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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-05-25 - 1982-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Protocol for an oral chronic toxicity and carcinogenicity study with Imazalil base-R 23979 in rats.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
Protocol for an oral chronic toxicity and carcinogenicity study with Imazalil base-R 23979 in rats.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
EC Number:
252-615-0
EC Name:
1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
Cas Number:
35554-44-0
Molecular formula:
C14H14Cl2N2O
IUPAC Name:
(±)-1-(β-allyloxy-2,4-dichloro-phenylethyl) imidazole / (±)-allyl 1-(2,4-dichlorophenyl)-2-imidazol-1-ylethyl ether
Test material form:
solid: crystalline
Details on test material:
- Physical state: crystalline solid
- Appearance: Yellow to brown crystalline solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D 4103, dated October 14, 1980
- Purity: 98.1%
- Appearance: slightly yellow to brown crytalline mass (soldified oil)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at room temperature.
- Stability under test conditions: Unlimited
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Test animals

Species:
rat
Strain:
Wistar
Remarks:
SPF (Cpb:WU; Wistar random)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: 3.5 weeks at delivery
- Weight at study initiation: 35 to 50 grams.
- Fasting period before study: no
- Housing: Conventional conditions, five per sex per cage, in suspended, stainless steel cages, fitted with wire mesh floors and fronts.
- Diet (e.g. ad libitum): Institute's powdered basal diet. Diet was available ad libitum.
- Water (e.g. ad libitum): Automatic watering system, ad libitum. Because of malfunction of the system in weeks 1 and 15, water was also supplied in glass botles which were filled daily with fresh tap water except for the weekend.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1°C
- Humidity (%): 40-70%
- Air changes (per hr): 8 - 10
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours dark (light between 06.00 a.m.and 6:00 p.m.)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The top-dose diet was prepared first; the lower dose diets were prepared by diluting it with stock diet. Homogeneity was achieved by mixing in a mechanical blender (Stephan) for 2 minutes.
- DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the test diets were prepared once every two weeks. Approximately once a month samples were taken from each of the diets immediately after preparation, and subsequently stored at -20°C.
- Mixing appropriate amounts with (Type of food): The test item was added to the diet at various levels to provide concentrations of 0, 25, 100 or 500 ppm.
- Storage temperature of food: no data

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From the batches of diets prepared on May 24, 1982 and July 26, 1982, five samples, taken at different places in each feed container, were analysed for the content and the homogeneous distribution of the test item in the diets. One sample of each test diet of the batch of July 26 was re-analysed after storage at room temperature for 4 weeks to obtain information on the stability of the test item in the diets.
The set of samples taken on September 6, 1982 were analysed to control the Imazalil content.
Duration of treatment / exposure:
6 months
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
1.25 mg/kg bw/day (nominal)
Remarks:
25 ppm in diet
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
100 ppm in diet
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
400 ppm in diet
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The test item was administered in the diet at 0, 25, 100 or 400 ppm. The levels were selected on the basis of the results of a carcinogenicity study in rats (experiment 667-79-04.20) conducted by the sponsor.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The general condition and behaviour of all animals were checked daily. All signs of ill health or reaction to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Before exposure, then weekly for the first 12 weeks and subsequently once every two weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food intake was measured per cage (5 animals) weekly. The efficiency of food utilization was calculated over the period of rapid growth (week 0-10) and expressed as gram weight gain per gram food consumed.

FOOD EFFICIENCY: YES
Food intake was measured per cage (5 animals) weekly. The efficiency of food utilization was calculated over the period of rapid growth (week 0-10) and expressed as gram weight gain per gram food consumed.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males: Day 174; Females Day 175
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Not specified
- How many animals: No data
- The following hematology parameters were determined: hemoglobin, packed cell volume, red blood cells, thrombocytes, white blood cells, differential white blood cell count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy (on day 183 for males and 184 for females)
- Animals fasted: Yes. Overnight fasting.
- How many animals: All
- The following clinical biochemistry parameters were determined: glucose (plasma), total protein, albumin, haptoglobin, alkaline phoshatase (ALP), glutamic-oxalacetic-transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactate dehydrogenase (LDH), urea, creatinine, bilirubin total, cholesterol, inorganic phosphate, calcium (Ca), chloride (Cl), potassium (K), sodium (Na)

