Climbazole

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976-11-03 to 1976-11-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed in a manner similar to OECD TG 412 and was scientifically sound and is well documented. However, compared to today's standards, it shows shortcomings in the number of organs and parameters examined.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

, shortcomings in the number of organs and parameters examined

GLP compliance:

no

Remarks:

pre-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)
- Exactly identification: SPF-Albino rats, Wistar II
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 162-164 g, females: 164-166 g
- Fasting period before study: No data
- Housing: Macrolon cage Type III (5 animals per cage)
- Diet: Altromin-R-standard feed, ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): No data
- Air changes (per h): Artifical air circulation
- Photoperiod (h dark / h light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: Ehtanol/polyethylene glycol 400 (1:1)

Remarks on MMAD:

MMAD / GSD: 97% of the particles: 1.0 +/- 0.5 µm
3% of the particles: < 5.0 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Dynamic inhalation apparatuses (Kimmerle & Eben (1973, Arch Toxicol 30:115)
- Method of particle size determination: Measurement with cascade impactor

VEHICLE
- Composition of vehicle: Ethanol/polyethylene glycol 400
- Concentration of test material in vehicle: 50%
- Purity of vehicle: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atomized spray from the inhalation air was adsorbed on cotton wool and then eluted with benzene. The active substance was determined by gas chromatography with the help of a thermo-ionical nitrogen detector.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week, 3 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
17.2 (13.5-21.0) mg/m3
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
44.3 (39.5-48.0) mg/m3
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
104.7 (92.7-133.5) mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Animal assignment: randomly
- Negative control: ethanol/polyethylene glycol (20mL/m3)

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 24 h after the last exposure (directly after section)
- Groups that were examined: All dose groups + negative control group, 5 males and 5 females per group

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 24 h after the last exposure (directly after section)
- Groups that were examined: All groups, 5 males and 5 females per group
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- Parameters examined: Haematocrit; haemoglobin; No. of erythrocytes, leukocytes, and thrombocytes; differential haemogram

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 24 h after the last exposure (directly after section)
- Groups that were examined: all groups, 5 males and 5 females per group
- Animals fasted: No
- Parameters examined: GPT (Glutamate-Pyruvate-Transaminase), GOT (Glutamate-Oxalacetate-Transaminase), alkaline phosphatase, urea, creatinine, and glucose

URINALYSIS: Yes
- Time schedule for collection of urine: After the last exposure the urin was collected over 6 h during daytime
- Groups that were examined: All groups, 5 males and 5 females per group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: pH; content of glucose, protein, blood, bilirubin, and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All groups
- Battery of functions tested: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Examined material: Liver, kidney, adrenal glands, heart, lung, thyroid glands, lymph nodes, spleen, testes, ovaries, trachea, larynx, oesophagus, stomach, and smears of bone marrow

Statistics:

The statistical evaluation of the results was performed according to the Wilcoxon rank-sum test (Wilcoxon (1947) Biometrics 3:119).

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 Details on results

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see Details on results

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
The body weight increase of the males, treated with the highest dose of crinipan (MEB 6401) was significantly decreased after 1 week of exposure until the end of the test (see Tables 1 and 4a). The body weights of the female rats were all in accordance with the physiological norm.

HAEMATOLOGY

The Nos. of leukocytes were significantly increased in males treated with 17.2 and 44.3 mg/m3. The evaluation of the differential haemograms did not show any sign of a percental shift in males of the 17.2 mg/m3 dose group; however, in the 44.3 mg/m3 group, a slight increase in the No. of segmented cells and a decrease in the No. of lymphocytes was observed. In female rats a significant increase in the No. in erythrocytes and haemoglobin in the group treated with 44.3 mg/m3 and a significant decrease in the No. of thrombocytes in the highest dose group was observed. Furthermore, an increase in the No. of lymphocytes and a decrease in the No. of segmented neutrophils were determined in the lowest dose group (17.2 mg/m3) (see Table 2).

CLINICAL CHEMISTRY

The values of GOT, GPT, and urea were all in the range of the physiological norm. However, a significant decrease of the creatinine values were observed in all treated male rats. In female rats, the values of alkaline phosphatase were decreased (compared to the controls) in the 44.3 mg/m3-dose group and the glucose values were significantly decreased in the two highest dose groups (see Table 3).

