[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2019 - January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

EC Number:

209-269-0

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Cas Number:

564-20-5

Molecular formula:

C16H26O2

IUPAC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Healthy adult rats (Rattus norvegicus) of Wistar strain (RccHan: WIST) were obtained from the Animal Breeding Facility, Jai Research Foundation. Female rats were nulliparous and non-pregnant. At the initiation of acclimatisation, rats were 11-12 weeks old. Care was taken to avoid mating of siblings.
Rats were maintained in an environment-controlled room at a temperature of 22 ± 3°C and relative humidity of 30 to 70 per cent. The photoperiod was 12 hours light and 12 hours darkness, fluorescence light hours being 06.00 - 18.00 hours.
Throughout the experimental period, rats were housed in 2 or 3 rats/sex/cage. During the mating period, rats were housed in groups of 2 rats/cage (one male plus one female). Enrichment material (wooden chew blocks) was provided to all animals. Mated female rats were caged individually and nesting material was provided at near parturition. During the study, rats were housed in solid floor polypropylene rat cages. Each cage (42.7x28.7x19.8 cm) was fitted with a stainless steel top grille having provision for a polypropylene water bottle with a stainless steel drinking nozzle. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material.
Cages were placed on 5 tier racks. Cages and enrichment material were changed minimum twice a week. Cages were arranged in such a way that possible effects due to cage placement were minimised.
Rats were fed ad libitum with standard rodent diet (Teklad Certified Global 16% Protein Rodent Maintenance Diet, procured from Envigo Laboratories, Inc., USA). The rats were provided RO water ad libitum (Reverse Osmosis water filtration system) in polypropylene bottles.
Every food consignment received is accompanied by a certificate of analysis of nutrient content from the food supplier. The quality of food and water is monitored.
A larger number of rats than necessary for the study was ordered to permit the selection of individuals before start of the treatment. A total of 34 male and 34 female rats were selected for acclimatisation. The body weight variation of the animals was within ±20% of the mean body weight at the beginning of the acclimatisation. Rats were received into the experimental room and acclimatised for 5 days prior to pre-treatment oestrous cycle evaluation. During the acclimatisation period, rats were observed daily twice for mortality and morbidity, and at least once for clinical signs.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Tween 80 and 0.75% CMC in ratio 1:99

Details on exposure:

Tween 80 and 0.75 % carboxymethylcellulose (CMC) in a ratio of 1:99 were selected as a vehicle based on the solubility check. CMC was prepared at least once in a week and was stored at room temperature.
This study was conducted as a limit test. Hence, following dose levels were selected based on sponsor’s recommendation: 0 and 1000 mg/kg. b. wt./day. for control and limit dose group, respectively.
The dose formulations were prepared by mixing the test item with a vehicle. It was thoroughly mixed using magnetic stirrer in order to administer a homogeneous preparation.
Test dose formulation and vehicle were administered to male and female rats daily for seven days a week, at approximately the same time each day. The dose-volume for the administration was 10 mL/kg body weight. The doses were adjusted according to the most recently recorded body weight. The control group received vehicle alone. The first day of dosing was designated as treatment day 1 for each animal.

Details on mating procedure:

During the mating period, male and female rats were mated in a 1:1 ratio to obtain the F1 pups. Each morning, female rats were examined for the presence of sperm and the ‘day 0’ of pregnancy was recorded. ‘Day 0’ of pregnancy was defined as the day on which sperm were observed in the vaginal smear of rats.
For each mating, each female rat was placed with a male rat from the same dose level (1:1 mating) until mating occurred or 2 weeks had elapsed. Acyclic females were assumed as pregnant after completion of two week mating period. A detailed record of the mating pairs was maintained in the raw data. Care was taken to avoid mating amongst siblings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the active ingredient (a.i.) in the vehicle was determined prior to initiation of treatment using a validated analytical method (GC-FID). Based on the result, dose formulations were stable up to 8 days, hence dose formulations were prepared minimum once in a week.
The active ingredient (a.i.) concentration and homogeneity of the test item was analysed once before initiation of treatment and twice during the treatment period. Two sets of samples (4 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from limit dose whereas from control only one aliquot. One set of samples was used for analyses and the other set of samples was stored at 2 – 8 °C. On each occasion, the mean concentration was determined and compared to the nominal value. The acceptance criteria were ±15% deviation from the nominal value and the %CV < 10. The samples were analysed using a validated analytical method (GC-FID).

Duration of treatment / exposure:

Dosing of both the sexes was initiated at least 2 weeks prior to mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice (until approximately 80% of the females had delivered). In total, male rats were dosed for 43 days. Female rats were further dosed during pregnancy and up to post-partum day 14.

