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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 January 2019 – 21 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 January 2019 – 21 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (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 were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of 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.
Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.
Duration of treatment / exposure:
Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.
Frequency of treatment:
Once a day
Details on study schedule:
F1 animals were not mated.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
G3 - Mid dose
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose + G4R - High dose Recovery Group
No. of animals per sex per dose:
Main Group: 12
Recovery Group: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) 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 main group 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 (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment).
Oestrous cyclicity (parental animals):
Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.
Sperm parameters (parental animals):
Parameters examined in all parental male in the control and high dose groups: testis and epididymis weight. Also, a detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.
Also, blood samples were collected for measurement of serum T4 levels on the following schedule:
1. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
2. Two pups per litter (same sex) at termination (lactation day 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of 36 days of treatment.
- Maternal animals: All surviving animals on lactation day 14
- Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on lactation day 4 (those pups selected for blood collection) and on lactation day 13 (the rest of pups)
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.
Reproductive indices:
See table 2 below.
Offspring viability indices:
See table 2 below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.

There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and 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):
The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. 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):
Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. 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 tested dose group recovery group animals of either sex conducted at termination.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is 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:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males the assessment of T4 in blood samples from the dams was not performed.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects were detected during examination of spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no changes observed in the copulatory interval and number of conceiving days at groups G2 and G3 when compared with vehicle control group. However, an increased copulatory interval was noted at group G4 when compared with vehicle control group. This change is considered as treatment related due to occurrence of treatment related clinical signs and reduced mean body weights at this dose group during the treatment period.

There were no changes observed in the gestation length, litter delivered, number of pups delivered, sex ratio, live birth index at groups G2 and G3 when compared with vehicle control group. However, a treatment related statistically significant reduction in mean gestation length at group G4 was noted when compared with vehicle control group. This observation is considered as treatment related due to observed clinical signs during the treatment period at this dose level.
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental general (systemic) toxicity
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Increased copulatory interval and gestation length observed at high dose tested (600 mg/kg bw/day).
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental general toxicity and sexual function and fertility
Remarks on result:
other: No adverse effects were found at any dose tested.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no changes noted in daily observation of pups at all the tested and vehicle control groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no mortalities of pups during the 13-day lactation period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in mean pup weight of either sex during lactation period at all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean male and female pup weight on lactation day 4 at groups G2 and G4; statistically significant increase in mean female pup weight on LD 7 at group G4 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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the pups sacrificed.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum Thyroxine (T4) Levels: There were no effects found in serum Thyroxine (T4) hormone levels noted at lactation day 13 pups. As no effects were noted in lactation day 13 pups the assessment of T4 in blood samples from the day 4 pups was not performed.
Anogenital Distance of Pups on Lactation Day 4: There were no treatment related changes observed in mean pup ano-genital distance ratio of either sex during measured on LD 4 at all the tested dose groups when compared with vehicle control group. However, statistically significant decrease in mean male and female mean pup ano-genital distance ratio at groups G3 and G4 was noted when compared with vehicle control. These changes cannot be considered as treatment related effects as the obtained values are within historical control range and also no treatment related effects noted in mean pup weight on the day of measurement of ano-genital distance.
Nipple Retention in Male Pups on Lactation Day 13: There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Development of the offspring
Remarks on result:
other: No treatment related effects were observed at the highest dose tested (600 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Development of the offspring
Remarks on result:
other: No adverse effects were observed at any dose level tested.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 4. Summary of vaginal smear examination for determination of oestrus cyclicity

Group & Dose
(mg/kg body weight/day)

Total No. of Females

No. of Females with Regular Oestrus Cyclicity during

Pre-mating and Mating

No. of Females confirmed with pregnancy

 

No. of Females with Regular Oestrus Cyclicity on Lactation Day 14

No. of Females with Irregular Oestrus Cyclicity during

Pre-mating, Mating and on Lactation day 14

G1 & 0

12

12

10

10

0

G2 & 100

12

12

11

11

0

G3 & 300

12

12

10

10

0

G4 & 600

12

12

10

10

0

Table 5. Summary of cohabitation record

Group & Dose
(mg/kg body weight)

Copulatory Interval

(Days)

 

Conceiving Days

(1 to 5)

Conceiving Days

(6 to 14)

 

G1 & 0

Mean

8.17

n

5

7

±SD

6.37

n

12

%

41.7

58.3

 

G2 & 100

Mean

8.50

n

4

8

±SD

5.96

n

12

%

33.3

66.7

 

G3 & 300

Mean

8.58

n

5

7

±SD

6.14

n

12

%

41.7

58.3

 

G4 & 600

Mean

10.42

n

3

9

±SD

5.81

n

12

%

25.0

75.0

Table 6. Summary of gestation length and delivery data record

Group & Dose
(mg/kg body weight)

Gestation Length (Days)

Litter Size

(No.)

Live Pups (No.)

Dead Pups (No.)

Sex Ratio (M/F) at Birth

Live Birth Index
(%)

Total (No.)

Male

(No.)

