1,4:5,8-Dimethanonaphthalene, 2-ethylidene-1,2,3,4,4a,5,8,8a-octahydro-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2010 - 18 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered reliable on the basis that it was conducted according to an appropriate OECD Test Guideline, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The study report includes certificates of GLP compliance for both sites involved in the study, issued by the MHRA.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(3R,6S,9E)-9-ethylidenetetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene; (3S,6R,9E)-9-ethylidenetetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene

EC Number:

700-518-3

Cas Number:

38233-76-0

Molecular formula:

C14H18

IUPAC Name:

(3R,6S,9E)-9-ethylidenetetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene; (3S,6R,9E)-9-ethylidenetetracyclo[6.2.1.1³,⁶.0²,⁷]dodec-4-ene

Details on test material:

- Name of test material (as cited in study report): ETD
- Physical state: Transparent Liquid
- Analytical purity: 100%
- Lot/batch No.: M0901
- Expiration date of the lot/batch: 31 December 2011
- Storage condition of test material: Refrigerated (approximately +4°C) and protected from light.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: Males: 287 - 318 g; females: 168 - 208 g
- Fasting period before study: No
- Housing: 5 animals per cage except during mating (1 male and 1 female per cage) and females after mating (1 female per cage). Cages were polycarbonate, except during mating in which polycarbonate cages with stainless steel floors were used.
- Diet (e.g. ad libitum): Free access to a standard rodent diet.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%):40 - 70% RH
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: From: 21 December 2010 To: 07 February 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance, ETD, was prepared for administration as a series of emulsions in the vehicle. The required amount of test material was weighed into a suitable container. Starting with the lowest concentration, approximately 50 % of the final volume of the vehicle was added to the test material and magnetically stirred. The required volume was made up with vehicle and mixed using the magnetic stirrer until homogenous.

All formulations were prepared weekly and stored refrigerated.

VEHICLE
- Concentration in vehicle: 2.5, 10, and 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1-to-1
- Length of cohabitation: Up to 15 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The achieved concentrations of ETD in formulations was analysed in the first and last weeks of the study.

Samples were extracted and diluted initially in Tetrahydrofuran (THF), with a final dilution in THF/Methanol (25/75 v/v) for analysis by HPLC-UV.

Prior to the first analysis, the method was validated for formulations at 1 mg/mL and 100 mg/mL (overall mean recovery value = 101.4%, CV = 1.04%, n=16). Stability and homogeneity / resuspendability of the formulations at 1 mg/mL and 100 mg/mL was demonstrated over 24 hours at ambient temperature, and for up to 15 days at refrigerated (nominally +4°C) storage.

Mean analysed concentrations of ETD in formulations were found to be within the acceptable limits (+10/-15% of the nominal concentration), confirming acurate formulation.

Duration of treatment / exposure:

All F0 (parental animals) were dosed from 15 days before pairing / mating, until day 7 of lactation.
F1 generation animals were not dosed.

Frequency of treatment:

Animals were dosed once per day, 7 days per week, at approximately the same time each day.

Details on study schedule:

- Age at mating of the mated animals in the study: 12 to 13 weeks (10 to 11 weeks at the start of treatment).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
12.5 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
200 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Visual inspections at least twice daily, and observations of the animals and their cages at least once per day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, and at days 0, 7, 14, and 20 after mating, and days 1 and 7 of lactation for F0 females.

BODY WEIGHT: Yes
- Time schedule for examinations: Males weighed weekly throughout study. Females weighed weekly until mating was detected, then on days 0, 6, 13, and 20 after mating and days 1 and 7 of lactation.

Oestrous cyclicity (parental animals):

For 15 days before pairing, vaginal smears were taken daily from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
clinical signs, litter size, sex ratio, and bodyweight

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals; males were killed after confirmation that a second mating was not required.
- Maternal animals: All surviving animals; F0 females were killed on Day 7 of lactation. Females that failed to produce a viable litter were killed on Day 25 after mating. Females whose litter died before Day 7 of lactation were killed on the day the last offspring died. F1 offspring were killed on Day 7 of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera, which were examined in situ.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:

Epididymides*, prostate, kidneys, seminal vesicles (with coagulation gland), ovaries*, testes*, pituitary, and uterus with cervix and oviducts (*denotes cases where bilateral organs were weighed individually).

Postmortem examinations (offspring):

For F1 offspring surviving to scheduled termination, animals were examined externally for gross abnormality.

Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined as detailed above. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Description (incidence and severity):

Test substance intake: Not applicable as test material was admiistered by oral gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Two males, nos. 39 and 40, receiving 200 mg/kg/day were killed for reasons of animal welfare on Day 2 and 8 of treatment respectively. Terminal signs included limited use and tremors of hindlimbs for both males, with male no. 39 also showing underactive behaviour, piloerection, prostration, red staining perigenital area and irregular breathing. Male 39 was found to have a distended urinary bladder containing red fluid, distended ureters, pale kidneys and an accentuated liver lobular pattern when examined at necropsy and microscopic examination showed that animal no. 39 found haemorrhage of the blood vessels in the walls of the urinary bladder and ureters. There were no macroscopic findings at necropsy of male 40 and the only microscopic findings were cortical tubular vacuolation and cortical tubular hyaline droplets in the kidneys. It was considered that both deaths were probably treatment related because of the common observation of tremors and loss of use of the hind limbs.

There were no signs at routine physical examination that could be related to treatment with ETD.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During Week 1 of treatment males receiving 200 mg/kg/day showed a transient group mean loss in bodyweight (p<0.01). Subsequent weight gain was essentially similar to Controls but animals remained lighter than controls throughout. Bodyweight gain at 12.5 or 50 mg/kg/day was similar to, or slightly greater than, that of the Controls. During the two week treatment period before pairing for mating, bodyweight gain for females receiving ETD was unaffected by treatment. During gestation weight gain at both 50 and 200 mg/kg/day was low compared with control (80 % of control values for both) although group mean maternal weights at Day 1 post partum were not significantly affected by treatment. Weight gain between Day 1 and 7 of lactation was slightly lower than control at these dose levels so that maternal weights at Day 7 of lactation were significantly lower than control. Bodyweight and bodyweight change for females at 12.5 mg/kg/day was unaffected by treatment with ETD during gestation and lactation.

During Week 1 of treatment males receiving 200 mg/kg/day had low food consumption (74% of Control) when compared with the Controls; however during the second week of treatment the food consumed for males at this dose level was slightly high when compared with Controls (109% of Controls). Food consumption for males at 12.5 and 50 mg/kg/day and for females at all dose levels for the two weeks before pairing was unaffected by treatment with ETD. During the first week of gestation females receiving 50 or 200 mg/kg/day showed marginally, but significantly, lower food consumption when compared with the Controls; subsequent food consumption was similar to the Controls. At 12.5 mg/kg/day food consumption during gestation was unaffected by treatment. At 50 and 200 mg/kg/day food consumption during lactation was significantly low when compared with the Controls, whilst food consumption at 12.5 mg/kg/day was essentially similar to Controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles showed that females receiving 200 mg/kg/day had a higher incidence of 5 day cycles when compared with the Controls (p<0.01). Five of the 10 females were affected and the majority of extended cycles occurred in the first week of treatment. Oestrous cycles for females receiving 50 or 12.5 mg/kg/day were unaffected by treatment with ETD.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Pre-coital interval was unaffected by treatment with all animals showing positive evidence of mating within 4 days of pairing at the first oestrus.
Gestation length and gestation index was unaffected by treatment with ETD.
Mating performance and fertility was unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
After six weeks of treatment males that received 50 or 200 mg/kg/day had significantly high kidney weights when compared with the Controls (p<0.01: 112% and 120% of control values). At 200 mg/kg/day males also had significantly lower seminal vesicle weights (p<0.05: 81% of Control values) but in the absence of effects upon the other accessory sex organs this effect is considered unlikely to be of biological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic examination of males after 6 weeks of treatment and females on Day 7 of lactation did not reveal any findings that could be related to treatment with ETD.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Changes related to treatment with ETD were seen in the kidneys, predominantly in males which received 200 mg/kg/day. These consisted of medullary tubular dilatation, medullary tubular casts, cortical tubular hyaline droplets and cortical tubular basophilia.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

female

2

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: Based on a minor change in the oesterous cycles, and a significantly low number of implantations and reduaced littler size.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
One female at 200 mg/kg/day had no implantations (not pregnant) and the numbers of implantations and subsequent litter size was statistically significantly lower than control. Implantation counts and litter size were marginally low at 50 mg/kg/day but there were no effects at 12.5 mg/kg/day. Offspring survival and sex ratio at all dose levels were unaffected by parental treatment with ETD.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings that could be attributed to parental treatment.
At scheduled termination on Day 7 of age all offspring external macroscopic examination did not reveal any abnormalities.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Within this study, there was a minor change in the pattern of oestrous cycles at 200 mg/kg/day, and a significantly low number of implantations and reduced litter size. Effects at 50 mg/kg/day were minimal and it was concluded that the NOAEL was 50 mg/kg/day for reproductive performance when ETD was administered orally to the Wistar rat.