1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)-1H-imidazolium hydrogen sulphate

Endpoint summary

Description of key information

Repeated dose toxicity - oral: The NOAEL of imazalil sulfate is determined to be 5 mg/kg body weight/day (100 ppm in the diet) based on read across to a key K1 chronic oral (diet administration) toxicity study in male and female rats carried out with imazalil base-R 23979 over a period of 26 weeks (6 months).

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

 

Repeated dose toxicity - dermal: A key K1 study in male and female New Zealand White rabbits in a repeated dose dermal toxicity study was conducted similar to OECD 410. The test item when administered dermally for three weeks to rabbits at doses 10, 40, and 160 mg/kg body weight/day was well tolerated and without mortality up to the highest dose tested. A barely perceptible irritation of the skin was present in all dosed groups. The presence of swollen red foci on the treated skin was related to the sesam oil vehicle. No systemic toxic effects were observed except for some slightly changed urine variables in males dosed at 160 mg/kg body weight/day. Histological examinations of the skin, kidneys, liver and lungs of rabbits dosed at 160 mg/kg body weight/day did not reveal test item related changes. The NOAEL is this study can be established at at least 160 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

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

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.)

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

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.

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.

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

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

Key result

Critical effects observed:

no

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.

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.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

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.

Justification for type of information:

Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

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%

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

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

Key result

Critical effects observed:

no

Conclusions:

No reliable repeated dose toxicity study with the test substance. Data generated with the substance imazalil is used for endpoint coverage. 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. The same is assumed correct for imazalil sulphate.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-09-26 - 1990-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: G3A561
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in closed containers as delivered
- Stability under test conditions: Known

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

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The New Zealand White rabbit was selected because this species is currently used for dermal irritation studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stock-farm of Buyens, Lichtaart, Belgium
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: no data
- Weight at study initiation: 2.180 to 2.80 kg
- Fasting period before study: no data
- Housing: Housed individual in suspended stainless steel walls and a wire-mesh bottom and front door.
- Diet (e.g. ad libitum): Huybrechts pelleted rabbit food, administered in self-raising hoppers, ad llibitum.
- Water (e.g. ad libitum): Tap water administered via automatic drinking nipples, ad libitum.
- Acclimation period: One-week

DETAILS OF FOOD AND WATER QUALITY:
The food was prepared on the basis of an open formula of Janssen with guanranteed analysis by the manufacturer. The food did not contain antibiotics, chemotherapeutics or prophylatic agents.
The water was controlled by the Governmental Laboratory: Provinciaal Instituut voor Hygiene, Afdeling Waterkontrole, Kroneburstraat 45, 2000 Antwerpen.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air conditoned room
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Type of coverage:

semiocclusive

Vehicle:

other: sesam oil

Remarks:

Batch number: 25951

Details on exposure:

TEST SITE
- Area of exposure: Shaved back
- % coverage: no data
- Type of wrap if used: porous gauze dressing
- Time intervals for shavings or clipplings: no data

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was gently removed with lukewarm water and dried with soft cloth.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): no data
- Constant volume or concentration used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, restraint boxes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item in the solution was analyzed.
The solution was prepared on September 28, 1990 and analysed on October 2, 1990 and October 15, 1990.

Duration of treatment / exposure:

6 hours exposure, 21 days of dosing

Frequency of treatment:

5 days/week

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low group

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Medium group

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

High group

No. of animals per sex per dose:

5 males and 5 females/group, 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based upon the available information of a dose range finding study in rabbits. In the preliminary irritaiton study, the test article was given to rabbis for 4 consecutive days at 63, 250 and 1000 mg/kg. No irritation was noted at 63 mg/kg whereas slight erythema was observed at 250 and 1000 mg/kg. After 4 days severe skin lesions were seen at 250 and 1000 mg/kg and at autopsy a slight to moderate hepatotoxicity was observed.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- All rabbits were observed for waning health, abnormal behavior or unusual appearance, occurence of untoward clinical effects and manifestation of toxic and pharmacological response, moribundidty and mortality.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dailiy, one hour after the 6 hour exposure period.
- the average of all scores for erythema and oedema per dosage group gave the dermal irritation index according to Draize

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured initially and weely during the study period.

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

FOOD EFFICIENCY:
- 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All rabbits
- The following hematology parameters were determined: haematocrit, haemoglobin, red and white blood cell count, thrombocyte count, normoblasts and red blood cell indices (mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration). A differential count was carried out for: haematocrit (paced cell volume), haemoglobin, red and white blood cell count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, white blood cell count, thrombocyte count, differential count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of study
- Animals fasted: No data
- How many animals: All rabbits
- The following clinical biochemistry parameters were determined: sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides; phospholipids, blood urea nitrogen, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactic dehydronase and cholinesterase.