ORGAN WEIGHTS

Significant, but concentration-independent and randomly occurring differences in organ weights of the treated animals compared to that of the control group were observed in several organs. Males, treated with the lowest dose of crinipan showed increased absolute kidney weights and males treated with the highest dose showed decreased absolute weights of the hearts and decreased relative weights of lungs, livers, and testes (see Table 4a). In case of the female rats, animals of the highest dose group showed significantly increased absolute and relative weights of livers and adrenal glands. Concentration-dependently increased relative weights of thyroid glands were observed in male rats, treated with >/= 44.3 mg/m3 (see Table 4b).

HISTOPATHOLOGY: NON-NEOPLASTIC
Light-microscopically no pathological findings indicating for an organ-harming effect of crinipan (MEB 6401) were observed. The above-mentioned clinical findings (significant different weights of livers and adrenal glands) observed in the animals treated with the highest dose of crinipan were not microscopically verified. Some minor random findings was observed, as can be seen in Table 5.
In the smears of bone marrow, the following findings were observed in the treated animals compared to the control: In the highest dose group a slight increase in segmented leukocytes and a slight decrease of basophile cells. The fraction of eosinophil cells varied. Alltogether, there were hardly any differences observable.

Effect levels

Dose descriptor:

NOAEC

Effect level:

44.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly decreased body weight in males and slight changes in organ weights in males and females, without any histopathological alterations.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Summarized body weight (bw) data of male rats

test week			0	1	2	3
Dose [mg/m^3]	0	mean bw	164	179	196	208
	17.2	mean bw	163	180	201	213
		p<0.05	-	-	-	-
		p<0.01	-	-	-	-
	44.3	mean bw	164	175	191	202
		p<0.05	-	-	-	-
		p<0.01	-	-	-	-
	104.7	mean bw	162	169	183	194
		p<0.05	-	+	-	+
		p<0.01	-	+	+	+

 

Table 2 Summarized results of the examination of the haematological examinations (only parameters included with significantly

different values in any of the treated groups compared to the control)

Parameter (unit)			No. of leukocytes (mio/µL)	Haemoglobin (G/100 mL)	Segmented cells (% of all blood cells)		Lymphocytes (% of all blood cells)
Sex (male (m); female (f)			m	w	m	f	m	f
Dose (mg/m^3)	0	mean w	8.5	14.8	8.6	13.8	90.6	85.6
	17.2	mean w	10.0	14.3	14.8	6.2	84.8	93.6
		p<0.05	-	-	-	-	-	-
		p<0.01	+	-	-	+	-	+
	44.3	mean w	11.6	15.4	19.2	14.4	80.4	84.8
		p<0.05	-	-	+	-	+	-
		p<0.01	+	+	+	-	+	-
	104.7	mean w	9.7	15.3	12.8	15.2	86.2	83.6
		p<0.05	-	-	-	-	-	-
		p<0.01	-	-	-	-	-	-

Table 3 Summarized results of the clinico-chemical examinations (only parameters included the values of which were

significantlydifferentin any treated group compared to the control group) 

Parameter (unit)			Creatinine (mg/100 mL)	ALP* (mU/mL)	Glucose (mg/100 mL)
Sex (male (m); female (f)			m	f	f
Dose [mg/m^3]	0	mean w	0.77	257	97.4
	17.2	mean w	0.66	249	91.2
		p<0.05	+	-	-
		p<0.01	+	-	-
	44.3	mean w	0.67	225	90.7
		p<0.05	+	-	+
		p<0.01	+	+	+
	104.7	mean w	0.60	267	89.8
		p<0.05	+	-	-
		p<0.01	+	-	+

* ALP: alkaline phosphatase

Table 4a Summarized data of organ weights (w) for male rats (only organs included with significantly different weights in any of

the treated groups compared to the control)

Organ			Body weight gain		Thyroid glands		Heart		Lung		Liver		Kidney
Relative(r) / absolute (a) organ weight			a	r	a	r	a	r	a	r	a	r	a	r
Dose [mg/m^3]	0	mean w (g)	208	208	*	5	647	*	*	411	*	3735	1271	*
	17.2	mean w (g)	213	213	*	5	683	*	*	424	*	3886	1358	*
		p<0.05	-	-	*	-	-	*	*	-	*	-	-	*
		p<0.01	-	-	*	-	-	*	*	-	*	-	+	*
	44.3	mean w (g)	202	292	*	6	609	*	*	427	*	3906	1268	*
		p<0.05	-	-	*	-	-	*	*	-	*	-	-	*
		p<0.01	-	-	*	+	-	*	*	-	*	-	-	*
	104.7	mean w (g)	194	194	*	6	592	*	*	445	*	4186	1228	*
		p<0.05	+	+	*	-	-	*	*	-	*	+	-	*
		p<0.01	+	+	*	+	+	*	*	+	*	+	-	*

