Butanoic acid, 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-indenyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2001 - 23 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

441-420-8

EC Name:

-

Cas Number:

113889-23-9

Molecular formula:

C14H20O2

IUPAC Name:

tricyclo[5.2.1.0²,⁶]dec-3-en-8-yl butanoate; tricyclo[5.2.1.0²,⁶]dec-4-en-8-yl butanoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-6 weeks
- Weight at study initiation:
Males: 139-183 g
Females: 134-158 g
- Housing: In groups of five, under standard laboratory conditions
- Diet (e.g. ad libitum): Ad libitum, pelleted diet
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material prepared at appropriate concentrations as a solution in dried Arachis oil BP.

VEHICLE
- Concentration in vehicle: 7.5, 75 and 500 mg/mL (low, intermediate and high dose group)
- Amount of vehicle (if gavage): 2 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test formulation and were analysed for concentration of Cyclobutanate by gas chromatography (GC) using an external standard technique.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 150, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were determined in a 14-day repeated dose range-finding toxicity study in rats
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Recovery period
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and wherever possible 5 hours after dosing (twice daily during recovery period)
- Cage side observations: overt signs of toxicity, ill-health or behavioural change

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 7, 14, 21 and 28, and for the recovery group Day 35 and 42.

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

WATER CONSUMPTION: Yes, by visual inspection for overt changes
- Time schedule for examinations: Daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 (non-recovery animals) and Day 42 (recovery animals)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All
- Parameters checked:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices:
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 (non-recovery animals) and Day 42 (recovery animals)
- Animals fasted: No
- How many animals: All
- Parameters checked:
Urea
Inorganic phosphorus (P)
Glucose
Gamma glutamyltranspeptidase (y-GT)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Triglycerides (Tri)
Chloride (Cl-)
Total cholesterol (Chol)
Calcium (Ca++)
Total bilirubin (Bili)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (non-recovery animals) and Week 6 (recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked:
Volume
Ketones
Specific Gravity
Bilirubin
pH
Urobilinogen
Protein
Reducing Substances
Glucose
Blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of treatment and on Days 7, 14, 20 and 25. During week 4 functional performance tests together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All animals
- Battery of functions tested: behavioural assessment / motor activity / forelimb/hindlimb grip strength / sensory reactivity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full external examination, macroscopic abnormalities were recorded

ORGAN WEIGHTS: Yes, recorded for:
Adrenals, brain, epidymides, heart, kidneys, liver, ovaries, spleen, testes, thyroids

HISTOPATHOLOGY: Yes, see below for which tissues (* = microscopic examination):
Adrenals*
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)*
Bone & bone marrow (sternum)*
Brain (including cerebrum, cerebellum and pons)*
Caecum*
Colon*
Duodenum*
Epididymides
Eyes
Gross lesions
Heart*
Ileum*
Jejunum*
Kidneys*
Liver*
Lungs (with bronchi)*
Lymph nodes (cervical and mesenteric)*
Muscle (skeletal)
Oesophagus*
Ovaries*
Pancreas
Pituitary
Prostate*
Rectum*
Salivary glands (submaxillary)
Sciatic nerve*
Seminal vesicles*
Skin (hind limb)
Spinal cord (cervical)*
Spleen*
Stomach*
Testes (with epididymides)*
Thymus*
Thyroid/parathyroid*
Trachea*
Urinary bladder*
Uterus*

Statistics:

- Dose response relationships: linear regression and one-way ANOVA (incorporating Levene's test for homogeneity of variance)
- If homogenous pairwise comparisons: Dunnett's test
- Recovery group data: Two-tailed t-test (incorporating Levene's test for homogeneity of variance)
- If unequal variances: non-parametric methods Kruskal-Wallis ANOVA and Mann-Whitney 'U' test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

but not toxicologically significant

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
High-dose group: increased salivation from day 6 (recovery apparent).

BODY WEIGHT AND WEIGHT GAIN
Mid-dose group: significant reduction in last week of treatment (females). No dose-relationship observed, therefore not considered of toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Higher incidence of globular accumulations of eosinophilic material in the tubular epithelium in male rats of high and mid-dose group (hydrocarbon nephropathy). Regression of this condition was seen in the 14 days of recovery. All remaining morphological changes were commonly observed in laboratory rats and therefore not considered to be of toxicological importance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects are seen. The non-adverse effects seen is alpha-hydrocarbon nephropathy

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, no adverse effects were observed in female rats up to the highest dose group. In males, hydrocarbon nephropathy was observed histopathologically in the 150 and 1000 mg/kg bw/day dose groups. A NOAEL of 1000 and 15 mg/kg bw/day was established for female and male rats, respectively. However, it should be noted that hydrocarbon nephropathy is a male-rat specific effect, which is not observed in human. For human risk assessment, this effect should therefore not be taken into account.

Executive summary:

This study (OECD 407) was performed to determine the toxicity of cyclobutanate in rats after repeated exposure. Male and female rats were exposed to 0, 15, 150 or 1000 mg/kg bw/day of cyclobutanate for 28-days. Also two satellite groups were included that were allowed to recover for 14 days. Mortality, clinical signs, body weight, food consumption and organ weights were recorded. Hematology, clinical chemistry and urinanalys, gross pathology and histopathology were performed.

An increase in salivation from day 6 onwards was observed in the high-dose group animals, recovery was apparent in the 14 day period after treatment. This effect is usually considered attributable to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity. In the mid-dose group females a significant reduction of body weight was observed in the last week of treatment. As no dose-relationship was observed, this finding was not considered of toxicological importance. A higher incidence of globular accumulations of eosinophilic material in the tubular epithelium was observed in male rats of the high and mid-dose group (hydrocarbon nephropathy). Regression of this condition was seen in the 14 days of recovery. All remaining morphological changes were commonly observed in laboratory rats and therefore not considered to be of toxicological importance.

Under the conditions of this study, no adverse effects were observed in female rats up to the highest dose group. For males, hydrocarbon nephropathy was observed histopathologically in the 150 and 1000 mg/kg bw/day dose groups. Therefore, a NOAEL of 1000 and 15 mg/kg bw/day was established for female and male rats, respectively. However, it should be noted that hydrocarbon nephropathy is a male-rat specific effect, which is not observed in human. For human risk assessment, this effect should therefore not be taken into account.