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

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2004-03-31 to 2006-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MAFF GLs: 12-Nousan No. 8147

Version / remarks:

2000, 2001

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

Specific details on test material used for the study:

colorless powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU, SPF-bred

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females were nulliparous and not pregnant.
- Age at study initiation: about 7 weeks.
- Housing: rats were kept conventionally and individually in polycarbonate cages Type HI h on low-dust wood granulate.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 5%
- Air changes: > 10 times per hour.
- Photoperiod: 12 hours rhythm.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

blended with diet

Details on exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION
The test substance was mixed in the diet at the appropriate concentrations at room temperature and maximally used over the stability period determined by analytical investigations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of the study analytical determinations of stability and homogeneity in diet were performed. During the study analytical determinations of concentration and homogeneity of the test substance in diet were performed at the beginning, once in the mid-time and at the end of the study.

Duration of treatment / exposure:

13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

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:

12/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:
Dose levels were selected according to the results obtained in previous dietary exposure studies in rats.

In these studies, no compound-related clinical signs were observed at dietary levels including 10000 ppm in a subacute, and sub-chronic study, 5000 ppm in a chronic study, and 10000 ppm in two multi-generation studies. In summary, no treatment related clinical signs were observed in these studies and there was no evidence of neurotoxicity at dietary levels of 10000 ppm. Therefore, in the present study, the limit dose of 1000 mg Pencycuron/kg body weight/day was tested.

In the above-described studies the concentration of 10000 ppm was considered as to correspond to a dose of 1000 mg test substance/kg body weight, which is stated as limit dose in the guidelines. Contrariwise, based on food consumption and body weight measurements of Wistar rats in the 90-day studies, the dose of 1000 mg/kg/day corresponds to approximately 15000 ppm. Based on these results, the proposed doses for the present study were 0, 500, 2500, and 15000 ppm for males and females.

Examinations

Observations and clinical examinations performed and frequency:

1- Morbidity and Mortality:
Inspections on mortality and morbidity of the animals were done twice daily (once daily on weekends and public holidays).

2- Clinical Signs and Open Field Observations:
A clinical examination (with handling) without observation outside the home cage was made once daily on days on which neither a careful clinical examination including observation outside the home cage in a standard arena, nor a FOB investigation was performed. Any clinical signs and abnormalities including changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, changes in response to handling, stereotypes and bizarre behavior were recorded with time of onset, location and grading.

In addition, in weeks without FOB an observation outside the home cage in a standard arena (open field observation) was made once a week.

If animals became ill, they were set apart, observed more frequently and sacrificed prematurely, if death seemed imminent.

3- Determination of Body Weights:
The body weights of the individual experimental animals were determined just before the first treatment and weekly thereafter up to scheduled necropsy. Body weights were also recorded immediately before the scheduled necropsies, for calculation of relative brain weight.

4- Determination of Food, Water and Active Ingredient Intake:
Food/water intakes were determined weekly individually from the start of the study, based on the difference between the weight of the food/water made available and the weight of that non consumed. The consumption per animal per day and per kg body weight per day was determined from these primary data.

5- Ophthalmology
Ophthalmological examination was made prior to administration of the test substance on all animals of all groups. If necessary, animals showing effects on eyes were replaced at this term. At termination of the study, the examination was made on all animals of the control and high dose group.

Neurobehavioural examinations performed and frequency:

Functional Observational Battery and Motor Activity Testing:
All animals that were assigned to the study were tested using the functional observational battery including grip strength measurements and motor activity on five occasions: once during the week before initiating exposure and again during weeks 2, 4, 8 and 13. On the test day, the appropriate animals were placed in the correct sequence that had been established for testing on that day. Animals were then transferred to the room where testing took place and allowed to acclimate with minimal disturbance for at least 15 minutes until testing. The dose group identification was concealed prior to testing to ensure that testing would be conducted without knowledge of the group assignment. Sets of 12 animals (maximum) were evaluated individually using the FOB and then, approximately one hour after the testing of the last animal in the set had been finished in the FOB, all 12 rats were placed individually into the mazes for measurement of their activity.

Each week testing was staggered over two days for each sex to accommodate the schedule for behavioral testing. Males and females were tested on separate days, with the open field and mazes cleaned during the ensuing interval to reduce the residual scent from the other sex.

