4-allylveratrole

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-allylveratrole

EC Number:

202-223-0

EC Name:

4-allylveratrole

Cas Number:

93-15-2

Molecular formula:

C11H14O2

IUPAC Name:

1,2-dimethoxy-4-(prop-2-en-1-yl)benzene

Test material form:

liquid

Specific details on test material used for the study:

Name of Test Item : Methyl Eugenol
Chemical Name (IUPAC) : 4-allyl-1,2-dimethoxybenzene
Physical Appearance (with color) : Pale yellow liquid
Batch No. : 40003010419
CAS No. : 93-15-2
Purity (as per Certificate of Analysis) : 99.24%
Batch Produced by
(Name and address) : Yasho Industries Limited, Plot no. 2514/2515,
Phase IV, G.I.D.C., Vapi - 396195, Gujarat,
India
Date of Manufacture : Feb 2019
Date of Expiry : Jan 2021
Storage Conditions : Ambient (21 to 29 oC)
Test Item Code by Test Facility : D819-001

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum 10 to 11 weeks
- Weight at study initiation: (P) Males: 241 and 298 g; Females:180 and 222 g
- Fasting period before study: No
- Housing: in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill
i. Pre mating
Per cage two animals of the same sex and group was housed.
ii. Mating
During mating, two animals (one male and one female) of the same group was housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear and/or vaginal plugs (Day 0 of pregnancy),
the mated pairs were separated. Males were housed with their former cage mates while females were
housed individually. Sterilized paper shreds will be provided as a nesting material from gestation day 20 onwards.
Maximum of three animals of same sex will be housed for recovery group animals.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum throughout the study
- Water (e.g. ad libitum): ad libitum, Deep bore-well water passed through reverse osmosis unit was provided.
- Acclimation period: 4 - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day.
The required quantity of test item was weighed into a clean glass beaker and there by adding little v
olume of vehicle into the beaker, mixed well with glass rod and transferred into the measuring cylinder
and the beaker was rinsed using a small volume of vehicle and the volume was transferred to me
asuring cylinder. The rinsing procedure was repeated until entire quantity of the test item formulation
was transferred into a measuring cylinder. Finally, the volume was made up to the required quantity
with vehicle to get a desired concentration of 5, 10 and 30 mg/mL of test item for low, mid and high d
ose groups respectively as mentioned in the table below.
The test formulations will be maintained under stirring conditions using magnetic stirrer to maintain ho
mogeneity of the test item formulations.
Group Group Description Dose(mg/kg body weight) Conc.(mg/mL) Dose Volume (mL/kg body
weight) Quantity of Test Item (mg) Volume made up with Vehicle (mL)
G1/G1R Vehicle Control 0 0 10 - 50
G2 Low dose 50 5 10 250 50
G3 Mid dose 100 10 10 500 50
G4/G4R High Dose 300 30 10 1500 10
VEHICLE
- Amount of vehicle (if gavage): 10ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical
Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods
detailed in the Study Plan No. BIO-ANM 1382 and the results were presented in the report. Sampling
and analysis of formulations were performed during week 1 and week 4/5 of the treatment. The sa
mples were collected in duplicates from top, middle and bottom layers from low, mid and high dose c
oncentrations and in duplicates from single layer from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Privat
e Limited for dose formulation analysis. One set of aliquot of each formulation was analyzed. The s
econd aliquot was stored as a backup purpose at established stability conditions. Formulations were
considered acceptable, if mean results are within the range of 85 to 115% of the nominal concentra
tion and the relative standard deviation (% RSD) was ≤10%.
If the dose formulation analysis results failed to meet these criteria, the analysis was repeated using
second set of samples or samples of subsequent preparation during the treatment period.

Duration of treatment / exposure:

