Climbazole

Endpoint summary

Description of key information

The following four repeated dose toxicity studies were performed in rats with crinipan; in two studies each the test item was administered via the oral route or via the inhalation route.
1. Sub-acute (28-day repeated) oral toxicity study in male Wistar rats: LOAEL 50 mg/kg bw/day
2. Sub-chronic (90-day repeated) oral toxicity study in male and female rats: NOAEL 45 mg/kg bw/day)
3. Sub-acute (21-day repeated) inhalation toxicity study in male and female rats (1976): LOAEC 69.1 mg/m3
4. Sub-acute (21-day repeated) inhalation toxicity study in male and female rats (1977): NOAEC 44.3 mg/m3
No dermal studies were performed with crinipan.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

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).

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.

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.

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").

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'

Critical effects observed:

not specified

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A OECD TG 408 and GLP compliant study was available.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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.

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

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

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

Remarks:

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

Remarks:

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

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

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).

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.

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.

Critical effects observed:

not specified

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

44.3 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The following 4 repeated dose toxicity studies with crinipan were performed in rats; in two studies each, the test item was administered via the oral route or via the inhalation route.

1.  Sub-acute (28-day repeated) oral toxicity study in male Wistar rats (Bayer/Symrise 1975046)

2.  Sub-chronic (90-day repeated) oral toxicity study in male and female rats (Bayer/Symrise 1978006)

3.  Sub-acute (21-day repeated) inhalation toxicity study in male and female rats (Bayer/Symrise 1976029)

4. Sub-acute (21-day repeated) inhalation toxicity study in male and female rats (Bayer/Symrise 1977005)

 

From these studies, a NOAEL of 15 mg/kg/d via the oral route, and a NOAEC of 44.3 mg/m³ via the inhalation route were established in rats.

 

Repeated dose oral toxicity study:

Two repeated dose toxicity studies (28-day and 90-day) were performed in rats with crinipan.

28-day repeated oral toxicity study

The toxicity of crinipan was evaluated in a 4-week repeated dose oral study in male Wistar rats. The test item was administered daily by gavage as a suspension in 0.5% aqueous Tylose at dose levels of 50 and 100 mg/kg/d. The examinations performed included clinical observations, food and water consumption, body weight gain, and clinical chemistry as well as gross pathology and histopathology. After the first and fourth week, animals of the high-dose group showed a reduced body weight gain as compared to control animals. Clinico-chemical analyses showed that blood glucose levels were significantly elevated in the high-dose group at day 23 of the study, as compared to controls. GPT levels were significantly elevated in the low-dose group at the end of the study, as compared to controls. Levels of aminopyrine-N-demethylase were elevated in animals of both treated groups. Levels of the test item in blood on the second day were 2.6 and 2.8 µg/mL in the low-dose group and the high-dose group, respectively. The respective values on day 23 were 3.75 and 3.10 µg/mL. At the end of the study, the test compound was not detectable in blood from the animals at 24 h after the last application. Elevated liver weights were observed in both treated groups, as well as elevated weights of the thyroid glands in the high-dose group. Necropsy at the end of the study revealed a pale discoloration with clear delineation of the lobes in the livers of 5 animals from each treated group. Histopathological examinations revealed some degree of intermediate-peripheral or diffuse fatty degeneration in the animals from both treated groups. No effects were observed in testes, adrenal glands, and thyroid glands.

In summary, this study showed that daily oral administration of crinipan at dose levels of 50 and 100 mg/kg/d to male Wistar rats did not cause any obvious detrimental effects in the test animals, but detailed analyses revealed degenerative changes in the liver which occurred at both dose levels examined. Thus, a clear-cut no NOAEL value could not be established from this study; however NOAEL should be <50 mg/kg/d for rats.

90-day repeated oral toxicity study

The toxicity of crinipan was evaluated after daily oral application for 90 d (3 months) in male and female Wistar rats. The test animals (each 15 males and females) received daily oral doses of crinipan at 5, 15, and 45 mg/kg/d in 0.5% aqueous Tylose via gavage. The various endpoints were examined and recorded in a similar manner as done in the 28-d repeated dose toxicity study. The animals were found to tolerate the treatment with the test item without the occurrence of any clinical symptoms; treatment-related mortality was not observed. In males treated with the test item at 45 mg/kg/d, a reduction in body weight gain was observed which became statistically significant from the 10th week of treatment onwards. The results of the haematological and clinico-chemical analyses as well as the results of the gross pathological and histopathological examinations did not reveal any indication of treatment-induced injury of the blood, the kidneys, the liver, or other organs. At the highest dose level, evidence of a stimulation of the microsomal enzyme system was obtained in both males and females. Based on these findings it could be argued that a NOAEL of 45 mg/kg body weight per day could be considered the NOAEL but taking a conservative approach and in line with the effects observed in the reproductive toxicity studies, a NOAEL of 15 mg/kg body weight per day is considered appropriate.

Repeated dose inhalation toxicity study:

Two sub-acute (each 21 -day) repeated dose toxicity studies with crinipan were performed in rats. They differed only in dose levels administrated of crinipan (17.2, 44.3, and 104.7 mg/ m³ in one study, and 69.1, 144.4, and 377.1 mg/ m³ in another study), hence both studies are discussed together.

