Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2019 - 30 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
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

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

IN-LIFE DATES:
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on 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 volume 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 measuring 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 dose groups respectively as mentioned in the table below.
The test formulations will be maintained under stirring conditions using magnetic stirrer to maintain homogeneity 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

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- If no evidence of copulation is obtained after 14 days, the animals will be separated, re-mating of females with proven males of the same group was tried for an additional week.
- After successful mating each pregnant female was caged (how): housed individually in a plastic cage
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 samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquot of each formulation was analyzed. The second 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 concentration 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 sacrifice 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
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Remarks:

actual ingested
Dose / conc.:
100 mg/kg bw/day
Remarks:

actual ingested
Dose / conc.:
300 mg/kg bw/day
Remarks:

actual ingested
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 evidenced by decreased total protein and albumin concentrations in the 300 and 1000 mg/kg groups; liver 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 significantly 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 increased 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 item 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
Parental animals: Observations and examinations:
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 throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount 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 obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. 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 animals 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 observation from the first scheduled sacrifice of dams. The organs were collected and Relative organ weights 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 tissues 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 (parental animals):
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 pseudopregnancy. Estrous cyclicity was also be monitored on the day of sacrifice for females.
Sperm parameters (parental animals):
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.
Litter observations:
PARAMETERS EXAMINED
The day of littering was considered as lactation day 1.
The number of pups born (dead and live) in a litter, sex and external examination observations were recorded at birth.
Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded.
The ano-genital distance of each pup was measured on postnatal day 4 (lactation day 4).
The number of nipples/areolae in male pups were counted on postnatal day 13 (lactation day 13).
Fertility index for dams, sires and pup survival index and sex ratio at birth were calculated.

GROSS EXAMINATION OF DEAD PUPS:
The pups was sacrificed on lactation day 13 and the sacrificed pups and dead pups (if any) were examined for gross abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):
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


Postmortem examinations (offspring):
SACRIFICE
The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups (if any) were examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of the organs specified above

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table above were prepared for microscopic examination and weighed, respectively.
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 in the table.

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

Reproductive indices:
Mating (%): Number of females mated x 100
Number of females paired

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females x 100
Number of females mated

Gestation index (%): Number of females with living pups on Day 1 x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born x 100
Total number of uterine implantation sites

Lactation index (%): Number of live offspring on Day 13 after littering x 100
Offspring viability indices:
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100
Number of live pups at First Litter Check

Viability index (%): Number of live offspring on Day 4 before culling x 100
Number live offspring on Day 1 after littering

Lactation index (%): Number of live offspring on Day 13 after littering x 100
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 treatment 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 cohabitation 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 concurrent 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 concurrent control groups.

However, the following statistically significant changes were noted in clinical chemistry parameters when 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 (performed 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
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
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no irregularities observed in the oestrus cyclicity of females at any of the tested dose and vehicle control main group females during pre-mating and mating treatment periods.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no changes observed in the copulatory interval and number of conceiving days at any of the tested dose main group females when compared with vehicle control group females.
Gestation Body Weight and Percent Change in Gestation Body Weight
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at any of the tested dose group females when compared with vehicle control group females.

Feed Consumption during Gestation Period
There was no test item related changes observed in the mean gestation feed consumption at any of the tested dose group females when compared with vehicle control group females. However, statistically significant reduction in mean gestation feed consumption at all the tested dose group females was noted during GD 14 to 20 when compared with vehicle control group females. This observation can be considered as incidental and unrelated to treatment due to lack of consistency and also there were no changes noted in mean body weight and percent change in body weight during this period.

Lactation Body Weight and Percent Change in Lactation Body Weight
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at any of the tested dose group females when compared with vehicle control group females.

Feed Consumption during Lactation Period
There was no changes observed in the mean lactation feed consumption at any of the tested dose group females when compared with vehicle control group females.

Gestation Length and Delivery Data
There were no changes observed in the gestation length, number of pups delivered, sex ratio and live birth index at any of the tested dose group litters when compared with vehicle control group litters.

Litter observation during Lactation Period
There were no effects observed in litter observation parameters [number of live pups, sex ratio and pup survival index] at any of the tested dose group litters when compared with vehicle control group litters during lactation period.

Uteri Observations
There were no changes observed in mean number of implantations per litter and no test item related resorptions and pre/post-natal loss per litter at all the tested dose group litters when compared with vehicle control group litters.

Serum Thyroxine (T4) Levels - Adult Males
There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males when compared with vehicle control group males.

Pup observation during Lactation Period
There were no clinical signs and mortalities were noted during daily observation of pups at all the tested and vehicle control group litters during lactation period.

Pup Weight
There was no treatment related changes observed in mean pup [both male and female] weight per litter during post-natal period at all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean male pup weight on post-natal day 1 at group G2 and G3; statistically significant increase in mean male pup weight on post-natal day 4 at group G2 and G4; statistically significant increase in mean female pup weight on post-natal day 1 at group G2 and G3 were noted when compared with vehicle control. These changes are considered incidental and not treatment related as the obtained weights are within historical control data and also the changes are not dose dependant.

Ano-genital Distance of Pups
There was no treatment related changes observed in mean pup [both male and female] ano-genital distance ratio per litter measured on post-natal day 4 at all the tested dose groups when compared with vehicle control group.

Nipple Retention in Male Pups on Lactation Day 13
There were no occurrences of nipples examined in male pups on post-natal day 13 at any of the tested dose group litters and vehicle control group litters.

Serum Thyroxine (T4) Levels - PND 13 Pups
There was no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group post-natal day 13 pups.


Key result
Dose descriptor:
NOAEL
Remarks:
(Systemic)
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level tested
Key result
Dose descriptor:
NOAEL
Remarks:
(Reproduction)
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level tested
Key result
Dose descriptor:
NOAEL
Remarks:
Development
Effect level:
> 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest level tested
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Clinical signs
The nature and incidence of the clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered not to affected by treatment and therefore of no toxicological relevance.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Body weights
Body weights of pups were not considered to be affected by treatment.

Sex ratio
Sex ratio was not considered to be affected by treatment.

Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Areola/nipple retention
Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Clinical biochemistry (T4 levels)
Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.

Macroscopy
No macroscopic findings were noted among pups that were considered to be related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
(Development)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose tested
Reproductive effects observed:
no
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 recoveryto 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 the animals. Ophthalmological examination was carried out once before treatment for all animals, towards end of the dosing period for group G1 and G4 animals and towards the end of recovery period for group G1R and G4R animals. Neurological/functional examination was performed for five randomly selected animals from each main group per sex towards the end of the dosing period and for all the recovery group 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 five randomly 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 main group 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification