1,4:5,8-Dimethanonaphthalene, 1,2,3,4,4a,5,8,8a-octahydro-, (1R,4S,4aR,5S,8R,8aS)-rel-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 March to 1 Novemebr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2R,3S,6R,7S,8S)-tetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene

EC Number:

700-678-4

Cas Number:

1076-12-6

Molecular formula:

C12H16

IUPAC Name:

(1R,2R,3S,6R,7S,8S)-tetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene

Test material form:

liquid

Details on test material:

- Substance type: Organic
- Physical state: Clear Liquid
- Analytical purity: ca 100%

- Lot/batch No.: M0904
- Expiration date of the lot/batch: 31 December 2013

- Storage condition of test material: 2-8↨°C on receipt; subsequently transferred to ambient temperature and protected from light storage and purged with nitrogen after each use, within three days of reciept.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reputable laboratotory animal supplier
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: Males: 343 to 395 g; Females: 237 to 278 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body (and floor) with a stainless steel mesh lid; changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: certified pelleted diet, ad libitum
- Water (e.g. ad libitum): potable water from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance TCD was prepared for administration as a series of graded concentrations in the vehicle, corn oil.

Starting with the lowest concentration (2 mg/mL), approximately 50 % of the final volume of vehicle was added to the required amount of pre-weighed test substance and magnetically stirred until dissolved. The solution was made up to final volume with vehicle and mixed using a magnetic stirrer until homogeneous. Higher concentrations (6 and 18 mg/mL) were prepared in ascending order of concentration using the same method.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity - before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix.

Achieved concentration - samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Analysis of the concentration of test substance in the fomulation ws undertaken by gas chromatograhy using external standard calibration. The method was validated prior to use.

Duration of treatment / exposure:

At least 4 weeks (28 days)

Frequency of treatment:

Once daily at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

45 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on results for 78 day preliminary study
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included:.evidence of ill health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: males/females, daily for week 1-2; twice weekly for week 3 onwards. After mating females were observed on days 0, 6, 13, and 20 of gestation and on days 1 and 6 of lactation.

-BODY WEIGHT: yes
- Time schedule for examinations: males - each week including day of commencement of treatment and necroscopy; females - each week inclusing day of commencm,ent of treatment, the following mating days 0, 6, 13, and 20 after mating and on days 1, 4 and 7 of lactation.


FOOD CONSUMPTION: yes
- Time schedule: males - weekly except during mating (week 3)

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
-Time schedule: week 2 prior to pairing

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 2 prior to pairing

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 5 of treatment, females during Days 4-6 of lactation

- Dose groups that were examined: five lowest numbered males in each group and the five lowest numbered surviving lactating females in each group during Days 4-6 of lactation.

- Battery of functions tested: approach response, touch response, auditory startle reflex, tail pinch response, grip strength, motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Grip strength, motor activity, body weight, food consumption, clinical pathology, organ weights:

Parametric analysis
Bartlett's test for variance homogeneity (Bartlett 1937)
Williams’ test (Williams 1971, 1972)
Dunnett's test (Dunnett 1955, 1964)
Shirley's test (Shirley 1977)
Steel's test (Steel 1959)
Fisher’s Exact tests (Fisher 1973)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mortality at the top dose level

Mortality:

mortality observed, treatment-related

Description (incidence):

mortality at the top dose level

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

low bodyweight and weight gain at the top amd mid dose dose level

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high white blood cell count at top dose level

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

low plasma bile acid in males at top dose level

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Low motor activity in females, during Lactation, at the top dose level

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

males - liver, prostate high; females- adrenal and brain low

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

cortical hypertrophy in females, all dose groups. Involution/atrophy in females at 15 and 45 mg/kg/day

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Two females were killed for welfare reasons on Days 10 or 11 of the pre-mating period.
One female (45mg/kg/day) had a slight convulsion on Day 6, and was killed for welfare reasons on Day 11, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical hypertrophy). The factor contributory to death was poor clinical condition.

One female (45mg/kg/day) had a moderate convulsion on Day 6, showed slight tremor on Day 7, and was killed for welfare reasons on Day 10, after exhibiting a marked convulsion at one to two hours after administration. Macroscopic examination revealed dark kidneys and small thymus. Following microscopic examination, significant findings were seen in the spleen (decreased cellularity of the red pulp) and the adrenals (cortical
hypertrophy). The factor contributory to death was poor clinical condition.

There were no general clinical signs and no signs during the arena observations related to treatment with TCD.

