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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Remarks:

Sub-acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-09-20 to 1992-03-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

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:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age and body weight: At the start of the study, the male animals exhibited a mean initial weight of 3.03 (2.74 - 3.21) kg, and the females of 2.98 (2.39 - 3.38) kg. Based on these body weights, the age of the animals was around 10 - 16 weeks.
Animal housing: During the adaptation and study periods, the animals were individually kept under conventional conditions in Cellidor rabbit cages. It was not necessary to change the cages during the study period. The stool trays were changed twice weekly. During the six-hour exposure periods, the animals were confined in restraining racks which prevented the formulation from being rubbed off or orally ingested.
Rooms: All animals used in this study were housed in the same animal room.
Cleaning, disinfection and pest control: The animal room was cleaned each day and disinfected once monthly with Zephirol® (100 g Zephirol® contains 10 g benzalkonium chloride as active ingredient). Contamination of the diet or cages and experimental animal contact with the disinfectant were avoided during this work. No pest control measures were taken in the animal rooms.
Room climate conditions
The room climate in the stall was set as follows.
Room temperature 22° ± 2° C
Humidity (relative) About 50 %
Light/dark cycles 12 hours; artificial lighting from 6 AM to 6 PM
Air exchanges About 10 times per hour
Occasional deviations from these standards took place, for example due to cleaning of the animal room. They had no perceptible effect on the study routine.
Feeding: The food was provided to the animals each day from automatic rabbit food dispensers. Tap water from watering bottles was provided for unlimited consumption. Polycarbonate bottles with a capacity of about 700 ml were used; the tap water met drinking water standards. Records of the analyses to monitor adherence to the drinking water specification are filed.

Administration / exposure

Vehicle:

other: The test substance was formulated with a 2 % v/v Cremophor EL emulsion in sterile physiological saline solution to afford a suspension prior to each treatment.

Details on exposure:

The back and flanks of the rabbits were shaved one day before initiating treatment. The hair which grew back during the study was shaved off the skin areas scheduled for treatment twice weekly.
Four-ply gauze patches measuring 11 x 12 cm were placed on the shaved dorsal skin of the experimental animals, and the test substance formulations in a treatment volume of 2 ml/kg body weight evenly applied and distributed over these using a syringe. The gauze patches were then turned over and fixed using surgical tape. The treatment area measured 11 x 12 cm, and thus corresponded to more than 10 % of the skin area of the rabbits. The fixation dressings and gauze patches were removed, and the treatment area cleaned with soap and water after an exposure period of six hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The 12.5 % and 25 % formulations were kept homogeneous by agitation on a magnetic stirrer during the treatment. The 50 % formulation was homogenized in a mortar before and during the treatment.
The stability and homogeneity of the test substance in the treatment formulations were analytically confirmed. The active ingredient levels in the formulations were checked and confirmed once during the study.

Duration of treatment / exposure:

The rabbits were dermally treated with the test substance once daily in this manner. During the first two weeks of the study, the animals were treated on five working days, and during the third week on the weekend as well. The female animals were treated for one day longer than the males.

Frequency of treatment:

Once daily. During the first two weeks of the study, the animals were treated on five working days, and during the third week on the weekend as well. The female animals were treated for one day longer than the males.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: Not specified.
- Rationale for animal assignment: Random.
- Fasting period before blood sampling for clinical biochemistry: Not specified.
- Rationale for selecting satellite groups: Not relevant.
- Post-exposure recovery period in satellite groups: Not relevant.

Examinations

Observations and examinations performed and frequency:

General examinations
The appearance and behavior of the animals were monitored at least once daily during the study period.
Food consumption
The individual food intakes were determined once weekly and the mean daily food intakes calculated. The times stated in the tables refer to the end of the pertinent feeding week.
Body weights
The animals were weighed before initiating the study and after 7, 14 and 22/23 days; the postexposure observation groups were also weighed after 21, 28 and 36 days.
Testing for local skin tolerance
The treated skin areas were inspected for redness before initiating the study and 24 hours after each treatment.
Redness was graded on the following scale, which is based on guidelines published by the U.S. Department of Agriculture and on the Draize test.
No redness = 0
Very slight redness = 1
Moderate redness = 2
Marked redness = 3
Severe (deep red) redness = 4
Swelling was assessed by measuring the skinfold thickness at the center of the treatment area with a Cutimeter on the following days: 0, 2, 6, 9, 13, 16, 20 and 23 (last day female animals only). A measurement was made at two points of the treatment area in each case; the mean figure calculated from the two individual results is listed in the tables.
Laboratory tests
Laboratory tests were performed on blood samples taken from the rabbits before initiating treatment, after the three-week treatment phase and following the two-week post exposure observation period. The blood required for the tests was withdrawn from the ear veins of fasted animals. Specific hematology and clinical chemistry parameters were determined.

Sacrifice and pathology:

Necropsy
One day after the last treatment, and following the postexposure observation period, the appropriate groups of rabbits were sacrificed by exsanguination under deep anesthesia, necropsied and examined for gross pathology.
The absolute and relative organ weights of the brain, thyroid, heart, lung, liver, kidneys, adrenals, spleen, testes, and ovaries were determined. The following organs or organ specimens were fixed in Bouin' s solution for histological examinations; untreated and treated skin, thyroid, heart, lung, liver, kidneys, spleen, adrenals, testes, epididymis, ovaries, uterus, sternum and organs exhibiting macroscopic changes. In addition, liver and kidney organ specimens were fixed in 10 % formalin-calcium solution.
Histopathological examinations
Thin Paraplast sections were prepared from the organs or organ specimens fixed in Bouin's solution to the extent shown in the tables of the Histopathology Report, stained with hemalum-eosin and histopathologically examined. Kidneys were also stained using the periodic acid-Schiff reaction (PAS).

