acute oral toxicity study with rats and mice and on the maximum no effect level of 1000 mg/kg from an unspecified one-month sub-acute oral toxicity study with rats and mice.
- Rationale for animal assignment (if not random): random.
- Rationale for selecting satellite groups: not relevant.
- Post-exposure recovery period in satellite groups: not relevant.

Examinations

Observations and examinations performed and frequency:

General behaviour and mortality:
Throughout the experimental period physical appearance, general behaviour and habit were observed daily. When dead animals were found during the experiment, they were dissected to find the cause of death.
Body weight:
During the experimental period test animals were weighed weekly.
Food consumption, active ingredient intake and food efficiency:
The amounts of food consumed by test animals were measured 3 times a week, and the feed was renewed each time. Active ingredient intake was calculated from the total amount of food consumption and food efficiency from total amount of food consumption and body weight gain.
Terminal examination:
(a) Hematological examination:
All the animals that survived at the end of the experiment were dissected by thoracic incision under slight narcotization with ether and blood was taken from. The blood collected was used for the hematological examination mentioned below.
Hematocrit volume (Ht value): Microcapillary tube
Hemoglobin content (Hb value): Cyanmethemoglobin method
Count of erythrocytes:/ Count of leucocytes:/ Count of thrombocytes: Micro cell counter.
Differential blood picture: Microscopical examination
The mean corpuscular hemoglobin concentration (MCC):/ The mean corpuscular volume (MCV):/ The mean corpuscular hemoglobin (MCH): Calculation from Ht value, Hb value and count of erythrocytes.
Clinical chemical examination
The following items of the clinical chemical examination were performed with the remainder of blood samples used for the above hematological examination.
Glucose
Lactic dehydrogenase (LDH)
Blood urea nitrogen (BM)
Glutamic-pyruvate transaminase (GPT)

Sacrifice and pathology:

Autopsies
Immediately after the blood samples were taken, all the animals that survived were dissected by abdominal incision to macroscopically examine alternations in the shape, color, location, etc. of each organ. Then the following organs were removed and weighed, and the relative organ weights were calculated from their final body weights: brain, submaxillary gland, thymus, heart, lung, liver, kidney, spleen, adrenals, and gonads (testes, ovaries)
In addition, the following organs and tissues were removed for histopathological examination: pituitary, thyroid, pancreas, stomach, duodenum, jejunum, mesentric lymph nodes, urinary bladder (prostate), skeletal muscle and bone marrow
Histopathological examination
The above organs or tissues were fixed in buffer formalin and routainly processed for histopathological samples.

Statistics:

Student's t-test was employed to assess the significance of the inter-group differences and the evidence of a response to the administration of the compound could be suggested when the data gave p<0.05 For the evaluation of total food consumption, the analysis of variance was employed with equal number by two-way classification. The statistical significance observed among dose groups was assessed by Turkey’s Q-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

As the result of daily observation of general "behaviour and occurrence of intoxication of test animals, they were in good health and any alternations in gloss of coat and feces, etc. were not observed throughout the experimental period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One dead animal was found in males of 400 ppm dose group after 71 days of feeding. In this mouse remarkable change of body weight was not observed before death, and as discovery of the body was delayed, the cause of death is not identified.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences between dose groups and the controls and therefore, in the body weight the effect of the compound was not observed in mice up to the highest dose group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The total food consumption was significantly increased in male mice of 2,000 ppm and 10,000 ppm groups and in female mice of 400 ppm group. The increase of total food consumption was not attributed to the administration of the compound because it was not related to sex of mice and any changes were not observed in body weight gain and food efficiency.

Food efficiency:

no effects observed

Description (incidence and severity):

In food efficiency there was no significant differences between any dose group and control group.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The hematological examination performed at the end of the experiment using blood collected from hearts of mice under ether anaesthesia. No significant differences were observed between any dose group and control group in male and female mice.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In GPT value an increase was observed in male mice of 2,000 ppm, a decrease in males of 10,000 ppm. Glucose value was significantly decreased in males of 2,000 ppm and 10,000 ppm, and in females of 80, 400 and 10,000 ppm dose groups. Significant increase was observed in LDH value of males of 2,000 ppm and 10,000 ppm groups, and in BUN value of male mice of 2,000 ppm. As the changes of the values of GPT, LDH and BUN were neither sex-related nor dose-related, they were not considered to be affected by the administration of the compound. Only in glucose value of both male and female mice some changes were seen and particularly in both sex of 10,000 ppm a decrease was observed. It is difficult to decide whether these changes were attributable to the administration of the compound because inclusion of 4% clay and 1% compound in the diet should be taken into consideration.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

