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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1989 - Jan 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Pesticide Assessment Guidelines, Subdivisoin F, § 83-1, pages 107-117 NTIS, Nov. 1982 and revised edition, Nov. 1984

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

406-850-2

EC Name:

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

Cas Number:

133855-98-8

Molecular formula:

C17 H13 Cl F N3 O

IUPAC Name:

1-{[(2R,3R)-3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach, Germany
- Age at study initiation: 35 days
- Weight at study initiation: 189 g males (175 - 202 g); 148 g females (132 - 161 g)
- Fasting period: 16-20 h before sacrifice
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 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): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: January 26 (administration period began) 1989 To: January 24, 1991 (last day of sacrifice)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral 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 analysis of the test substance in the food was carried out using HPLC.

Duration of treatment / exposure:

24 months

Frequency of treatment:

continous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 ppm

Remarks:

males: 1 mg/kg bw/day, females: 2 mg/kg bw/day, all animals: 2 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

150 ppm

Remarks:

males: 6 mg/kg bw/day, females: 9 mg/kg bw/day, all animals: 8 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

750 ppm

Remarks:

males: 32 mg/kg bw/day, females: 44 mg/kg bw/day, all animals: 38 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 500 ppm

Remarks:

males: 67 mg/kg bw/day, females: 89 mg/kg bw/day, all animals: 78 mg/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The selection of doses was based on the following studies: A 4-week study in rats via diet with concentrations of 4000 ppm, 1000 ppm, and 250 ppm, the first 3-month feeding study in rats via diet using 800 ppm, 270 ppm, 90 ppm, and 30 ppm, a second 3-month feeding study in rats using 2000 ppm, 1500 ppm, 1000 ppm, and 500 ppm.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: not fasted

Positive control:

not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at leat once daily
- Cage side observations checked: state of health

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

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week during the first 14 weeks. Thereafter, at 4-week intervals and at the end of the study

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. Thereafter, at 4-week intervals and at the end of the study

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: Yes
- Time schedule for examinations: before start and end of study
- Dose groups that were examined: both sexes from control and maximum dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after about 3, 6, 12, 18 and 24months after the start of the study.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets count (PLT), differential blood count, reticulocytes (RETI), thromboplastin time (Hepato Quick's test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after about 3, 6, 12, 18 and 24months after the start of the study.
- Animals fasted: No
- How many animals: all animals
- Parameters checked: Alanine aminotransferase, aspartate aminotrasferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: after about 3, 6, 12, 18 and 24months after the start of the study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Complete necropsies were performed on all rats.
- Terminal body weight, weights of adrenal glands, brain, liver, kidneys, and testees were determined from all animals which survived to scheduled necrospy

HISTOPATHOLOGY: Yes
- 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, jejunum, kidneys, liver, lungs, lymph node (mandibular, mesenteric), ovaries, pancreas, parathyroid glands, pituitary gland, 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 abnormalities.

Statistics:

ANOVA, Dunnett's test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

The mortality rate of the male and female animals was not affected by admnistration of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Retarded body weight gain in the males and at the end of the study reduced body weight in the males of about 9% and in the females of about 9%
- 750 ppm: In males, during the second half of the study body weights turned to comparable values to control and at the end of the study did not show statistically significance or biological relevance

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Reduced food consumption in males of about 12% and in females of about 7% at the end of the study
- 750 ppm: In males, slight decrease in food consumptionoccurred until the 86th week and then became comparable to the control group until the end of the study, where no biologically evident deviation was noted.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The remainders of the pupillary mambrane are incomplete involution of vessels, which are evident for the nutrition of the eye during the normal embryonic development. They have no influence on the general development, general state and the normal life span of the affected animals. Due to this, this finding is without any pathological or substance-related relevance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Decrease in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), platelets, and alkaline phosphatase in both sexes. Decrease in mean corpuscular hemoglobin concentration (MCHC) and thromboplastin time in females. These changes are assessed to be substance related and are due to a slight hemotoxic potential of the test substance.
- 750 ppm: Decrease in platelets in both sexes. Decrease in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thromboplastin time, and alkaline phosphatase in females
- 150 ppm: Decrease in platelets in males

