Climbazole

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977-02 to 1977-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to a method comparable to the former OECD guideline 408 and was performed prior to the implementation of GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

Male and female Wistar rats (each 15 males and females) received daily oral doses of the test item at dose levels of 5, 15, and 45 mg/kg in 0.5% aqueous Tylose via gavage for 3 months (90 d). Animals were observed for general symptoms and food and water consumption as well as body weight gains were recorded. Additionally, blood and urine samples were analyzed after 6 weeks and at the end of the study, and the animals were subjected to gross pathology and histopathology after termination of the study.

GLP compliance:

no

Remarks:

the study was conducted prior to the implementation of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Winkelmann (Borchen, Germany)
- Age at study initiation: 42-49 d
- Weight at study initiation: Males: 119-122 g, females: 115-118 g
- Housing: Animals were housed in Makrolon cages (type II) on dust-free wood granulate.
- Diet: Altromin R powder (Altromin GmbH, Lage, Germany), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2°C

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tylose

Details on oral exposure:

VEHICLE
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

Animals were treated for 3 months (90 d).

Frequency of treatment:

Animals were treated daily (7 d per week) for 3 months (90 d).

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15, and 45 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

15 males and 15 females were used per dose level.

Control animals:

yes, concurrent vehicle

Details on study design:

Male and female animals were seperately assigned to 3 body weight groups (light, intermediate, and heavy) and were then randomised.

Positive control:

No positive control was used.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD ANd WATER CONSUMPTION:
- Food and water comsumption was weekly recorded by back-weighting.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 6 weeks and 3 months
- Anaesthetic used for blood collection: Yes (diethyl ether)
- How many animals: 5 males and 5 females from each of the groups
- Parameters examined: Numbers of erythrocytes, leukozytes, thrombocytes, and reticulocytes, haemoglobin content, heamatocrit, differential haemogram, MCH, and MCV

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 6 weeks and 3 months
- Anaesthetic used for blood collection: Yes (diethyl ether)
- How many animals: 5 males and 5 females from each the control an the high-dose group
- Parameters examined: Alkaline phosphatase (ALP), glutamate-oxaloactetate-transaminase (GOT), glutamate-oxaloactetate-transaminase (GPT), creatinine, urea, glucose, cholesterol, bilirubin, total protein content, triglycerides in the blood, triglycerides in the liver

URINALYSIS: Yes
- Time schedule for collection of urine: At 6 weeks and 3 months
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: semiquantitative: Glucose, blood, protein, pH, ketone bodies, bilirubin and urobilinogen; quantitative: protein; microscopic examination of the urine sediment after centrifugation

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS:
The following organs of animals that died during the study or were sacrificied at study termination were weighed: Thyroid gland, thymus, heart, lung, liver, spleen, kidneys, adrenal glands, and testes or ovaries.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals that died were subjected to necropsy. At termination of the study, all other animals were sacrificed and examined by necropsy.

HISTOPATHOLOGY: Yes
- Histopathological examinations were performed on 10 animals each of the control and the high-dose group. The following organs were examined: Aorta, eyes, small intestine (doudenum, jejunum, ileum, and colon), femur, brain, bladder, heart, testes, pituitary gland, liver, lung, lymph nodes, stomach, spleen, epididymes, adrenal glands, kidneys, oesophagus, skeletal muscles, sternum, thymus, trachea, ovaries, pancreas, prostate, seminal vesicles, thyroid gland, and uterus as well as all tissues that showed macroscopic changes.

Other examinations:

not applicable

Statistics:

Arithmetic group mean values, standard deviations as well as upper and lower confidence limits for p = 0.05 and p = 0.01 were calculated. Values for the treated groups were compared to those of the control group and differences were tested for significance using the U-test of Mann, Whitney, and Willcoxon for p = 0.05 and p = 0.01. These calculations were performed on an IBM-370/145 computer.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
Males of the high-dose group showed a slight reduction in body weight gain, which became significant (p<0.05) from the 10th week onwards (see the image attached below).

WATER CONSUMPTION
Water consumption was slightly increased in males of the high-dose group (see table 1 in section "Any other information on results including tables").

HAEMATOLOGY
The haematologic examinations did not reveal any significant differences from the untreated control except for a statistically significant (p<0.01) reduction in the number of erythrocytes observed in males of the high-dose group at the end of the study (see table 2 in section "Any other information on results including tables"). This reduction in erythrocyte counts was not considered treatment-related, as the observed values still fell wthin the naturally occurring variation (i.e. 6.50 to 8.82 mill. erythrocytes/mL, as determined in 89 rats of the same age as the test animals).

