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

Endpoint summary

Description of key information

Studies of repeated dose oral toxicity for pencycuron are available in the rat (90-day, 2-year), mouse (90-day, 2-year) and dog (12-month).  A 21-day repeated dose dermal toxicity in the rabbit is also available.

Test Species/Type	Results	Assessment	Reference
None - 4-week dietary toxicity study in the rat (100, 1000, 10000 ppm) with 4-week and 9-week recovery periods	A NOAEL could not be determined for this study due to methodological deficiencies	Not specified.	Inukai et al (1978)
None - 4-week dietary toxicity study in the mouse (100, 1000, 10000 ppm) with 4-week and 9-week recovery periods	A NOAEL could not be determined for this study due to methodological deficiencies	Not specified.	Inukai et al (1978)
None - 21-day dermal study in rabbits (50, 250 mg/kg bw/d)	A NOAEL could not be determined for this study due to methodological deficiencies	Not specified.	Flucke and Gröning (1981)
OECD 410 - 21-day dermal study in rabbits (250, 500, 1000 mg/kg bw/d) with a 14-day recovery	A NOAEL of 1000 mg/kg bw/d was determined for this study in the absence of any clear effects at the high dose level	Supporting study	Diesing (1992)
None - [Formulation study] 21-day inhalation toxicitiy study in the rat (0.022, 0.088, 0.446 mg/L)	A NOAEC could not be determined for this study; effects were attributed to product co-formulants.	Not specified.  The product study is not relevant for active substance risk assessment.	Thyssen and Mohr (1981)
None, pre-dates OECD 408 but broadly comparable - 13-week dietary toxicity study in the rat (80, 400, 2000, 10000 ppm)	A NOAEL of 2000 ppm (120 mg/kg bw/d was determined for this study, based on liver findings at 10000 ppm.	Supporting study	Inukai et al (1978)
None, pre-dates OECD 408 but broadly comparable - 13-week dietary toxicity study in the mouse (80, 400, 2000, 10000 ppm)	A NOAEL of 400 ppm (65 mg/kg bw/d was determined for this study, based on liver findings in females at 2000 ppm and in both sexes at 10000 ppm.	Key study	Inukai et al (1978)
OECD 409 - 12-month chronic toxicity study in the dog (100, 1000, 10000 ppm)	A NOAEL of 10000 ppm (277 mg/kg bw/d) was determined for this study in the absence of any effects of treatment at the top dose level	Supporting study	Bathe et al (1983)
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)
Non-guideline - Position paper considering liver histopathology in the rat chronic tox/carc study	The paper concludes that there is no liver carcinogenicity in the rat study, and supports an overall NOAEL of 500 ppm for liver effects	- concludes that there is no liver carcimogenicity	Rinke (2005)
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)
OECD 424 - Sub-chronic neurotoxicity study in the rat (500, 2500, 15000 ppm)	The NOAEL for this study was 2500 ppm (181/275 mg/kg bw/d) based on increased marginally increased motor and locomotor activities at 15000 ppm.  Gait abnormalities were seen in all treated groups but were not considered adverse.	Supporting study	Schladt and Lawrence (2006)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

The study pre-dates OECD 408 but is broadly comparable

Deviations:

not specified

GLP compliance:

no

Remarks:

Older study, predates mandatory GLP

Limit test:

no

Specific details on test material used for the study:

White crystals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

JCL-SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4-week old
- Weight at study initiation: males and females weighted 116 g and 106 g, respectively.
- Housing: individually housed in a five-box cage of stainless steel with wire net.
- Diet: powder feed, ad libitum.
- Water: tap water ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 25 ± 3°C
- Humidity: 55 ± 7%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Pencycuron was mixed with powder feed CE-2

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Pencycuron was mixed with powder feed CE-2
- Storage temperature of food: normal temperature.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuously

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

80 ppm

Dose / conc.:

400 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

5/sex/group

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the experimental period physical appearance, general behaviour and habit were observed daily. When dead animals were found during the experiment, they were dissected to find the cause of death.

