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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2017 - May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This study was conducted using rats (Rattus norvegicus), Wistar Hannover strain, males and females with age about 8 weeks. The body weight variation not exceed ± 20% of the average weight for each sex.
The supplier was CEMIB - Centro Multidisciplinar para Investigação Biológica na Área de Ciências de Animais de Laboratório.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were identified, during the acclimation and treatment periods, with traces on the tail using a permanent marker. Each box was identified with a colored card containing the following information: study number, Bioagri code, box number, group, dose, number of animals, sex, Study Director, treatment start date and scheduled necropsy date.
During acclimation and treated period, the animals were housed in up to 2 individuals per sex per box, in polypropylene boxes with metal grids and lining material (autoclaved wood shavings). Box exchanges were performed twice a week for all animals. The boxes were distributed on the shelves to ensure uniform ventilation and brightness throughout the study.
The temperature of the experimental room was 21-25 dC and humidity 42-70%. The ambient air was renewed 10 to 20 times per hour and a 12-hour light/dark cycle was maintained.
The animals received a conventional diet for laboratory animals (irradiated) and filtered and autoclaved drinking water supplied by SABESP (São Paulo State Basic Sanitation Company), both ad libitum throughout the study. Feed, water, and wood shavings were analyzed for environmental contaminants. The diet was analyzed for nutritional composition.

An additional amount of animals beyond that required for conducting the study was acquired to allow selection and replacement of individuals prior to initiation of treatment. Thus, a total of 24 males and 24 females were purchased from a suitable external supplier. Upon receipt, these animals were housed in boxes (2 animals/sex/box) and kept in quarantine for at least 7 days. During this period, the animals were observed daily for morbidity and mortality. Animals showing signs of abnormality were not used in the study. After quarantine, the animals were acclimated to the experimental conditions for at least 5 days.

The randomization of the animals was performed on the first day of the acclimation period. Prior to randomization, the animals were weighed and submitted to a clinical examination. The required amount of animals was selected according to health status and body weight of each animal (individual weights were within ± 20% of the mean body weight of each sex). The animals were randomly assigned to the experimental groups. The allocation of animals that were not used for this study was recorded in the raw data. On the day before the pre-exposure period, the animals were re-weighed and a detailed clinical examination was performed.

This study has been conducted in compliance with the principles for animal welfare and humane use and care of laboratory animals. Whenever possible, procedures were designed to avoid or minimize discomfort, stress and pain to animals. The animal care procedures were conducted in accordance with the principles of Diretriz Brasileira para o Cuidado e a Utilização de Animais em Atividades de Ensino e Pesquisa Científica – DBCA and of the Guide for the Care and Use of Laboratory Animals.

Route of administration:

oral: gavage

Details on route of administration:

Oral administration is the intended route for human exposure and therefore recommended by regulatory agencies. The oral route is the route of administration recommended by OECD 407 (2008). Gavage was chosen as an accurate and reliable method for dosing.

Vehicle:

other: acetone 25% diluted in olive oil

Details on oral exposure:

Preparation of the sample in water formed precipitate. Thus, evaluating the chemical information of Test Item, the organic solvent (acetone) diluted in olive oil was chosen as a vehicle. The solution test performed homogeneous, without precipitate, after the use of the new vehicle.
For each dosage group, the appropriate amount of the test item was weighed into a specific apparatus. The vehicle was added in sufficient quantity to achieve the desired concentration. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals. The volume of doses and vehicle did not exceed 2mL/100g body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After preparation of test solutions, an aliquot of each dose evaluated were analyzed by chromatography to determine the effective concentration of the active ingredient (a.i.) in the solution. The acceptance limit should be less than ± 20%.

Duration of treatment / exposure:

28 days treatment
18-19 days additional observation period without treatment in satellite groups

Frequency of treatment:

7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

satellite group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

satellite group

No. of animals per sex per dose:

6 males and 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

The limit dose was selected based on information from other toxicity studies with the test substance.

Positive control:

N/A

Observations and examinations performed and frequency:

During the treatment period, all animals were observed for mortality and morbidity, daily, two times a day (in the beginning and at the end of each day).

The observation period was 28 days for limit and control groups, on the other hand the animals in a satellite groups (limit and control) were observed for 18-19 days without treatment to detect delayed occurrence or persistence or recovery from toxic effects.

General clinical observations was made at least twice daily and all animals will observe for mortality and morbidity. Once before the first exposure and once a week detailed clinical observations was made in all animals and preferably at the same time of day on each occasion. These observation were made outside the home cage and it include but not limited of changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were recorded.

