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

Carcinogenicity

Description of key information

Carcinogenicity study in rats:

The chronic toxicity/oncogenicity studies with epoxiconazole include a 24-month Wistar rat chronic feeding study, a 24-month Wistar rat oncogenicity study and an 18-month C57BL mouse feeding study; they basically confirmed the signs of toxicity seen in subchronic rat and mouse studies.

In the carcinogenicity study in rats, slight decrements in food consumption and body weight gain resulted in slightly to moderately lower body weights for males and females at 750 ppm and 1500 ppm after 24 months of dietary administration of epoxiconazole. The liver was identified as a target organ exhibiting non-neoplastic changes such as weight increases, hepatocellular hypertrophy and increased incidence of eosinophilic and mixed cell foci (males) at doses of 750 ppm and 1500 ppm. Decreased fatty change was seen in the females dosed with 150 ppm and higher, while increased fatty change was noted in males at 1500 ppm. Increased incidences of adrenal gland cortex neoplasms were observed in 1500 ppm males and females.

A dose-related increase in the number of females with ovarian cysts was found at 150 ppm and above while increased incidences of ovarian theca granulosa cell tumours were seen at 750 ppm and 1500 ppm.

Neoplastic change were also observed in the liver: adenoma and carcinoma in males. In the chronic toxicity study, in the carcinogenicity study and in both studies together, the incidence of hepatocellular adenoma in males of the high dose group lies within the range of historical controls, whereas the incidence of hepatocellular carcinomas is above the historical control range. Mechanistic studies have been performed to assess the mechanism of action of hepatic tumor formation in rats (see report attached to IUCLID section 13.2). The results of these studies show that these tumors are of limited relevance for humans.

Decreased incidences of neoplasms were noted for the testes (Leydig cell tumours) in 1500 ppm males, the adrenal gland medulla (phaeochromocytomas) in 1500, 750 and 150 ppm males and the pituitary gland (adenomas) in the 1500 ppm females. The findings are considered indicative of an effect on the synthesis or availability of steroid hormones and induction of liver enzymes.

The NOAEL in this study was 30 ppm for females (2 mg/kg bw) based on the incidence of ovarian cysts and 150 ppm for males (6 mg/kg bw) based on liver changes.

 

Carcinogenicity study in mice:

In the carcinogenicity study in mice, treatment with epoxiconazole resulted in a decrease in body weight gain of males and females at a food concentration of 200 ppm and above. The target organ was the liver as indicated by an increased organ weights in high dose and intermediate dose females (1000 ppm and 200 ppm) and males (500 ppm and 200 ppm). Moreover, histopathological changes such as hypertrophy, hyperplasia and focal necrosis were present in high dose males and females. An increased incidence of eosinophilic liver cell foci was seen in high dose males and females as well as in 200 ppm males. There was a test substance-related increase in the incidence of liver neoplasia in the 1000 ppm females and 500 ppm males.

Three males (6%) of dose group 5 (500 ppm) showed hepatocellular adenomas. This incidence lies within the range of historical controls. The incidence of hepatocellular carcinomas (66%) in males of dose group 5 is clearly above the historical control range, whereas the occurrence of hepatocellular carcinomas (6%) in dose group 4 (200 ppm) lies within the range of historical controls. The numbers of hepatocellular adenomas and hepatocellular carcinomas in females of dose group 5 (1000 ppm) lie clearly above the historical control range. Mechanistic studies have been performed to assess the mechanism of action of hepatic tumour formation in mice (see attachment to this endpoint summary). The results of these studies show that these tumours are of limited relevance for humans.

The NOAEL in this study was 5 ppm, equivalent to 0.7 mg/kg bw in males and 0.9 mg/kg bw in females (0.8 mg/kg bw for both sexes combined) in the main groups.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1989 - Feb 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Qualifier:

according to guideline

Guideline:

other: Pesticide Assessment Guidelines, Subdivision F, § 83-2, pages 117 - 125, NTIS, Nov. 1982 and revised edition, Nov. 1984

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies (Japan/Maff, 1985)

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, FRG
- Age at study initiation: 35 days
- Weight at study administration: males (161 g - 192 g); females (138 g - 166 g)
- Fasting period before sacrifice: 16 - 20 hours
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days


DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical as well as for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm illumination)

IN-LIFE DATES: From:Jan 30, 1989 To: Feb 27, 1991

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): regular intervals
- Mixing appropriate amounts with (Type of food): Kliba maintenance diet rat/mouse/hamster 343 meal
- Storage temperature of food: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The correctness of the concentrations was checked by taking samples of each concentration at the start of the study and thereafter at about 3-month intervals and these samples were sent to the analytical laboratory. The content of the test substance was determined by HPLC. The analysis of the test substance in the food was carried out using HPLC.

Duration of treatment / exposure:

24 months

Frequency of treatment:

continuous

Post exposure period:

none

Dose / conc.:

30 ppm (nominal)

Remarks:

about 2 mg/kg bw

Dose / conc.:

150 ppm (nominal)

Remarks:

about 7 mg/kg bw

Dose / conc.:

750 ppm (nominal)

Remarks:

about 40 mg/kg bw

Dose / conc.:

1 500 ppm (nominal)

Remarks:

about 80 mg/kg bw

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The selection of doses was based on results of a 4-week study, where rats received the test substance in doses of 250, 1,000, and 4,000 ppm (NOEL 250 ppm), a first three-months feeding study in rats receiving the test substance in doses of 30, 90, 270, and 800 ppm (NOEL 90 ppm), and a second three-months study in rats receiving the test substance in doses of 500, 1,000, 1,500, and 2,000 ppm (no NOEL determined). Results of all studies indicate that the maximum tolerated dose for male and female rats in the long-term study is 1,500 ppm.
- Rationale for animal assignment (if not random): random

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Mortality and moribundity: twice daily (Mon - Fr)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Day 728 of the study was the last day on which the findings of all animals were assessed as group findings. Thereafter, any changed findings were recorded until each specific day of necropsy in the relevant individual data sheets. The entries in the computer system were done analogously, i.e. after day 728 only changes to o1d findings or additiona1 findings were recorded, but not such ones which had been noted before and remained unchanged until the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 14 weeks, monthly thereafter and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: once a week for one week during the first 14 weeks, thereafter: for one week at 4-week intervals and at the end of the study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Yes (16-20 hrs)
- How many animals: all animals
- Parameters checked: Differential blood count

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Complete necropsies were peroformed on all rats. The terminal body weight and the weights of adrenal glands, brain, liver, kidneys, and testes were determined from all rats which survived to scheduled necropsy. Representative tissue specimens of the following organs were sampled and fixed in 4% formaldehyde: adrenal glands, aorta, bone (sternum, femur including femorotibial joint), bone marrow (sternum, femur), brain, cecum, coagulation glands, colon, duodenum, epididymides, esophagus, eyes, female mammary gland, heart, ileum, jejunum, kidneys, liver, lung, lymph node (mandibular, mesenteric), ovaries, pancreas, parathyroid glands, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, and all organs or tissues with macroscopic abnormalties.

HISTOPATHOLOGY: Yes
- After fixation and histotechnical processing of the tissues, light microscopic examination was performed according to:
- All animals affected: All gross lesions
- All animals of control and 1500 ppm: brain, pituitary gland, thyroid gland, parathyroid glands, thymus, trachea, heart, aorta, mandibular salivary glands, sublingual salivary glands, pancreas, epididymides, coagulation glands, prostate gland, seminal vesicles, esophagus, stomach, duodenun, jejunum, ileum, cecum, colon, rectum, urinary bladder, menteric lymph nodes, mandibular lymph nodes, sciatic nerve, sternum (with marrow), femur (with marrow, joint), skeletal muscle, female mammary gland, skin, spinal cord (3 levels), eyes
- All animals from all test groups and control: lungs, liver, spleen, kidneys, adrenal glands, testes/ovaries, uterus/cervix

Statistics:

ANOVA, Dunnett's test

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: only in males retarded body weight gain, but in both sexes reduced body weights at the end of the study (in males about 10% and in females about 18%)
- 750 ppm: only in males retarded body weight gain, but in contrast only in females reduced body weights at the end of the study of about 12%