URINALYSIS: Yes
- Time schedule for collection of urine: End of study, all animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- The following parameters were determined: pH, proteins, glucose occult blood, acetone bodies, bilirubin and urobilinogen, creatinine, specific gravity, sediment.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Organ weights: Yes
The following organs from all rabbits sacrified at the end of the study were dissected free of fat and weighed: adrenals, brain, heart, kidneys, liver, lungs, pancrease, gonads, spleen, thymus, thyroids.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On completion of the treatment period, all surviving rabbits were anaesthetized with Nembutal (sodium pentobarbital 30 mg/kg IV) and sacrificed by exsanguination of the carotid artery.
- All necropsied were performed as soon as possible after sacrifice at the end of the study and any macroscopic pathological changes were noted.
- Samples of the following tissues and organs were collected from all animals at necropsy and preserved in 10% buffered formalin: adrenals, aorta, bone (tibia, fibula, rib), bone marrow, brain, cecum, cervix, colon, duodenum, epididymides, esophagus, eyes (& contiguous Harderian gland), optic nerve, gall bladder, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin), lymph nodes (mesenteric, mandibular), mammary gland area, nasopharynx, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual, parotid), sciatic nerve, seminal vesicles, skeletal muscle (diaphram, psoas muscle), skin (treated, untreated), spinal cord - thoracal, spleen, sternum, stomach, testes, thymus, thyroid (incl. parathyroids), tongue, trachea, urinary bladder (infused with formalin), uterus, vagina, all tissues showing abnormality.

HISTOPATHOLOGY: Yes
The treated and untreated skin, kidneys, liver and lungs of steh vehicle and high dosed rabbits were examined afater routne sampling and porcessing. The tissues were trimmed, embedded, sectioned and stained with hematoxylin-eosin.

Statistics:

Mortality: Chi-square test for pairwise comparison with control according to Siegel (two-tailed, Yates' correction for continuity).
Mann-Whitney U test for pairwise comparison with control according to Siegel (two-tailed, correction for ties.) was used for the analysis of the following results: Body weight, Haematology, Serum analysis, Urinalysis, Organ weights, Gross pathology, Histopathology.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No drug or dose related side effects in each group.
One female (104) of the 10 mg/kg dosed group occasionally showed a slight waste of food during day 19.
One male (41) of the 40 mg/kg dosed group showed a transient diarrhea during the last week of dosing.
One female (125) of the 40 mg/kg dose group occasionally showed a slight waste of food during days 12 and 13.
One female (125) of the 160 mg/kg dose group occasionally showed a slight waste of food during days 14, 15 and 19.
No behavior side effects were noted in all groups.
In the vehicle dosed group and in the 10 mg/kg dosed roup, a slight to moderate presence of red stippples on the skin was noted in all rabbits during the last 2 weeks of the study. The same was seen in the 40 mg/kg dosed group, but to a lesser extend and only during the last week of dosing. In the 160 mg/kg dosed group only 2 rabbits showed these skin changes during the last week.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

- Vehicle dosed group: No erythema or oedema was noted during the 21-day study. The dermal irritation index showed no dernal irritation (index: 0.00). In two females (82 and 84), slight scaling of the skin was noted on day 5.
- 10 mg/kg dosed group: Slight erythema was noted in male No. 21 on day 5 and male No. 24 on days 5 to 7.
The dermal irritaion index showed a barely perciptible irritaion close to zero (index: 0.01). Slight scaling of the skin was noted in male No. 21 days 5 to 7, No. 22 on day 5 and No. 25 on days 5 to 7 and in female No. 104 on day 5.
- 40 mg/kg dosed group: Slight erythema was noted in females No. 121 on days 4 to 6, No. 122 on days 5 to 6, No. 124 on day 5 to 7 and No. 125 on days 5 and 6. The dermal irritation index was 0.02. Sligjht to moderate scaling was noted in 2 males No. 43 and 44 and 4 females No. 121, 122, 124 and 125 during days 4 to 7.
- 160 mg/kg dosed groups: Sligjht to moderate erythema was noted in males No. 61 (day 5), No. 63 (days 4 to 6), No. 64 (days 5 to 7 and day 14) and No. 65 (days 4 to 7) and in females No. 142 (days 4 to 6) and No. 145 (days 4 to 7).The dermal index was 0.05. Slight to moderate scaling in nearly all 160 mg/kg dosed rabbits during days 4 to 7.

