(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Endpoint summary

Description of key information

In conclusion, the short-term oral toxicity of epoxiconazole is characterized by effects on the liver in all three species tested. At high dose levels, liver function was impaired resulting in signs of liver toxicity. At lower dose levels, only liver weight increases were seen. In rats and mice, also the adrenal gland was a target organ. Histopathology demonstrated lipoid deposits as well as regressive transformation in female rats. In dogs, minimal to moderate hypertrophy of the proximal tubular epithelial cells of the kidneys was observed. At very high dose levels in the 4 -week studies in rats and mice, signs of anaemia were noted.

The overall NOAEL of 1.6 mg/kg bw/d for short-term toxicity of epoxiconazole was chosen from the 12-month feeding study in dogs, which constituted the most sensitive species.

Concerning long-term toxicity, it can be concluded that, in both species tested (chronic toxicity and carcinogenicity study in rats, carcinogenicity study in mice), one of the target organs for epoxiconazole-induced toxicity is the liver as indicated by increases in organ weights and histopathological changes. Clinical chemistry changes that are likely to be associated with alterations of liver function and adverse effects on red blood cell parameters were observed in rats. In addition, the studies identified male and female gonads as (possibly secondary) targets of epoxiconazole toxicity. Inhibition of enzyme(s) involved in the synthesis of steroid hormones and an induction of liver enzymes are considered the possible mechanisms that affect the endocrine system.

Key value for chemical safety assessment

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 1991 (first study), Nov 1991 (second study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

Nov 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

none given

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae, Biberach/Riss, FRG
- Age at study initiation: not specified
- Weight at study initiation: male: 253-283 g (first study), 264-280 g (second study); female: 218-241 g (first study), 211-224 g (second study)
- Fasting period before sacrifice: about 16 h
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: The feed batches used in the study were tested for impurities. The drinking water has been regularly assayed for chemical contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12 (illumination: 6 am to 6 pm)

IN-LIFE DATES: From: May 27, 1991 (first study) and Nov 18, 1991 (second study) To: Jun 26, 1991 (first study), Dec 18, 1991 (second study)

Type of coverage:

semiocclusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Tylose (cleaned sodium-carboxymethylcellulose in aqua bidest.)

Details on exposure:

TEST SITE
- Area of exposure: dorsal and dorsolateral parts of the trunk
- % coverage: at least 10% of the bodu surface
- Type of wrap if used: (4 layers of absorbent gauze and an elastic dressing).
- Time intervals for shavings or clipplings: at least thrice a week. Only the first clipping was carried out at least 24 hours before the first application.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 ml/kg
- Concentration (if solution): 1.000; 400 and 100 mg/kg body. Volumes of test substance preparations and solvent (0.5% solution of Tylose CB 30.000 in aqua bidest.) to be applied were calculated for each animal once a week on the day of body weight determination.
- Constant volume or concentration used: yes
- Preparation interval: daily

VEHICLE
- Tylose CB 30.000
- Amount(s) applied (volume or weight with unit): 2 ml/kg
- Concentration (if solution): 0.5%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To verify the correctness of the concentrations and the homogeneity of the test substance preparations, samples of each concentration were sent for analysis at the beginning of each study section. The content of the active ingredient was determined by HPLC.

Duration of treatment / exposure:

3 weeks

Frequency of treatment:

6 h per day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: An acute oral toxicity study in the rat, an acute dermal toxicity study in the rat, an acute dermal irritation/corrosivity study to the intact dorsal skin of the white rabbit, and an acute irritation to the eye of the white rabbit
- Rationale for animal assignment (if not random): random
- Other: Treatment had been performed in two consecutive study sections. In the first study section 1,000 mg/kg body weight and in the second study section 400 and 100 mg/kg body weight have been administered. In both study sections respectively, a control group for comparison was used.