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- 1500 ppm: Sporadic food reductions in males and females were assessed as not substance-related but incidentally.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The changes in the white blood cell count found in the prematurely sacrificed animals of both sexes in all test groups are spontaneous, incidental, or age-dependent and cannot be associated with the test substance administered. At the end of the study no prominent differences were observed in the red blood cell count of male and female animals of the control and highest dose group (1,500 ppm). Moreover, no substance-related deviations were detected in the red blood cell count of the prematurely sacrificed animals of both sexes.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: increased absolute (females) and relative (both sexes) liver weight
- 750 ppm: increased relative liver weight in females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: macroscopically increased incidence of masses and cysts of the ovaries and decreased incidence of masses of the pituitary gland in both sexes
- 750 ppm: macroscopically increased incidence of masses and cysts of the ovaries and decreased incidence of masses of the pituitary gland in females
- 150 ppm: macroscopically increased ovarian cysts and decreased incidence of pituitary gland masses in the females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Increase in incidence and severity of hepatocellular hypertrophy in both sexes and of eosinophilic and mixed cell foci of hepatocellular alteration in males increased incidence and severity of fatty changes in the liver in males, while a decrease in females was noted
- 750 ppm: Increase in incidence and severity of hepatocellular hypertrophy and of eosinophilic and mixed cell foci of hepatocellular alteration in males. Increase in incidence and severity of hepatocellular hypertrophy and of eosinophilic and mixed cell foci of hepatocellular alteration in males. Decrease in incidence and severity of fatty changes in the liver in females
- 150 ppm: decrease in incidence and severity of fatty changes in the liver in females

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Increased benign theca granulosa cell tumors and slightly increased combined incidence of gonadal stromal tumors in females. Increased cortical neoplasms of the adrenal glands in females and decreased medullary neoplasms of the adrenal gland in the males. Decreased incidence of pituitary gland adenomas, while the incidence of hyperplasia increased in the females. Decreased incidence of Leydig cell tumor of the testes. Slight increase in the incidence of hepatocellular liver tumors in male rats; statistically not significant but above historical control range.
- 750 ppm: increased benign theca granulosa cell tumors in the females. Decreased incidence of medullary neoplasms of the adrenal glands in males
- 150 ppm: decreased incidence of medullary neoplasms of the adrenal glands in males

Details on results:

Other findings which may be secondarily related to treatment were recorded in the adrenal glands, heart, kidneys, lungs, mammary glands, ovaries, and uterus/cervix. In the adrenal glands, the incidence and severity of cellular hypertrophy in the adrenocortex increased in males of group 2 and females of group 4. The incidence and severity of nodular hyperplasia of the adrenocortex increased in males and females of group 4.
In the heart, the severity of myocytolysis/myofibrosis decreased in males of groups 3 and 4. In the kidneys, the incidence and severity of chronic progressive nephropathy increased in males of group 2
and females of groups 2, 3 and 4.
In the lungs, aggregates of alveolar macrophages increased in incidence in males and females of group 4. The severity of this finding increased in females of group 4.
The incidence and severity of perivascular cuffing were marginally increased in males of group 4. In the mammary glands, the incidence and severity of alveolar secretion and glandular hyperplasia decreased in females of all treated groups. Galactoceles decreased in incidence in groups 2, 3 and 4, and in severity in groups 3 and 4.
In the ovaries, senile involution of the gonads decreased in incidence in groups 3 and 4. The severity of this finding decreased in groups 2, 3 and 4. Ovarian cysts, predominantly luteal cysts, were more frequently recorded in groups 2, 3, and 4 than in controls.
In the uterus, the incidence and severity of senile endometrial atrophy decreased in groups 1 and 4. The incidence and severity of squamous cell hyperplasia of the portio vaginalis uteri decreased in group 4.
All other histopathological findings recorded in this study were considered to be incidental.
There were no substance-related findings in the 30 ppm group.

Relevance of carcinogenic effects / potential:

It is concluded that the long-term administration of the test substance did result in increased numbers of ovarian tumors at 1,500 ppm and 750 ppm and adrenocortical tumors in females at 1,500 ppm. The observed increase in neoplasms in the ovaries and adrenals of females should be assessed with respect to pronounced toxicity and the induction of steroid hormonal imbalance in these animals. The neoplastic effect is considered to be the result of cellular proliferation induced by stimulating steroid hormones. As the test substance is devoid of a genotoxic potential, the hormone-induced neoplastic response will have no relevance at (low) dose levels which do not result in changes of the ovaries and adrenals. No increased incidence of neoplastic lesions was observed in males. The no observed adverse effect level for non-neoplastic lesions is 150 ppm in males and 30 ppm in females.