BODY WEIGHT: Yes
- Time schedule for examinations: During the experimental period test animals were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The amounts of food consumed "by test animals were measured twice a week, and the feed was renewed each time. Active ingredient intake was calculated from the total amount of food consumption and food efficiency from total amount of food consumption and body weight gain.

URINALYSIS: Yes
In the 1st, 2nd and 3rd month after the start of the experiment, urine was collected from these rats which were kept in a metabolic cage for 18 hours, and the amounts of glucose, protein, occult blood, pH, urobilinogen and ketone body in the urine were measured with Uro-combistix. Urinary sediment was microscopically examined after centrifugation of 2 ml of collected urine.

HAEMATOLOGY: Yes
On all the animals which survived at the end of the experiment, blood was taken from post-caval vein of the rats with an injector treated with an anticoagulant under slight anestyesia with ether. The blood collected was used for the hematological examination mentioned below.

Hematocrit volume (Ht value) : Microcapillary tube.
Hemoglobin content (Hb value) : Cyanmethemoglobin method.

Count of erythrocytes: Micro Cell Counter.
Count of leucocytes: Micro Cell Counter.
Count of thrombocytes: Micro Cell Counter.

Differential blood picture : Microscopical examination

the mean corpuscular hemoglobin concentration (MCC): Calculation from Ht value, Hb value and count of erythrocytes.
the mean corpuscular volume (MCV): Calculation from Ht value, Hb value and count of erythrocytes.
the mean corpuscular hemoglobin (MCH): Calculation from Ht value, Hb value and count of erythrocytes.

CLINICAL CHEMISTRY: Yes
On the rats which survived at the end of the experiment, blood was taken from post-caval vein with an injector, Blood collected was used for determination of BUN and GPT. Collected blood was directly centrifuged. The serum obtained was used for the following examinations:

Total protein: biuret reaction.
Albumin: HABCA method.
Calcium: OCPC method.
Creatinine: Jaff method.
Cholesterol: Iiebermann-Burchard method.
Glucose: Somogyi-Nelson method
Blood urea nitrogene (BUN): Fearon method.
Uric acid: Folin method.
Alkaline phosphatase (ALP): Bessey-Lowry method.
Lactic dehydrogenase (LDH): Waker-Amador method.
Glutamic-oxaloacetate transaminase (GOT): Kurmen method.
Glutamic-pyruvate transaminase (GPT): Kurmen method.
A/G ratio: Calculation from the total protein and albumin.

Sacrifice and pathology:

AUTOPSIES:
Immediately after the blood samples were taken, all the animals that survived were dissected by thoracic and abdominal incision to macroscopically examine alternations in the shape, color, location, etc. of each organ. Then the following organs were removed and weighed, and the relative organ weights to body weights were calculated:

Brain, pituitary, thyroids, submaxillary gland, thymus, heart, lung, liver, kidneys, spleen, adrenals and gonads (testes, ovaries).

HISTOPATHOLOGY: Yes
The following organs and tissues were removed for histopathological examination:
Pancreas, stomach, duodenum, small intestines, mesenteric lymph nodes, urinary bladder, muscle and bone marrow.

The above organs or tissues were fixed in 10% buffer formalin and routainly processed for histopathological samples.

Statistics:

The statistical treatment of the values obtained from the experimental results was performed by Student's t-test to compare dosed animals with the controls.

Clinical signs:

no effects observed

Description (incidence and severity):

As the result of daily observation of general behaviour of test animals, they were in good health and any alternations in gloss of coat and feces, etc. were not observed throughout the experimental period. And there were no differences in general behaviour compared with the controls.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In male rats there were no differences between dose groups and control group, but in female rats of 2,000 ppm and 10,000 ppm dose groups the body weight gain was significantly decreased compared with the controls.

While in 2,000 ppm dose group decreasing tendency was found in the first half of the experimental period but it was not observed at the end of the experiment, in 10,000 ppm dose group body weight gain was decreased from the 9th week after the start of the experiment. Though the cause of such different body weight change is unknown, the minimum effect level on body weight gain seems to be 2,000 ppm. In the rats of 80 and 400 ppm dose groups any significant differences were not observed compared with the controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Immediately after the start of the experiment (1 and 2 weeks after) the average food consumption was slightly decreased in males of 2,000 and 10,000 ppm groups compared with the controls, but from the 4th week on, it did not differ from the controls.