Males: The males will be treated for two weeks pre-mating, during mating and up to the day before sa
crifice during the post-mating period (total of at least completion of 28 days of treatment).
Females: The females will be treated for a two week pre-mating period, during mating, pregnancy
(gestation) and up to lactation day 13 after which the pups will be sacrificed on lactation day 13.
Females (dams) will be sacrificed on lactation day 14 after overnight fasting (water allowed).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Main groups:
12 males and 12 females per dose
Recovery Groups
5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the test item Methyl Eugenol administered to rats in a repeated dose 90-
Day oral toxicity in rodents induced erythrocyte microcytosis in 300 mg/kg males and 1000 mg/kg
males and females, and thrombocytosis in 100, 300 and 1000 mg/kg; caused an increase in serum
alanine aminotransferase and sorbitol dehydrogenase activities in 100, 300 and 1000 mg/kg and
bile acid concentration in 300 and 1000 mg/kg; induced hypoproteinemia and hypoalbuminemia evi
denced by decreased total protein and albumin concentrations in the 300 and 1000 mg/kg groups; live
r weights of 100, 300, and 1000 mg/kg males and 300 and 1000 mg/kg females and testis weights of
1000 mg/kg males were significantly increased; increased incidences of liver lesions occurred in 300
and 1000 mg/kg males and females and hepatocellular adenoma occurred in one 1000 mg/kg male.
The incidences of atrophy and chronic inflammation of the mucosa of the glandular stomach were s
ignificantly increased in rats administered 300 and 1000 mg/kg, incidences of cortical hypertrophy of
the adrenal cortex were significantly increased in 100, 300, and 1000 mg/kg males and 1000 mg/kg
females and incidences of cytoplasmic alteration of the submandibular salivary glands were increas
ed in rats administered 30, 100 and 1000 mg/kg.
Based on the above information, the doses of 0, 10, 30 and 100 mg/kg body weight were selected
as control, low, mid and high dose groups for dose range finding study [BIO-CTX 067]. The test ite
m Methyl Eugenol did not reveal any treatment related effects at all the tested doses [10, 30 and 100
mg/kg body weight/day] when administered for a period of 14 consecutive days by oral (gavage) to
Sprague Dawley rats.
Based on the results obtained from dose range finding study [BIO-CTX 067], there were no treatment
related effects noted up to 100 mg/kg body weight in a 14 day exposure period in both males and
females. However, the main group males will be dosed for a period of at least 28 days, main group
females and recovery group animals will be dosed approximately for a period of 50 days. Hence, the
doses of 0, 50, 100 and 300 mg/kg body weight has been selected as control, low, mid and high dose
groups for the current definitive study.

Positive control:

Not needed

Examinations

Observations and examinations performed and frequency:

Clinical Signs of Toxicity and Mortality/Morbidity
At least once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

Detailed Clinical Examination
On day 1 before treatment and weekly thereafter (may vary by ± 1 or 2 days) during treatment. Signs
noted included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of
secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and
unusual respiratory pattern.

Body Weight
At receipt, on the first day of dosing, at least weekly thereafter and at termination. The females were
weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during the
lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, at
least weekly thereafter and at termination.

Feed Consumption
Cage wise feed consumption was measured for main group animals once a week during premating
and weekly once for main group males during the post mating period. Feed consumption was not
measured during the mating period for both males and females. Thereafter, feed consumption for
females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4,
4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals once a week throu
ghout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amo
unt of feed offered and left over in each cage and the number of rats per cage.

Ophthalmological Examination
Once before treatment for all animals, at the end of the dosing period for males (Treatment Day 34)
and during the lactation period for females (Lactation Day 13) of vehicle control and high dose main
group animals and during the last week for the recovery group animals (Day 64). The examination
was not extended to lower dose groups as there were no changes noted in high dose group animals.

Estrous Cyclicity
Estrous cycles were monitored during the acclimatization to evaluate its normal estrous cyclicity (4 to
5 days). Only females with normal oestrous cyclicity were selected for the treatment. Vaginal smears
were monitored daily from the beginning of the treatment period until evidence of mating. When obt
aining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseu
dopregnancy. Estrous cyclicity was also monitored on the day of sacrifice for females.
Recovery group females were not evaluated for oestrous cyclicity.

Neurological/Functional Examination
Performed for five males and five females, randomly selected from each group, towards the end of
the dosing period for males (day 35) and during the lactation period for females (lactation day 13).
Neurological/Functional examination was performed for both recovery group animals towards the end
of the recovery period (day 63).

Clinical Pathology
Blood samples were collected from all the main group animals for measurement of serum T4 levels
on the following schedule:
a. Two pups per litter on lactation day 4
b. All dams at termination (lactation day 14)
c. Two pups per litter (same sex) at termination (lactation day 13)
d. All adult males, at termination (after completion of at least 28 days of treatment).
The clinical pathology (haematological and clinical biochemistry) examinations was conducted in
five males and five females randomly selected from each main group and from all recovery group
animals.
Urine were collected from five randomly selected males of each main group and for all recovery ani
mals at termination.