Frequency of treatment:

7 days a week

Details on study schedule:

On postnatal day four, the size of each F1 litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four female pups per litter. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (e.g., six males and two females) was performed.
Adjustments were not done for litters of eight pups or less.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

limit dose

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

See below

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

Rats were observed daily, twice, for mortality and morbidity.

Rats were observed daily, twice, for any visible clinical sign.

Body weight of all-male rats was recorded on the first day of dosing and at weekly intervals thereafter.
Bodyweight of all female rats was recorded on the first day of dosing and at weekly intervals during pre-mating and mating period. During the gestation period, female rats were weighed on gestation day 0, 7, 14, and 20. During the lactation period, female rats were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, 14, and on day of terminal sacrifice. Parturition day ‘0’ was defined as the day on which the female littered.
On the day of fasting, body weights of all surviving rats were recorded. Body weights of all the rats were also recorded on the day of sacrifice.

The food consumption was determined by differentiating the weight of food input and leftover.
Food weights of male rats were determined weekly during the pre-mating and post-mating period.
In female rats, during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on days 0, 7, 14, and 20. During the lactation period, food weights were measured at least on days 0, 4, 7, and 14 and on day of terminal sacrifice. Additional food was offered as and when required.
Food consumption was not measured during the mating period.

At the time of terminal sacrifice, blood (approximately 1 mL) was collected from all parental rats/sex/group under isoflurane anaesthesia by orbital plexus puncture. Rats were deprived of food overnight (water allowed) prior to blood collection. Blood samples were collected in vials without anticoagulant for thyroid hormone analysis.
Serum thyroid hormones (T4, TSH, and T3) levels were analysed from parental male and female rats.
Serum thyroid hormones (T3, T4) were analysed at JRF using validated bioanalytical and TSH ELISA methods.

Oestrous cyclicity (parental animals):

Estrous cycle length and pattern were evaluated by vaginal smears of individual female rat during the pre-treatment period of two weeks. Vaginal smear was monitored daily since the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant rat, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.

Sperm parameters (parental animals):

N/A

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Pups which died during lactation were weighed and subjected to a post-mortem examination.
AGD of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13.
Individual pup body weights were recorded on postnatal days 0, 4, 7, and 14.

Blood was collected from at least two surplus pups/litter on postnatal day 4 (when feasible) and at least two pups/litter on the day of terminal sacrifice through decapitation. Blood samples were collected in vials without anticoagulant for thyroid hormone analysis. Pup blood was pooled by litter for thyroid hormone analyses.
Serum thyroid hormones (T4, TSH, and T3) levels were analysed from terminal sacrificed pups (PND 14). Serum thyroid hormone, (T4) analysis was performed on PND 4 (T4) pups.
Serum thyroid hormones (T3, T4) were analysed at JRF using validated bioanalytical and TSH was analysed by ELISA methods.

Postmortem examinations (parental animals):

All rats were sacrificed by carbon dioxide asphyxiation except one dam which died after blood collection (not recovered after isoflurane anaesthesia).

Gross necropsy was conducted on all rats under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities.

Male rats were sacrificed after approximately 80% of female animals have delivered. Female rats were sacrificed on postpartum day 15. Females who had not delivered by day 25 post-coitum were sacrificed on post-coitum day 25.

At the time of sacrifice or death during the study, all adult rats were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to external reproductive genitals which were examined for signs of altered development.

A number of implantation sites were recorded for each dam. Implants were not present in any of the animal sacrificed on gestation day 25.

At the time of sacrifice, following organs/ tissues of rats were excised, weighed and fixed inappropriate fixatives for microscopic examination: Testes
Epididymis
Prostate, Seminal vesicles with Coagulating gland (as a whole)
Levator ani plus bulbocavernosus muscle complex (LABC)
Cowper’s glands
Glans penis
Paired ovary (wet weight)
Uterus including cervix
Thyroid with parathyroid (post-fixation)

The following organs and tissues of rats were fixed and stored in medium:
- Uterus with the cervix, prostate, LABC, Cowper's gland, glans penis and seminal vesicles with coagulating gland (as whole), and thyroid with parathyroid were preserved in 10% neutral buffered formalin.
- Paired ovaries, testes and epididymis were preserved in Bouin’s fixative. The tunica albuginea was gently and shallowly punctured at both the poles of the organ with a needle to permit rapid penetration of the fixative.
- Thyroid with parathyroid from parent rats were collected on the day of terminal sacrifice and preserved in 10% neutral buffered formalin.