Mortality:

no mortality observed

Description (incidence):

All rabbits survived the study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain was comparable between groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A slightly lower (p<0.05) food consumption was noted in the 160 mg/kg dosed males during week 2 of the study. This decrease however was of transient nature and therefore of minor importance.

Food efficiency:

not examined

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):

Haematocrit, haemoglobin and red blood cells levels were comparable between all groups.
There was a statistically significant (p<0.05) decrease in males dosed at 160 mg/kg but this decreased was within the physiological range.
Thromocyte levels were comparable between all groups.
Red blood cell indices and differential counts were comparable between all groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sodium: There was a statistically significant decrease in males dosed at 160 mg/kg (p<0.05) and in females dosed at 40 mg/kg (p<0.01). However, the values were within the physiological range.
The following clinical biochemistry parameter values were comparable between all groups: potassium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides; phospholipids, blood urea nitrogen, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactic dehydrogenase and cholinesterase.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No relevant adverse effects in males dosed at 10 and 40 mg/kg bw and in females dosed at 10, 40 and 160 mg/kg bw/day.
Creatinine: Statistically significant (p<0.01) decrease in males dosed at 160 mg/kg.
Specific gravity: Statistically significant (p<0.01) decrease in males dosed at 160 mg/kg.
Urobilinogen: Statistically significant (p<0.05) decrease in males dosed at 160 mg/kg. Statistically significant (p<0.05) decrease in females dosed at 40 mg/kg, but not in the 160 mg/kg dosed females.
All other examined parameters were comparable betweeen groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No behavior side effects were noted in all groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects in any of the groups.
Heart: Statistically significant (p<0.05) decrease relative weight in females dosed at 40 mg/kg.
Pancrease: Statistically significant (p<0.05) decreased absolute weight in males dosed at 40 mg/kg.
Brain: Statistically significant increased absolute weight in males dosed at 40 mg/kg (p<0.01) and in males dosed at 160 mg/kg (p<0.05).
The recorded differences were not great and the values remain within the physiological range.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects on major organs and tissues in each group. The incidence of swollen red foci on the skin was higher in the vehicle dosed group and in rabbit dosed at respectively 10 and 40 mg/kg when compared to the 160 mg/kg dosed group. Therefore this phenomenon can be related to the vehicle used in this study (i.e. sesam oil).
The other gross pathology observations are common findings in a normal rabbit population and therefore of no importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects were noted in the 160 mg/kg bw/day dosed groups when skin, kidneys, liver and lungs were compared to the vehicle group. The organs of the low and medium dosed groups were not examined since no significant histologcal modifications were observed in the high dosed group as compared to the vehicle dosed group.
Comparable findings in vehicle and 160 mg/kg dosed groups for the following organs: kidney, liver and lungs.
Skin, treated: No prominent increase of histological changes was observed between vehicle and 160 mg/kg dosed groups. A tendency for a decrease of inflammatory reaction was observed in the 160 mg/kg dosed groups, as characterized by a decrease of granulocytes in dermis and /or hypodermis (males and females) and decreased incidence of acanthosis, hyperkeratosis and epidermal crusts (males).

Skin, untreated: No prominent increase of histological changes was observed between vehicle and 160 mg/kg dosed groups, except for a slight decrease of granulocytes in dermis and /or hypodermis in the 160 mg/kg dosed male rabbits.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Results of analytical verification.:

The test item in the solutions was analyzed.

The solution was prepared on September 28, 1990 and analysed on October 2, 1990.

Nominal concentration 0.5; Analysed concentration 0.490

Nominal concentration 2; Analysed concentration 01.950

Nominal concentration 8; Analysed concentration 7.77

The solution was prepared on September 28, 1990 and analysed on October 15, 1990.

Nominal concentration 0.5; Analysed concentration 0.481

Nominal concentration 2; Analysed concentration 01.940

Nominal concentration 8; Analysed concentration 7.85

The results showed that the concentration and stability of the test item in solutions applied with the concept of the study.

Conclusions:

The test item when administered dermally for three weeks to rabbits at doses 10, 40, and 160 mg/kg body weight /day was well tolerated and without mortality up to the highest dose tested. A barely perceptible irritation of the skin was present in all dosed groups. The presence of swollen red foci on the treated skinwas related to the sesam oil vehicle. No systemic toxic effects were observed except for some slightly changed urine variables in males dosed at 160 mg/kg body weight/day. Histological examinations of the skin, kidneys, liver and lungs of rabbits dosed at 160 mg/kg body weight/day did not reveal test item related changes.