Other examinations:

Enzyme determinations were performed with samples of the liver following necropsy of the animals.

Statistics:

The arithmetic group means, and standard deviations were calculated from the food, body, and organ weights, the hematology and clinical chemistry results and the dermal findings. The values in the dose groups were compared to those in the appropriate control group using the two-tailed U-test of Mann and Whitney and Wilcoxon.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The appearance and behavior of the dose group animals did not differ from those of the control group rabbits during the treatment period. One female in the 1000 mg/kg body weight dose group ( animal no. 60) exhibited soft stool on the 14th day of the study. The female 500 mg/kg bodyweight dose group animal no. 49 favored its left rear leg from the sixth to the 24th day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A reticulocyte count could not be performed on Day -1 due to inadequate staining.
The leukocyte count relative to the animals used as the control group was depressed in the male dose group 3 animals at the start of the treatment (Day -1); the percentage of lymphocytes was depressed in the dose group 3 females, and the fraction of pseudo- eosinophils increased.
The following significant differences between the control and dose groups were found at the end of the treatment.
Dose group 1 males : Number of erythrocytes with Heinz inclusion bodies increased
Dose group 3 females : MCV value reduced
These differences, as well as the statistically significant deviations noted prior to initiation of the treatment, may be regarded as the consequence of incidental inhomogeneous distribution of the results rather than as an effect of the treatment. A dose relationship was absent.
The mean erythrocyte volume (MCV) in the female animal dose group was reduced relative to that of the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The differences found at the end of the study period were minor and were devoid of biological relevance. In all cases, only individual parameters were affected in isolated groups; a dose relationship was absent.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly reduced relative and absolute liver weights compared to those of the control group were determined in the female dose group 1 animals, but only a reduced relative weight in dose groups 2 and 3. The relative kidney weight in the female dose group 3 animals was reduced compared to that in the control group. This result is devoid of toxicological relevance since no dose relationship or equivalent findings in the male animals were present.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Local skin reactions

No redness, swelling or other skin reactions occurred in either the control group or any of the three dose groups.

The differences in skinfold thickness relative to the pertinent control group found statistically significant for individual dose groups on a few days should not be considered a result of the treatment: the control group figure lay above that of the dose group in all cases; inflammatory swelling was thus absent.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in the triglyceride level relative to the control group was determined in the male animals of dose groups 2 and 3.
Renal alterations were observed in both the control and the dose groups. These represented changes in the color or surface appearance (yellow, glazed or crateriform zones), which experience shows are attributable to an encephalitozoon infection.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The P-450 level in the dose group 3 females lay below that of the control group. No toxicological relevance is attached to these findings; the differences were minor, and only one sex was affected in each case.

A slight increase in the level of the monooxygenase cytochrome P-450 was determined in the dose group animals of both sexes relative to the control group. Since an equivalent effect was not present at the end of the treatment phase, this finding - as well as the marginal increase in 0-demethylase activity in the female animals - is devoid of toxicological significance.

Details on results:

The test substance treatment was tolerated by the rabbits without symptoms at levels up to the accepted threshold dose for dermal studies (1000 mg/kg body weight). The body weight and food consumption data, the hematology and clinical chemistry parameters determined, and the macroscopic, microscopic and gravimetric assessments of the internal organs of the rabbits afforded no evidence for test substance-related systemic effects. The test substance was also locally tolerated without skin reactions at levels up to the highest dose.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Clinical chemistry

-	Beginning of Treatment	End of Treatment
Dose group 1 males	Sodium level elevated	-
Dose group 2 females	-	Potassium level elevated
Dose group 3 males	Bilirubin level depressed; chloride level elevated	Bilirubin level depressed
Dose group 3 females	Phosphate P level elevated	Potassium level elevated

Applicant's summary and conclusion

Conclusions:

In the absence of treatment-related effects, the NOAEL for this study is 1000 mg/kg bw/d.

Executive summary:

The local and systemic toleration of Pencycuron  by rabbits was tested in a subacute dermal toxicity study. The study was conducted according to the OECD Guideline for Testing of Chemicals No. 410 and the EPA Pesticide Assessment Guidelines 82-2.
The test substance was formulated with a 2 % v/v Cremophor EL emulsion in sterile physiological saline solution to afford a suspension. Over a period of about three weeks, the test substance was applied to the shaved skin of the back and flanks of the rabbits on a total of 18 (males) or 19 (females) days and left there for six hours. The treatment volume was 2 ml/kg body weight.
The following doses were administered:
Control group 0 mg/kg b.w. (control formulation)
Dose group 1 250 mg/kg b.w. (12.5 % formulation)
Dose group 2 500 mg/kg b.w. (25 % formulation)
Dose group 3 1000 mg/kg b.w. (50 % formulation)
Five male and five female animals made up each group. In addition, a satellite group treated at 1000 mg/kg body weight and a further control group were observed for 14 days after treatment to check for the persistence or reversibility of possible toxic effects.
No test substance-related symptoms were determined. The body weights and food intakes of the dose group animals did not differ from those of the controls during or after the three-week test period. Neither the clinical chemistry, hematology and organ gravimetry tests which were performed, nor the macroscopic and microscopic assessments of the internal organs afforded any evidence for treatment-related effects.
No skin reactions occurred in the vicinity of the treatment areas.
Rabbits with intact skin thus tolerated repeated dermal exposure to Pencycuron at levels up to the accepted threshold dose for dermal studies (1000 mg/kg body weight) without perceptible local or systemic reactions under the present conditions.