The histopathologic evaluation of this study revealed treatment-related neoplastic and non-neoplastic lesions in both sexes. The increased incidence of benign ovarian neoplasms in females of groups 3 and 4 (750 ppm, 1500 ppm) and predominantly benign neoplasms in the adrenal cortex of females of group 4, as well as the decreased incidence of neoplasms in the adrenal medulla, pituitary gland, and testes are considered to reflect an endocrine dysfunction in the affected animals.
This assumption is further supported by certain nonneoplastic findings indicative of impaired adrenocorticotropic, mammotropic, or reproductive hormonal regulation (viz. increased adrenocortical hypertrophy and nodular hyperplasia, ovarian cysts, decreased secretory activity, alveolar hyperplasia, galactoceles, senile atrophy, cervical squamous cell hyperplasia).
Whilst the relatively high proportion of luteinized theca-granulosa cell tumors among treated animals may have resulted from a direct antagonistic effect of the test article, it could also be due to a disturbance of hypothalamic-hypophyseal interactions. The latter hypothesis is supported by the decreased number of adenomas in the anterior hypophysis and of interstitial Leydig cell tumors in the testes. It seems also worth mentioning, with respect to a supposed dysregulatory pathogenesis of the ovarian tumors, that some of them clearly evolved within persisting corpora lutea and that a great number of cysts in the poly­ cystic ovaries were of luteal or gin. These findings are compatible with a de­ creased luteolytic activity in these rats.
As in previous studies, the administration of the test article did also result in nonneoplastic liver lesions. The incidence and severity of hepatocellular hypertrophy in males of groups 3 (750 ppm) and in both sexes of group 4 (1500 ppm) were remarkably increased and of a sufficient degree of severity to explain the increased liver weights in animals of group 4. It was also confirmed that a correlation exists between the degree of severity of hepatocellular hypertrophy and the topography of hypertrophic cells within the liver plates. In the less severe stages, hypertrophic liver cells were confined to centrolobular or mid­zonal areas of the liver plates, whereas a rather diffuse distribution was seen in the most severe stages. There was no correlation between the degree of hypertrophy and the increased number or size of eosinophilic or mixed cell foci of hepatocellular alteration. Although the pathophysiological mechanism of this proliferation of endoplasmic reticulum cannot be inferred from this study, it may be speculated that the remarkable hypertrophy of liver cells could also interfer with the metabolic pathways of steroid hormones.
An inverse relationship between males and females was seen in incidence and severity of fatty change. This finding increased in males of group 4 but decreased in females of all treated groups. Again, sex-specific metabolic pathways could play a role.
The treatment-related lesions in heart, kidneys, and lungs are considered to be of minor toxicological significance.
All other findings, recorded at a differing incidence or severity in treated and control groups, are considered to be incidental. It is concluded from this study, that the long-term administration of the test article did not result in increased numbers of ovarian or adrenocortical tumors in females, when given at nominal concentrations of 30 ppm and 150 ppm. The administration of the test article did not cause toxicologically significant non-neoplastic lesions at concentrations of 30 ppm and 150 ppm.

Dose descriptor:

NOAEL

Remarks:

non-neoplastic lesions

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: 6 mg/kg bw

Dose descriptor:

NOAEL

Remarks:

non-neoplastic lesions

Effect level:

30 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: 2 mg/kg bw

Dose descriptor:

NOAEL

Remarks:

neoplastic lesions

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

750 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

750 ppm

System:

endocrine system

Organ:

adrenal glands

ovary

pituitary gland

testes

Treatment related:

yes

Dose response relationship:

yes

Table 1: Evaluation of neoplasms - absolute numbers

Sacrifice	F1
Sex	M/F
Group	0	1	2	3	4
Animals in selected Group	50/50	50/50	50/50	50/50	50/50
Number of animals with:
Neoplasms	49/49	39/49	39/49	43/48	46/48
1 Primary Neoplasm	10/14	11/11	14/18	22/17	17/13
2 and > primary neoplasms	39/35	28/38	25/31	21/31	29/35
Number of animals with:
Benign neoplasms	44/47	34/48	37/48	40/46	42/46
Benign neoplasms only	30/31	26/32	29/38	35/35	28/37
Malignant neoplasms	19/18	13/17	10/11	8/13	18/11
Malignant neoplasms only	5/2	5/1	2/1	3/2	4/2
Systemic neoplasms	1/2	4/1	1/1	1/1	2/-
Metastasized neoplasms	3/3	-/2	2/4	1/3	2/1
Total number of:
Primary neoplasms	134/115	88/115	89/103	82/106	100/114
Benign neoplasms	112/89	75/97	77/90	72/92	81/101
Malignant neoplasms	22/26	13/18	12/13	10/14	19/13
Systemic neoplasms	1/2	4/1	1/1	1/1	2/-
Metastasized neoplasms	3/3	-/2	2/4	1/3	2/1