Table 4a Summarized data of organ weights (w) for female rats (only organs included with

significantly different weights in any of the treated groups compares to the control)

Organ			Liver		Adrenal glands
Relative(r) / absolute (a) organ weight			a	r	a	r
Dose [mg/m^3]	0	mean w (g)	6250	3529	50	28
	17.2	mean w (g)	6279	3557	54	31
		p<0.05	-	-	-	-
		p<0.01	-	-	-	-
	44.3	mean w (g)	6145	3582	53	31
		p<0.05	-	-	-	-
		p<0.01	-	-	-	-
	104.7	mean w (g)	6962	4058	60	35
		p<0.05	-	+	-	+
		p<0.01	+	+	+	+

Table 5 Summarized results from the histopathological examinations (only organs included with findings

different from the control in any of the treated groups)

Dose (mg/m^3)	Animal No.	Organ, taken from male (m) or female (f) animals
		Larynx		Trachea		Heart		Lung
		m	f	m	f	m	f	m	f
Control	1	Oe+	o	o	Ez +	o	o	o	Ez 1
	2	o	o	Ez +	o	o	o	Sch +	o
	3	o	o	o	o	o	o	Gran +	o
	4	o	o	o	o	o	N 1	o	pbRu +
	5	o	o	o	o	o	o	pvRu 1	pbRu +
17.2	1	o	o	o	o	o	o	o	o
	2	o	o	o	o	o	o	o	o
	3	o	o	o	o	o	o	Gran 1	o
	4	o	o	o	o	o	o	o	E +
	5	o	o	o	o	o	o	Gran 1	o
44.3	1	o	o	o	o	o	o	o	o
	2	o	o	o	o	o	o	o	o
	3	o	o	o	o	o	o	o	pbRu +
	4	o	o	o	o	N +	o	o	o
	5	o	o	o	o	o	o	o	Gran 1
104.7	1	o	o	o	o	o	o	o	o
	2	o	o	o	o	o	o	o	pbRu 1
	3	Ez +	o	o	Ez 1	o	o	o	pbRu 1
	4	o	o	Ez +	o	N +	o	o	E +
	5	Ez +	o	Ez +	o	o	o	o	o

Abbreviations:

Ez = inflammatory-cellular infiltration; Gran = histocyte granuloma; N = scarring; oe =oedema; pbRu = peribronchial rounded cell infiltrates

Applicant's summary and conclusion

Conclusions:

A 3-week subacute inhalation study in Wistar rats was carried out using exposure at 17.2, 44.3, and 104.7 mg/ m3 in dynamic inhalation chambers for 6 h/d, 5 d/wk. Slightly reduced body weights were noted in males of the high dose group and males and females of the high dose groups showed weight changes in some organs. No histopathological alterations were observed. A NOAEC of 44.3 mg/ m3 was derived.

Executive summary:

The subacute inhalation toxicity of crinipan was tested in a 3-week study in rats using three different test concentrations (17.2, 44.3, and 104.7 mg/ m3 air) in dynamic inhalation chambers, in which animals were exposed for 6 h/d, 5 d/wk. During the treatment period the neurobehaviour and body weights of the animals were daily recorded. After the last exposure, haematological and clinico-chemical parameters of the animals were determined and urine analysis was performed. In addition, histopathological examinations were done after section of the animals. Finally, a statistical evaluation of the results was done.

Body weights of males were slightly reduced, reaching statistical significance in the high dose group, but the reduction was >10% compared to controls. No body weight effect was seen in females.

The examination of haematological parameters revealed a significant, but not dose-related increase in the number of leucocytes in males of the low and mid dose groups.

The histopathological evaluation of the bone marrow smears yielded almost no differences between the treated and the control animals.

The clinico-chemical examinations yielded significantly decreased values of creatinine for all male rats which were not considered toxicologically relevant and were within the normal range (0.53-1.05 mg/100 mL) for male Wistar-II rats of this age. Mid dose females showed slightly decreased values for alkaline phosphatase and blood glucose (also in high dose females). All glucose values were within the physiological range (lowest average value: 89.9 mg/100 mL). These findings were therefore not considered toxicologically relevant.

 

High dose males showed significantly decreased absolute (but not relative) heart weights, and relative weight increases of testes, lung, liver and thyroids, while females had increased absolute and relative liver and adrenal weights.

At the mid dose significant increase of relative thyroid weights occurred in female rats only. In the absence of absolute weight changes, similar effects in males and the absence of any histopathological alterations, this effect was not considered toxicologically relevant.

In conclusion, a NOAEC of 44.3 mg/ m3 was derived.