URINALYSIS: Yes
- Time schedule for collection of urine: Day 177
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes. On Day 177, all rats were deprived of water for 24 hours and of food for 16 hours. Urine was collected from individual animals during the last 16 hours of the deprivation period.
- The following parameter were determined for individual samples: volume, density.
The following semi-quantitative observations were made in pooled sampes (one sample/sex/group): appearance, pH, protein, glucose, occult blood, ketones, uribilinogen, bilirubin, sediment: erythocyes, leucocytes, epithelial cells, amorph material, crystals, casta, bacteria, sperm cells and worm eggs.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Sacrifice: On day 183 all male rats and on day 184 all females rats were killed in such a way that on the average the time of killing for each group was about the same. The animals, killed under ether anaesthesia by bleeding the aorta were examned grossly for pathological changes. The following organs were weighed: adrenals, brain, heart, kidneys, liver, lungs (with mainstem bronchi), ovaries, pancreas, pituary, spleen, testes, thymus, thyroid (with parathyroids).

HISTOPATHOLOGY: Yes
Detailed microscopic examinations were carried out on all male and female rats of the top-dose group and on all control rats. The following organs and tissues were examined: adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, cervical lymph nodes, coagulating glands, colon, duodenum, epididymides, external auditory canal (Zimbal glands), extra orbital lachrymal glands, eyes, heart, ileum, jejunum, kidneys, larynx, liver, lungs with mainstem bronchi, mammary gland (female only), mesenteric lymph nodes, nostrils (nasal cavity), oesophagus, ovaries, pancreas, paratid salivary glands, pituary, preputial glands, prostate, rectum, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin (flank), spinal cord (two levels), spleen, sternum (with bone marrow), stomach (cardia, fundus and pylorus), sublingual salivary glands, submaxillary salivary glands, tongue, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus with cervix.
They were embedded in paraffin wax and sections were cut at 5 µm; the sections were stained with haematoxylin and eosin. Microscopic examination of the kidneys were extended to the males and females of the mid-and low-dose group.
Statistics:
Data on body weight, food intake, red blood cell parameters, volume and density of urine, clinical chemistry values and organ weights were evaluated by analysis of (co-)variance followed by multiple comparison tests (Dunnett) or the L.S.D. test (for food intake and food efficiency). Total and differential white blood counts were analysed by the mann/Whitney U-test. The histopathological changes were examined by the Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related changes in general condition or behaviour were noted in any of the animals employed in the study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of the males in the top-dose group were slightly, but not statistically significantly, lower than those of the controls throughout the study. Otherwise there were no consistent changes in growth rate in any of the groups.
The relatively low terminal body weights in all test groups and in controls are attributed to weighing the animals after overnight fasting prior to autopsy.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no noticeable changes in food intake in any of the groups, other than those in week 1 and 15. The later changes were considered artifacts attributable to the failure of the automatic watering system.
(In week 1 and 15, the automatic drinking water system malfunctioned, which resulted in irregulaties in growth rate, food intake and food conversion efficiency in the affected cages.)
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
There were no consistent changes in food conversion efficiency in any of the groups, other than those in week 1 and 15. The later changes were considered artifacts attributable to the failure of the automatic watering system.
(In week 1 and 15, the automatic drinking water system malfunctioned, which resulted in irregulaties in growth rate, food intake and food conversion efficiency in the affected cages.)
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was an isolated, slight increase in haemoglobin concentration in females of the low-dose group. Total white blood cell count was relatively high in both sexes of all dose groups, but the differences with the controls were statistically significant in males of the top-dose group only and there was no evidence of a dose-related response. Moreover, the values in controls were relatively low as compared to the values of historical controls at the same stage (males 13.8 +/- 13.6; females 11.4 +/- 4.0). Futhermore, there were no changes of any significance in differential white blood cell count. Therefore, no toxicological significance is attached to these slight changes in total white blood count.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Lactate-dehydrogenase activity was increased in females of the top-dose group.
Plasma urea level was relatively high in females of the low- and top-dose group but not in the mid-dose group, therefore this finding is considered fortuitous and not related to treatment. Glucose concentration was increased in females of all dose groups, but there was not dose-reponse relationship.
An isolated increase in haptoglobin concentration was found in males of the mid-dose group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes in any significance in the values for the volume and density of the urine.
The composition of the urine, with respect to appearance, pH, protein, glucose, ketones, occult blood, urobilinogen, bilirubin and microscopy of the urinary sediment was comparable in all groups.
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):
In the top-dose group statistically significant increases were seen in the relative weight of the kidneys in males, and in the absolute- and relative weights of the kidneys and liver in females.
The absolute weight of the thymus and the relative weight of the lungs were slightly, though significantly increased in females of the top-dose group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross examination at autopsy did not reveal any abnormality that could be ascribed to the administration of the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The presence of a thymoma in the thymus of one low-dose female was the only tumour found.
The degree and incidence of all histopathological changes observed were similar in the test groups and the controls or the lesions occurred only in a single animal. None of these abnormalities, which are common finds in rats of this strain and age could, therefore, be ascribed to the feeding of the test item.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Results of analytical verification:

The mean actual levels of Imazalil in the batches of diets prepared on May 24, 1982 and July 26, 1982 were lower than the intended levels in the mid-and top-dose diet. In the batches prepared on September 6, 1982 the actual levels were lower than the intended levels in all dose groups.

The high coefficient of variation in the low-and mid-dose diets preapred on May 24, 1982, is indicative of a poor homogeneity of the test item in the diets. A better homogeneity was found in the diets prepared later on.

There was no noticable loss of Imazalil after a storage period of 4 weeks at room temperature.

Applicant's summary and conclusion

Conclusions:
The feeding study of Imazalil base-R-23979 to rats at levels of up to 400 ppm resulted in some minor changes in the top-dose group. No significant effects on general health, behaviour, survival, food intake, food efficiency, haematology and urinalysis. The slight differenes seen did not suggest a relationship with treatment.

Growth of male rats of the top-dose group was somewhat lower than that of the control animals but food intake and food efficiency were not noticeably different.

The increased plasma LDH activity, seen only in females of the top-dose group, was not associated with any histopathological abnormality. Since, moreover, there is a large variation in normal values for this unspecific parameter no toxicological significance is attached to this finding.

The increased weight of the lungs in females ofo the top-dose group ws not accomplished by treatment-related microscopic changes. Therefore, it is doubtful whether any toxicological significance may be attached to this finding.

The increased weight of the lungs in females of the top-dose group was not accompanied by treatmtne-related microscopic changes. Therefore, it is doubtful whether any toxicological signifiance may be attached to this finding.

The increased relative weights of the kidneys in the top-dose group were not accompanied by detecable changes in urine parameter or in macroscopic or microscopic pathology of the kidneys. So, there is at the present time no explanation for these findings. Further evaluation will be awaited until the results of the 18 and 30 months studies are available.

Thienpont et. al. (1981) reported that Imazalil fed at dietary levels of 800 ppm to rats for 2 years caused slight liver changes consisting of slight centrilobular swelling, an increased amount of glycogen in the centrilobular area and an increased fatty surcharge of the hepatocytes. In the 6-month study a dietary level of 400 ppm induced neither similar alterations nor any other treatment-related histopathological changes in the liver, although the liver weight was increased in females at this dietary level.

Based on the results of the study, the no-effect level for imazalil base R-23979 was 100 ppm in rats after 6-month oral exposure. This level is equivalent to a nominal intake of about 5 mg/kg b.w./day. No major abnormalities were observed at the next higher level of 400 ppm. The test substance is therefore classified as STOT RE 2 according to the CLP Regulation.