And there were no .significant differences in average amount of food consumption during the whole experiment between the males of dosed groups and those of control group. In females, no significant differences were observed in average food consumption compared with the controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In food efficiency calculated from food consumption and body weight gain, significant decrease was seen in female rats of 10,000 ppm group and decreasing tendency was seen in males of the same dose group. No significant changes were observed in food efficiency of 2,000 ppm and lower dosed groups compared with the controls.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the amount of urea nitrogen, no significant alternations were found in any groups. Some slight differences in the values of GPT activity were statistically seen between dose groups and control group, but they were within the physiological range.

In the total amount of protein of female rats of 400 2,000 and 10,000 ppm groups, significant difference was seen compared with the controls but was considered to be within the physiological range. Changes observed in other items besides creatinine, that is, in albumin of the rats of 80, 2000 and 10000 ppm, calcium of those of 80 and 400 ppm were also within the range of the physiological range.

The amount of creatinine in females provided an indication of a dose-related increase while that in males showed a dose-related decrease. However, these changes were not consistent and histopathological examination of kidney on both male and female rats showed no abnormalities.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The brain weights of male rats of 80 and 400 ppm dose groups showed a significant decrease compared with the controls. In the thymus, spleen and pituitary weights of male rats of 400 ppm dose group, significant differences were seen compared with the controls, but they were not considered to be dose-related. The liver weight of male rats provided an indication of significant increase in 10,000 ppm group and also of slight increase in 2,000 ppm dose group compared with the controls, and therefore, it is considered that the inclusion of Pencycuron affected the liver weight. Out of the relative organ weights of male rats, only relative liver weight showed an increase with the increase of dietary concentrations.

For female rats, shown a significant increase was seen in brain and adrenal weights in 10,000 ppm dose group, and a decrease was observed in heart, adrenal and pituitary weights in 80 ppm dose group and in spleen weight in 80 and 2,000 ppm dose groups compared with the controls. As to relative organ weights, in the rats of 10,000 ppm dose group which had decreased body weights, the relative brain, submaxillary gland, heart, liver, adrenal and pituitary weights were significantly increased and the relative liver and lung weights were also increased in the rats of 2,000 ppm group. Among the above changes in dose group, only the relative liver weight showed a dose-related changes.

As mentioned above, only the liver weight was affected by the administration of Pencycuron in male rats of 10,000 ppm group and in females of 2,000 ppm or more dose groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Males) One rat of control group and one of 2,000 ppm do3e group had an atrophic testis. In two rats of 400 ppm dose group kidney edema was found. Others did not show any noted changes. (Females) In one rat of 400 ppm dose group and two rats of 10,000 ppm dose group kidney edema was found.
Any changes that seemed to be related to the inclusion of Pencycuron in the diet were not observed in both male and female rats.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

1- Liver: Minimal change of nuclear polymorphism, abnormal distribution of chromatin in the nucleus and irregular nucleus in size were seen in a large proportion of the male and female rats from 10,000 ppm.

2- Kidney: Deposits of hyaline-like substance in the tubule were seen in a large proportion of the male and female rats of all dose and control groups. This change was commonly seen in the laboratory rats and there was no evidence of dose-related effects of the tested compound.

3- Spleen: Minimal deposits of pigment were seen in a large proportion of the male and female rats from all dose and control groups. This change was commonly seen in the laboratory rats and was not considered to be of toxicological significance.

4- Heart: Minimal granulation was seen in the myocardium of a small numbers of rats. This change was not related to the administration of the compound.

5- Testis: An atrophy was seen in a male rat of the control.