Detailed histopathological examination of testes, epididymis and ovaries were performed in high limit and control dose group rats.
Detailed testicular histopathological examination (paraffin embedding and transverse sections of 4-5µm thickness) was conducted with special emphasis on stages of spermatogenesis and histopathology of interstitial cell structure. The testes were evaluated to identify treatment-related effects such as retained spermatids, missing germ cell layer or types, multinucleated giant cells or sloughing of spermatogenic cells into lumen. Examination of intact epididymis was included the caput, corpus and cauda, which was accomplished by evaluation of longitudinal section. The epididymides were evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS) and hematoxylin staining were used for examination testes and epididymis.
Histopathological examination of ovaries was detected for treatment-related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increase/decrease corpus luteam, ovarian degeneration/atrophy and stromal cell proliferation.

Postmortem examinations (offspring):

Pups of PND 14 were sacrificed by carbon dioxide asphyxiation. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed through intraperitoneal administration of thiopentone sodium.

Gross necropsy was conducted on all rats under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities.

Pups were sacrificed on postpartum day 14.

At the time of sacrifice or death during the study, all pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to external reproductive genitals which were examined for signs of altered development.

Pups which were found dead on PND 0 were subjected to gross examination and portion of lung was immersed in water for confirmation of status (stillbirth or dead). Pups which were found dead were observed for the presence of a milk band. Pups without gross lesion were discarded after examination.

Thyroid with parathyroid was excised, collected, weighed and preserved from one male pup and one female pup/litter on day of terminal sacrifice. Thyroid with parathyroid from 1 male and 1 female pup per litter wherever applicable and from parent rats were collected on the day of terminal sacrifice and preserved in 10% neutral buffered formalin.

Histopathological examination was not performed for thyroid from pups (PND 14).

Statistics:

The statistical analysis was carried out for parameters using validated software developed at JRF.
Data such as body weight, body weight gain, food consumption, organ weight, organ weight ratio were subjected to Shapiro-Wilk’s test for checking normality wherever applicable, followed by Student’s t-test. If the data did not meet the normality, then data was transferred to the log form to check the normality again. If log transfer data did not meet the normality, the Mann-Whitney test was performed to calculate significance. Litter size was subjected to a non-parametric test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis. Non-pregnant rats were not subjected to statistical analysis.
Count data (like litter size, number of implants and duration of gestation) were subjected to non-parametric analysis. The non-parametric data (mortality rate, pregnancy rate, male fertility index, female fertility index, live birth index, survival index, gestation index, parturition index, etc.) were analysed using a Chi-Square test.
Flags for significant difference between control and treated groups (single arrow for p≤0.05 and double arrows for p≤0.01) were given in tables along with the footnote.

Reproductive indices:

Prenatal loss, postnatal loss, mating index, fertility index, gestation index, parturition index

Offspring viability indices:

Pup survival index, live birth index, AGD normalised, male sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight and mean body weight gain of male and female rats belonging to the test item group were comparable with that of the respective control group. Except during treatment days 15-22, the male mean body weight gain was statistically significantly increased when compared with that of the control group. Increase in mean body weight gain is toxicologically insignificant, hence considered as unrelated to treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic lesions (degeneration, atrophy and sperm stasis) observed in testes were at a lower rate of occurrence with unilateral and minimal to mild severity. These lesions were present in treated and control group animals. Hence, those lesions were considered to be spontaneous or incidental in nature and not treatment-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male fertility index, the number of implants and mating index of test item group was comparable with that of the control group. The duration of gestation days was comparable among test item treated group with that of the control group.
Statistically significant decrease in female fertility index, gestation index, parturition index, percentage of pregnant animals was observed, however, changes are within the historical range of performing lab, hence considered as unrelated to treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects attributed to the test item were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

During the lactation period, no treatment-related effect on mortality and mortality index was observed in the test item treated group when compared with that of the control group. Except, a statistically significant decrease in the mortality index of female, and composite of male and female pups from test item group was observed during PND 0 – 4, when compared with that of the control group. This decrease in mortality index has no toxicological significance.

Body weight and weight changes:

no effects observed

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:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects attributed to the test material were observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of present study, it is concluded that Sclareolide at 1000 mg/kg b. wt./day (limit dose) did not produce any treatment-related effect on systemic, performance of parent male and female rats and growth of pups. Hence, the No Observed Adverse Effect Level (NOAEL) for parental toxicity, reproductive, fertility toxicity and for offspring toxicity was 1000 mg/kg b. wt./day.