Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2011 - 23 August 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline 408 and under GLP conditions. Reliability 2 is assigned because of its use as read across

Justification for type of information:

The information is used for read across to Cyclabute.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

EC Number:

911-369-0

Molecular formula:

C12H16O2

IUPAC Name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

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, United Kingdom
- Age at study initiation: 6-8 weeks
- Weight at study initiation:
Males: 194-257 g
Females: 161-206 g
- Housing: Polypropylene cages with stainless steel mesh lids and softwood flake bedding (except for urine collection: metabolic cages)
- Diet (e.g. ad libitum): Ad libitum, ground diet
- Water (e.g. ad libitum): Ad libitum (mains drinking water)
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): at least 15

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Monthly (dietary admixtures stable for at least 6 weeks at -18 °C)
- Mixing appropriate amounts with (Type of food): Basal laboratory diet
- Storage temperature of food: -18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of selected dietary admixtures were analysed for homogeneity and concentration prior to use by gas chromatography using an external standard technique.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuously in the diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200, 2000, 6000, 20000 mg/kg feed
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
15.3, 154.9, 464.1, 1504.6 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: 14-day rangefinding study
- Rationale for animal assignment: Random
- Section schedule rationale (if not random): No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked: overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes (signs of functional/behavioural toxicity)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 7 and at end of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Hb, RBC, Hct, MCH, MCV, MCHC, WBC, Neut, Lymph, Mono, Eos, Bas, PLT, CT, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 7 and at end of study
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, ASAT, ALAT, AP, creatinine, total cholesterol, total bilirubin, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: Week 7 and at end of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, specific gravity, ketones, bilirubin, pH, protein, glucose, urobilinogen, blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12 only
- Dose groups that were examined: All dose groups
- Battery of functions tested: motor activity, forelimb/hindlimb grip strength, sensory reactivity to auditory, visual and proprioceptive stimuli

OESTROUS CYCLE: Yes
- Time schedule: during weeks 6&7 and weeks 12&13
- Method: daily vaginal smear was stained with giemsa stain, stage of oestrus was examined microscopically

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Examinations: full external and internal examination

ORGAN WEIGHTS: Yes
- Tissues measured: adrenals, brain, epdididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid/parathyroid, thymus, uterus and cervix

HISTOPATHOLOGY: Yes
- Dose groups that were examined: control and high dose group, except for kidneys, liver and adrenals (all groups)
- Tissues examined: Adrenals, Oesophagus, Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Cervix, Salivary glands (submaxillary), Coagulating gland, Sciatic nerve, Colon, Seminal vesicles, Duodenum, Skin (hind limb), Epididymides, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes, Jejunum, Thymus, Kidneys, Thyroid/parathyroid, Liver, Tongue, Lungs (with bronchi), Trachea, Lymph nodes (cervical and mesenteric), Urinary bladder, Mammary glands, Uterus, Muscle (skeletal), Vagina
- Staining: haematoxylin & eosin, Mallory's Heidenhain (for male kidney)

Other examinations:

After necropsy:
- Thyroid hormone assessment: blood samples collected for analysis of thyroid hormone, in case pituitary-thyroid axis effects identified
- Sperm assessment: 1. weighing of testis and epidydimis (excl. connective tissue), 2. analysis of homogenisation resistant spermatids in testis (DNA specific fluorescent staining), 3. determination of number of motile, progressively motile and non-motile sperm, 4. morphometric analysis (eosin staining), 5. analysis of homogenisation resistant spermatids in cauda epidydimis (eosin staining)

Statistics:

Group mean values and SD used for reporting. For each variable, the most suitable transformation of data was found, the use of possible covariates was checked and the homogeneity of means was assessed with ANOVA, ANCOVA or Bartlett's test.

Tests to demonstrate statistical significance:
- Williams Test for parametric values and Shirley Test for non-parametric values
- Stepwise Dunnett (parametric) or Steel (non-parametric) in case no dose response found (non-homogeneity of means)
- Pair-wise tests: Student t-test (parametric) or Mann-Whitney U test (non-parametric)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

overall reduction in body weight gain in highest dose group for both sexes, minor effects in other dose groups

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduction in food consumption throughout the study in highest dose group

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

adverse effect in highest dose group (related to reduced body weight gain)

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Highest dose males: decreased chloride, sodium, bile acid, alanine aminotransferase and aspartate aminotransferase, increased cholesterol. No toxicologically relevant effects in females.