From the results of the histopathological examination, minimal changes of nuclear polymorphism, abnormal distribution of chromatic in the nucleus and irregular nucleus in size were noted in the liver of the male and female rats from the highest dosage. These changes were not qualitative change and it is concluded that influence of Pencycuron on rats liver could be of ignorance. Some alternations were seen in the kidney, spleen, heart and testis of rats but they were not associated with the administration of Pencycuron.

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Equivalent to 120 mg/kg/day

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Equivalent to 27 mg/kg/day

Conclusions:

From the results of this study, it is concluded that the minimum poisoning level are 10,000 ppm for males and 2,000 ppm for females, that is, the maximum no-effect level of Pencycuron administered to rats are 2,000 ppm for males and 400 ppm for females.

Executive summary:

Male and female rats of SD strain were fed with the diet containing Pencycuron at the dietary concentrations of 0, 80, 400, 2,000 and 10,000 ppm for 14 weeks. At the end of 14-week experiment all the animals were sacrificed for various examinations. Five male and five female rats of respective dose groups were used for an intermediate examination in 4 weeks and 8 weeks after the start of the feeding experiment.

Any changes in general behaviour and appearance of the test animals were not observed during the experiment. No dead animal was observed through the experiment.

Suppressed tendency of the body weight increase, lower body weight gain and average body weight were observed from the 9th week of the experiment in female rats of 10,000 ppm dose group, end in females of 2,000 ppm group the body weight gain was suppressed from the first week.
There were no changes attributable to administration of the compound in body weight change, body weight gain and average body weight of male rats of any dose groups.

The average food consumption in males of 10,000 ppm group was increased in 1, 2 and 3 weeks after administration. However, no significant differences were seen in the total food consumption through the whole experimental period among all dose groups of male and female rats. Lover food efficiency was observed in females of 10,000 ppm-dose group.

As the result of haematological analyses carried out in 4 and 8 weeks after administration and at the end of the experiment, any influence by administration of the compound was not found in any dose groups of male and female rats.

The administration of the compound did not affect the differential blood counts in percentage of lymphocyte, neutrocyte, monocyte, eosinocyte and basophil leukocyte.

No dose-related differences were observed in both males and females in the clinical biochemical examination performed in 4 and 8 weeks after administration and at the end of the experiment.

There was an increasing tendency of liver weight in male and female rats of 10,000 ppm dose group, but any changes attributable to administration of the compound were not observed in other organ weights.

As the result of urine analysis performed in 4 and 8 weeks after administration and at the end of the experiment, no changes attributable to administration of the compound were observed.

The urinary sediment examined in 4 and 8 weeks after administration and at the end of the experiment showed that any changes which were considered to be attributed to administration of the compound were not observed.

From the results of histopathological examination, minimal changes were noted in the liver of the male and female rats from the highest dosage. These were not considered to be qualitative changes and it is concluded that the effect of Pencycuron on rat liver could be of ignorance.

 

From these results, no effect levels in the rats fed with the diet containing Pencycuron for 14 weeks were 2,000 ppm in males and 400 ppm in females, and the active ingredient intakes corresponding to these dietary concentrations were 120 mg/kg/day in males and 27 mg/kg/day in females.

Reference 2

Endpoint:

chronic toxicity: oral

Remarks:

Combined chronic dietary toxicity/ carcinogenicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

reference to same study

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-04-21 to 1983-05-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Remarks:

It was not specified on what basis the doses were selected. Haematological and clinical chemistry examination were performed before the treatment and 1, 2, 3, 4, 5, 6, 8, 10 and 12 months after initiation of treatment.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

Pencycuron was weighed in an Erlenmeyer flask on a Mettler balance. The nominal concentrations of the test compound were mixed with the microgranulated dog feed. Before pelleting, water was added to each mixture at a ratio of 1:10 (v/v) to ensure pellet, quality. The pellets were air-dried.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the concentration of the test material, its stability and homogeneity in the diet was performed in the analytical laboratories of RCC

Duration of treatment / exposure:

360 days minimum

Frequency of treatment:

Once/day, 7 days a week

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- Dose selection rationale: not specified.
- Rationale for animal assignment (if not random): not specified.
- 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.

