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

Carcinogenicity

Description of key information

Although not a REACH information requirement, the carcinogenicity of pencycuron has been investigated in GLP- and guideline-compliant studies in the rat and mouse in order to meet EU and international data requirements for the assessment of pesticide active substances.  These studies do not provide any evidence that pencycuron is carcinogenic.  The 2010 EFSA Conclusion on pencycuron concludes (based on the same data) that pencycuron has no carcinogenic potential.  The lowest relevant NOAELs for carcinogenicity are stated to be 18 mg/kg bw/d for the 2-year rat study; 43 mg/kg bw/d for the 2-year mouse study.

Test Species/Type	Results	Assessment	Reference
OECD 453 - Combined chronic dietary toxicity/ carcinogenicity study in the rat (50, 500, 5000 ppm)	A NOAEL of 500 ppm (18 mg/kg bw/d) was determined for this study, based on liver and lung effects.  There was no evidence of carcinogenicity.	Supporting study	Shirasu et al  (1981)
OECD 453 - Combined chronic dietary toxicity/ carcinogenicity study in the mouse (50, 500, 5000 ppm)	A NOAEL of 500 ppm (43 mg/kg bw/d) was determined for this study, based on liver in males.  There was no evidence of carcinogenicity.	Supporting study	Shirasu et al  (1981)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Remarks:

Combined chronic dietary toxicity/ carcinogenicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-11-30 to 1981-11-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

ICR

Remarks:

CRJ:CD-1 (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: the treatment was commenced at 5 weeks of age.
- Housing: the mice were housed in aluminum cages with wire-mesh floors (width 215 mm x depth 330 mm x height 180 mm) in groups of 4 animals each. The mice were kept in a barrier-sustained (BS) animal room.
- Diet: Pulverized Diet M, ad libitum.
- Water: ad libitum.
- Acclimation period: one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 1 °C
- Humidity: 55 ± 5%
- Air changes: completely ventilated, 14 times per hour (all fresh air system).
- Photoperiod: Illumination was provided for 14 hours per day

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSES:
Pencycuron was incorporated into the basal diet to obtain the dietary levels of 0, 50, 500, and 5,000 ppm. The dose levels were determined on the basis of the results obtained from a 4-week preliminary feeding study.
Preparation of test diets was performed twice a week as follows: pencycuron was firstly mixed with a part of the basal diet in a mortar and then the mixture was stirred with the basal diet to obtain the prescribed concentration for each dose level by a mixer.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously

No. of animals per sex per dose:

80/sex/dose (including 10/sex/dose for one interim kill)

Control animals:

yes

Details on study design:

The dose levels were determined on the basis of the results obtained from a 4-week preliminary feeding study.

Observations and examinations performed and frequency:

Blood samples of ten animals of each sex from each dose group after 52 weeks and at the termination of treatment were taken for haematological and blood biochemical analysis.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 52 and at termination.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 / sex / dose group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 52 and at termination.
- Animals fasted: No data
- How many animals: 10 / sex / dose group

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 51 and 103.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

Sacrifice and pathology:

Pathological examinations were performed on 10 animals per dose group and sex after 52 weeks and on all surviving animals at the termination of treatment.

Statistics:

Data of body weight, hematology, blood biochemistry, and organ weights were subjected to analysis of variance followed by Student's t test to determine the statistical significance of the results between the control group and the treated groups. Chi-square test was used for the histological data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in any of the test groups of either sex.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no significant differences between the treated groups and the controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 5,000 ppm group, a significantly decreased body weight gain was observed in males. Body weight changes in females in the 5,000 ppm group and in other treated groups of both sexes were comparable to those in the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no marked differences in food consumption between the treated groups and the control group.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no marked differences in food efficiency between the treated groups and the control group.