And there were no differences in general behaviour compared with the controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative liver weight showed an increase in males of 10,000 ppm and in females of 2,000 ppm. In females of 10,000 ppm relative liver weight was also increased. The absolute and relative adrenal weight showed a decrease in males of 2,000 ppm, and an increase was observed in absolute spleen weight of males of 10,000 ppm and in relative spleen weight of those of 2,000 ppm and 10,000 ppm. For female mice, the absolute and relative weights of submaxillary glands were decreased in 80, 2,000 and 10,000 ppm groups and those of thymus were also decreased in 80, 400 and 2,000 ppm dose groups. As to kidney of females, absolute and relative weights in 10,000 ppm and relative weight in 2,000 ppm showed a decrease, respectively. As mentioned above, the absolute and relative organ weights of dose groups indicated a significant change at random compared with the controls. However, these changes are hardly considered to be attributable to the administration of NTN 19701 because sex-relation and dose-relation were not clearly seen. However, it is considered that in general organ weights of female mice were more influenced by the administration of the compound than males. The liver weight showed a dose-related increase both in male and female mice of 400 ppm and more dose groups compared with the controls, and a significant increase seen in 10,000 ppm group was considered to be attributed to the administration of the compound.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings were as follows:
Liver: Minimal change of irregular nucleus in size and abnormal distribution of chromatin were seen in some male and female mice of the highest dose group. This change is considered to be associated with the administration of the compound but was not qualitative change.
Kidney: Deposits of hyaline-like substance were noted in a large proportion in male and female mice of all dose and control groups. This change was commonly seen in the laboratory mice and was not attributable to the administration of the compound.
Spleen: Minimal deposits of pigment were noted in a large proportion in males and females of all dose and control groups. This change was commonly seen in the laboratory mice and has no toxicological significance.
Adrenal: Minimal to medium fatty degenerations in the medulla were seen in large proportion in females of all groups.
Other organs: Minimal droplets of fat in the myocardium, minimal foci of cell infiltration in the submaxillary gland and moderate foci of cell infiltration in the kidney. These were seen in a few animals.

Histopathological findings: neoplastic:

not examined

Details on results:

Oral exposure of mouse to Pencycuron for 90 days resulted mainly in effects on the liver. The changes of the weights of the submaxillary glands and thymus were actually observed in all female dose groups but were however not always statistically significant. As there was no clear dose relation, effects on these organ weights were not considered related to treatment. The effects on spleen weight in males observed at all dose levels are probably due to the relatively low average of their control group. Also, in the female group spleen weight is rather variable, in a not treatment-related fashion. Therefore, the effect on the spleen weight in males is not considered to be treatment related. The changes in kidney weight observed in females at doses of 2000 mg/kg food and higher are considered related to treatment. In view of the relatively low decrease at 2000 mg/kg food and in absence of histopathological findings, the kidney effect is not considered to be adverse in this dose group. From a dose level of 400 mg/kg and higher statistically significant increase of the (relative) liver weight was observed in the females. In view of the relatively low increase at 400 mg/kg food and in absence of histopathological findings, the liver effect is not considered to be adverse in this dose group. In the males only at the highest dose level of 10000 mg/kg food an increase in the (relative) liver weight was observed. In addition, microscopical changes in the liver included minimal change of polymorphism in the nucleus and abnormal distribution of chromatin in the nucleus of males and females at a dose level of 10000 mg/kg food. One female also showed these microscopical changes at dose level 2000 mg/kg food, but as they are regularly observed in mice with varying incidence this particular finding is considered incidental.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 65 mg/kg bw/d

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the NOAEL is set at 400 mg/kg (equal to 65 mg/kg bw/d), based on liver effects first observed in females and at higher doses also in males.

Executive summary:

The male and female mice of ICR strain were fed with the diet containing NTH 19701 at the dietary concentrations of 0, 80, 400, 2,000 and 10,000 ppm for 13 weeks. At the end of 13-week experiment all the animals were sacrificed for various examinations.
Any changes in general behavior and appearance of the test animals were not observed during the experiment. Only one female mouse of 400 ppm dose group died during the experiment.
The administration of the compound did not affect the body weight gain of male and female mice fed with NTN 19701 at the concentrations up to 10,000 ppm during 13-week experiment.
An increasing tendency of the average food consumption was observed in males of 2,000 ppm and 10,000 ppm dose groups.
Any dose-related differences among all the males and females of the dose groups were not detected in hematocrit value, hemoglobin content, erythrocytes count, leucocytes count and thrombocytes count and MCH, MCV and MCC values measured in hematological analysis.
The administration of the compound did not affect the differential blood counts in percentage of lymphocyte, neutrocyte, monocyte, eosinocyte and basophil leucocyte of all the male and female mice of the dose groups.
According to the measurement of blood glucose, lactic dehydrogenase (LDH), glutamic-pyruvate transaminase (GPT) activity and blood urea nitrogen, an increase was observed in LDH and GPT activity of male mice of 2,000 ppm and 10,000 ppm dose groups.
In male mice of 10,000 ppm dose group, the average and relative liver and spleen weights were increased. Significant decrease was observed in those of kidneys of females of 10,000 ppm.
From the results of histopathological examination, minimal changes were noted in the liver of the male and female mice from the highest dosage. These were not considered to be qualitative changes and it is concluded that the effect of pencycuron on mice liver could be of ignorance.
From the above-mentioned results, no effect levels to the mice fed with the diet containing NTN 19701 for 13 weeks were 400 ppm in males and females, and the active ingredient intakes corresponding to this dietary concentration were 52 mg/kg/day and 65 mg/kg/day, respectively.