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:

effects observed, treatment-related

Description (incidence and severity):

kidney effects in males in the highest dose group. No toxicologically relevant effects in females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

all males: hyaline droplets in kidney, hepatocellular hypertrophy, vacuolation of zona fasciculata (adrenals). No toxicologically relevant effects in females.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No toxicologically relevant effects observed.

BODY WEIGHT AND WEIGHT GAIN
Reduction in overall body weight observed in all animals in the highest dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Reduction in food consumption observed in animals of the highest dose group throughout the study.

FOOD EFFICIENCY
Adverse effects observed in highest dose group (related to reduced body weight gain).

WATER CONSUMPTION
No treatment related effects observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment related ocular effects detected.

HAEMATOLOGY
No toxicologically significant effects detected.

CLINICAL CHEMISTRY
Males
Week 7 - 6000 ppm: reduced chloride concentration
Week 7- 20000 ppm: reduced chloride concentration and aspartate aminotransferase levels, increased cholesterol levels
Week 13 - 6000 ppm: reduced aspartate aminotransferase and alanine aminotransferase levels
Week 13 - 20000 ppm: reduced chloride concentration, sodium concentration and aspartate aminotransferase levels, alanine aminotransferase levels, bile acid levels, increased cholesterol levels
Females
No effects observed in all treatment groups

URINALYSIS
No treatment related effects observed

NEUROBEHAVIOUR
No treatment related effects were observed in behavioural assessment, functional performance test, and sensory reactivity assessment.

ORGAN WEIGHTS
Males
All treatment groups: Increased kidney weight (both absolute and relative)
20000 ppm: increased adrenal weight and relative liver weight

Females
No toxicologically significant effects observed.

GROSS PATHOLOGY
Three males at the highest dose group has pale kidneys. No toxicologically significant macroscopic findings were detected in females.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males
Hyaline droplet nephropathy in kidneys observed in all treated males. in liver, centrilobular to midzonal hepatocellular hypertrophy was observed at minimal severity degrees at 2000, 6000, and 20000 ppm. In adrenals, increased incidence in vacuolation of the zona fasciculata was observed in all treatment groups.

Females
No toxicologically significant changes were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicological significant effects observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. In males, hyalin droplet nephropathy was observed in the kidneys, which is a rat-specific effect that is not relevant for humans. In females, no toxicologically significant effects were observed. The NOAEL could be established as the highest dose tested, 20000 ppm or 1500 mg/kg bw/day, under the conditions of this test.

Executive summary:

In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. The dose levels were 0, 200, 2000, 6000, and 20000 ppm, corresponding to 0, 15.3, 154.9, 464.1, and 1504.6 mg/kg bw/day, respectively. Clinical signs, functional observations, body weight change, dietary intake, and water consumption were monitored during the study. During week 7 and at the end of the study, haematology, blood chemistry and urinalysis were evaluated. Furthermore, oestrus cycle assessment was performed on female animals between week 6 and 7 and week 12 and 13.Opthalmoscopic examination was performed at the start and end of the study in control and high-dose animals. At the end of the study, all animals were subjected to gross necropsy examination and histopathological examination of tissues was performed on animals from control and high-dose. Furthermore, sperm assessment was performed on males at necropsy.

Reduced overall body weight was observed in male and female animals in the highest dose group, correlated with reduced food consumption and adverse effects on food efficiency, indicating decreased food palatability. At the highest dose level, reduced chloride concentration, sodium concentration, aspartate aminotransferase levels, alanine aminotransferase levels, bile acid levels, and increased cholesterol levels were observed in male animals. The observed changes in aminotransferases, bile acid and cholesterol can be explained by the reduced food consumption. Changes in chloride and sodium concentrations may be explained by the observed kidney effects in males. Increased kidney weights were also observed in males, as well as hyalin droplet nephropathy, which is a rat-specific effect that is not relevant for humans. In females, no toxicologically significant effects were observed in clinical chemistry, organ weights or histopathology. Therefore the NOAEL could be established as the highest dose tested, >=20000 ppm or >=1500 mg/kg bw/day, under the conditions of this test.