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Data from the related sustance Imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: G3A561
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Known

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

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

No reliable repeated dose toxicity data via dermal application is available for the test item. Data generated with the structurally related substance Imizalil is used for endpoint coverage. The test item when administered dermally for three weeks to rabbits at doses 10, 40, and 160 mg/kg body weight /day was well tolerated and without mortality up to the highest dose tested. A barely perceptible irritation of the skin was present in all dosed groups. The presence of swollen red foci on the treated skinwas related to the sesam oil vehicle. No systemic toxic effects were observed except for some slightly changed urine variables in males dosed at 160 mg/kg body weight/day. Histological examinations of the skin, kidneys, liver and lungs of rabbits dosed at 160 mg/kg body weight/day did not reveal test item related changes.
The same is assumed to be correct for imazalil sulphate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

160 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-09-26 - 1990-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: G3A561
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in closed containers as delivered
- Stability under test conditions: Known

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

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The New Zealand White rabbit was selected because this species is currently used for dermal irritation studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stock-farm of Buyens, Lichtaart, Belgium
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: no data
- Weight at study initiation: 2.180 to 2.80 kg
- Fasting period before study: no data
- Housing: Housed individual in suspended stainless steel walls and a wire-mesh bottom and front door.
- Diet (e.g. ad libitum): Huybrechts pelleted rabbit food, administered in self-raising hoppers, ad llibitum.
- Water (e.g. ad libitum): Tap water administered via automatic drinking nipples, ad libitum.
- Acclimation period: One-week

DETAILS OF FOOD AND WATER QUALITY:
The food was prepared on the basis of an open formula of Janssen with guanranteed analysis by the manufacturer. The food did not contain antibiotics, chemotherapeutics or prophylatic agents.
The water was controlled by the Governmental Laboratory: Provinciaal Instituut voor Hygiene, Afdeling Waterkontrole, Kroneburstraat 45, 2000 Antwerpen.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air conditoned room
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Type of coverage:

semiocclusive

Vehicle:

other: sesam oil

Remarks:

Batch number: 25951

Details on exposure:

TEST SITE
- Area of exposure: Shaved back
- % coverage: no data
- Type of wrap if used: porous gauze dressing
- Time intervals for shavings or clipplings: no data

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was gently removed with lukewarm water and dried with soft cloth.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): no data
- Constant volume or concentration used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, restraint boxes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item in the solution was analyzed.
The solution was prepared on September 28, 1990 and analysed on October 2, 1990 and October 15, 1990.

Duration of treatment / exposure:

6 hours exposure, 21 days of dosing

Frequency of treatment:

5 days/week

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low group

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Medium group

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

High group

No. of animals per sex per dose:

5 males and 5 females/group, 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based upon the available information of a dose range finding study in rabbits. In the preliminary irritaiton study, the test article was given to rabbis for 4 consecutive days at 63, 250 and 1000 mg/kg. No irritation was noted at 63 mg/kg whereas slight erythema was observed at 250 and 1000 mg/kg. After 4 days severe skin lesions were seen at 250 and 1000 mg/kg and at autopsy a slight to moderate hepatotoxicity was observed.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- All rabbits were observed for waning health, abnormal behavior or unusual appearance, occurence of untoward clinical effects and manifestation of toxic and pharmacological response, moribundidty and mortality.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dailiy, one hour after the 6 hour exposure period.
- the average of all scores for erythema and oedema per dosage group gave the dermal irritation index according to Draize

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured initially and weely during the study period.

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

FOOD EFFICIENCY:
- 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All rabbits
- The following hematology parameters were determined: haematocrit, haemoglobin, red and white blood cell count, thrombocyte count, normoblasts and red blood cell indices (mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration). A differential count was carried out for: haematocrit (paced cell volume), haemoglobin, red and white blood cell count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, white blood cell count, thrombocyte count, differential count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of study
- Animals fasted: No data
- How many animals: All rabbits
- The following clinical biochemistry parameters were determined: sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides; phospholipids, blood urea nitrogen, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactic dehydronase and cholinesterase.