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (before and after exposure)

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily (about 30 min after removal of the dressing)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 21; Males and females first study June 26, 1991; males and females second study Dec 18, 1991
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 21; Males and females first study June 26, 1991; males and females second study Dec 18, 1991
- Animals fasted: No
- How many animals: all
- Parameters checked: sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, ALT, AST, ALP, GGT

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- the exsanguinated animals were necropsied and assessed by gross pathology.
- Organ weights from all animals: liver, kidneys, spleen, testes and adrenal glands

HISTOPATHOLOGY: Yes
- Organs examined for all test groups and all animals: treated skin, normal skin, liver, kidneys, spleen
- all gross lesions: all animals affected per group

Statistics:

Wilcoxon test, Kruskal-Wallis-H-test, Mann-Whitney-U-test, Kruskal-Wallis

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

No signs of irritation on the treated skin could be observed in test or control animals.
Adhesive tape caused mechanical skin lesions beside the treated area.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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):

- 1000 mg/kg bw/day: slight decrease in red blood cells and hematocrit in the peripheral blood in males

No substance—induced changes were observed in the clotting analyses of both sexes

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No substance-induced changes were observed in the enzyme activities of both sexes. No substance—induced changes were observed in the clinicochemical examinations of both

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: significantly increased absolute mean liver weights in male and female rats; significantly increased absolute mean kidney weight in female rats, without a morphological equivalent
- 400 mg/kg bw/day: significantly increased relative mean liver weight in female rats, without a morphological equivalent

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: slight centrilobular liver cell hypertrophy in male rats

Details on results:

The application of 1000 mg/kg body weight to the skin resulted in a treatment related increase of the absolute liver weight in male female rats. In males, a slight (grade 2) hypertrophy of the hepatocytea is in accordance with
the weight increase. Although no morphological alteration was noted in the females, the weight increase most likely represents a treatment related effect, too.
In contrast to this, the observed significant increase of the absolute weight of the testes does not represent a treatment related effect, as two control animals had exceptional low testes weights — due to spontneous tubular atrophy - and hence the mean value of dose group 1 was compared with a too low mean valuein the concurrent control group 0.
Although no morphological observation was made that may explain the significantly increased mean absolute kidney weights in the female rats of dose group 1000 mg/kg, it can not be excluded that this observation represents a marginal toxic effect. Its biologic relevance, however, is of little if any importance. No treatment related effects were seen in the treated skin.
In the untreated skin of the abdomen of one control and four treated females of dose group 1000 mg/kg, crust formation was noted grossly in the abdominal region where the adhesive fleece of the semiocclusive dressing stuck to the skin. This gross lesion proved to be hyperkeratosis, focal acanthosis, crust formation, focal necrosis, and/or dermal fibrosis histopathologically. None of these findings represent a treatment related effect as they are only linked with the technical procedure used for the application of the test article to the treated
site of the skin.
In the second part of the study (control group 00 and dose groups 3 [100 mg/kg body weight] and 2 [400 mg/kg body weight]), the only possible treatment related effect might be traced from the significantly increased relative live: weight of the female rats of dose group 2 (400 mg/kg body weight). However, no microscopic finding was noted that may refer to the increased weight. Moreover, the application
of 1000 mg/kg body weight to the skin did not result in significantly increased relative liver weights in the female rats. Therefore, the significance of this observation is of little if any biological relevance.
The increased absolute kidney weights in the females of dose group 3 are a by chance observation as the mean of this group is lower than that one of dose group 2 which does not show a statistically significant deviation from the concurrent control group 00.
All other gross lesions or microscoPic findings are regarded to have occurred incidentally and they could not be related to the application of the test article.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Table 1: Absolute weights of test groups 0 (control) and 1 (1000 mg/kg bw/day)

ABSOLUTEWEIGHTS- MEANVALUES (FEMALE)-GROUPS 0AND1

 

Sacrifice				group	Fl
				Sex	F
Dose				group	0	1
Body weight			g	M	220.22	228.5
				SD	8.327	16.833
				n	5.	5.
Liver		g		M	7.278	8.788*
				SD	0.44	1.348
				n	5.	5.
Kidneys		g		M	1.876	2.076**
				SD	0.107	0.094
				n	5.	5.
Spleen		g		M	0.54	0.526
				SD	0.02	0.121
				n	5.	5.
Adrenal	glands	mg		M	103.6	111.
				SD	11.739	7.517
				n	5.	5.

ABSOLUTE WEIGHTS - MEAN VALUES (MALE) - GROUPSOAND1

 

Sacrifice			group	Fl
			Sex	M
Dose			group	0	1
Body weightg			M	291.44	306.56
			SD	16.477	11.225
			n	5.	5.
Liver	g		M	10.142	11.428**
			SD	0.702	0.169
			n	5.	5.
Kidneys		g	M	2.486	2.622
			SD	0.186	0.118
			n	5.	5.
Testes		g	M	2.844	3.108*
			SD	0.237	0.178
			n	5.	5.
Spleen		g	M	0.594	0.682
			SD	0.074	0.064
			n	5.	5.
Adrenal	glands	mg	M	86.8	80.4
			SD	14.255	12.502
			n	5.	5.

 

Wilcoxon-Test (two-sided)

* P<=0.05 ** P<=0.01

 Table 2: Relative weights of test groups 00 (control), 3 (100 mg/kg bw/day), and 2 (400 mg/kg bw/day)

 

RELATIVE WEIGHTS - MEAN VALUES (FEMALE) GROUPS 00, 3 AND2

 

Sacrifice group					Fl
Sex					F
Dosegroup					00		3	2
Body weight		M		100.		100.		100.
			SD
			n	5.		5.		5.
Liver		%	M	3.394h		3.544		3.686**
			SD	0.063		0.241		0.186
			n	5.		5.		5.
Kidneys			M	0.877		0.921		0.903
			SD	0.015		0.049		0.097
			n	5.		5.		5.
Spleen		%	M	0.265		0.293		0.254
			SD	0.032		0.039		0.048
			n	5.		5.		5.
Adrenal	glands	%	M	0.049		0.051		0.053
			SD	0,005		0.004		0.009
			n	5.		5.		5.

 

h P<=0.05			:Kruskal-Wallis-H-Test
* P<=0.05**	P	<=0.01	:Kruskal-Wallis-H-+Wilcoxon-Test
two-sided

 

 

 

Additional information

4-week oral toxicity:

The 4-week short-term oral toxicity of epoxiconazole was investigated in rats, mice, and dogs.

In rats, a dose of 4000 ppm resulted in a severe reduction in body weight and induced an anaemic effect with compensatory reactions. Clinico-chemical changes indicating liver toxicity were seen at 1000 ppm and higher, however, already at the lowest dose tested (250 ppm), gamma-GT induction in the liver and a decrease in triglycerides were observed. Target organs were the liver and the adrenals. Hepatocellular hypertrophy was observed at doses of 1000 ppm and higher. Electron-microscopy showed a severe proliferation of the smooth endoplasmatic reticulum. In the adrenals, a weight decrease was seen in high-dose females. Histopathology showed lipoid deposits in the cortical cells of males, while in the 4000 ppm females a regressive transformation of the outer cortical zone of the adrenals was noted. Based on this study, no NOAEL could be set.

In mice, the dose level of 4000 ppm resulted in a reduction of body weight development in the males. Signs of a weak anaemic effect were also noted at this dose level. Clinico-chemical and pathological investigations show that the liver was the target organ. Liver weight increases were seen at all dose levels. Hepatocellular hypertrophy was seen in males and females of the 1000 ppm and 4000 ppm groups as well as in males of the 250 ppm group, i.e. the lowest dose administered. Adrenal weights at 4000 ppm were increased. Based on this study, a NOAEL could not be set.

Overall, epoxiconazole showed comparable effects in rats and mice. NOAELs were not obtained in both studies.