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 1989 - Mar 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Qualifier:

according to guideline

Guideline:

other: Pesticide Assessment Guidelines, Subdivision F. § 83-2, page 117-125 NTIS, revised edition, Nov 1984

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies (Japan/Maff, 1985)

GLP compliance:

yes

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

Since the liver is the main target organ of the test substance's toxicity, it was important to choose a mouse strain with a low background of spontaneous liver changes to be able to discriminate between substance-related and spontaneous lesions. To avoid a possible bias of the results due to fluctuations of spontaneous nature, the C57BL mouse rather than the B6C3F1 mouse was chosen.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, D-W8741, Sulzfeld, FRG
- Age at study initiation: 49 days
- Weight at study initiation: 23 g (males satellite and main group); 19 g (females satellite and main group)
- Fasting period before study: 20 hrs before sacrifice
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

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)

OTHER:
- Before beginning of the study and thereafter at about 6-month interval, rooms were fully disinfected
- Virological status of animals was checked over the entire study period. Serum samples were tested for the following antibodies: MHV (mouse hepatitis virus), Reo 3 (Reo virus, type 3), TEMV (Theiler's encephalomyelitis virus), PVM (pneumonia virus of mice), Sendai virus, MVM (minute virus of mice)


IN-LIFE DATES: From: August 15, 1989 To: March 20, 1991

Route of administration:

oral: feed

Vehicle:

other: via diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 4-week interval
- Mixing appropriate amounts with (Type of food): Kliba maintenance diet rat/mouse/hamster
- Storage temperature of food: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations were checked by taking samples of each concentration at the start and thereafter at about 3-month interva1s up to the end of the study and these samp1es were sent to the analytical laboratory. The content of the test substance/food mixtures was determined by HPLC.

Duration of treatment / exposure:

52 weeks (satellite groups), 78 weeks (main groups)

Frequency of treatment:

continuous

Dose / conc.:

1 ppm (nominal)

Remarks:

about 0.12 and 0.22 mg/kg bw in males and females, resp.; about 0.16 mg/kg bw combined

Dose / conc.:

5 ppm (nominal)

Remarks:

about 0.69 and 0.92 mg/kg bw in males and females, resp.; about 0.91 mg/kg bw combined

Dose / conc.:

200 ppm (nominal)

Remarks:

about 28.1 and 42.4 mg/kg bw in males and females, resp.; about 36 mg/kg bw combined

Dose / conc.:

500 ppm (nominal)

Remarks:

males only; about 70.4 mg/kg bw

Dose / conc.:

1 000 ppm (nominal)

Remarks:

females only; about 205.4 mg/kg bw

No. of animals per sex per dose:

50 (main group), 10 (satellite group)

Control animals:

yes, plain diet

Details on study design:

- Toxicokinetic data
- Dose selection rationale: Doses were chosen based on a 4-week feeding study with animals receiving the test substance to 4000, 1000, and 250 ppm, a first 3-month feeding study with mice receiving the test substance in doses of 30, 90, and 270 ppm, and a second 3-month with doses of 7.5, 15, 30 ppm and a further 3-month feeding study using 1000, 500, 250, 125, and 7.5 ppm. Based on the investigations observed in the prelimiary study, the following dose levels were chosen for the 78-week feeding study: 1 ppm: as an expectable no adverse effect level; 5 ppm: as a concentration with possible marginal toxic effects or as another no adverse effect level; 200 ppm: as a concentration with expectable toxic effects; 500 ppm (male)/1000 ppm (female): expectable toxic effects without any adverse effect on the normal life-span.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: none

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 14 weeks and thereafter generally at 4-weekly intervals and before sacrifice of satellites animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Once a week during the first 14 weeks and thereafter generally at 4-weekly intervals and before sacrifice of satellites animals

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after about 12 months (satellite)
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes (20 hrs)
- How many animals: all animals
- Parameters checked: White blood count and red blood count

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Virology: 5 male and 5 female rats were killed shortly after arrival, after about 1, 6, 12, and 18 months after beginning of the study and their serum samples were investigated for specific antibodies (see above)

- Animals of the satellite groups were subjected to an interim kill after 52 weeks; the surviving mice in the main groups were sacrificed after about 78 weeks.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were necropsied and assessed by gross pathology. The weight of the anesthetized animals as well as the weights of liver, kidneys, heart, brain, spleen, adrenal glands and testes from all animals sacrificed at scheduled dates were determined.