Observations and examinations performed and frequency:

Symptoms Signs of local and/or systemic toxicity daily a.m. and p.m.
Mortality Daily a.m. and p.m.
Body weight Weekly
Food consumption Daily, but reported weekly
Eye examination Pretest, after 3, 6, 9 and 12 months with Heine-Bifocal Ophthalmoscope (miroflex type)
Hearing test Pretest, after 3, 6, 9 and 12 months (simple noise test)

Clinical laboratory investigations for hematology, biochemistry and urinalysis were performed on 6 male and 6 female dogs per group during pretest and after 1, 2, 3, 4, 5, 6, 8, 10 and 12 months of treatment. The blood samples were drawn from the jugular vein between the hours of 07.00 and 09.30 a.m. to reduce the biologic variability due to circadian rhythms. Blood samples from each animal were aliquoted into individual specimen vials containing the anticoagulant (EDTA-K3 for performing hematological measurements, Na-Citrate for coagulation testing and Li-Heparin for biochemical analysis). Liver tissue removed at the time of sacrifice was weighed, rinsed in ice-cold saline (0.9 %), blotted dry, and immediately frozen in liquid nitrogen, and stored at -20 degrees centigrade until analyzed. Urine for analysis was obtained by catheterization. The quantitative assay of blood and urine parameters was performed under Quality Control conditions.

Sacrifice and pathology:

Necropsies were performed by experienced prosectors under the supervision of the RCC pathologist. The beagles were exsanguinated after intravenous injection of T61. The following organs were weighed: brain, heart, liver, kidneys, lung, pituitary gland, adrenal glands, thyroid gland (with parathyroid), testes, ovaries.
Hepatic cytochrome P-450 content, N-demethylase activity and hepatic triglyceride concentrations were measured at termination of the treatment

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Spontaneous vomiting and diarrhoea were seen in dogs of all groups during the treatment period. In some cases, variocolored feces (light brown to reddish) or mucus traces were evident. Local transient alopecia and mild erythema of the visible skin were observed in some dogs of all groups. The abdominal region as well as the inside of the hind legs were mostly affected.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain of the animals of all treated and control groups was generally comparable

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of all treated males was comparable. The food consumption of the controls was slightly decreased during weeks 1 to 12 and weeks 25 to 52. The food consumption of the females of group 2 was comparable to the controls during the test period, but was slightly reduced in group 3 from week 16 to term and in group 4 during weeks 1, 10 to 21 and 30 to 52

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Isolated lesions of the cornea, such as small opaque spots, were noted in some animals of all groups. These findings were considered to be spontaneous; they are occasionally observed in the beagle stock used. Hyaloid foci, arranged in a circular pattern on both lens and one greyish structure on the right discus opticus was observed in dog no. 16 (group 3) at the 9- and 12-rnonth examination. Dog no. 37 (group 3) showed a light greyish spontaneous papillary structure at the right discus opticus at the 12-rnonth examination.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some intergroup differences with statistical significance were found to occur for some biochemical parameters, nevertheless, these changes were considered to be reflections of normal biological variability with no toxicological relevance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

HEARING TEST No impairment of the hearing was evident in groups 1 to 4.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: The increase of absolute liver and heart weights paralleled the body weight differences between control and treated groups (relative weights showed no relevant differences). The thyroid weights were increased in treated groups (also the relative organ weights).
Females: There were no relevant differences between the groups.
The organ weight to brain weight ratio shows in males of group 3 higher ratios for heart, liver and kidneys; this is due to lower absolute brain weights in this group. The thyroid ratio is higher in treated males when compared with the controls. The females failed to show differences in this ratio.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of treatment-unrelated changes were encountered in various organs at all dose levels. They consisted mainly of small indurated and discolored foci in the lungs, and of multifocal capsular retractions in the spleen.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lungs: Multifocal chronic suppurative bronchopneumonia, slight to moderate in degree, was observed in a number of beagles at all dose levels, occasionally with intraalveolar nematodes. In several cases, mild multifocal interstitial fibrosis was also encountered.
Spleen: At all dose levels, sclerosiderotic foci were observed that corresponded to the multifocal capsular retraction seen at necropsy.
Other organs/tissues: A number of common and minor lesions were encountered in various organs of most beagles.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Measurements of hepatic cytochrome P-450 content at the end of the experiment resulted in slight but significant increase in male and female dogs of the middle and high dose groups.