URINALYSIS: Yes
- Time schedule for collection of urine: End of study, all animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- The following parameters were determined: pH, proteins, glucose occult blood, acetone bodies, bilirubin and urobilinogen, creatinine, specific gravity, sediment.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Organ weights: Yes
The following organs from all rabbits sacrified at the end of the study were dissected free of fat and weighed: adrenals, brain, heart, kidneys, liver, lungs, pancrease, gonads, spleen, thymus, thyroids.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On completion of the treatment period, all surviving rabbits were anaesthetized with Nembutal (sodium pentobarbital 30 mg/kg IV) and sacrificed by exsanguination of the carotid artery.
- All necropsied were performed as soon as possible after sacrifice at the end of the study and any macroscopic pathological changes were noted.
- Samples of the following tissues and organs were collected from all animals at necropsy and preserved in 10% buffered formalin: adrenals, aorta, bone (tibia, fibula, rib), bone marrow, brain, cecum, cervix, colon, duodenum, epididymides, esophagus, eyes (& contiguous Harderian gland), optic nerve, gall bladder, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin), lymph nodes (mesenteric, mandibular), mammary gland area, nasopharynx, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual, parotid), sciatic nerve, seminal vesicles, skeletal muscle (diaphram, psoas muscle), skin (treated, untreated), spinal cord - thoracal, spleen, sternum, stomach, testes, thymus, thyroid (incl. parathyroids), tongue, trachea, urinary bladder (infused with formalin), uterus, vagina, all tissues showing abnormality.

HISTOPATHOLOGY: Yes
The treated and untreated skin, kidneys, liver and lungs of steh vehicle and high dosed rabbits were examined afater routne sampling and porcessing. The tissues were trimmed, embedded, sectioned and stained with hematoxylin-eosin.

Statistics:

Mortality: Chi-square test for pairwise comparison with control according to Siegel (two-tailed, Yates' correction for continuity).
Mann-Whitney U test for pairwise comparison with control according to Siegel (two-tailed, correction for ties.) was used for the analysis of the following results: Body weight, Haematology, Serum analysis, Urinalysis, Organ weights, Gross pathology, Histopathology.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No drug or dose related side effects in each group.
One female (104) of the 10 mg/kg dosed group occasionally showed a slight waste of food during day 19.
One male (41) of the 40 mg/kg dosed group showed a transient diarrhea during the last week of dosing.
One female (125) of the 40 mg/kg dose group occasionally showed a slight waste of food during days 12 and 13.
One female (125) of the 160 mg/kg dose group occasionally showed a slight waste of food during days 14, 15 and 19.
No behavior side effects were noted in all groups.
In the vehicle dosed group and in the 10 mg/kg dosed roup, a slight to moderate presence of red stippples on the skin was noted in all rabbits during the last 2 weeks of the study. The same was seen in the 40 mg/kg dosed group, but to a lesser extend and only during the last week of dosing. In the 160 mg/kg dosed group only 2 rabbits showed these skin changes during the last week.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

- Vehicle dosed group: No erythema or oedema was noted during the 21-day study. The dermal irritation index showed no dernal irritation (index: 0.00). In two females (82 and 84), slight scaling of the skin was noted on day 5.
- 10 mg/kg dosed group: Slight erythema was noted in male No. 21 on day 5 and male No. 24 on days 5 to 7.
The dermal irritaion index showed a barely perciptible irritaion close to zero (index: 0.01). Slight scaling of the skin was noted in male No. 21 days 5 to 7, No. 22 on day 5 and No. 25 on days 5 to 7 and in female No. 104 on day 5.
- 40 mg/kg dosed group: Slight erythema was noted in females No. 121 on days 4 to 6, No. 122 on days 5 to 6, No. 124 on day 5 to 7 and No. 125 on days 5 and 6. The dermal irritation index was 0.02. Sligjht to moderate scaling was noted in 2 males No. 43 and 44 and 4 females No. 121, 122, 124 and 125 during days 4 to 7.
- 160 mg/kg dosed groups: Sligjht to moderate erythema was noted in males No. 61 (day 5), No. 63 (days 4 to 6), No. 64 (days 5 to 7 and day 14) and No. 65 (days 4 to 7) and in females No. 142 (days 4 to 6) and No. 145 (days 4 to 7).The dermal index was 0.05. Slight to moderate scaling in nearly all 160 mg/kg dosed rabbits during days 4 to 7.

Mortality:

no mortality observed

Description (incidence):

All rabbits survived the study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain was comparable between groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A slightly lower (p<0.05) food consumption was noted in the 160 mg/kg dosed males during week 2 of the study. This decrease however was of transient nature and therefore of minor importance.

