cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 Feb 2000 to 23 Mar 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Cas Number:

94361-06-5

Molecular formula:

C15H18ClN3O

IUPAC Name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

SPF

Sex:

male/female

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: 1% CMC and 0.1% Tween 80.

Details on exposure:

A dosing volume of 5 mL/kg was used. The test item was applied to the clipped dorsal area and held in contact with the skin with gauze patches. Patches were covered with aluminum foil and fastened with adhesive tape.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week for the first 3 weeks, and everyday thereafter

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

System:

other: hepatobiliary and endocrine systems

Organ:

liver

thyroid gland

Treatment related:

yes

Any other information on results incl. tables

MORTALITY AND CLINICAL SIGNS

There were no deaths in the test and no treatment-related clinical signs were observed. Detailed clinical observations did not result in any additional treatment related findings. There were no signs of irritation at the skin application site.

Ophthalmoscopic examination revealed no treatment-related findings. No alterations of the pupillary reflex nor the eyelids and only one animal of dose group 1000 mg/kg bw/d presented with a grade 1 crust around the eye from week 3 to 4.

 

BODY WEIGHT

Body weight and body weight gain in all dose groups were not affected by treatment. There were no differences among the control and treated groups in food consumption or food utilisation efficiency.

 

HAEMATOLOGY

Differences among the groups in the haematology parameters were seen in elevated values among the 1000 mg/kg bw/d animals for haemoglobin concentration distribution width (RDW) and increased prothrombin time. Analysis of WBCs revealed that 3 males at the high dose had elevated levels of WBCs. There was no apparent increase in total WBC levels in females, however analysis of individual parameters revealed an apparent reduction in the level of basophils at 100 and 1000 mg/kg bw/d. There was an increase in the level of monocytes (significant), leukocytes (not sig.) in females at the high dose.

 

Table 1. 28-day dermal rat - affected haematology parameters.

	Males				Females
Dose level (mg/kg bw/d)	0	10	100	1000	0	10	100	1000
haemoglobin concentration distribution width (mmol/L)	1.49	1.63	1.51	2.02**	1.19	1.26	1.28	1.63##
leukocyte count (G/L)	0.07	0.08	0.07	0.08	0.05	0.06	0.07	0.09*
prothrombin time (rel. 1)	0.86	0.93	0.88	1.11**	1.13	1.15	1.30	1.49**
relative monocytes	2.41	2.31	2.65	3.70**	2.53	2.16	2.39	3.39*
Basophils (x10^-3)	5	3.4	4.4	3.9	3.7	4.1	2.4	2.6
absolute large unstained cells	0.07	0.08	0.07	0.08	0.05	0.06	0.07	0.09*

 

CLINICAL CHEMISTRY

A slight but non-significant reduction in glucose levels was noted in the males at the high dose, whereas in the females there was a significant reduction at 100 mg/kg bw/d
only. Treatment-related changes in clinical chemistry parameters included dose dependent increase in globulin levels, which was significant in males (1000 mg/kg bw/d) and females (at 100 and 1000 mg/kg bw/d). In relation to globulin levels there was a significant reduction in the ratio of Albumin /Globulin in both males and females at the high dose. Analysis of blood protein revealed an apparent dose-dependent increase in total protein in both males and females, which only attained significance in the high dose females. Cholesterol levels were increased in 100 and 1000 mg/kg bw/d females and were raised in dose dependent manner in males but never achieved significance.

Treatment-related changes in electrolytes were seen in lower chloride levels in 100 and 1000 mg/kg bw/d animals of both sexes, lower potassium levels in 1000 mg/kg bw/d females and higher sodium levels in 1000 mg/kg bw/d males. Males in the 1000 mg/kg bw/d group showed increased activity for aspartate and alanine aminotransferase. Both males and females displayed a reduction in the levels of bilirubin at all doses in particular at the high dose, significant reduction in males only.

 

Table 2. 28-day dermal rat - affected blood chemistry parameters.

	Males				Females
Dose Level (mg/kg bw/d)	0	10	100	1000	0	10	100	1000
Glucose (mmol/L)	7.55	8.39	7.91	6.36	6.15	6.61	5.01*	5.97
total bilirubin (µmol/L)	1.81	1.48	1.49	1.06##	1.71	1.59	1.57	1.43
protein (g/L)	66.0	66.7	67.1	68.4	63.7	65.7	67.1*	69.2**
albumin (g/L)	31.8	32.1	32.0	31.0	31.2	32.3	32.1	31.6
globulin (g/L)	34.1	34.6	35.1	37.4##	32.4	33.4	35.1**	37.6*
albumin/globulin ratio	0.93	0.93	0.91	0.83**	0.97	0.97	0.92	0.84**
cholesterol (mmol/L)	1.54	1.52	1.67	1.82	1.41	1.64	2.01**	1.96**
sodium (mmol/L)	143.1	143.2	143.2	144.3*	141.7	141.9	141.8	143.0
potassium (mmol/L)	3.80	4.04	3.83	3.95	3.49	3.37	3.37	2.91**
chloride (mmol/l)	98.2	97.8	96.9*	96.1**	99.3	98.9	96.8*	96.4*
aspartate aminotransferase (U/L)	84.3	77.1	79.7	100.5	107.0	97.7	102.1	100.9
alanine aminotransferase (U/L)	45.5	42.5	45.7	75.2##	42.8	41.2	43.6	46.2

* = p ≤ 0.05, ** = p ≤ 0.01, ANOVA + Dunnett; # = p < 0.01, Kruskal-Wallis + Dunn

 

ORGAN WEIGHTS

Organ weight analysis revealed increased absolute and relative liver weights in 1000 mg/kg bw/d animals of both sexes. Mean absolute liver weights of 1000 mg/kg bw/d males and females were 20 and 26 % higher than their respective controls, and relative liver weights were similarly 25 and 28 % higher. All other organ weights were comparable among the control and treated animals.

 

Table 3. 28-day dermal rat - affected absolute and relative organ weights. 

		Males				Females
Dose Level (mg/kg bw/d)		0	10	100	1000	0	10	100	1000
body	absolute^a	309.0	295.8	305.8	295.3	217.1	217.6	214.9	214.0
liver	absolute	13.7	14.2	14.4	16.4**	10.2	9.87	10.5	12.8**
	relative^b	44.3	47.9	46.9	55.5**	46.8	45.3	48.9	59.9**

a All absolute weights in grams

b Relative organ weights = % of body weight (g/g); ** = p ≤ 0.01, ANOVA + Dunnett

 

GROSS PATHOLOGY

Macroscopic examination at the end of the treatment period revealed an enlarged liver in one 1000 mg/kg bw/d male and one 1000 mg/kg bw/d female. The only other findings in the test animals (renal pelvic dilatation in one 1000 mg/kg bw/d male and one 100 mg/kg bw/d female, small seminal vesicles in one 10 mg/kg bw/d male) were not considered to be related to treatment.

Histopathology revealed treatment-related changes in the skin application site, liver, spleen and thyroid. The incidence and/or severity of acanthosis were increased in 100 and 1000 mg/kg bw/d males and females. Acanthosis is associated with increased insulin levels in the blood. At both 100 and 1000 mg/kg bw/d there was a corresponding drop in serum glucose levels.

Centrilobular hepatocellular hypertrophy was seen in 100 and 1000 mg/kg bw/d males and 1000 mg/kg bw/d females. Splenic haemosiderosis was noted in 1000 mg/kg bw/d males and 100 and 1000 mg/kg bw/d females. The incidence of thyroid follicular hypertrophy was increased in 100 and 1000 mg/kg bw/d animals of both sexes, relative to the respective controls. A variety of other changes that are commonly seen in rats of this strain and age were noted, but the incidence, distribution and morphological appearance of these findings did not give any indication of a treatment-related association.

 

Table 4. 28-day dermal rat - treatment-related microscopic findings.

		Males				Females
Dose level (mg/kg bw/d)		0	10	100	1000	0	10	100	1000
no. of tissues examined		10	10	10	10	10	10	10	10
skin application site	incidence	4	5	9	10	4	3	4	5
acanthosis	average grade	1.3	1.4	1.3	1.6	1.0	1.0	1.3	1.4
liver	incidence	0	0	7	10	0	0	0	5
centrilobular hepatocellular hypertrophy	average grade	-	-	1.0	1.8	-	-	-	1.0
spleen	incidence	0	0	0	9	2	1	5	10
haemosiderosis	average grade	-	-	-	1.0	1.0	1.0	1.0	1.3
thyroid gland	incidence	5	5	8	9	5	5	8	9
follicular hypertrophy	average grade	1.6	1.4	1.3	1.8	1.0	1.0	1.1	1.7

Applicant's summary and conclusion

Conclusions:

Dermal treatment of Wistar rats with the test substance at 0, 10, 100 and 1000 mg/kg bw/d for 4 weeks was well tolerated by the rats at all dose levels tested. There were no signs of overt toxicity. No treatment-related body weight changes were noted in any dose group. Dose levels of 100 and 1000 mg/kg bw/d elicited systemic effects noted as changes in a few hematology and blood chemistry parameters. Significantly increased liver weights were recorded in males and females of the high dose groups. A treatment-related epidermal acanthosis was found in dose groups 100 and 1000 mg/kg bw/d animals as a local reaction to the treatment at the application site, and cenlrilobular hepatocellular hyperthrophy was observed in dose groups 100 and 1000 mg/kg bw/d males and dose group 1000 mg/kg bw/d group 4 females. Hemosiderosis in the spleen was noted in dose group 1000 mg/kg bw/d males and dose groups 100 and 1000 mg/kg bw/d females. Hypertrophy of thyroid follicular epithelium was seen in animals of dose groups 100 and 1000 mg/kg bw/d. The higher protein levels, the increased activities of ALAT and ASAT, and the hypertrophy of the thyroid are indicative of a possible enzyme induction. These findings point to the occurrence of adaptive metabolic processes. Due to the adaptative nature of all observed effects, they were not considered to be adverse. Based on these observations, the NOEL was defined at 10 mg/kg bw/d, and the NOAEL was determined at 1000 mg/kg bw/d.