HISTOPATHOLOGY: Yes
- Subsequently, the following organs were fixed and examined by light microscopy:
- All test groups: lungs, liver, spleen, kidneys, adrenal glands, uterus with cervix uteri, testes/ovaries, gallbladder
- Control and high doses only: brain, pituitary gland, thyroid glands (with parathyroid glands), thymus, trachea, heart, pancreas, prostate, seminal vesicle, coagulation gland, epididymides, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and mandibular), peripheral nerve (sciatic nerve), sternum with marrow, salivary glands (mandibular and sublingual gland), bone marrow (femur), skin, female mammary gland, skeletal muscle, eyes and Harderian glands, spinal cord (cervical, thoracic, lumbar cord), aorta, femur (with joint)
- Only affected animals: all gross lesions

Statistics:

ANOVA, Dunnett's test, Student's t-test, Kruskal-Wallis, Wilcoxon-test, Fischer's exact test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 1000 ppm: Females revealed signs of extended abdomen and reduced general state in a higher incidence than the other dosed and control group animals.
The four doses did not adversley affect the general condition of males.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

The mortality rate of male and female mice was not affected by the administration of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

SATELLITE GROUP
- 1000 ppm (females) and 500 ppm (males): retarded body weight gain in males and at the end after about one year reduced body weights in males of about 19-22% as well as in females of about 22-24% in comparison to the two control groups. Significant reduction of terminal body weight in both sexes.
- 200 ppm: retarded body weight gain in the males and at the end of the study slightly reduced body weights in both sexes in comparison to the control groups. Statistically significant decrease of the terminal body weight in males

MAIN GROUPS
- 1000 ppm (females) and 500 ppm (males): Clearly reduced body weight throughout the study and by the end 15-20% and 17-20% reduction in males and females, respectively. Significant reduction of the terminal body weight in both sexes.
- 200 ppm: retarded body weight gain in the males but at the end of the study slight reduced body weights in both sexes in comparison to the control groups statistically significant decrease of the terminal body weight in males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

SATELLITE GROUP
- 1000 ppm (females) and 500 ppm (males): reduced food consumption in males and more pronounced in females

MAIN GROUP
- 1000 ppm (females) and 500 ppm (males): male and female mice showed reduced food consumption during the first year; but during the last few months of the study no clear substance-related effect was evident

Food efficiency:

no effects observed

Description (incidence and severity):

In summary, it can be stated that there was no substance related impairment of the food efficiency in any group.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

SATELLITE GROUPS
No adverse effect observed

MAIN GROUPS
The deviations observed in isolated cases in the treatment groups of the males are not considered to be substance related. These differences are assessed as being incidental, spontaneous or age dependent. Individual prematurely sacrificed animals showed pathognomic changes in the blood which were found both in the controls and in the treatment groups. Thus, changes are considered to be incidental or age-dependent and are not due to the test compound administered.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes in behavior in any dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

SATELLITE GROUP
- 1000 ppm (females) and 500 ppm (males): Significant increase of absolute and relative liver weights in both sexes
- 200 ppm: statistically significant increase of the absolute liver weights in females only and relative liver weights in both sexes

MAIN GROUPS
- 1000 ppm (females) and 500 ppm (males): Significant increase of absolute and relative liver weights in both sexes
- 200 ppm: statistically significant increase of the absolute and relative liver weights in females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

SATELLITE GROUP
Only a few gross lesions were noted. Among these, the following were regarded to be of interest.
In the liver, differently colored foci were reported in one male mouse ot group 1 ppm, in another male mouse of group 5 ppm, and in two males of group 500 ppm, while no male mouse of the two control groups had such a finding, macroscopically. In female mice, foci of this kind occurred in one animal of group 5 ppm, and in five females of dose group 1000 ppm, while again no foci were reported from any of the control animals. One female mouse of group 1000 ppm had an enlarged liver.
In the males, a most likely congenital deformation of the sternum was recorded in two animals of group 5 ppm, in one male mouse of group 200 ppm and in five males of group 500 ppm, whereas none of the controls had such an observation.
All other gross lesions noted in the satellite groups were spontaneous in origin and could not be related to the application of the test article.