Dietary administration of epoxiconazole to Beagle dogs over a period of 4 weeks (range-finding study), led to distinct liver damage in addition to pronounced clinical signs (e.g. lack of appetite of various intensity, pronounced weight loss, vomiting) and associated clinicochemical, gross-pathological and histopathological findings (cachexia, atrophy of isolated organs). In particular the increased enzyme levels, the reduced cholesterol levels and the reduction in the total protein, albumin and triglyceride levels as well as the increased relative liver weights, the cloudy swelling and acute dystrophy of the liver are clear evidence of liver damage.

No NOAEL could be determined.

13-week oral toxicity

The 13-week oral toxicity of epoxiconazole was studied in rats, mice and dogs.

In rats, after administration of epoxiconazole for 13 weeks at dietary dose levels of 30, 90, 270, and 800 ppm, clinico-chemical investigations revealed a test substance related increase in serum gamma-glutamyl transferase activity in high dose females as well as increased liver homogenate gamma-glutamyl transferase activity in the upper two male dose groups and the female high-dose group. Additionally, cholesterol was significantly increased in high dose females and triglycerides were raised in all treated male and in female low- and mid-dose groups, but the latter changes occurred in a non-dose-related way, did not achieve statistical significance (at least, this was not stated) and were therefore not considered as treatment-related. Organ weights determination revealed an increase in relative liver weight in high dose males as well as increased relative and absolute liver weights in females. The increased absolute liver weights in females of the 90 and 270 ppm groups were not considered to be test substance related as there was no change in relative liver weight and because the body weight of the females of these groups was clearly higher than that of the controls. In addition, at least at 90 ppm, the increase was not accompanied by histopathological changes. Both absolute and relative adrenal weights were slightly reduced in all treated groups, but more clearly so at the upper two dose levels. Histopathological examinations confirmed the liver as the target organ. The main finding was a predominantly centrilobular hypertrophy of hepatocytes, especially developed in the 800 ppm test group, being diffuse in single females. This finding held responsible for the increase in the absolute (females only) and relative (males and females) liver weights in this test group. There were likewise indications of a centrilobular hepatocellular hypertrophy to be found in five males and seven females of the 270 ppm group, which mostly did not exceed grade 1 (minimal).

The overall NOAEL for the 13-week dietary administration in rats is 90 ppm, equivalent to 7 mg/kg bw/d in males and 8 mg/kg bw/d in females.

In two 13-week studies using B6C3F1 mice, liver toxicity was induced at 270 ppm in males as evidenced by clinico-chemical changes in most, as well as a mild degeneration in hepatocytes of some males. Reduction of serum protein levels suggested impaired liver function in the highdose males and females as well as in the 90 ppm males. The reduction of triglycerides and cholesterol in both sexes from the lowest dose level of 30 ppm indicates that the test substance has an effect on lipid metabolism, possibly due to enzyme induction. This conclusion was based on the increased (relative) liver weights at all dose levels in both sexes and the histopathological findings (centrilobular hypertrophy of hepatocytes) at 90 and 270 ppm. Effects at 30 ppm were confirmed for male mice in a second 13-week study using lower doses; NOAEL was set at 15 ppm (RSS of supporting study not reported in IUCLID).

In another 13-week study using C57BL mice, reduced body weight gain was noted in males at 500 ppm and 1000 ppm. Haematological and clinico-chemical changes were similar to the findings with B6C3F1 mice and became obvious at a dose level of 125 ppm. For the target organ liver, increased weights were seen at doses of 125 ppm and higher. Increased serum enzymes indicated liver toxicity at 1000 ppm. Histopathologically, this correlated with liver cell degeneration in 1000 ppm males and females as well as in 500 ppm males. Due to the dose selection, the NOAEL in this study was 7.5 ppm; i.e. 2 mg/kg bw/d in males and females, and a LOAEL of 125 ppm; i.e. 26 mg/kg bw/d in males and 37 mg/kg bw/d in females. However, as the signs of toxicity in both strains of mice were very similar, it can be assumed that the NOAEL of 15 ppm for the B6C3F1 mice is also valid for the C57Bl strain.