Food efficiency:

not examined

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):

Haematocrit, haemoglobin and red blood cells levels were comparable between all groups.
There was a statistically significant (p<0.05) decrease in males dosed at 160 mg/kg but this decreased was within the physiological range.
Thromocyte levels were comparable between all groups.
Red blood cell indices and differential counts were comparable between all groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sodium: There was a statistically significant decrease in males dosed at 160 mg/kg (p<0.05) and in females dosed at 40 mg/kg (p<0.01). However, the values were within the physiological range.
The following clinical biochemistry parameter values were comparable between all groups: potassium, chloride, calcium, inorganic phosphate, total protein, albumin, haptoglobin, glucose, cholesterol, triglycerides; phospholipids, blood urea nitrogen, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactic dehydrogenase and cholinesterase.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No relevant adverse effects in males dosed at 10 and 40 mg/kg bw and in females dosed at 10, 40 and 160 mg/kg bw/day.
Creatinine: Statistically significant (p<0.01) decrease in males dosed at 160 mg/kg.
Specific gravity: Statistically significant (p<0.01) decrease in males dosed at 160 mg/kg.
Urobilinogen: Statistically significant (p<0.05) decrease in males dosed at 160 mg/kg. Statistically significant (p<0.05) decrease in females dosed at 40 mg/kg, but not in the 160 mg/kg dosed females.
All other examined parameters were comparable betweeen groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No behavior side effects were noted in all groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects in any of the groups.
Heart: Statistically significant (p<0.05) decrease relative weight in females dosed at 40 mg/kg.
Pancrease: Statistically significant (p<0.05) decreased absolute weight in males dosed at 40 mg/kg.
Brain: Statistically significant increased absolute weight in males dosed at 40 mg/kg (p<0.01) and in males dosed at 160 mg/kg (p<0.05).
The recorded differences were not great and the values remain within the physiological range.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects on major organs and tissues in each group. The incidence of swollen red foci on the skin was higher in the vehicle dosed group and in rabbit dosed at respectively 10 and 40 mg/kg when compared to the 160 mg/kg dosed group. Therefore this phenomenon can be related to the vehicle used in this study (i.e. sesam oil).
The other gross pathology observations are common findings in a normal rabbit population and therefore of no importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant adverse effects were noted in the 160 mg/kg bw/day dosed groups when skin, kidneys, liver and lungs were compared to the vehicle group. The organs of the low and medium dosed groups were not examined since no significant histologcal modifications were observed in the high dosed group as compared to the vehicle dosed group.
Comparable findings in vehicle and 160 mg/kg dosed groups for the following organs: kidney, liver and lungs.
Skin, treated: No prominent increase of histological changes was observed between vehicle and 160 mg/kg dosed groups. A tendency for a decrease of inflammatory reaction was observed in the 160 mg/kg dosed groups, as characterized by a decrease of granulocytes in dermis and /or hypodermis (males and females) and decreased incidence of acanthosis, hyperkeratosis and epidermal crusts (males).

Skin, untreated: No prominent increase of histological changes was observed between vehicle and 160 mg/kg dosed groups, except for a slight decrease of granulocytes in dermis and /or hypodermis in the 160 mg/kg dosed male rabbits.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Results of analytical verification.:

The test item in the solutions was analyzed.

The solution was prepared on September 28, 1990 and analysed on October 2, 1990.

Nominal concentration 0.5; Analysed concentration 0.490

Nominal concentration 2; Analysed concentration 01.950

Nominal concentration 8; Analysed concentration 7.77

The solution was prepared on September 28, 1990 and analysed on October 15, 1990.

Nominal concentration 0.5; Analysed concentration 0.481

Nominal concentration 2; Analysed concentration 01.940

Nominal concentration 8; Analysed concentration 7.85

The results showed that the concentration and stability of the test item in solutions applied with the concept of the study.

Conclusions:

The test item when administered dermally for three weeks to rabbits at doses 10, 40, and 160 mg/kg body weight /day was well tolerated and without mortality up to the highest dose tested. A barely perceptible irritation of the skin was present in all dosed groups. The presence of swollen red foci on the treated skinwas related to the sesam oil vehicle. No systemic toxic effects were observed except for some slightly changed urine variables in males dosed at 160 mg/kg body weight/day. Histological examinations of the skin, kidneys, liver and lungs of rabbits dosed at 160 mg/kg body weight/day did not reveal test item related changes.