 

The sub-acute inhalation toxicity of crinipan (MEB 6401) was tested in a 3-week study in rats using different test concentrations (17.2, 44.3, 69.1, 104.7, 144.4, and 377.1 mg/m³) in dynamic inhalation chambers, where the aerosol can only be taken in with the breathing air. Within an exposure period of three weeks, the rats were exposed 6 h per day (15 times). 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. The determination of the body weights revealed significant decreases in body weight gain of males during the whole test period at ≥69.1 mg/m³. This effect was not seen in females, but a decline in the general condition was observed in the animals treated with the highest dose of crinipan. Significant and concentration-dependent differences in organ weights (compared to the control) occurred in the animal groups treated with >69.1 mg/m³ crinipan (livers of male rats, adrenal glands of female rats) and with >144.4 mg/m³ crinipan (livers of females, spleens of males). A significant increase of the relative weights of the thyroid glands occurred in female rats treated with 104.7 mg/m³ crinipan. However, neither the absolute mean weights were significantly increased nor male rats of these dose groups showed adequate findings. Rats from the 104.7 mg/m³ dosed group, showed significantly differences in several organs weights (e.g. increased absolute weights and relative weights of the livers in females or both sex, respectively; increased absolute and relative kidney weights of females) which indicates a toxic effect of crinipan. The examination of haematological parameters resulted in some differences between treated animals up to 104.7 mg/m³ and the control animals (e.g. significant increased no. of leukocytes in male rats), but all these differences occurred concentration-independently and randomly and could not be considered as treatment related. No macroscopically nor histological pathological changes indicating an organ-damaging effect occurred. The clinico-chemical examinations yielded in significantly decreased values of creatinine for male rats treated up to 104.7 mg/m³. However, these values were not considered to indicate a harmful effect (a disturbance of the kidney function always is indicated in case of increased creatinine values). Furthermore the determined creatinine values are all within the normal range (0.53-1.05 mg/100 mL) for male Wistar-II rats of this age. A randomly and concentration-independent decrease in the alkaline phosphatase in female rats of dose group 44.3 mg/m³ was observed, but could not be considered as treatment related. Only glucose values were significantly and concentration-dependently different from the control; female rats which received a dose of 44.3 mg/m³ crinipan or more showed significantly decreased values. However, all glucose values were within the physiological range (lowest average value: 89.9 mg/100 mL).

Based on the above-described 3-week sub-acute inhalation study in Wistar rats, it can be concluded that under the test conditions, treatment with crinipan caused already some significant adverse effects at the test dose of 69.1 mg/m³, however a test dose of 44.3 mg/m³ was very well tolerated by the animals and considered as harmless. This dose concentration of 44.3 mg/m³ could be considered as NOAEC for this study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was reliable and was conducted according to a method comparable to the former OECD guideline 408. While the subchronic study in rats revealed a NOAEL of 45 mg/kg bw/day (i.e. no adverse effects were observed at the highest dose tested), in a conservative approach and considering the parental effect levels observed in the reproductive toxicity studies a NOAEL of 15 mg/kg body weight per day was considered appropriate as a starting point for DNEL derivation.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The study appears to be scientifically sound and is well documented. Since the principles of GLP were published after the study was performed, the study is not GLP-conform and it was not conducted according to any guideline. However, it was performed in a manner very similar to OECD guideline No. 412 and identified a NOAEC

Justification for classification or non-classification

The substance does not have to be classified regarding systemic and target organ toxicity after repeated oral exposure according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) for the following reasons:

 

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. At the highest dose (considered as NOAEL) only a slight decrease of body weights in males (about 8.7% compared to controls) 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 was not accompanied by any histopathological or relevant clinical chemistry alterations. These are organ weight changes without evidence of organ dysfunction and without histopathological alterations do not constitute toxicologically significant or severe effects. Therefore, a dose level of 45 mg/kg/day did clearly not come close to an effect level requiring classification. It is considered unlikely that at a dose of 100 mg/kg/day would cause a severity level that would be considered classifiable. Also the acute oral toxicity studies in four different species did not reveal any organ changes in gross pathology.

 

Two 3-week subacute inhalation studies in Wistar rats were carried out using exposure levels of 69.1, 144.4 and 377.1 mg/m3 in the first and 17.2, 44.3, and 104.7 mg/m3 in the second study in dynamic inhalation chambers for 6 h/d, 5 d/wk.

At the highest dose significantly effects on several organ weights were noted (spleen, liver, adrenals, ovaries). However, no toxicologically relevant changes in clinical-chemical or haematological parameters or relevant histopathological alterations were identified in any of the dose groups.These are organ weight changes without evidence of organ dysfunction and without histopathological alterations do not constitute toxicologically significant or severe effects. Therefore, a dose level of 377 mg/m3 (0.4 mg/L) did clearly not come close to an effect level requiring classification. It is considered unlikely that at a concentration of 0.6 mg/L would cause a severity level that would be considered classifiable.

 

Given the low bioavailability via the dermal route, it can be considered unlikely that a STOT-RE classification would be required if a repeated dose dermal toxicity study was done.