In the fourth exposure week a functional observations was conducted to evaluate reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment.

Each animal was weighted before randomization, the previous day of treatment, once week over the treatment period, the previous day of necropsy (before fasting) and the day of necropsy (with animal in fasting).

Feed consumption was evaluated weekly for all animals during the treatment period. The average feed consumption was calculated per animal per day.

Hematological and biochemical analyzes were performed on all animals only at the end of the treatment period (on the day of the scheduled necropsy). The animals were fasted for a period of approximately 12 hours prior to blood collection.

For hematological evaluation, an automatic analyzer was used to evaluate the following parameters: haematocrit, haemoglobin concentration, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count and a measure of clotting potential such as prothrombin time, thromboplastin time, fibrinogen or platelet count be investigated at the end of the test period.

Biochemical analysis was accomplished through commercial colorimetric kits according to manufacturer's instructions and certain automated equipment. The following parameters were analysed: nitrogen urea, creatinine, total protein, albumin, total cholesterol, triglycerides, glucose, calcium, bilirubin, phosphate, chloride, sodium, potassium, aspartate aminotransferase, alanin aminotransferase, gamma-glutamyl transpeptidase, bile acids, cholinesterase.

The determination of thyroid hormones were based upon alterations of thyroid histopathology. Once there was no abnormalities in the histological evaluation, the specific hormones did not performed.

Sacrifice and pathology:

At termination, all animals were examined macroscopically for any structural abnormalities or pathological changes. Animals found dead or sacrificed moribund during the study were subjected to gross examination as soon as possible after death. If the necropsy cannot be performed immediately, the animals are kept in a refrigerator until the examination is performed.

The gross necropsy includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.

During the necropsy, a series of organs of all animals were collected, weighed and preserved. Organs pairs were weighed together. The ratios between organ weight and body weight were calculated. The followings organs were weighed: liver, testicles, prostate+seminal vesicles and coagulating glands, brain, uterus, kidneys, epididymides, heart, adrenals, thymus, spleen, ovaries.

At scheduled necropsy, the following organs of all animals were preserved in Neutral 10% Formalin Fixative: all gross lesions, kidneys, heart + aorta, brain, adrenals, thymus, liver, spleen, thyroid, trachea, lungs, lymph nodes, peripheral nerve (sciatic), skeletal muscle, bone, bone marrow, esophagus, spinal cord (thoracic and lumbar), eye, stomach, small (splender) and large intestines, cerebellum + pons, hypophysis, mammary gland.
And the following organs were preserved in Davidson’s fixative: testicles, epididymides, ovaries, uterus + cervix, prostate + seminal vesicles with coagulating glands, vagina, urinary bladder.

Other examinations:

The functional observation battery was performed on open field arena and the duration was 5 minutes. The parameters evaluated was CNS (Central Nervous System) excitation such as tremors, clonic and tonic convulsions and sterotypic behavior; and autonomic responses such as lacrimation, exophthalmia and piloerection. Additionally, they are also evaluated for spontaneous activity (hypoactivity or hyperactivity), affective response (freezing, grooming, defecation and urination), sensorial response (tail-pinch response), gait and posture, and righting reflex (raise the animals to 30 and 40 cm above the floor of the arena and back flip the rat in the air).

The above-mentioned parameters were measured as follows:
- Qualitative, presence or absence: CNS excitation and autonomic responses;
- Qualitative, normal or altered: gait and posture;
- Quantitative, time duration: freezing, grooming, tail-pinch response and righting reflex (time of postural straightening after free-fall);
- Quantitative, frequency: the spontaneous activity was based on the number of times the test system had moved, got up and went through the center of the arena. The open field consists of a circular arena divided into 19 parts, due to each time the test system stepped with the 4 paws inside a part was considered one locomotion;
- Quantitative, distance: righting reflex (measurement of plantar impression after free fall);
- Quantitative, quantity: defecation and urination.

Statistics:

GraphPad Prism 7 evaluated all parameters according to described below:
- Two Way Variance (ANOVA) and post-test of Bonferroni’s: body weight, feed consumption, functional observation battery (activity and autonomic response);
- T Parametric Test: clinical pathology parameters (CBC, WBC, coagulation and biochemistry) and functional observation battery (grooming, freezing and tail-pinch).
All tests was conducted of significance level was 5%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the 28-day exposure period, the following animal of control group showed abnormalities:
- one male: discretely nasal porphyria;
- one male: presented moderate pulmonary wheezing for 2 days;
- one female: presented pulmonary wheezing on the first 10 days of the study. However, absence of nasal and ocular secretions, without sialorrhea and with some intermittent sneezing. Some days presented salivation during the administration period;
- one female: presented pulmonary wheezing on the first 10 days of the study and salivation;
- one female salivated once in the last week of the study.

At the dose of 1000 mg/kg b.w., 5 of 6 females and all males presented periodical salivation, the one male presented alopecia on the first two weeks of the study and one male has a nasal porphyria for one day.

In the satellite control group one female presented pulmonary wheezing, punctually, twice for the first 10 days of the study and absence of nasal and ocular secretions, without sialorrhea.No abnormalities were observed in males of the control satellite group.
Salivation was observed in 5 of 6 females and all males of treated satellite group during the administration of test substance. The reversal occurred as soon as the administration of the test substance was completed, since for the next 10 days none of the animals, both males and females, presented no alteration.

Only salivation can probably be attributed to test item effects, however considering its nature, it is not considered as adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in control group died at the beginning of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistical difference was observed in treated males in comparison to the control group.
In treated females of regular group statistical difference (body weight decrease) was observed on days 21 and 28 of the study.
The females of satellite groups were evaluated until 46th day, and there was no statistical difference between the control and treated groups.

Body weight changes in females might have been related to treatment, however are not considered adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences were observed in treated males compared to the control group in the following parameters: erythrocytes (decrease), platelets (increase), HGB (decrease), VCM (increase).
Statistically significant differences were observed in treated females compared to the control group in the following parameters: HGB (increase), VCM (increase), HCM (increase).
After comparing the results to standard norms found in literature, it turned out the values of the control groups were slightly outside the range, while the values for treated group were within the norm.

No differences were observed in the parameters revelant for white blood cells system and clotting parameters between the treated and control animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences were found in treated males compared to the control with regards to the following parameters: glucose (decrease), cholesterol (increase), urea (decrease), sodium (increase), albumin (increase).
Statistically significant differences were found in treated females compared to the control with regards to the following parameters: cholesterol (increase), urea (increase), creatinine (increase), calcium (increase), phosphorus (increase), potassium (increase), ALT (increase), protein (increase), albumin (increase).

In the satellite groups, after the observation period, the following parameters were statistically significantly different in treated males compared with the control: sodium (increase), albumin (increase). No change was observed in the level of glucose, cholesterol and urea.
In the satellite groups, after the observation period, the following parameters were statistically significantly different in treated females compared with the control: creatinine (decrease). No change was observed in the level of cholesterol, urea, albumin, calcium, phosphorus, potassium, ALT and protein.

The statistically significant values were compared with normality values found in literature. The protein values were within the normal range. Only the values of albumin, glucose and sodium in the control group were outside the normal range. Therefore, for these three parameters, it was considered that the results were not of biological relevance.
For the cholesterol, creatinine, calcium, phosphorous parameters the values of the treated group were outside the range of normality. The values of urea, potassium and ALT were outside of the range for both groups, control and treated.

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statically significant change in treated males compared with the control was the decrease of the relative weight of brain.
In treated females compared to the control, statistically significant changes in relative weight of the following organs were observed: liver (increase), adrenals (increase), heart (decrease), thyroid/parathyroid (increase), spleen (decrease).

No statistically significant differences were found in satellite animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

During the animal necropsy the macroscopic evaluation showed the following alterations in males:
- Liver with lattice pattern (accentuated or moderate): in all animals of control group and 2 of 6 of treated group . 2 males of control group and 1 male of treated group, besides the already mentioned change, also presented hepatic lobe with well defined red / wine focal area. One male of treated group presented left cranial lobe with firm nodulation, reddish red.
- Kidneys: 2 of 6 animals of control group and 3 of 6 animals of treated group presented dilatation of the pelvis (hydronephrosis). Additionally, 1 of 6 animals of control group and 1 of 6 animals of treated group presented congestion in the medullar cut region.

In the satellite control group, 5 of 6 males and 2 of 6 females presented liver with lattice pattern evidenced accentuated. 3 of 6 males and all females presented alterations on kidneys. In satellite treated group, all males and all females presented liver with lattice pattern. 4 of 6 males and 2 of 4 females presented alterations on kidneys.

In females the following alterations were found:
- Liver with lattice pattern evidenced accentuated: 2 of 6 animals of control group and 2 of 6 animals in the treated group.
- Kidneys: 1 of 6 animals of control group and 1 of 6 animals of treated group presented dilatation of the pelvis. Additionally, 1 to 6 animals of control group presented congestion in the medullar cut region.

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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:

clinical biochemistry

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available