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

effects observed, non-treatment-related

Description (incidence and severity):

The 5,000 ppm group of both sexes showed a slightly higher values than did the controls throughout the testing period. The total intake (ml/mouse) in males and females was 17% and 19% more than those in the controls, respectively. Water consumption in other treated groups was comparable to the controls.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean corpuscular hemoglobin concentration (MCHC) was significantly increased in the 5,000 ppm group of both sexes and in males in the 500 ppm group after 104 weeks of treatment. But the values were within normal ranges for ICR mice of this strain.
In hematology, significant fluctuations were observed in some parameters in the 500 and 5,000 ppm groups of both sexes. But these changes were not considered to be related to the treatment, since there were no marked changes in any of the parameters and the values were within normal ranges for ICR mice of this strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 5,000 ppm group, males showed a significantly increased blood glucose level and females exhibited a significantly lower value of total protein after 52 weeks of treatment. Females in the 500 ppm group also showed a decreased value of total protein after 52 weeks of treatment. But these values were within normal ranges for ICR mice of this strain.
In blood biochemistry, significant fluctuations were observed in some parameters in the 500 and 5,000 ppm groups of both sexes. But these changes were not considered to be related to the treatment, since there were no marked changes in any of the parameters and the values were within normal ranges for ICR mice of this strain.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no findings in any of the treated groups of either sex.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males in the 5,000 ppm group showed an increased relative organ weight of the testis after 52 weeks and a decreased weight of the kidney after 104 weeks of treatment, which were considered to be associated with the decreased body weight gain.
Females in the 5,000 ppm group after 52 weeks of treatment showed significantly decreased absolute and relative organ weights of the ovary. However, this change was not considered to be related to the treatment, since there was no histological evidence corresponding to the change and the weight of ovary considerably fluctuates depending on the oestrous cycle.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related gross abnormalities were observed in any of the animals killed by design, killed in extremis and/or found dead

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 5,000 ppm group, incidence of diffuse hepatocellular swelling and degeneration was significantly increased in males, but not in females. Although this change was also observed in the control mice, the degree and extent of the lesions were more severe in males in the 5,000 ppm group than those in controls.
Histopathological examinations revealed that the incidence of diffuse hepatocellular swelling and degeneration was significantly increased in males in the 5,000 ppm group. This change was considered to be attributed to pencycuron, since the grade and extent of the lesions were also severer than those in the controls.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Other lesions including tumors observed in the liver were not related to the treatment. In addition, various neoplastic and non-neoplastic lesions were observed in other organs and tissues. But, there were no treatment-related changes in any of the test groups of either sex.
Although various neoplastic lesions were observed in the treated groups of both sexes, there were no treatment-related changes and the incidences and types of the tumors were comparable to those of the spontaneous neoplasms occurring in ICR mice of this strain.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 43 mg/kg bw/d

Dose descriptor:

LOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 465 mg/kg bw/d

Incidences of diffuse hepatocellular swelling and degeneration (number affected/number investigated)

Dose	0		50		500		5000		dr
	m	f	m	f	m	f	m	f
liver - diffuse hepatocellular swelling and degeneration	2-80 (+)	1/79^1 ^(++)	1/80 (+)	2/80 (+ - ++)	6/80 (+ - ++)	1/80 (+ - ++)	12/80^* (+ - +++)	4/80 (+ - ++)	m

dr dose related
¹ one female was killed in extremis in week 13

^* p < 0.05 (chi² test), (+) = slight, (++) = moderate, (+++) = severe

Conclusions:

Chronic oral administration of Pencycuron during 104 weeks to mice significantly decreased the body weight gain of males at a dose of 5000 ppm (statistically significant decreases in bw during the largest part of the study; decreases >10% in first half of study).
At 5000 ppm, the incidence of diffuse hepatocellular swelling and degeneration was significantly increased in males, but not in females. Although this change was also observed in the control mice, the degree and extent of the lesions were more severe in males at 5000 ppm than those in controls.
Since only at 5000 ppm severe diffuse hepatocellular swelling and degeneration was noted in males, and the increase in slight to moderate liver changes also occurred in males and females in the control group, the observed changes at 5000 ppm were considered toxicologically relevant. The increased incidence of diffuse hepatocellular swelling and degeneration found in males of the 500 ppm group was considered marginal. Based on the effects seen in the liver of males at a dose of 5000 ppm and the dose-related increase of these effects, the NOAEL was set at 500 ppm (equal to 43 mg/kg bw/d). Pencycuron did not show carcinogenic potential in mice.

Executive summary:

The chronic toxicity of pencycuron was studied in SPF ICR (CRJ: CD-1) mice of both sexes. Pencycuron was administered to each dose group of 80 males and 80 females at dietary levels of 0, 50, 500, or 5,000 ppm for a period of 104 weeks. Ten animals from each group of either sex after 52 weeks and all surviving animals at the termination of treatment were killed for hematological, blood biochemical and pathological examinations.