The functional observations closely followed the battery of tests described by Moser [Screening Approaches to Neurotoxicity. A Functional Observational Battery. J. Amer. Coll. Toxicol. 8(1), 85-93, 1989], with each animal tested individually. Scoring criteria and explicitly defined scales were used to rank the severity of observations that do not readily lend themselves to quantitation. The procedures used to determine landing foot splay and grip strength are based on established methods. The technicians conducted the FOB blind with respect to the animal's group assignment. Observations for all animals were routinely performed by the same observer in study week 0, 2 and 13 and by another observer in study week 4 and 8. Measurements (e.g., grip strength and foot splay) were performed by either the observer or a second person. Inter-observer reliability has been established in order to allow a second person to perform either the observations or measurements, ensuring the consistency of the results of each technician. Studies have been conducted with carbaryl and untreated rats to establish the sensitivity, reliability, and validity of these test procedures and to serve as a historical control. All data was entered into a computer; in the event the computer is not available, data were recorded manually and later entered into the computer. Due to technical reasons, for FOB additional FOB numbers were used.

The following observations/examinations were performed:
* Home cage observations: posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations, other (if present).
* Observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation, salivation, nasal discharge, stains, other (if present).
* Open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalizations, arousal, rearing, defecation, urination, other (if present).
* Manipulative test: pupil response, approach response, touch response, pupil size, auditory response, tail pinch response, righting reflex, forelimb and hindlimb grip strength, foot splay, body weight, body temperature (degrees Celsius), free text information (if necessary).

Motor and locomotor activities were measured by testing animals individually for 60 minutes in the figure-eight maze. The figure-eight maze was selected as an established and widely used automated activity device that can be used to detect both increases and decreases in activity. Each maze consists of a series of inter-connected alleys (approximately 10x10 cm in cross-section) converging on a central arena and covered by transparent plastic. Each maze has eight infrared emitter/detector pairs (three in each of the figure eight alleys and one in each of the blind alleys) to measure activity; each time a beam is interrupted, an activity count is registered. The floor of each maze rested above absorbent paper which was changed at the end of each day. A Columbus Instruments Universal Maze Monitoring System and a personal computer were used for automated data collection. Broad-spectrum background noise (approximately 70 ± 3dB(A)) was provided throughout the test to minimize acoustical variations during testing. A white noise generator was used to produce the background noise which was amplified by an audio-mixer amplifier and presented through two speakers that were positioned upside the test area. The uniformity of light intensity (66 ± 14 Lux) over each of the mazes was verified daily. Studies with untreated animals and with rats treated with reference substances that increase (triadimefon) and decrease (chlorpromazine) motor activity established the sensitivity, reliability, and validity of these test procedures.

Motor and locomotor activities were examined as activity for the 60-minute session and activity during each ten-minute interval. Motor activity was measured as the number of beam interruptions that occur during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the rat relocated in the maze and interrupted another beam. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and (histo)pathology:

* Necropsy:
All animals placed on study were subjected to a complete gross necropsy. The necropsy involved an examination of all organs (including the brain), body cavities, cut surfaces, external orifices, and surfaces. All gross abnormalities were recorded.

The first six males and six females at each dietary level were selected for perfusion and collection of tissues, with replacement, as necessary, when the perfusion was considered inadequate or in the event that animals died before study termination or were injured in a manner that would interfere with interpretation of study results.

These animals were deeply anesthetized using an intraperitoneal dose of pentobarbital (approximately 50 mg/kg) and then perfused via the left ventricle with a sodium nitrite (in phosphate buffer) flush followed by in situ fixation using Universal fixative (1.0% (w/v) glutaraldehyde and 3% (w/v) EM-grade formaldehyde in phosphate buffer. The eyes and the brain with the optic nerves, the Gasserian ganglia, the spinal column (with spinal cord, spinal nerve roots and cervical and lumbar dorsal root ganglia), both hindlimbs (with muscles and nerves), the physical identifier and organs with gross findings were taken from each animal and placed in 10% buffered formaldehyde.

The remaining animals, including ones that must be sacrificed prior to study termination, were necropsied after exsanguination under deep ether anesthesia. The organs were fixed in 10% formaldehyde.

* Brain Weights:
From all animals selected for perfusion and collection of tissues, the brain was weighed upon removal from the skull, prior to placement into formalin, and the brain:body weight ratio was calculated by normalization to 100 g body weight (individual brain weight divided by body weight times 100). The body weight used as a basis for this calculation was determined immediately prior to necropsy of the pertinent animal.

* Histopathology:
The fixed material was retained. The tissues from control and high-dose animals were examined.