CLINICAL CHEMISTRY
When evaluated at 6 weeks after inititation of the study, no significant differences in the parameters examined were noted, apart from a significant (p<0.01) reduction in creatinine levels observed in males of the high-dose group (see table 3 in section "Any other information on results including tables"). The reduction in creatinine levels were not regarded as a sign of kidney damage, as renal failure should result in an increase rather than in a decrease in creatinine levels, and the creatinine levels obseved in this study still fell wthin the naturally occurring variation (i.e. 0.52 to 0.88 mg/100 mL for males and 0.49 to 0.97 mg/100 mL for females, as determined in 135 untreated rats of the same age as the test animals).
When evaluated at the end of the study, the activity of alkaline phosphatase was significantly reduced in males and females of all treated groups (with p<0.05 for the low-dose and mid-dose groups and p<0.01 for the high-dose groups) (see table 4 in section "Any other information on results including tables"). This effect was found to be dose-related but was not considered an adverse effect, as the observed values still fell wthin the naturally occurring variation (i.e. 91 to 479 mU/mL, as determined in 135 untreated female rats of the same age as the test animals).
Furthermore, the activity of N-demethylase was significantly (p<0.01) increased in livers of males of the high-dose group, and the content of CYP-450 isoenzymes was significantly (p<0.05) increased in the livers of females of the high-dose group (see table 5 in section "Any other information on results including tables"). These changes were not considered adverse effects but the result of physiological adaptation processes or a result of the stimulation of the microsomal enzyme system.

URINALYSIS
When evaluated after 6 weeks after inititation of the study, urinary protein contents were significantly (p<0.05) elevated in males of the high-dose group (see table 6 in section "Any other information on results including tables"). However, no such observations were made during the evaluation at the end of the study. The elevation in urinary protein content was not considered indicative of kidney damage, as no other adverse renal effects were observed in this study and urinary protein content values generally show a wide variation.

ORGAN WEIGHTS
A significant (p<0.01) increase in liver weights was observed in females of the mid-dose and the high-dose group (see table 7 in section "Any other information on results including tables").

Effect levels

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Water intake of male rats

Dose (mg/kg/d)	Average water intake
	L/animal	mL/animal/d
0	2.32	25.48
5	2.45	26.89
15	2.39	26.21
45	2.51	27.56

Table 2: Numbers of erythrocytes in blood of male rats at the end of the study

Dose (mg/kg/d)	Number of erythrocytes (mill./µL)
0	8.62
5	8.41
15	8.54
45	7.94*

* = p<0.01

Table 3: Creatinine levels in blood of male rats at 6 weeks

Dose (mg/kg/d)	Creatinine levels (mg/100 mL)
0	0.73
5	0.67
15	0.63
45	0.58*

* = p<0.01

Table 4: Alkaline phosphatase activity and creatinine levels and in blood of male and female rats at the end of the study

Dose (mg/kg/d)	Alkaline phosphatase activity (mU/mL)		Creatinine levels (mg/100 mL)
	males	females	males	females
0	199	198	0.75	0.64
5	221	174*	0.71*	0.67
15	276**	171*	0.69	0.60*
45	233	147**	0.64**	0.58**

* = p<0.05; ** = p<0.01

Table 5: Content of N-demethylase in the livers of male rats and content of CYP-450 isoenzymes in the livers of female rats at the end of the study

Dose (mg/kg/d)	N-Demethylase (nMol/g liver)	CYP-450 isoenzymes (nMol/g liver)
	males	females
0	88	31
5	92	31
15	108	29
45	150**	37*

* = p<0.05; ** = p<0.01

Table 6: Content of protein in the livers of male rats after 6 weeks

Dose (mg/kg/d)	Protein (mg/100 mL)
0	68.5
5	71.1
15	74.9
45	105.9*

* = p<0.05

Table 7: Liver weigths of female rats

Dose (mg/kg/d)	Average liver weights (mg)
0	6925
5	7298
15	7720*
45	8232*

* = p<0.01

Applicant's summary and conclusion

Conclusions:

In a 90-day study, male and female Wistar rats received daily oral doses of the test item at dose levels of 5, 15, and 45 mg/kg in 0.5% aqueous Tylose. A NOAEL of 45 mg/kg bw/day was derived. At this dose only a slight decrease of body weights in males was observed. Females showed an increase in liver weight of less than 20% compared to controls at 45 and 15 mg/kg bw/day that - in the absence of any histopathological or relevant clinical chemistry alterations - was considered an adaptive response.

Executive summary:

Male and female Wistar rats (each 15 males and females) received daily oral doses of the test item at dose levels of 5, 15, and 45 mg/kg in 0.5% aqueous Tylose via gavage for 3 months (90 d). There was no effect on survival and no treatment-related clinical symptoms were observed. In males treated with the test item at a dose level of 45 mg/kg, a reduction in body weight compared to controls was observed which became statistically significant from the 10th week of treatment onwards. However, the body weight decrease was less than 0% compared to controls. No effects on body weight was found in females. At all doses there was a tendency for a reduced alkaline phosphatase activity in serum in females, while there was a slight increase in males. GOT and GPT transaminases were slightly lower in females of the low and mid dose groups. Creatinine tended to be lower (sometimes statistically significant) at all doses in males and females. In both sexes a slight increase in activity and protein content of Cytochrom P450 monooxygenases was found. Erythrocyte numbers were slightly, but statistically significantly reduced. In the absence of any corresponding histological findings, these effects on hematological and clinical chemistry parameters were not considered toxicologically relevant. The liver weights were significantly increased in females at the mid and high dose levels. However, increases were less than 20% compared to controls and in the absence of any histopathological alterations were considered to be an adaptive response. The results of the gross pathological and histopathological examinations did not reveal any indication of treatment-induced alterations. Based on these findings, a NOAEL of 45 mg/kg bw/day was derived.