5,000 ppm group: Male mice showed a significant decrease in body weight gain and a higher incidence of hepatocellular swelling and degeneration than did the controls. There were no treatment-related changes in female mice.

500 and 50 ppm groups: No treatment-related changes were observed in any of the groups of either sex.

Based on the results, the NOAEL of Pencycuron to ICR (CRJ: CD-1) mice was determined as 500 ppm (equal to 43 mg/kg bw/d).

Reference 2

Endpoint:

carcinogenicity: oral

Remarks:

Combined chronic dietary toxicity/ carcinogenicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-11-16 to 1981-12-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

After a one-week period of acclimatization, the animals were used in this study. At the initiation of the study, 80 males and 80 females were alloted to each dose group. They were housed in wire-meshed stainless cages in groups of 5 males and 5 females each. Each rack and cage were identified by numbered color tape, with the color indicating dose levels. Animals in a cage were individually identified by the marking of a part of the body with saturated picric acid solution in 70 % alcohol.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Pencycuron was incorporated into the basal diet at levels of 0, 50, 500 or 5000 ppm based on the results of the preliminary range finding studies. For each dose level, pencycuron was mixed with a part of the basal diet in a mortar. This mixture and the basal diet were stirred to obtain the test diet of prescribed concentration by a mixer.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

24 months

Frequency of treatment:

ad libitum

Dose / conc.:

0 ppm

Dose / conc.:

50 ppm

Dose / conc.:

500 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

56

Control animals:

yes

Details on study design:

- Dose selection rationale: based on range finding studies.
- Rationale for animal assignment (if not random): random.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly until week 27, then biweekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured on a cage basis twice a week for 8 cages per group during the treatment.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes. Water consumption was measured on a cage basis twice a week for 4 cages per group during the treatment.
- Time schedule for examinations: 2 / week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 27, 52 ( 53 in females ), 78 weeks and 104 weeks.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: 8. 10 at 104 weeks.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 27, 52 ( 53 in females ), 78 weeks and 104 weeks.
- Animals fasted: No data
- How many animals: 8. 10 at 104 weeks.

URINALYSIS: Yes
- Time schedule for collection of urine: 27, 52 ( 53 in females ) and 78 weeks.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

Sacrifice and pathology:

The animals were killed by incision of the diaphragm and vena cava for autopsy. The rest of the survivors after 104 weeks of treatment was also anesthetized with ether after collection of urine samples, and sacrificed by incision of- the posterior vena cava and diaphragm for autopsy. Autopsy of the surviving animals was performed at 728 (104 weeks), 729 (105 weeks) and 730 (105 weeks) days of treatment in males , and at 728 (104 weeks) , 729 (105 weeks) and 732 (105 weeks) days of treatment in females. Animals found dead during the testing period were autopsied as soon as possible after the discovery in order to minimize the autolysis, and those organs described above were sampled as possible for histopathological examination. After these organs and tissues were fixed in 10 % formalin, paraffin sections of these were processed by a conventional method, stained with hematoxylin and eosin, and subjected to microscopic examination. In addition, following stainings were used when necessary; azan staining and fat staining. For the histopathological examination, specimens of the following organs and tissues in addition to those weighed were preserved in 10 % formalin; salivary gland, esophagus, stomach, pancreas, duodenum, jejunum, ileum, cecum, colon, urinary bladder, epididymis, prostate, seminal vesicles, coagulating gland, uterus, eyes, intraorbital lacrymal gland, bone and bone marrow (sternum and femur), sciatic nerve, and gross lesions. In addition to the organs and tissues described above, the following organs and tissues were sampled as possible from the animals killed on schedule at 52 (53 in females) weeks of treatment and thereafter, or all animals subjected to moribund sacrifice in compliance with the guidelines proposed by Environmental Protection Agency of the United States of America; oral cavity (oral mucosa), tongue, pharynx, larynx, trachea, aorta (thoracic region), mammary gland (abdominal region), skin (lumbodorsal region), lymph node (cervical and mesenteric), head (including nasal cavity, paranasal sinus and middle ear), tibio-femoral joint, spinal cord (cervical, thoracic and lumbar regions).

Other examinations:

Urinalysis was carried out on the urine samples collected after 26 (25 in females), 51 (52 in females) 77 and 103 weeks of treatment by pressing the lumbodorsal region of the animals. Specific gravity of the urine was determined with a Hand Refractometer (Atago Optical Instrument Inc., Hon-cho, Itabashi-ku, Tokyo), and pH, protein, glucose, ketones and occult blood were examined by

Statistics:

Student's t-test was used for the quantitative analysis of significant differences between the treated and control groups in the parameters of body weight, hematology, blood biochemistry, and organ weight. Fischer's exact test was applied to estimate the significance of differences between the treated groups and the control group for the incidences of histological lesions and tumors.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormalities ascribed to pencycuron in any of the treated groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no differences between the treated groups and the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight Males in the 5000 ppm group showed a slight but a significant inhibition of body weight gain from 1 to 16 weeks after the start of the study. This change was not found thereafter. Mean body weights of females in this group were consistently lower than those of the control, which were significantly different throughout the test period. In the other treated groups, there were no marked differences from the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Calcium: Males in the 5000 ppm group showed a significant increase of calcium concentration at 27 and 52 weeks of treatment. Such a change was seen in males of the 500 ppm group only at 27 weeks of treatment. Total cholesterol: Females in the 5000 ppm group showed a significant increase of cholesterol concentration at 53, 78 and 104 weeks of treatment. Total protein: Male rats in the 5000 ppm group exhibited a significant increase in total protein concentration at 27, 52 and 78 weeks of treatment. This change was also observed in males of the 500 and 50 ppm, groups in relation to the dietary levels at 27 weeks of treatment. In these dosage groups, there were no change in protein fractions corresponding to the increase of total protein. GPT: Males in the 5000 ppm group showed a significant decrease of GPT at 27, 52 and 78 weeks of treatment, but this change was not seen at 104 weeks of treatment.
Besides these changes, significant fluctuations were seen in LDH in male treated groups at 27 and 52 weeks of treatment, glucose in 5000 ppm group males at 27 and 52 weeks of treatment, GOT in 5000 ppm group males at 27 and 52 weeks, and GOT in 5000 ppm group females at 53 weeks of treatment. However, these are not considered to reflect any specific toxic effect, since enzyme activities were altered with the time when the blood was kept at room temperature after sampling.
In the other parameters, there were no changes related to the dietary levels and / or the duration of treatment, although statistically significant deviations from the control values were seen occasionally in the treated groups.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no abnormalities in the rats treated with pencycuron in macroscopic examination.
In organ weight data, significant changes related to the dietary levels were found in the liver, kidney and adrenal.
Liver: Male rats in the 5000 ppm group showed a significant increase in absolute and relative weights of the liver at 27, 52 and 78 weeks of treatment.
Females in this group exhibited higher relative liver weights at each of the testing periods. In the 500 ppm group, significant increases of absolute and relative weights were observed in the liver of males at 27 and 78 weeks of treatment. In addition, a slight but not significant increase in the liver weight also were seen at 52 weeks of treatment.
Kidney: Female rats in the 5000 ppm. group showed a significant increase in relative weight of the kidney at 104 weeks of treatment.
Adrenal: Male rats in the 5000 and 500 ppm groups showed a significant increase in absolute and relative adrenal weights at 27 weeks of treatment.
This change was not seen thereafter.
In the other organs, there were no changes related to the dietary levels and / or the duration of treatment, although several significant fluctuations were seen in the treated groups.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In non-neoplastic lesions, several morphological abnormalities were detected in the liver and adrenal in males, and the liver and kidney in females of the 5000 ppm group. Details of the lesions in these organs were as follows: Liver: In the 5000 ppm group of both sexes, the incidences of hepatocellular hypertrophy, nodular hepatocellular hyperplasia and diffuse hepatocellular fatty change were significantly increased as compared with the controls. Hepatocellular hypertrophy was observed diffusely and characterized by anisokaryosis with cytoplasmic swelling of hepatocytes. But this lesion was generally very slight in degree. The incidence of this lesion showed a significant increase or increasing tendency as compared with those in the control groups throughout the test period. The highest incidence of this lesion in each stage was seen at 27 weeks of treatment. In males of this group, congestion of the liver was observed together with hepatocellular hypertrophy in all animals (8 cases) examined at 27 weeks of treatment. Significant increases in the total incidence of nodular hyperplasia and diffuse fatty change of hepatocyte were attributable to the higher incidence of these changes in the animals killed at termination of treatment. This nodular hepatocellular hyperplasia consisted of proliferative foci or areas extending to a few lobules and was accompanied by enlargement of the eosinophilic cytoplasm. Thus, the histological morphology of this lesion was considered to be very mild hyperplasia and could be clearly distinguished from hepatocellular adenoma. The term of "diffuse hepatocellular fatty change" included" multifocal changes as well as diffuse changes. This change was observed frequently in the area of nodular hepatocellular hyperplasia. Adrenal: In male rats of the 5000 ppm group, the incidence of diffuse cortical fatty change was significantly increased at 27 weeks of treatment. But there were no significant differences between the treated groups and the control group in the incidence of this lesion thereafter. Morphologically, this change was recognized as an appearance of large intracytoplasmic lipids in the cortical cells of zona fasciculata, and usually observed bilaterally. In females of this group, the incidence of this lesion was not significant as compared with that in the control. Kidney: In histopathological examination on the animals killed at termination, the incidence of nephrosis was significantly increased in females of the 5000 ppm group. This lesion is seen frequently in senile rats, and is characterized by sclerosis of glomerulus, atrophy or dilatation of renal tubules, and appearance of hyaline casts. In females of this group, the incidence of this lesion was not significant as compared with the controls. Besides these changes, a wide spectrum of different types of spontaneous lesions was observed in the treated and the control groups, but the incidence of such lesions in the treated groups showed no significant increases clearly attributable to the feeding of pencycuron.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no significant differences in the incidences, types and onset of neoplastic lesions between the treated groups and the control group.