Statistics:

The statistical evaluation of data related to body and organ weights as well as food and water intake were performed using SAS® routines.
Statistical evaluations on body weight and organ weight data were done using the Dunnett-test in connection with a variance analysis. A Kruskal-Wallis-Test with a Steel-Test were performed for analyzing data of food and water intake.
All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
For the statistical evaluation of samples drawn from continuously distributed random variables three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variables in question are approximately normally distributed with equal variances across treatments, the Dunnett test were used, if heteroscedasticity appeared more likely, a p value adjusted Welch test were applied. If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e., the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.
Statistical evaluation of FOB-data and motor activity were performed using software from SAS. With the exception of Bartlett's test, the level of significance will be set at p<0.05. In most cases data will be visually screened for potential effects prior to being subjected to statistical analysis.
See "Any other information on materials and methods incl. tables" for the rest of the statistics.

Results and discussion

Results of examinations

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain of males and females at 2500 and 15000 ppm was retarded during week one of the study. Since body weight gain of all male and female dose groups over the entire study period was similar to body weight gain of male and female control groups, the retarded body weight gain during week 1 is considered to be transient and not to be adverse. This effect could be possibly related to palatability.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake of females at 2500 ppm and both males and females at 15000 ppm was increased. In addition, water intake of females at 2500 and 15000 ppm was increased. The effects on food and water intake are considered not to be of a toxicologically relevant adverse nature.

Ophthalmological findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Several gait abnormalities were observed during the study, including spastic gait, high stepping gate, uncoordinated gait, stilted hindlimbs and walking on tiptoe. These effects were not observed in control animals; however, these effects were transient and occurred in the absence of concomitant findings such as hindlimb foot splay and histopathology findings. Furthermore, no clear dose-dependent relationship was observed for these effects, except for spastic gait in females in week 5. Therefore, the gait abnormalities are considered not to be of a toxicologically relevant adverse nature.

Both motor and locomotor activity appeared increased for both males and females of all dose groups during the study period, however, these changes did not occur in a dose-dependent magnitude. Furthermore, motor and locomotor activities were within the range of historical reference except for the activities of half of the females at 15000 ppm. Although achieving statistical significance at week 2 and 8, the increased activities in females at 15000 ppm did not progress in incidence or severity with time.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes in brain weights were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Several females at 15000 ppm developed distinct liver lobulation which was not examined histopathologically, since it was considered to be irrelevant for the assessment of neurotoxicity.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Macroscopic post mortem examination and histopathological examination of the central and peripheral nervous system did not reveal any abnormalities related to neurotoxicity.

Histopathological findings: non-neoplastic:

not examined

Details on results:

No mortality occurred. Body weight gain of males and females at 2500 and 15000 ppm was retarded during week one of the study. Since body weight gain of all male and female dose groups over the entire study period was similar to body weight gain of male and female control groups, the retarded body weight gain during week 1 is considered to be transient and not to be adverse. Food intake of females at 2500 ppm and both males and females at 15000 ppm was increased. In addition, water intake of females at 2500 and 15000 ppm was increased. The effects on food and water intake are considered not to be of a toxicologically relevant adverse nature.

Several gait abnormalities were observed during the study, including spastic gait, high-stepping gate, uncoordinated gait, stilted hindlimbs and walking on tiptoe. These effects were not observed in control animals; however, these effects were transient and occurred in the absence of concomitant findings such as hindlimb foot splay and histopathology findings. Furthermore, no clear dose-dependent relationship was observed for these effects, except for spastic gait in females in week 5. Therefore, the gait abnormalities are considered not to be of a toxicologically relevant adverse nature.

Both motor and locomotor activity appeared increased for both males and females of all dose groups during the study period, however, these changes did not occur in a dose-dependent magnitude. Furthermore, motor and locomotor activities were within the range of historical reference except for the activities of half of the females at 15000 ppm. Although achieving statistical significance at week 2 and 8, the increased activities in females at 15000 ppm did not progress in incidence or severity with time.

No treatment related effects were observed at ophthalmological examinations and no changes in brain weights were noted. Macroscopic postmortem examination and histopathological examination of the central and peripheral nervous system did not reveal any abnormalities related to neurotoxicity. Several females at 15000 ppm developed distinct liver lobulation which was not examined histopathologically, since it was considered to be irrelevant for the assessment of neurotoxicity.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Remarks on result:

other: Equivalent to: 181 mg/kg bw/day in males and 275 mg/kg bw/day in females. Gait abnormalities were seen in all treated groups but were not considered adverse.