Necropsy
The males were sacrificed after completion of at least 35 days of treatment, females were sacrificed
on lactation day 14 and recovery animals were sacrificed at least after completion of 14 days observ
ation from the first scheduled sacrifice of dams. The organs were collected and Relative organ wei
ghts were calculated against fasting body weight.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control and
high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads
and histopathology of interstitial testicular cell structure) including all macroscopically abnormal tissu
es of all animals, whether died during the experiment or sacrificed at termination.
All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of
3 to 4 micrometers and stained with hematoxylin and eosin. The testes was sectioned at 3 to 4
micrometers and stained with Hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS)
and hematoxylin stain. PAS stain aided spermatogenesis evaluation
Bone marrow smear (from one femur) was prepared at the time of necropsy. The bone marrow smear
was fixed in methanol and stained with Giemsa Stain.

Oestrous cyclicity
Estrous cycles was monitored during the acclimatization to evaluate its normal estrous cyclicity (4 to 5
days). Only females with normal estrous cyclicity was selected for the treatment. Vaginal smears was
monitored daily from the beginning of the treatment period until evidence of mating. When obtaining
vaginal/cervical cells, care will be taken to avoid disturbance of mucosa, which may induce pseudopr
egnancy. Estrous cyclicity was also be monitored on the day of sacrifice for females.

Sperm parameters
The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and eosin stain and
also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain will aid spermatogenesis
evaluation.

Sacrifice and pathology:

SACRIFICE
The animals were euthanized using deep Isoflurane anaesthesia/CO2 followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination. The males were sacrificed after completion of at least 28 days of treatment. The females were sacrificed on lactation day 14 and recovery animals were sacrificed at least after completion of 14 days observation from the first scheduled sacrifice of dams. The pups were sacrificed on lactation day 13

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy for all selected animals:
Testes
Epididymides
Levator ani plus bulbocavernosus muscle complex
Cowper’s glands
Glans penis
Eye
Liver
Kidneys
Adrenals
Thymus
Spleen
Brain (cerebrum, cerebellum and pons)
Heart
Ovaries
Prostate
Seminal vesicles with coagulation glands
Organs showing macroscopic lesions
All gross lesions
Spinal cord
Stomach
Duodenum
Jejunum
Ileum with Peyer’s patches
Caecum
Colon
Rectum
Thyroid along with parathyroidW
Trachea
Lungsa
Uterus with cervix
Urinary bladder
Mandibular Lymph nodes
Mesenteric Lymph nodes
Sciatic nerve
Bone marrow smear
Skeletal muscle
Femur bone
Vagina

HISTOPATHOLOGY
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) including all macroscopically abnormal tissues of all animals, whether died during the experiment or sacrificed at termination.

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons will be evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests and will be designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
The statistical analysis was followed, but not limited to the parameters, as mentioned below
Parameter Type of Analysis
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent Change in body weight (weekly body weights and gestation/lactation body weights)
• Feed consumption (weekly and gestation/lactation)
• Copulatory interval
• Gestation length
• Hematology
• Clinical chemistry
• Urinalysis (whichever applicable)
• Absolute/relative organ weights
• Functional Observation parameters (whichever applicable)
• Anogenital distance ratio
• Nipple retention (if any)
• Mean pup weight
• Serum T4 values Parametric - One-way ANOVA with Dunnett’s post test

• Implantations/dam
• No. of pups/dam
• Sex ratio
• Litter size
• No. of early resorptions
• No. of late resorptions
• Pups abnormalities (if any)
• Pre implantation loss
• Pre natal loss
• Post natal loss
• Post implantation loss Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated

• Pregnancy rate
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions Cross Tabs -Chi-square test/ Fischer's Exact Test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity at any of the tested and vehicle control group animals of either sex in both main and recovery groups throughout the experimental period.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity observed at any of the tested and vehicle control group animals of either sex in both main and recovery groups throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item related changes noted in mean body weight and percent change in mean body weight with respect to day 1 during experimental period at any of the tested dose group animals of either sex in both main and recovery groups throughout the experimental period when compared with
concurrent vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption during pre-mating and post-mating trea
tment period at any of the tested dose group males (main group) when compared with concurrent
vehicle control group. The feed consumption was not measured for main group males during cohabi
tation period.
There were no changes noted in mean feed consumption during pre-mating treatment period at any
of the tested dose group females (main group) when compared with concurrent vehicle control group.
The feed consumption was not measured for main group females during cohabitation period.
There were no changes noted in mean feed consumption during experimental period (both treatment
and recovery periods) at the high dose recovery group animals of either sex when compared with
concurrent vehicle control group.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in vehicle control (G1) and high dose (G4) group animals
of either sex during the ophthalmological examination conducted towards end of the dosing period.
Hence, the ophthalmological examination was not conducted for mid and low dose groups.
There were no ocular changes observed in vehicle control recovery (G1R) and high dose recovery
(G4R) group animals of either sex during the ophthalmological examination conducted at the end of
recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item related changes observed in haematology parameters at any of the tested
dose group animals of either sex in both main and recovery groups when compared with their conc
urrent control groups.
However, the following statistically significant changes were noted in haematology parameters when
compared with their concurrent control groups:
- increase in percent basophils and absolute monocytes at group G4R males;
- decrease in activated prothrombin time (APTT) at group G4R males;
- increase in HGB, HCT, MCV, PT and APTT at group G4R females;
These changes are considered as incidental and not treatment related, due to lack of dose
dependency and similar changes were not observed in other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item related changes observed in clinical chemistry parameters at any of the tested
dose group animals of either sex in both main and recovery groups when compared with their concur
rent control groups.
However, the following statistically significant changes were noted in clinical chemistry parameters wh
en compared with their concurrent control groups:
- increase in calcium and phosphorous levels at group G3 and G4 males;
- increase in cholesterol levels at group G4 males;
- decrease in glucose and total bilirubin levels at group G3 males;
- increase in sodium and potassium levels at group G4R males;
- decrease in total bilirubin levels at group G4R males;
- increase in globulin and potassium levels at group G4R females
These changes are considered as incidental and not treatment related, due to lack of dose
dependency and similar changes were not observed in other sex.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes observed in urinalysis parameters at any of the tested dose main group
males and high dose recovery group animals of either sex conducted at termination when compared
with their concurrent control groups. The urinalysis was not conducted for the main group females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular (hind limb foot splay), physiological observations, assessment of grip strength and assessment of motor activity at all the tested dose main groups (per
formed for five males and five females, randomly selected from each main group, towards the end of the dosing period) scheduled sacrifice) and recovery groups (performed for both recovery group animals towards the end of the recovery period when compared with concurrent vehicle control group animals.
However, statistically significant reduction in mean number of urination during open field examination at group G2, G3 and G4 males, statistically significant increase in length of hind limb foot splay at group G2 males, statistically significant reduction in hind limb grip strength at group G4R males, statistically significant increase in fore limb grip strength at group G4 females and statistically significant reduction in hind limb grip strength at group G2 females were noted when compared with concurrent vehicle control groups. These changes are considered as incidental but not treatment related
due to lack of dose dependency and no effects were noted in other functional observation battery parameters at these dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

TThere were no treatment related changes observed in the absolute and relative organ weights at
any of the tested dose group animals of either sex in both main and recovery group animals.
However, statistically significant increase in absolute and relative liver weight at group G4 females
; statistically significant increase in absolute testes weight and relative heart weight at group G4R
males; and statistically significant decrease in absolute adrenals weight at group G4R females were
noted when compared with concurrent vehicle control group. These changes are considered can be
considered as test item related as the previous 90-Day oral toxicity in rodents denoted with increased
liver weights at 300 and 1000 mg/kg. However, there were no gross or histopathological changes
were noted in liver at this dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All the adult animals from main and recovery groups and all the pups which were found dead at birth
and sacrificed at termination (on post-natal day 4 for blood collection and on post-natal day 13 for
terminal scheduled sacrifice) were subjected to gross pathological examination. There were no gross
pathological changes observed during necropsy in all of the adult animals and pups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no treatment related histopathological findings noticed in group G4 males and females.
The noted lesions in both vehicle control and high dose group males and females are considered as spontaneous and incidental. These lesions consisted of interstitial mononuclear cell infiltrate unilateral minimal (1 male from group G4), Ultimobranchial cyst (1 female from group G1), Ectopic Tissue Thymus in thyroid (1 female from group G1), Accessory cortical tissue in adrenals (1 female from group G4), Interstitium, Mononuclear cells infiltrate of prostate gland (1 male with minimal and 1 male with slight from group G1 and G4) and Concretions of prostate gland (1 male each from group G1 and G4).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related histopathological findings noticed in group G4 males and females.
The noted lesions in both vehicle control and high dose group males and females are considered as spontaneous and incidental. These lesions consisted of interstitial mononuclear cell infiltrate unilateral minimal (1 male from group G4), Ultimobranchial cyst (1 female from group G1), Ectopic Tissue Thymus in thyroid (1 female from group G1), Accessory cortical tissue in adrenals (1 female from group G4), Interstitium, Mononuclear cells infiltrate of prostate gland (1 male with minimal and 1 male with slight from group G1 and G4) and Concretions of prostate gland (1 male each from group G1 and G4).