Relevance of carcinogenic effects / potential:

No carcinogenic potential found.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 18 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 186 mg/kg bw/day.

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

A 24-month chronic toxicity study with pencycuron in Fischer rats was carried out by incorporating it into the diet at the levels of 0, 50, 500 or 5000 ppm. At 6, 12 and 18 months of treatment, 8 animals of either sex from each group were sacrificed to be examined. In general condition, there were no abnormalities ascribed to pencycuron in all treated groups. There were no differences between the treated and the control groups in mortality. Males in the 5000 ppm group showed a slight inhibition of body weight gain at the early stage of the study, but this change was not seen thereafter. Females in this group showed a slight inhibition of body weight gain throughout the test period. There were no differences between the treated and the control groups in the values of food consumption, water consumption and food efficiency. Accordingly, it was apparent that the inhibition of body weight gain seen in the 5000 ppm group of both sexes was not attributable to the decrease in food consumption, although., inhibition was not so evident to cause food efficiency decrease. Therefore, these findings might indicate that the inhibition of body weight gain was a toxic manifestation caused by the feeding of pencycuron. No abnormalities related to pencycuron were found in urinalysis and hematology.
In blood biochemistry, total cholesterol values in female rats of the 5000 ppm group were increased significantly at the late stage of the study. this change was considered to be associated with the higher incidence of nephrosis in this group compared with the controls at the same stage. A significant increase of total protein values was seen in males of the 5000 ppm group at 27, 52 and 78 weeks of treatment and also in males of the 50 and 500 ppm groups at 27 weeks of treatment. However , the changes of total protein in these groups were not considered to be a specific toxic effect of pencycuron, since the values were not so high as to exceed obviously the normal range of those in untreated rats ' and no consistent fluctuations were seen in any of protein fractions. Calcium values were significantly increased in males of the 5000 ppm group at 27 and 52 weeks of treatment and in males of the 500 ppm group at 27 weeks of treatment. In general, the values of calcium in serum varies between narrow limits. Therefore, slight variation exceeding the normal range is considered to be of great biological significance. However, higher values of total protein in serum are known to induce simultaneously higher calcium values, since calcium in serum not only exist as an ion but also is chelated with serum protein. In the present study, higher values of calcium in the higher dosage groups were seen concomitantly with the increase of serum protein. Therefore, the higher calcium values are considered to be a secondary effect due to the increased serum protein.
In organ weight data, males in the 5000 and 500 ppm group showed a significant increase in absolute and relative weight of the liver. The changes in these groups were prominent in the early stage of the study, but there were no differences in the liver weight between these groups and the controls at 104 weeks of treatment. Females in the 5000 ppm group revealed a higher values of relative liver weight at each of the examination time, and the degree of increase in the relative liver weight became more obvious at the latter stage. This increase of the liver weight in this female group might be attributable to the inhibition of body weight gain observed throughout the test period. However, a significant increase in the incidence of hepatocellular hypertrophy was observed in the 5000 ppm group of both sexes from the early stage of the study. Moreover, males of this group, killed by design at 27 weeks, exhibited congestion of liver. Therefore, the increases of the