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

Repeated dose toxicity: dermal

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

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: 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low group

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Medium group

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.