ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

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:

27 May - 25 Jun 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Version / remarks:

Adopted in 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

Adopted in 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-870-0

EC Name:

-

Cas Number:

163520-33-0

Molecular formula:

C18H17NO3

IUPAC Name:

ethyl 5,5-diphenyl-4,5-dihydro-1,2-oxazole-3-carboxylate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD (SD) BR

Details on species / strain selection:

The rat was chosen as the test system according to regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at receipt: approximately 42 days
- Body weight at receipt: males: 180 - 196 g, females: 188 - 207 g
- Housing: individually, in polycarbonate cages with mesh bases suspended over waste trays. The waste tray of each cage was lined with absorbent paper
- Diet: ground laboratory rodent diet, Modified SQC Expanded Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 May 1997 To: 25 Jun 1997

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: 1% (w/v) methyl cellulose in water

Details on exposure:

TEST SITE
- Area of exposure:dorsal area of the trunk
- % coverage: 10%
- Type of wrap if used: the test substance was held in contact with the skin on a 2 x 2 cm pad of porous gauze pad backed by aluminium foil which was held in place over the treatment site by elastic bandage
- Time intervals for shavings or clippings: clipping was repeated as necessary during the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test material was rinsed of with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 5 mL/kg bw, daily preparation of test suspension
- Concentration: 2, 20 and 200 mg/mL
- Constant concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analysed by extraction of AE F122006 from the suspensions by shaking with acetonitrile + methanol (90 + 10, v/v). The concentration of the test substance in the extract was measured by HPLC using UV detection.The mean results for the test suspension samples analysed and prepared for dosing were within the range 89.5 to 122.3% of nominal at the time of preparation (specified range +20% to -20% of nominal).

Duration of treatment / exposure:

28 days (males)
29 days (females)

Frequency of treatment:

5 days/week, excluding weekends

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of findings in an earlier dose ranging study conducted immediately prior to this study with 4 groups of 3 males dosed at 0, 250, 500 and 1000 mg test substance kg bw/day for 7 days, excluding the weekend. There were no treatment-related effects at 1000 mg/kg bw/day, equivalent to the accepted international regulatory limit dose, in this range-finding study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed each morning for abnormal behaviour, including neuro-muscular coordination and physical appearance. Animals were also observed in the afternoon on Mondays to Fridays except on public and Company holidays.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at randomisation, at the start of treatment, weekly thereafter and at
necropsy.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Haematocrit (HCT), White blood cells (WBC), Haemoglobin (HB), Neutrophils (NEUT), Red blood cells (RBC), Lymphocytes (LYMP), Mean corpuscular volume (MCV), Monocytes (MONO), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Platelets (PLT), Reticulocyte count (RET), Prothrombin time (PT), and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (Na), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (Cl), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), and Creatine kinase (CPK).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, organ weights of kidneys, adrenals, liver and testes were recorded. Following tissues were fixed in 10% neutral buffered formalin: kidneys, liver, ovaries, pinnae, skin (treated and untreated), testes, adrenals, and any other tissue showing macroscopic abnormalities.
HISTOPATHOLOGY: Yes, of kidney and liver sections and treated and untreated skin.

Statistics:

The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Test substance related clinical signs other than local dermal irritation were not evident.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, slight dermal irritation, as indicated by skin reddening at the application site, was seen in all animals from Week 2 till study termination. Slight sloughing, defined as shedding of the superficial layer of skin at the application site, was seen in 3/5 males and 3/5
females from Week 2 until Week 3 and 4, respectively.
At 100 mg/kg bw/day, slight dermal irritation was seen in 2/5 females from Day 4 and 11 to Day 6 and 19, respectively.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed. At the 1000 mg/kg bw/day dose level a mild decrease in cholesterol levels was observed in female animals compared with concurrent control, but this decrease was within the normal physiological range of control data.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: scabs at the application site in 2/5 female rats

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, 3/5 males and 5/5 females had mononuclear cell infiltration of the dermis to a moderate degree.
At 100 mg/kg bw/day, 2/5 males and 3/5 females had moderate cell infiltration.
At 10 mg/kg bw/day, 1/5 males had a mild infiltration. Because a similar lesion was observed in a control female this is considered not to be induced by application of the test compound.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

2

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no cell infiltration observed at doses <100 mg/kg bw/day

Remarks on result:

other: 0.5 mg/cm²/day, calculated based on a body weight of 200 g and a patch area of 4 cm²

3

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no