liver weights in the 5000 ppm group of both sexes were also considered to be related to these morphological changes induced by pencycuron. Similar increase of the liver weight in males of the 500 ppm group was seen concurrently with the increased liver weights in the 5000 ppm group, and the degree of these changes appeared to be dose-dependent, although no histological abnormalities corresponding to the increased liver weight were observed in the 500 ppm group. These evidences in males of the 500 ppm group might suggest that pencycuron exerted a very slight effect which was difficult to detect microscopically.
A significant increase in the relative kidney weight was observed in females of the 5000 ppm group at the late stage of the study. At this stage, the incidence of nephrosis was significantly increased in this female group. Although nephrosis is a spontaneous lesion which appears with increasing age, the degree of this lesion in the 5000 ppm group was more marked than that in the control. Besides these pathological findings, blood biochemical examination revealed higher values of cholesterol in this group at the late stage. These findings might indicate the possibility that pencycuron exert mild toxic effects in the kidney of females in the 5000 ppm group.
Absolute and relative weights of the adrenal in male rats treated with more than 500 ppm showed higher values as compared with the controls at 27 weeks of treatment. Histological examination revealed a significant increase in the incidence of cortical fatty change in the adrenal of males of the 5000 ppm group which were sacrificed at this stage. The chemical intake in the treated groups during the first 27 weeks of treatment was greatly higher than that in the later stage of the study, reflecting an increased food intakes in this growth stage. Therefore, increases of lipid droplets in the cortical zone of the adrenal and the adrenal weight might be toxic changes induced by pencycuron.
However, these changes had not been observed with prolongation of the feeding. Similar changes were also reported in the animals subjected to the condition of stress. From these points of view, causal relationship between the changes of the adrenal and the feeding of pencycuron could not be ascertained. A significant increase in the incidences of nodular hepatocellular hyperplasia in the liver was seen in males and females of the 5000 ppm group at the late stage of the study. This change is known to occur spontaneously with increasing age, and are clearly distinguished from hepatocellular adenoma. In this study, this hyperplasia was not severe enough to produce the increased liver weight, and the areas of this change were usually limited in a few lobules. In addition to this change, hepatocellular hypertrophy also occurred in this group at a significantly high incidence from the early stage. This hypertrophy may imply that pencycuron exert a mild toxic effect to the hepatocyte. Therefore, it is assumed that the hepatocellular hypertrophy may be related to the occurrence of hepatocellular hyperplasia.
As for neoplastic lesions, there were no differences between the treated groups and the control group in the incidences, types and onset of the tumor. Neoplastic lesions observed in this study were similar to those in the control rats in the other chronic toxicity studies conducted at this Institute and also to those in Fischer rats from published literatures.

Conclusions:

Acceptability
The study is considered acceptable. The adequacy of the top dose was further investigated. A statistical significant decrease in body weight was noted in males in week 1-16, but the reduction was not more than 3%. In females a statistically significant decrease in body weight was noted during the entire study (decreases >10% during the second half of the study). Although the effect on body weight is limited in males, in combination with the effects on liver and kidney (increases in relative liver weights were 10-20% in both sexes during several stages of the study) the top dose in this study is considered adequate.