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Increase in gamma-glutamyltransferase in both sexes. Increase in urobilinogen in the urine of both sexes. Increase in albumin in males. Increase in cholesterol, total protein and globuline in females. Decrease in triglyceride concentration in both sexes.
- 750 ppm: Increase in gamma-glutamyltransferase and albumin in males. Increase in cholesterol and globulins in females. Decrease in triglycerides in females.
- 150 ppm: Decrease in triglycerides in females, increase in cholesterol in females

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- 1500 ppm: Discoloration of the urine specimens in females. Increased urobilinogen levels in both sexes, which suggestes a liver dysfunction

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 and relative liver weights in both sexes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

As compared with controls, the incidences of ovarian cysts and thickening of the cervix were higher in groups 150, 750, and 1500 ppm. However, there was no clear dose relationship. In males, the incidence of testicular masses was remarkably lower in all treated groups.

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
- 750 ppm: Increase in incidence and severity of hepatocellular hypertrophy in both sexes

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All neoplastic lesions recorded in this study were considered to be of a spontaneous nature.
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.

Details on results:

The slight decrease in food consumption until the 86th week and the slight retardation of the body weight gain in the males dosed 750 ppm is probably induced by the test substance, but both changes improved during the further course of the study and at the end of the study these parameters were comparable to the respective controls. Thus, these changes were assessed as signs of minor or no toxicological relevance.
At histopathological examination, non-neoplastic treatment-related findings of toxicological significance were recorded in the liver in the form of hepatocellular hypertrophy. As compared with controls, the incidence and severity of hypertrophy were increased in males and
females of groups 750 and 1500 ppm.
Further, as compared with controls delayed age-related involution of the female gonads and less advanced age­ related lesions in adrenal glands, heart, kidneys and uterus/cervix were recorded in males and females of dose groups 750 and/or 1500 ppm, respectively.
All other histopathological findings recorded in this study were considered to be incidental.

It is concluded from this study, that the long-term administration of the test article did not result in oncogenic effects in rats when given via the feed at nominal concentrations of 30, 150, 750, and 1,500 ppm. All neoplastic lesions recorded in this study were considered to be of a spontaneous nature. Malignant hepatocellular neoplasms were recorded at a higher incidence in males of the dose group 1500 ppm. This difference, however, did not prove to be statistically significant. The combined incidence of benign plus malignant hepatocellular
neoplasms across the groups in males was 6, 2, 3, 4, 9, in females 0, 1, 1, 0, 0 for control, 30 ppm, 150ppm, 750 ppm, 1500 ppm, respectively. Theca-granulosa cell tumors were more frequently recorded in treated groups at an incidence of 0, 0, 1, 3, 3. This difference did not prove to be statistically significant. The administration of the test article did not cause any nonneoplastic histopathologic lesions at concentrations of 30 ppm and 150 ppm.

Effect levels

Dose descriptor:

NOAEL

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

750 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

750 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Table 1: Neoplastic lesions on the liver

		Group 0	Group 1	Group 2	Group 3	Group 4
Liver -adenoma Males	n N	6 20	2 20	1 20	3 20	5 20
Liver – carcinoma Males	n N	0 20	0 20	2 20	1 20	4 20
Ovaries – theca-granulosa cell tumor	n N	0 20	0 20	1 20	3 20	3 20

N = number of animals examined; n = number of animals affected

Table 2: Absolute weights, summary

		Group 0	Group 1	Group 2	Group 3	Group 4
Kidneys	M N	4.239 17	4.296 14	4.141 13	3.866* 19	3.813* 16
Liver	M N	19.906 17	19.819 14	20.537 13	20.547 19	25.566* 16
Testes	M N	5.069 17	3.802* 14	4.073 13	4.113 19	3.808* 16

M = Mean number in g, N = number of animals examined, p = <= 0.05