MAIN GROUPS
Macroscopically, in the liver there were masses in one male of group 200 ppm and 20 males of group 500 ppm. In females, masses were seen in each one animal of control groups, in two animals of group 1 ppm, in each one animal of groups 5 ppm and 200 ppm, and in 25 animals of group 1000 ppm.
Different colored foci occurred in six males of control, in four males of the second control, five males of group 1 ppm, four males of group 5 ppm, seven males of group 200 ppm, and in 33 males of group 500 ppm.
In females, different colored foci were seen in three animals of control, two animals of the second control, four animals of group 1 ppm, seven animals of group 5 ppm, three animals of group 200 ppm, and 23 animals of group 1000 ppm.

All other macroscopically diagnosed changes - comprising granular surface, enlarged, prominent acinar pattern, focal constriction, discoloration, torsion, adhesion, and reduced organ size - occurred only in a few individual animals.
In the kidneys, cysts were seen in eight males of control, each ten males of the second control and 1 ppm, 14 males of group 5 ppm, 11 males of group 200 ppm and 16 males of group 500 ppm.
In females, cysts occurred in three animals of control, six animals of the second control, five animals of group 1 ppm, six animals of group 5 ppm, two animals of group 200 ppm, and 13 animals of group 1000 ppm.
The organ size of the kidneys was reduced in two males of control, one male of the second control, two males of group 1 ppm, three males of group 200 ppm, and five males of group 500 ppm.
In females, kidneys with reduced organ size were seen in one animal of control, each four animals of the second control and 1 ppm, two animals of group 5 ppm, three animals of group 200 ppm, and nine animals of group 1000 ppm.
In males, enlarged kidneys occurred in one male of control, three males of the second control, four males of group 1 ppm, three males of group 5 ppm, and in six males of group 500 ppm.
In females, kidneys with granular surfaces were present in one animal of control, each two animals of groups 1 ppm, 5 ppm, and 200 ppm, and in six animals of group 1000 ppm.
The incidence of all other findings in the kidneys was comparable between control animals and treated animals.

In males, a most likely congenital deformation of the sternum was seen in two animals of control, one animal of the second control group, seven animals of group 1 ppm, six animals of group 5 ppm, eight animals of group 200 ppm, and six animals of group 500 ppm.

All other gross lesions noted were spontaneous in origin and could not be related to the application of the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

SATELLITE GROUP
- 1000 ppm (females) and 500 ppm (males): Diffuse or centrolobular hypertrophy of hepatocytes in both sexes. Eosinophilic foci in the liver of two males. Increased deposition of amyloid in testes, and in liver, glandular stomach, duodenum, cecum, colon, thyroid glands, parathyroid glands, and heart of females. Decrease of lipid storage in the liver of both sexes.
- 200 ppm: Centrolobular hypertrophy of hepatocytes in males

MAIN GROUPS
- 1000 ppm (females) and 500 ppm (males): increased incidences of focal necrosis, focal hyperplasia, and foci of cellular alteration (especially the eosinophilic type) in both sexes. Increased deposition of amyloid in testes, and in liver, kidneys, gastrointestinal tract (excluding the forestomach), ovaries, uterus, spleen, thyroid glands, parathyroid glands, heart, mandibular lymph nodes, pancreas, mandibular salivary glands, and adrenal glands of females
- 200 ppm: slightly more eosinophilic foci of cellular alteration in the liver of males (4 animals)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MAIN GROUPS
- 1000 ppm (females) and 500 ppm (males): increased number of hepatocellular carcinomas of the liver in both sexes (each 33 animals). Increased number of hepatocellular adenomas in the liver in both sexes (3 males, 5 females)

Other effects:

no effects observed

Description (incidence and severity):

The virological examinations carried out in the serum of the sentinel animals showed that no infection with mouse hepatitis virus, reovirus type 3, Theiler's encephalomyelitis virus, pneumonia virus of mice, Sendai virus, or minute virus of mice occurred during the study.