The overall NOAEL in these is considered to be 15 ppm, i.e. 4 mg/kg bw/d for males and 5 mg/kg bw/d for female mice.

In a 13-week study in dogs, clinico-chemical examinations revealed, at the high dose (800 ppm) only, increased alanine aminotransferase activity in a single high dose male, increased alkaline phosphatase activity in females as well as reduced cholesterol and total protein in males. These findings suggest the induction of some liver toxicity, although histopathological examination of the liver did not result in test substance related findings. In the kidneys, at 800 ppm and 200 ppm most of the males displayed minimal to moderate hypertrophy of the proximal tubular epithelial cells. At 800 ppm 1 female dog was also found with this lesion. The NOAEL was 200 ppm, i.e. 7.3 mg/kg bw/d for female dogs and 50 ppm, i.e. 1.9 mg/kg bw/d for males.

12-month oral toxicity

In the 12-month dog feeding study, a dose level of 1500 ppm was shown to be too high as three animals died, most likely due to liver damage. The clinico-chemical changes observed also indicated the liver to be the target organ. In one male of the 500 ppm group, a moderate hepatitis was noted. In addition, the test substance showed an anaemic effect in males of all dose groups. During peer-review of EFSA, an overall NOAEL of 1.6 mg/kg bw/d was proposed for both sexes (see attached EFSA report in IUCLID section 13.2).

In a further 12 -month dog study carried out using lower dose levels, no treatment-related effects were observed in this study, which was performed with male animals only, therefore the NOEL was 40 ppm (1.1 mg/kg bw/d), the highest dose tested.

An overall value of 1.6 mg/kg bw/d was proposed for both sexes as a NOAEL in dogs.

Chronic toxicity study in rat

In the chronic toxicity study in rats, the test substance reduced body weight development in males and females of the 1500 ppm group. A transient reduction of body weight development was seen in 750 ppm males. Food consumption was also affected in these groups. A reduction in some red blood cell parameters was observed at the high dose level in males and females as well as in females at 750 ppm. Platelets were reduced at 150 ppm and above in males and at 750 ppm and above in females. Clinical chemistry findings indicating the liver as a target organ were seen predominantly at 1500 ppm, the most sensitive parameter being a reduction of triglycerides at 150 ppm and above in females. Liver weights were noticeable increased only at the high dose level whereas an increase in the incidence and severity of hepatocellular hypertrophy was seen already at 750 ppm. Females at 750 and 1500 ppm exhibited increased cyst formation of the ovary. The incidence of some tumour types identified in the carcinogenicity study (see IUCLID 7.7) to be treatment-related were also slightly increased in the 2-yr chronic toxicity study. Males: slight increase of hepatocellular carcinoma at 1500 ppm; females: slight increase of ovarian tumours (granulosa) in all doses; but the changes to the control groups were not statistically significant. The NOAEL in this study was 30 ppm, equivalent to 1 mg/kg bw in males and 2 mg/kg bw in females (2 mg/kg bw for both sexes combined).

3-week dermal toxicity

In the 21-day dermal study in rats at 1000 mg/kg bw/d, reduced red blood cells and haematocritwere noted, confirming the potential anaemic effect of epoxiconazole. Increased absolute and relative liver weights (males and females), with slight centrilobular hepatocellular hypertrophy (males) was seen in 1000 mg/kg bw/d. At 400 mg/kg bw/d, both absolute and relative liver weights in females were increased, however, these findings were not accompanied by any histopathological changes and were, therefore, considered to represent an adaptive process rather than an adverse effect. There were no signs of local irritation. The NOAEL was 400 mg/kg bw/d.

Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, as the leading health effect is liver toxicity, leading to a classification for carcinogenicity. The information is therefore conclusive but not sufficient for classification.