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Data from the related sustance Imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: G3A561
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Known

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

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

No reliable repeated dose toxicity data via dermal application is available for the test item. Data generated with the structurally related substance Imizalil is used for endpoint coverage. The test item when administered dermally for three weeks to rabbits at doses 10, 40, and 160 mg/kg body weight /day was well tolerated and without mortality up to the highest dose tested. A barely perceptible irritation of the skin was present in all dosed groups. The presence of swollen red foci on the treated skinwas related to the sesam oil vehicle. No systemic toxic effects were observed except for some slightly changed urine variables in males dosed at 160 mg/kg body weight/day. Histological examinations of the skin, kidneys, liver and lungs of rabbits dosed at 160 mg/kg body weight/day did not reveal test item related changes.
The same is assumed to be correct for imazalil sulphate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Additional information

Repeated dose toxicity: chronic (6 months) oral toxicity

A key K1 chronic oral toxicity study in male and female rats carried out with imazalil base-R 23979 over a period of 26 weeks treated at levels of 0, 25, 100 or 400 ppm in the diet (equivalent to about 1.25, 5 and 20 mg imazalil/kg body weight/day) was used to assess the repeated dose oral toxicity of imazalil sulfate (Lina, 1983).

General health, behaviour, survival, growth, food intake, food efficiency, haematology, clinical chemistry and urinalysis were not adversely affected by the feeding of imazalil base R-23979.

In the high dose group, the relative weights of the kidneys in males and the relative weights of the kindeys, liver and lung in females were increased compared to the control group.

Gross and microscopic examination at autopsy did not reveal any abnormality attributable to the feeding of the test substance.

Based on the absence of adverse effect at 5 mg/kg body weight a NOAEL for Imazalil sulfate of 5 mg/kg body weight was assigned.

Additional supporting studies were identified, but not entered as separate study summaries:

• Van Deun et al. (1994): A three-month oral mechanistic toxicity study with one month interim sacrifice in male and female Swiss mice carried out with imazalil base-R 23979 treated at levels of 0, 50, 200 and 600 ppm in the diet was used to assess the repeated dose oral toxicity of imazalil. Imazalil was well tolerated. Test item treatment did not result in mortality, clinical abnormalities and adverse effects on body weights and food consumption.

The dose of 50 ppm in the food was well tolerated and did not result in any adverse effects in males and females after one month or three months dosing; Therefore the dose of 50 ppm is considered to be no-adverse effect level (NOAEL) in this study. This dose level corresponded with a calculated mean test article intake of 11.6 and 13.6 mg/kg bw in males and females respectively.

At 200 ppm in the food, slight toxicity was noted. The relevant findings were changed organ weight in males (increased liver weights) and histological changes in the liver in males and/or females (centrilobular clearer aspect, large hepatocytic vacuoles and small hepatocytic vacuoles).

Male and female mice dosed at 600 ppm in the food showed moderate, drug-related toxicity in males and females, as evidenced by altered serum parameters (increased alkaline phosphatase), organ weight changes (increased liver weights in both sexes), presence of dark livers and various histological changes in the liver (cfr. above, as well as individual cell necrosis and diffuse hepatocytic swelling).

In addition, electron microscopy revealed an increased number of lipids droplets in mainly periportal hepatocytes in the liver of the 600 ppm dosed animals. This corresponds with the increase in the hepatocytic vacuolation observed microscopically. Furthermore, the rough endoplasmic reticulum (RER) of those hepatocytes showing a large number of lipid droplets, had a morphology different from the RER in hepatocytes of control animals. Whereas under normal conditions, the RER in hepatocytes tends to be arranged in parallel stack of cisternae, in this case the RER appeared under the form of small vesicles diffusely spread over the cytosol.

  

• Van Deun et al. (1996a): A three-month oral dose range finding study with one month interim sacrifice in male and female rats carried out with imazalil base-R 23979 treated at levels of 0, 200, 400 and 800 ppm in the diet was used to assess the repeated dose oral toxicity of imazalil. Imazalil was well tolerated. It did not result in mortality, abnormal clinical or ophthalmological observations, altered food intake, changed urinary parameters or gross pathology deviations.

The dose of 200 ppm in the diet did not result in any adverse effects in males and females either after one or three months of dosing. Therefore 200 ppm was considered as the non-adverse-observed-effect level in this study. This dose level corresponded to an average imazalil-intake during the entire study period of 15.8 and 18.7 mg/kg body weight very day in males and females, respectively.
The dose of 400 ppm in the diet resulted in increased liver weights in male and female rats after one month of dosing and increased adrenal weights in female rats after 3 months of dosing.
The dose of 800 ppm in the diet goes to moderate, drug-related toxicity, as evidence by body weight and weight gain decrease in males, haematology and serum deviations in males and females as well as liver and adrenal weight increases.

The liver weight increases were associated with histological changes, i.e. some more pronounced hepatocytic swelling in male rats and an increased incidence of large vacuoles in female rats.

The adrenal weight increases were associated in some females with swelling of the cortical cells.

