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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-08-03 to 1990-02-27

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:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

The study was performed in accordance with OECD 414 (1981), except the macroscopic examination of the dams, which was not carried out. The dams were killed on day 28 of pregnancy and the foetuses removed by caesarean section.

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:

Chinchilla

Details on test animals or test system and environmental conditions:

Species Rabbits, Chinchilla hybrid
Reasons for selection This system has been selected as standard non-rodent species (according to guidelines) and is internationally recognized for this type of investigation.
Initial age (at mating) between 4 and 6 months
Initial body weight (at test start) 2409 - 4187 g
Acclimation 1 week under test conditions, minimum.
Number of animals 64 mated females, 16 per group
Identification Individually numbered on the inner surface of the pinna by an indelible felt tip pencil.
Accommodation During the whole experiment the animals were housed individually in a "Ehret-Battery".

CONDITIONS:
The experiment was conducted under optimal hygienic conditions (OHC).
The animal room was air-conditioned with:
temperature: 22 +/- 2 degrees centigrade,
relative humidity, 55 +/- 10 %, and
light cycle: 12 hours/day
changes of air approx. 12 times/hour.
Neither insecticides nor chemicals were applied in the animal room with the exception of a disinfectant for floor and wall cleaning once a week.
DIET:
The diet consisted of pelleted standard Kliba 341 rabbits maintenance diet, ad libitum, defined for contaminant level. Certificates for the analyses of contaminants are attached.
WATER:
Tap water was available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: As vehicle distilled water with 0.25 % cremophor EL* (BASF) was applied; all groups 8 ml/kg body weight. * Cremophor EL is a emulgator with a particular good physiological digestibility.

Details on exposure:

Route of administration Oral by intubation. As vehicle distilled water with 0.25 % cremophor EL was applied; all groups 8 ml/kg body weight.
Reason for selection International guidelines recognize the efficacy of this administration method.
Preparation of the pencycuron suspension The suspension of the pencycuron was prepared daily by homogenizer and/ or magnetic stirrer. The vehicle was 0.25 % cremophor in distilled water.
Stability of the pencycuron
Prior to the initiation of the study, a chemical analysis of the stability of the pencycuron in the suspension was performed.
Concentration of the pencycuron suspension
Determination of concentration of the pencycuron suspension was performed during the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical tests for determination of the stability and concentration of the applied suspension were performed during the study. The analytical methods used to confirm the stability of the test and control material were supplied by the sponsor.

Details on mating procedure:

Females were mated with males of proven fertility in the ratio of 1 male 1 female. If practicable, each female was mated twice~ the second time as soon as possible by the same male within about 1 hour. The day of observed mating was day 0 after mating.

Duration of treatment / exposure:

Once daily.

Frequency of treatment:

From day 6 through day 18 after mating.

Duration of test:

30 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

15

Control animals:

yes

Details on study design:

- Dose selection rationale: Preliminary study. The dose levels were chosen based on a preliminary range-finding study (reference not given). In that study concentrations of 100, 300 and 1000 mg/kg body weight were applied and no dose-related effects on dams and foetuses were found.
- Rationale for animal assignment (if not random): Random.

Examinations

Maternal examinations:

Body weight daily; The individual body weight gain was defined as the difference between weights on the first day of treatment and the day of necropsy, respectively. Maternal body weight on the day of necropsy was corrected by subtracting the weight of the gravid uterus from the total body weight. Non-gravid females were excluded from the calculation of the average weight gain.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 of pregnancy and day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- 6, 11, 15, 19, 24 and 28 of pregnancy

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: not specified.

Ovaries and uterine content:

Termination of the study
On day 28 of pregnancy, dams were killed by cervical dislocation and fetuses removed by caesarean section.
The investigations of dams and fetuses were performed in accordance with international standard procedures. Following examination of the organs, ovaries (number of corpora lutea) uterus and uterus contents, the fetuses were identified by uterine position and removed weighed and examined for external abnormalities.

Fetal examinations:

The fetuses were submitted to one of the following procedures individually.
1) Evaluation of the situs of the body cavities (thorax, abdomen , pelvis), sex were noted.
2) Investigations of the cephalic viscera and brain according to the slicing technique of Wilson. Heads of fetuses were fixed in a mixture of trichloroacetic acid and formal.
3) Skeletal investigations of the trunks of fetuses including limbs after clearing in potassium hydroxide and staining with alizarine red; slightly modified technique of Dawson·
Archivation of slices and skeletals
1) Slicing technique of Wilson: After investigations, slices of the fetuses heads are preserved in a mixture of ethyl alcohol and glycerine in the ratio 1,3, one fetus/bottle.
2) Alizarine red skeletal staining: The individual fetuses are preserved in plastic bags after investigation.

Statistics:

Mean values and standard deviations were applied, whenever feasible. For the sex ratio of fetuses, the 2 x 2 chi-square test was applied.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No toxic signs or clinical symptoms were evident in the females of all groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The treatment with the pencycuron caused no mortality. The death of one female in the high dose group (day 20 p. c.) is considered a random occurrence. Macroscopic examination did not reveal the cause of death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Similar mean body weights in all groups during the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Similar mean food consumption in all groups during the treatment period and also during the periods without treatment.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Details on maternal toxic effects:

No treatment related differences between the means of all groups. The differences existing were within the normal range of biological deviations for animals of this strain and age.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related differences between the means of all groups. The higher mean body weight of the high dose group fetuses is caused by a smaller number of fetuses per dam.