Relevance of carcinogenic effects / potential:

The number of males with neoplasms, especially the number of males with 2 and more than 2 neoplasms, the number of males with benign neoplasms, with malignant neoplasms and the total number of benign and malignant neoplasms was increased in group 500 ppm.
For males that died or that were sacrificed prematurely, the number of animals with neoplasms, with benign or malignant neoplasms and the total number of benign and malignant neoplasms was comparable between control groups and group 500 ppm.
In females, the number of animals with neoplasms, the number of animals with malignant neoplasms and the total number of malignant neoplasms was increased in group 1000 ppm.
For animals that died or that were sacrificed in a moribund state, there were no relevant differences between control groups and group 1000 ppm.

The increased incidence of hepatocellular carcinomas observed at 500 and 1000 ppm is assessed as being the result of a proliferative stimulus on the liver due to a marked induction of the P450 enzyme system at these excessively high dose levels (i.e. beyond the maximum tolerated dose [MTD]). Hepatic P450 enzyme inducers exert only in mice an oncogenic effect on the liver. No increased incidence of tumors was seen at 200 ppm.
In conclusion, a carcinogenic effect on the liver of mice was observed at the high dose level in this study. This effect is assessed to be the result of a proliferative stimulus on the liver due to a marked induction of the P450 enzyme system. As the test substance is devoid of a mutagenic potential and a carcinogenic response was only seen at dose levels higher than the maximum tolerated dose, it is concluded that the test substance has no carcinogenic potential at dose levels below MTD.

Dose descriptor:

NOAEL

Effect level:

5 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 0.7 mg/kg bw in males and 0.9 mg/kg bw in females (0.8 mg/kg combined)

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Table 1: Incidences of hepatocellular adenomas and hepatocellular carcinomas in main groups

F1	Group 0 Control	Group 1 Control	Group 2 1 ppm	Group 3 5 ppm	Group 4 200 ppm	Group 5 500/1000 ppm
Hepatocellular adenomas -       Males -       females	0 0	0 0	0 0	0 0	0 0	3 5
Hepatocellular carcinomas -       males -       females	1 0	0 1	0 1	0 1	3 1	33 33

The incidences of hepatocellular tumors in males and females of group 5 are considered treatment-related and are statistically significant at 0.001 level using Fisher's exact test. The incidences in all other treated groups did not differ statistically significantly from the controls and are within the range known for this strain of mice. Therefore, they are considered not to be treatment-related.

Table 2: Incidences of toxic alterations in the liver

F1	Group 0 Control	Group 1 Control	Group 2 1 ppm	Group 3 5 ppm	Group 4 200 ppm	Group 5 500/1000 ppm
Necrosis -       Males -       females	3 2	2 1	1 2	2 2	6 4	7 5
Hyperplasia -       males -       females	0 0	0 0	0 0	0 0	0 0	2 5
Foci -       males -       females	3 1	0 0	3 1	2 0	5 1	21 16
Eosinophilic foci -       males -       females	1 0	0 0	0 0	0 0	4 0	19 14

Mode of Action Analysis / Human Relevance Framework

Some studies have been performed to elucidate the mode of action of epoxiconazole on both the synthesis of steroid hormones and liver enzyme induction. These studies are summarized in the attachment to this endpoint summary. In the absence of any evidence for a genotoxic potential of epoxiconazole, the available data points to a non-genotoxic mechanism for tumor induction, which is associated with a threshold dose. This means that a dose level can be defined at which there is no carcinogenic risk.

For further elucidation of the mechanism underlying the tumour formation in the rodent liver, a comprehensive suite of toxicological studies in vitro and in vivo has been completed, investigating effects in liver cells from rats, mice (normal and CAR/PXR knockout male and female mice), and from human liver cells. In both animal and cell culture studies, liver cytochrome P450 (Cyp2B1, Cyp2B2 and Cyp3A1 in rats and Cyp2B and Cyp3A in WT mice) were induced, together with increase of the correspondent enzyme activity, and cell proliferation. In contrary, these findings were not seen in knockout mouse or in human cell

cultures. This demonstrates that the mode of action of the tumour formation is due to a CAR/PXR activation, like phenobarbital, and are therefore of doubtful relevance for humans.

For a review of mode of action studies available, please see the MoA report attached to this IUCLID chapter.

Justification for classification or non-classification

Epoxiconazole is classified for carcinogenicity cat.2 (H351) according to Regulation (EC) 1272/2008 Annex VI