 

• Van Deun et al. (1996b): Imazalil base, when given to Hannover Wistar rats at dietary doses of 800, 1600, 2400 and 3200 ppm resulted in various drug-related and dose-dependent changes.
Waste of food, decreased body weight gain and decreased food consumption were noted at all dose levels. Haematological changes were noted in males from 1600 ppm onwards (decreased monocytes) and in females at almost all dose levels (increased red blood cells, decreased mean cell volume and increased mean cell haemoglobin concentration). Serum alterations were noticed at all dose levels in both sexes (especially decreased levels of aspartate aminotransferase).
The liver was selected as the target organ at all doses, as demonstrated in males by increased related weights in both sexes, by macroscopic changes (dark livers and more pronounced lobulation), by microscopic changes (hepatocellular hypertrophy and hepatic fatty vacuolization) or by electron microscopic changes (lipid-loaden hepatocytes).
When waste of food was taken into account, test article intake at the various dose levels was calculated to be 64.4, 129, 181 and 252 mg/kg bw in male rats and 78.8, 150, 236 and 333 mg/kg bw in female rats.
Drug-related effects were seen dose-dependently, however a stabilization of effects was noted between 1600 and 2400 ppm in males and between 800 and 1600 ppm in females, indicating a saturation of absorption by the liver probably between the dose level of 100 and 150 mg/kg bw/day. 

• Verstraeten et al. (1993): In a 90-day toxicity study, imazalil (purity not specified) was administered in the diet to groups of 10 albino Swiss mice/sex/dose admixed with the diet at concentrations of 0, 200, 400, or 800 ppm, equivalent to 0, 1.2, 5.0, and 20 mg/kg bw per day, for 3 months. General health and behaviour were checked daily, body weight was measured weekly, and food intake was measured weekly. Blood for haematological and clinical chemical investigations was collected at the end of the study. At termination of the study, the animals were examined post mortem, and organ weights, gross and micropathological examinations were carried out of livers from all animals after dissection.

No adverse effects were seen at 200 ppm, expect for slightly decreased aspartate aminotransferase and cholesterol values in females and a vacuolar degeneration of hepatocytes in males. In the 400 ppm dosed mice, toxicity was characterized by some altered serum parameters and by histological modifications of the liver (vacuolar degeneration and centrilobular swelling). These effects were also, but more pronounced, present at 800 ppm. In addition, a lower body weight in females was noted at this dose whereas also increases in some blood parameters were retained as drug-related effects.

 

 • Verstraeten et al. (1989):

Imazalil base (R 23979) when given orally to dogs for 12 months, was well tolerated and without mortality up to 20 mg/kg. At 1.25 and 2.5 mg/kg, no drug related adverse effects were noted. Dosing at 20 mg/kg resulted in toxicity characterized by slightly softened faeces, salivation, vomiting, decreased appetite and a lowered body weight gain. Serum analysis revealed a slight decrease of calcium and a marked increase of alkaline phosphatase. Liver weight was increased but without histological changes. Most of these finding were similar to those observed in a chronic toxicity study in dogs (Ex. No. 370).

Repeated dose toxicity: subchronic (21 days) dermal toxicity

A key K1 single repeated dose dermal toxicity study similar to OECD 410 was performed on New Zealand White Rabbits exposed to Imazalil vehicle in Sesam oil at 0, 10, 40 and 160 mg/kg bw by dermal exposure. Clinical signs, food consumption, body weights, haemotology, serum analysis, and organ weights were recorded periodically during the acclimatization and treatment periods The animals were sacrificed at the end of the study period, necropsied and examined post mortem.

No mortality occurred during the study period.

No drug or dose related side effects in all groups. The dermal irritation index showed no irritation for the vehicle dose group and barely perceptible irritation for all dosed groups. The presence of red stipples on the treated skin of the vehicle and to lesser extent in the treated groups was related to the vehicle.

No adverse effects on body weight and body weight gain in all groups.

No relevant adverse effects were observed for haematology and serum analysis in all groups.

No relevant adverse effects in urinalysis results were observed in males dosed at 10 and 40 mg/kg body weight/day and in females dosed at 10, 40 and 160 mg/kg body weight/day. In males dosed at 160 mg/kg a test item related decreased of creatinine, specific gravity and urobilinogen was noted.

No adverse effects in organs weights were observed in all groups.

Justification for classification or non-classification

According to the criteria of the CLP Regulation and based on the available data with a NOAEL of 5 mg/kg bw, Imazalil sulfate should be classified as STOT RE 2 toxicant.