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects observed at the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Methyl Eugenol were established:
Systemic NOAEL: 300 mg/kg bw
Reproduction NOAEL: 300 mg/kg bw
Developmental NOAEL: 300 mg/kg bw

Executive summary:

The test item, Methyl Eugenol was evaluated for possible adverse effects following repeated oral administration to the main group males for a period of 35 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period 50 days with a 14 days recovery to evaluate systemic toxicity end points, effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle [0.5% w/v Carboxy Methyl Cellulose], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 50, 100 and 300 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability of test item formulations in 0.5% w/v Carboxy Methyl Cellulose was established before initiation of the treatment at the concentrations of 5 mg/mL and 30 mg/mL for 6 hours at room temperature. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main and recovery group animals were observed once daily for clinical signs, twice daily for mortality and morbidity, weekly once for detailed clinical examination. The body weights and feed consumption (except during cohabitation for main group animals) was recorded once weekly for all theanimals. Ophthalmological examination was carried out once before treatment for all animals, towardsend of the dosing period for group G1 and G4 animals and towards the end of recovery period for groupG1R and G4R animals. Neurological/functional examination was performed for five randomly selectedanimals from each main group per sex towards the end of the dosing period and for all the recoverygroup animals towards the end of the recovery period. The clinical pathological parameters such as haematology, clinical chemistry and urinalysis (except for main group females) were conducted for fiverandomly selected animals from each main group per sex and from all the recovery group animals of both the sex at termination. The serum thyroxine hormone (T4) levels were estimated for all the main group males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.

All the main group males and females were evaluated for its mating and fertility index and all the maingroup litters were evaluated for its gestation and parturition index.All the main group females were evaluated for oestrus cyclicity during pre-mating / cohabitation period. The pre-coital interval (copulatory interval) was calculated for each litter. The body weight and feed consumption was recorded for all the females during gestation and lactation periods till day 14 after parturition. The gestation length, number of live pups born, sex ratio, live birth index and pup survival index per litter was observed for all the females from each main group. The total number of implantations per litter was noted during necropsy and the pre-/post-natal losses per litter was calculated. The pups were observed once daily for clinical signs and external examinations till termination, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for ano-genital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method.

 

Systemic toxicity

There were no clinical signs of toxicity and no mortality/morbidity were noted during the experimental period all the tested dose main and recovery group animals of both the sex. There were no effects noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all the tested dose group animals of both the sex. There were no ophthalmological changes noted in G4/ G4R group animals and no changes noted in neurological/functional examination at all the tested dose group animals of both the sex. There was no treatment related changes noted in absolute or relative organ weights at all the tested dose group animals of both the sex. The clinical pathology end points such as haematology, clinical chemistry and urinalysis did not reveal any test item related changes. The serum thyroxine (T4) hormone level estimation in main group males did not reveal any changes at all the tested dose groups. There were no macroscopic changes noted during necropsy at all the tested dose group animals of both the sex and no microscopic changes noted during histopathological examination conducted for group G4 animals of both the sex.

 

Reproduction toxicity

For reproduction toxicity end points, there were no test item related effects noted in mating and fertility index of both main group males and females in any of the tested dose groups. There was no test item related effects were noted in gestation and parturition index of litters from any of the tested dose groups. For maternal toxicity end points, there were no irregularities in oestrus cyclicity, no effects on copulatory interval and no test item related changes in mean body weight, percent change in mean body weight gain and mean feed consumption during gestation and lactation periods noted at any of the tested dose group females. There were no changes observed in the gestation length, number of live pups born, sex ratio, live birth index and pup survival index at any of the tested dose group litters. There were no changes noted in number of implantations per litter and no test item related pre-/post-natal losses were noted in any of the tested dose group litters.

 

Developmental toxicity

For developmental toxicity end points, there were no clinical signs and no test item related mortalities noted in any of the tested dose group pups. There was no test item related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in both the sex at all the tested dose group litters. There were no occurrences of nipples examined in male pups on post-natal day 13 at any of the tested dose group litters. The serum thyroxine (T4) hormone level estimation in post-natal day 13 pups from each litter did not reveal any changes at all the tested dose groups. There were no gross pathological changes noted during scheduled sacrifice of each pup from all the tested dose group litters.