Details on results:

The observed decrease in food consumption in the female high dose group is not considered adverse, as it is not statistically significant and did not result in a reduced body weight gain. The difference in absolute liver weight between the male mid and high dose group on the one hand and the male control group on the other, are most likely provoked by differences in body weight, already present at the start of the experiment. Differences in relative liver weight are small and not statistically significant. Also considering that no histopathological changes have been found in the liver, it is concluded that there are no clear adverse effects on this organ weight. The clear, dose related increase in cytochrome P-450 content of both male and female livers is an adaptive reaction and is not considered adverse since it is not associated with other, probably adverse, liver effects.

Key result

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Equivalent to 277 mg/kg bw/d

Conclusions:

As no adverse effects of treatment were observed, the NOAEL of this study is set at 10000 ppm (277 mg/kg bw/d).

Executive summary:

The oral chronic toxicity of Pencycuron was investigated in Beagle dogs. Animals were daily dosed 0, 100, 1000, 10000 ppm Pencycuron in food for 360 days.
No clinical and/or pharmacological signs were seen with the exception of spontaneous vomiting and diarrhoea in animals of all groups during the study.
There were no deaths during the study.
The food consumption of the treated male dogs and controls as well as the female dogs of group 2 was comparable. The food consumption of the female dogs of groups 3 and 4 was slightly reduced.
The body weight gain of the treated animals was comparable to that of the controls.
Eye examination and auditory perception showed no treatment-related findings.
Hematological, biochemical and urinalysis results at each of the investigation intervals did not show any differences between groups. Hepatic cytochrome P-450 content in liver homogenate mas slightly increased in the middle and high dose groups at the end of the study. In the absence of relative liver weight increase as well as histomorphologic liver findings, this increase in cytochrome P-45Q is not considered to reflect a toxic change in liver metabolism. It is interpreted as a reversible adaptive change.
Significant organ weight differences were calculated for treated males, particularly increased thyroid weights and ratios. The difference can be accounted in biological terms for pure chance because corresponding values were received in other experiments (within laboratory variation).
This assessment is supported by the negative histopathological results.
No treatment-related gross and histopathological lesions were found.

In conclusion, the NOAEL of this study is set at 10000 ppm (277 mg/kg bw/d). 

Reference 4

Endpoint:

chronic toxicity: oral

Remarks:

Combined chronic dietary toxicity/ carcinogenicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

reference to same study

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Unspecified to February 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

None, pre-dates OECD 408 but broadly comparable.

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

ICL-ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: feed

Details on route of administration:

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

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Continuous

Dose / conc.:

0 ppm

Dose / conc.:

80 ppm

Dose / conc.:

400 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

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

Observations and examinations performed and frequency:

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

Sacrifice and pathology:

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

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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

Food efficiency:

no effects observed

Description (incidence and severity):

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

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Details on results:

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

Key result

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 65 mg/kg bw/d

Key result

Critical effects observed:

no

Conclusions:

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

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

18 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Studies are guideline-compliant or guideline-comparable (Klimisch score 1 or 2)

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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

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

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.

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.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

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.

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

no

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

A guideline-compliant study is available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

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

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

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.

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.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

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.

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

no

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

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.

Endpoint conclusion

Dose descriptor:

NOAEL

1 000

Study duration:

subacute

Species:

rabbit

Quality of whole database:

A guideline-compliant study is available

Justification for classification or non-classification

The NOAEL in the 90-day rat study is 2000 ppm (120 mg/kg bw/d), based on effects seen at the LOAEL of 10000 ppm. In a 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 ppm (equal to 181 mg/kg bw/d in males and 275 mg/kg bw/d in females). Consequently, it can be concluded that pencycuron does not cause serious toxicity at dose levels relevant for classification.  Classification for STOT-RE is therefore not proposed.