Any other information on results incl. tables

Results summary

Dose (ppm food)	0		500		2500		15000		dr
	m	f	m	f	m	f	m	f
Mortality	no mortality
Clinical signs^1 - Spastic gait (week 5-6) - High-stepping gait (week 6-10) - Uncoordinated gait (week 9-10)	0/12 0/12 0/12	0/12 0/12 0/12	2/12 2/12 1/12	2/12 4/12 1/12	3/12 3/12 2/12	3/12 1/12 0/12	5/12 1/12 3/12	9/12 6/12 0/12	m,f
Body weight gain					dc^2	d^2	dc^2	dc^2	m,f^2
Food intake						i^3	ic	ic	m,f
Water intake						ic		ic	f
Functional observation battery^4 - Spastic gait (week 5) - Stilted hindlimbs (week 8^5) - Walking on tiptoe (week 13)	0/12 0/12 0/12	0/12 0/12 0/12	1/12 4/12 1/12	2/12 8/12^* 0/12	3/12 3/12 0/12	3/12 3/12 0/12	5/12 2/12 0/12	9/12 5/12 0/12	m,f
Motor activity - Motor activity - Locomotor activity								i^6 i^7
Ophthalmology	no treatment-related findings
Brain weights	no treatment-related findings
Pathology macroscopy microscopy	no neurotoxicity-related findings^8 no treatment-related findings

dr dose related
dc/ic statistically significantly decreased/increased compared to the controls

d/i decreased/increased, but not statistically significantly compared to the controls
1 Hair loss, a thin haircoat, an injury and/or a wound were also observed among animals.
2 During week 1 only, for the remaining study duration, body weight gain was similar for controls and dose groups.
3 Achieving statistical significance over Days 50-57 only.
4 No treatment-related findings were observed for other functional observations such as grip strength, foot splay and reflex testing.
5 Also observed in week 4 for one male and one female at 500 ppm and in week 13 for one male at 2500 ppm and one female at 15000 ppm.
6 Achieving statistical significance in week 2.
7 Achieving statistical significance in week 8.
8 5/12 females at 15000 ppm developed distinct liver lobulation, which was not examined histopathologically.
* statistically significant (p≤0.05)

Applicant's summary and conclusion

Conclusions:

In this sub-chronic dietary neurotoxicity study, several treatment-related effects were observed which are considered not to be of a neurotoxicologically relevant adverse nature. Based on the increased motor and locomotor activities in females at 15000 mg/kg food, the NOAEL is set at 2500 mg/kg food (equal to 181 mg/kg bw/day in males and 275 mg/kg bw/day in females).

Executive summary:

Groups of 12 male and 12 female Wistar rats of the strain HsdCpb:WU were administered Pencycuron at target concentrations of 0, 500, 2500 and 15000 ppm for 13 weeks in the diet.
Averaged over the study period the following doses of Pencycuron were taken up daily: 34.94, 180,64, and 1167.60 mg/kg body weight in males and 51.16, 274.80, and 1835.54 mg/kg body weight in females. Whereas the high dose in males only slightly (about 16%) exceeded the proposed limit dose of 1000 mg/kg, in females the high dose exceeded the proposed limit dose nearly twice.
The test substance was homogeneously distributed and chemically stable for at least 15 days in the diet under room temperature as well as deep frozen at -18°C, and its concentration in the formulation agreed to the target values within defined limits.
Animals were observed twice a day (once daily on weekends and public holidays) for mortality and morbidity. A clinical examination (with handling) without observation outside the home cage was made once daily on days on which neither a careful clinical examination including observation outside the home cage in a standard arena, nor a functional observational battery (FOB) investigation was performed. FOB and motor activity investigations were performed before the start of the study and in week 2, 4, 8, and 13. Body weights and food and water consumption were determined weekly. Furthermore, ophthalmological investigation was performed. A gross necropsy with brain weight determination and sampling of selected tissues was performed. From the sampled tissues the entire brain and spinal cord, eyes/retinae/optic nerves, gastrocnemius muscle, spinal nerve root/dorsal ganglia, Gasserian ganglia and peripheral nerves (sciatic, tibial, and sural) were microscopically examined.
No animal died throughout the entire study period in both sexes.
Body weight development was retarded at the start of the study in both sexes at 2500 ppm and above. This was a transient effect as the gain in body weight over the whole study period was comparable for the different dose groups in males and females, respectively. Therefore, this effect was considered not to be adverse.