Conclusions
A reduced body weight in the high dose group (5000 ppm) of both sexes was seen. As this reduction could not be attributed to changed food consumption, as this was normal as compared to the control group, it was considered to be related to treatment. In females, a significant increase of total cholesterol was observed in the high dose group (5000 ppm). In both males and females, some kidney effects were observed at the high dose level. The liver weights in males and females were increased at the high dose level of 5000 ppm. Liver weight increase was also observed in males at 500 ppm in week 27 and 78, but occurred not consistently throughout the study period and were therefore not considered toxicologically relevant. The increased liver weights at 5000 ppm were associated with histopathological lesions (hypertrophy, hyperplasia) in the liver (see further evaluation below). In males, a dose related increase in bronchitis/brochiolitis was observed, which clearly reached statistical significance in the highest dose group. As these lung effects were restricted to males, even at the highest dose, and were not observed in any other study, they may be a chance finding. Based on the observed liver effects at 5000 ppm, the NOAEL is set at 500 ppm (18 mg/kg bw/d). Pencycuron did not show carcinogenic potential in rats.

Non-neoplastic lesions in the liver included hepatocellular hypertrophy, nodular hepatocellular hyperplasia and diffuse hepatocellular fatty change. An increased incidence was clearly noted at 5000 ppm. At 500 ppm, the observed incidence in nodular hepatocellular hyperplasia was considered comparable to controls. Based on an overall evaluation of the data, it can be concluded that the observed incidence in nodular hepatocellular hyperplasia at 500 ppm, should be considered foci of hepatocellular alteration, eosisnophilic type, and occurred within the expected ranges.

Executive summary:

This combined chronic toxicity/carcinogenicity study with pencycuron was carried out in specific pathogen free (SPF) rats of Fischer strain. Pencycuron was administered orally by incorporating it into the basal diet for a period of 24 months at the following 4 dietary levels; 0, 50, 500 or 5000 ppm. At the initiation of the study, 80 males and 80 females were alloted to each dose group. At 6, 12 and 18 months of treatment, 8 animals of either sex from each group were sacrificed to be examined. At the termination of the study, all surviving animals were similarly handled. During the test period, moribund or dead animals were examined promptly after discovery.
In the 5000 ppm group, a significant decrease of body weight gain was seen only at the early stage of the study in males and throughout the test period in females. While significant fluctuations in blood biochemical parameters were detected at each of the testing periods in both sexes, higher total cholesterol values attributable to pencycuron were observed in females at the late stage of the study. Significant increases of absolute and relative weights of the liver were seen in males from the early to middle stage. In addition, absolute and relative weights of the adrenal were significantly increased in males. In females, the relative organ weights were increased in the liver at each of the testing periods and in the kidney at the late stage. In histopathological examination, the incidence of hepatocellular hypertrophy was significantly increased in both sexes at the early stage. Congestion of the liver was. observed together with the hypertrophy in all males examined at this stage. The incidences of nodular hyperplasia and diffuse fatty change of hepatocyte in males and females were significantly higher than those in the controls at the late stage. In addition, there were significantly higher incidences of diffuse cortical fatty change of the adrenals in males at the early stage and of nephrosis in females at the late stage.
In males of the 500 ppm group, increases of absolute and relative liver weights were seen from the early to middle stage of the study. Males also exhibited higher values of absolute and relative adrenal weights. There were no histological abnormalities related to the feeding of pencycuron in both sexes.
Animals in the 50 ppm group showed no abnormalities.

The NOAEL was 500 ppm and there was no evidence of carcinogenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

18 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The carcinogenicity of pencycuron has been investigated in GLP- and guideline-compliant studies in the rat and mouse.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not required in the absence of any evidence of genotoxicity or carcinogenicity for pencycuron.

Justification for classification or non-classification

Although not a REACH information requirement, the carcinogenicity of pencycuron has been investigated in GLP- and guideline-compliant studies in the rat and mouse in order to meet EU and international data requirements for the assessment of pesticide active substances. These studies do not provide any evidence that pencycuron is carcinogenic. The 2010 EFSA Conclusion on pencycuron concludes (based on the same data) that pencycuron has no carcinogenic potential. The lowest relevant NOAELs for carcinogenicity are stated to be 18 mg/kg bw/d for the 2-year rat study; 43 mg/kg bw/d for the 2-year mouse study.  Furthermore, it is noted that pencycuron did not show any genotoxic potential in an appropriate battery of tests in vitro and in vivo, covering all endpoints.

In the absence of any indication of carcinogenicity, classification for carcinogenicity is not required for pencycuron in any category under CLP.