BODY WEIGHT AND WEIGHT GAIN
Overall mean body weight gain (Week 0-5) was slightly high in males treated at 45 mg/kg/day (X 1.3), when compared with Control and was high in females at 15 or 45 mg/kg/day, with relationship to dose (X 1.7 or 1.9, respectively), during the two week pre-mating period.
When compared with Control, body weight gain during gestation was not affected by treatment; however, maternal body weight gain was low in treated females (5, 15 or 45 mg/kg/day) during the first four days of lactation (X 0.36, 0.18 or 0.18, respectively) and overall lactation body weight gain (Days 1 to 7) was X 0.77, 0.50 or 0.70, respectively.

FOOD CONSUMPTION
Overall food consumption of males at 45 mg/kg/day was marginally high in Weeks 4 and 5 (X 1.14), when compared with Control.
Overall food consumption of females treated at 15 or 45 mg/kg/day was slightly low during lactation (X 0.88 or 0.84, respectively).

HEMATOLOGY
Overall white cell and concomitant neutrophil, lymphocyte, eosinophil, basophil, monocyte and large unstained cell counts were high in both sexes treated at 45 mg/kg/day, when compared with Control.

CLINICAL CHEMISTRY

There were no differences in plasma enzyme activity in Week 2, when compared with Control.

In males at 45 mg/kg/day, plasma bile acid concentration was markedly low (X 0.34) and cholesterol concentration was slightly high.

NEUROBEHAVIOUR
Sensory reactivity findings in both sexes were considered to be unaffected by treatment. Motor activity scores for males in Week 5 were considered to be unaffected by treatment. Motor activity in females, during Lactation Days 4-6, treated at 45 mg/kg/day were markedly low.

ORGAN WEIGHTS
Mean adjusted liver weight was low, without dose-relationship, in males receiving 5, 15 or 45 mg/kg/day and mean prostate weight was low at 45 mg/kg/day; mean adrenal weight was high in females at 5, 15 or 45 mg/kg/day (X 1.16, 1.19 or 1.27) and mean brain weight was marginally high, without dose-relationship, in females at 15 or 45 mg/kg/day (both X 1.06), when compared with Control.


GROSS PATHOLOGY
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Increased cortical hypertrophy was seen in females at all dose groups. Involution/atrophy was seen in females at 15 and 45 mg/kg/day. Increased incidence and severity of cortical tubules with hyaline droplets was seen in males at 45 mg/kg/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Two females treated at 45 mg/kg/day had convulsions on Day 6, one of which showed slight tremor on Day 7 and both were killed for welfare reasons after having a marked convulsion on Day 10 or 11. A third female at this dose had a single, moderate convulsion on Day 3 and a fourth female had a convulsive episode on Day 4 of lactation. No other animals were noted to have convulsive episodes, despite extending the post dose observation regimen to the times after dosing that the convulsions occurred. The cause of the convulsions/tremor in these four females is not known. Chin rubbing, with or without salivation was seen in the majority of animals at 45 mg/kg/day and in three females at 15 mg/kg/day during the study, this was considered to be attributable to the palatability of the test substance.

At 45 mg/kg/day, overall body weight gain (Weeks 0-5) was slightly high and food consumption was slightly high during Weeks 4 and 5 in males and body weight gain during the two-week pre-mating period was high in females, which was also seen in females treated at 15 mg/kg/day, but these are considered not to be toxicologically significant. Body weight gain and food consumption were not affected by treatment during gestation. Body weight gain was low during lactation and food consumption was slightly low at 45 or 15 mg/kg/day.

There were some intergroup differences in haematology and blood chemistry parameters but they did not correlate with any microscopic pathology changes and were therefore considered to be of no toxicological significance.

Motor activity scores in females receiving 45 mg/kg/day were markedly low and the forelimb grip strength of both sexes receiving 45 mg/kg/day was marginally low, but both were within the Historical Control Data range. Oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index, litter size, sex ratio or survival of the offspring were not affected by treatment at 45 mg/kg/day. At 45 mg/kg/day, mean offspring bodyweights on Day 1 of age were low and remained low, to Day 7 of age and one litter had six dead offspring, from 15 born; however mortality in other litters at this dose was within acceptable limits. There were no macroscopic findings related to treatment. Microscopic investigation revealed minimal cortical hypertrophy in the adrenals from females in all treated groups, which correlated with the high mean tissue weight and involution/atrophy of the thymus from the majority of females at 45 mg/kg/day and a single case of thymic atrophy in females at 15 mg/kg/day, which were considered to be related to stress. Among males receiving 45 mg/kg/day, there was an increase in the incidence and severity of renal cortical tubules with hyaline droplets (alpha2 microgloulin nephropathy), which is occasionally seen in males following treatment with a hydrocarbon. Sensory reactivity and grip strength were unaffected by treatment at 45 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

For general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg/day.