Executive summary:

In this study which was conducted according to OECD TG 410 and followed GLP, the test item was applied at 0, 10, 100 and 1000 mg/kg bw/d under occlusive dressing to the clipped area of skin on the back of Wistar rats for a period of 4 weeks on an at least 5 day/week basis. The exposure period was 6 hours per day. The test item, suspended in vehicle, was administered at the selected dose levels to 10 males and 10 females per dose group. Control animals were treated with the vehicle only. Clinical signs, body weight, food consumption, and mortality were monitored throughout the study for all animals. Detailed careful clinical observations were performed in all animals once weekly, before the daily administration of the test item. Ophthalmological examination was made in all animals before treatment start, and in animals or the control and high dose groups towards the end of the treatment period. Hematological and blood chemistry analyses were performed at treatment end. At sacrifice, animals were examined macroscopically, and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues were examined microscopically.

All animals survived to the end of the treatment period. Clinical observations performed on a daily (general) or weekly (detailed) basis did not reveal any treatment-related findings, and no signs of local irritation were present. The dermal application of the test item had no influence on the animal's body weight development, and the mean food consumption in all treated groups was comparable to that of the control groups. Consequently, mean food utilisation efficiency in the treated groups, measured as body weight gain per 100 g food consumption was comparable to that of the control group, too. There were no treatment-related effects seen to the eyes. Higher mean values for hemoglobin concentration distribution width suggesting anisochromia of red blood cells and slightly higher monocyte counts were recorded for males and females at 1000 mg/kg bw/d, whereas higher large unstained cell counts were noted in females of the high dose group only. In addition, ma!es and females of the high dose group had significantly higher prothrombin activities. Significantly higher plasma globulin levels associated with lower albumin to globulin ratios and higher protein levels were recorded for males and females at 1000 mg/kg bw/d and ror females at 100 mg/kg bw/d. Treatment-related effects to plasma electrolytes were present as lower chloride values in males and females at 100 and 1000 mg/kg bw/d associated with minimally higher sodium values in mares at 1000 mg/kg bw/d and lower potassium values in females of the high dose group. In addition, males at 1000 mg/kg showed increased activities for alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and lower plasma bilirubin levels, while females at 100 and 1000 mg/kg bw/d had higher plasma cholesterol levels. The mean absolute/ relative liver weights were significantly increased in high dose males (+19.9 % / +25.3 %) and in females (+25.9 % / +28.0 %). At necropsy, enlargement in liver was recorded in one animal of either sex in dose group treated with 1000 mg/kg bw/d. This finding correlated with a treatment-related centrilobular hepatocellular hypertrophy seen microscopically. On microscopical examination, the following changes were considered treatment-related: Increased incidence and/or severity of acanthosis was observed at skin application site in dose groups 100 and 1000 mg/kg bw/d animals when compared with the conlrols; Centrilobular hepatocellular hypertrophy was found in dose groups 100 and 1000 mg/kg bw/d males and 1000 mg/kg bw/d females; Increased incidence of hemosiderosis was recorded in spleen of dose group 1000 mg/kg bw/d males and dose groups 100 and 1000 mg/kg bw/d females; Increased incidence of hypertrophy of thyroid follicular epithelium was seen in dose groups 100 and 1000 mg/kg bw/d animals when compared with controls.

Dermal treatment of Wistar rats with the test substance at 0, 10, 100 and 1000 mg/kg bw/d for 4 weeks was well tolerated by the rats at all dose levels tested. There were no signs of overt toxicity. No treatment-related body weight changes were noted in any dose group. Dose levels of 1000 and 100 mg/kg bw/d elicited systemic effects noted as changes in a few hematology and blood chemistry parameters. Significantly increased liver weights were recorded in males and females of the high dose groups. A treatment-related epidermal acanthosis was found in dose groups 100 and 1000 mg/kg bw/d animals as a local reaction to the treatment at the application site, and cenlrilobular hepatocellular hyperthrophy was observed in dose groups 100 and 1000 mg/kg bw/d males and dose group 1000 mg/kg bw/d group 4 females. Hemosiderosis in the spleen was noted in dose group 1000 mg/kg bw/d males and dose groups 100 and 1000 mg/kg bw/d females. Hypertrophy of thyroid follicular epithelium was seen in animals of dose groups 100 and 1000 mg/kg bw/d. The higher protein levels, the increased activities of ALAT and ASAT, and the hypertrophy of the thyroid are indicative of a possible enzyme induction. These findings point to the occurrence of adaptive metabolic processes. Due to the obvious adaptative nature of all observed effects, they were not considered to be adverse. Based on these observations, the NOEL was defined at 10 mg/kg bw/d, and the NOAEL was determined at 1000 mg/kg bw/d.