External malformations:

no effects observed

Description (incidence and severity):

No findings in all groups with exception of one fetus with partial hyperemic skin in the intermediate dose group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Vehicle control group: One fetus (0.8%), sternebrae 3-5 fused. One fetus (0.8%), distal part of all ribs irregular ossified (hyperplasia) One fetus (0.8%), rib 9+10, right, dorsal fusion.
200 mg/kg group: unilateral, one fetus (0.9%) , double rib.
600 mg/kg group: no findings.
2000 mg/kg group: one fetus (0.7%), rib shortened; one fetus (0.7 %), partial absence of a vertebra, rib shortened; one fetus (0.7%), split vertebra, rib(s) fused and/or partial absent or shortened.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Vehicle control group: Three fetuses, discolored (brown) lung. One fetus, discolored (white) liver, distal area. All fetuses of the same dam.
200 mg/kg group: no findings.
600 mg/kg group: no findings.
2000 mg/kg group: One fetus, aplasia of left ovary.

Other effects:

no effects observed

Description (incidence and severity):

Cephalic viscera investigations by Wilson Technique: No findings in all groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table showing the mean body weight gains during the treatment period.

Dose Group	Mean body weight gain Day 6 - 19 p.c.
vehicle control 0 mg/kg	306 g 9.7%
200 mg/kg	316 g 9.9%
600 mg/kg	361 g 11.4%
2000 mg/kg	288 g 8.9%

Table showing the mean reproduction data of dams.

Dose Group	Corpora lutea	Implantations	Living fetuses	Resorptions (embr.)	Resorptions (fetal)
vehicle control 0 mg/kg	11.2	9.6	8.33	0.53	0.73
200 mg/kg	10.64	8.86	8	0.14	0.71
600 mg/kg	10.88	10	9.19	0.38	0.44
2000 mg/kg	9.19	7.5	6.94	0.25	0.31

Table showing the mean body weights of fetuses (males and females).

Dose Group	Mean body weights of fetuses (males and females)
vehicle control 0 mg/kg	33.10 g = 100 %
200 mg/kg	35.00 g = + 5.7 %
600 mg/kg	33.30 g = + 0.6 %
2000 mg/kg	38.30 g = + 15.7 %

 

Applicant's summary and conclusion

Conclusions:

Acceptability
The number of pregnant animals in the control and the lowest dosing group is just below the minimum of 16 as requested in the now valid guideline (OECD guideline 414, 2001). These deviations are not considered to have major impact on the outcome of the study. The study is considered acceptable for evaluation.

Conclusions
The only effect of pencycuron found in this teratogenicity study in rabbits was an increased foetal weight in the highest dosing group. This effect was only significant if based on litter, not if based on individual animals. Therefore it is not considered as relevant. No treatment related maternal effects were found. Based on these findings both the NOAEL(maternal) and the NOAEL(developmental) are set at the highest concentration tested, 2000 mg/kg bw/day. No indication of a teratogenic potential of pencycuron in rabbits was found.

Executive summary:

PRELIMINARY STUDY
In the preliminary study, 100, 300, and 1000 mg/kg bw were applied. For each dose, two mated females were taken. The results were as follows: No death occurred. No clinical signs or symptoms were evident. No dose-related differences were found in body weight, food consumption, reproduction, malformations and/or anomalies by external investigations, situs investigations of body cavities.

CONCLUSION
Because there were no effects on dams and fetuses in all dose groups, the dose levels for the main study were doubled to 200, 600 and 2000 mg/kg body weight.

The purpose of this study was to assess the embryotoxic and/or teratogenic potential of pencycuron when administered orally to mated chinchilla rabbits (hybrids) at:
Group 1: 0 mg/kg bodyweight/day (vehicle control)
Group 2: 200 mg/kg bodyweight/day
Group 3: 600 mg/kg bodyweight/day
Group 4: 2000 mg/kg bodyweight/day
on days 6 through 18 after mating (16 mated female rabbits per group). These dosages were chosen because of results of the preliminary study.

DATA OF DAMS
Mortality No treatment-related death occurred in the females of all groups
Signs and symptoms No signs of toxicity or clinical symptoms were observed in the females of all groups.
Food consumption No treatment-related differences of food consumption means were evident between all groups.
Body weights No treatment-related differences in body weight means or body weight gain means were evident between all groups.
Reproduction data No treatment-related differences in the means of reproduction data were evident between all groups.

DATA OF FETUSES
Body weights No treatment-related differences of mean body weights were evident between all groups.
Malformations and/or anomalies by external investigations No treatment-related differences were evident between all groups.
Situs investigations of body cavities No treatment-related differences were evident between all groups.
Skeletal investigations No treatment-related differences were evident in all groups.
Cephalic viscera investigations by Wilson Technique No findings in all groups.
Assessment of pencycuron did not reveal any teratogenic or embryotoxic potency in chinchilla rabbits, under the described conditions of this study. The findings and differences observed were within the limit of biological variations of this rabbit strain.

CONCLUSION

The only effect of pencycuron found in this teratogenicity study in rabbits was an increased foetal weight in the highest dosing group. This effect was only significant if based on litter, not if based on individual animals. Therefore it is not considered as relevant. No treatment related maternal effects were found. Based on these findings both the NOAEL(maternal) and the NOAEL(developmental) are set at the highest concentration tested, 2000 mg/kg bw/day. No indication of a teratogenic potential of pencycuron in rabbits was found.