Female

(No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

22.20

11.60

11.60

4.50

7.10

0.00

0.00

0.00

0.83

100.00

±SD

0.42

3.31

3.31

2.76

2.64

0.00

0.00

0.00

0.91

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

22.73

10.82

10.82

6.09

4.73

0.00

0.00

0.00

1.55

100.00

±SD

0.65

2.52

2.52

3.18

1.68

0.00

0.00

0.00

1.10

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

22.40

12.00

12.00

6.10

5.90

0.00

0.00

0.00

1.33

100.00

±SD

0.52

2.31

2.31

2.47

2.33

0.00

0.00

0.00

1.06

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

23.40*

10.10

10.10

5.70

4.40

0.00

0.00

0.00

1.47

100.00

±SD

0.70

2.02

2.02

1.77

1.35

0.00

0.00

0.00

0.83

0.00

n

10

10

10

10

10

10

10

10

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.1. Summary of litter observation record during lactation period. LD1 to 4

Group & Dose
(mg/kg body weight)

No. of Live Pups At Birth

During LD 1 to 4

Sex Ratio (M/F) at

LD 4

No. of Survived Pups during

LD 1 to 4

Pup Survival

Index
(%)

LD1 to 4

Live Pups (No.)

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

11.60

11.60

4.50

7.10

0.00

0.00

0.00

0.83

11.60

100.00

±SD

3.31

3.31

2.76

2.64

0.00

0.00

0.00

0.91

3.31

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

10.82

10.82

6.09

4.73

0.00

0.00

0.00

1.55

10.82

100.00

±SD

2.52

2.52

3.18

1.68

0.00

0.00

0.00

1.10

2.52

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

12.00

12.00

6.10

5.90

0.00

0.00

0.00

1.33

12.00

100.00

±SD

2.31

2.31

2.47

2.33

0.00

0.00

0.00

1.06

2.31

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

10.10

10.10

5.70

4.40

0.00

0.00

0.00

1.47

10.10

100.00

±SD

2.02

2.02

1.77

1.35

0.00

0.00

0.00

0.83

2.02

0.00

n

10

10

10

10

10

10

10

10

10

10

Table 7.2. Summary of litter observation record during lactation period. LD4 to 7

Group & Dose
(mg/kg body weight/day)

Live Pups (No.) on LD 4

 

 

 

 

 

 

 

 

 

Pups Sacrificed for Blood Collection on LD 4 (No.)

 

 

 

 

 

 

 

 

 

Live Pups (No.) on LD 4 after Sacrificed for Blood Collection

During LD 4 to 7

Sex Ratio (M/F) at LD 7

No. of Survived Pups during

LD 4 to 7

Pup Survival Index
(%) during

LD 4 to 7

Live Pups (No.)

Dead Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0  

Mean

4.50

7.10

11.60

0.00

1.30

1.30

4.50

5.80

10.30

4.50

5.80

10.30

0.00

0.00

0.00

1.57

10.30

100.00

±SD

2.76

2.64

3.31

0.00

0.95

0.95

2.76

2.39

2.54

2.76

2.39

2.54

0.00

0.00

0.00

2.70

2.54

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

6.09

4.73

10.82

0.00

1.18

1.18

6.09

3.55

9.64

6.09

3.55

9.64

0.00

0.00

0.00

3.31

9.64

100.00

±SD

3.18

1.68

2.52

0.00

0.98

0.98

3.18

2.16

1.69

3.18

2.16

1.69

0.00

0.00

0.00

3.65

1.69

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G3 & 300 

Mean

6.10

5.90

12.00

0.00

1.50

1.50

6.10

4.40

10.50

6.10

4.40

10.50

0.00

0.00

0.00

2.47

10.50

100.00

±SD

2.47

2.33

2.31

0.00

0.71

0.71

2.47

2.22

1.84

2.47

2.22

1.84

0.00

0.00

0.00

3.19

1.84

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

5.70

4.40

10.10

0.00

0.80

0.80

5.70

3.60

9.30

5.70

3.60

9.30

0.00

0.00

0.00

1.79

9.30

100.00

±SD

1.77

1.35

2.02

0.00

0.92

0.92

1.77

1.07

1.25

1.77

1.07

1.25

0.00

0.00

0.00

0.88

1.25

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 7.3. Summary of litter observation record during lactation period. LD7 to 13

Group & Dose
(mg/kg body weight)

Live Pups (No.) on LD 7

During LD 7 to 13

Sex Ratio

(M/F) at

LD 7

No. of Survived

Pups during LD 7 to 13

Pup Survival Index
(%) during

LD 7 to 13

Live Pups (No.)

Dead Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0

Mean

4.50

5.80

10.30

4.50

5.80

10.30

0.00

0.00

0.00

1.57

10.30

100.00

±SD

2.76

2.39

2.54

2.76

2.39

2.54

0.00

0.00

0.00

2.70

2.54

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

6.09

3.55

9.64

6.09

3.55

9.64

0.00

0.00

0.00

3.31

9.64

100.00

±SD

3.18

2.16

1.69

3.18

2.16

1.69

0.00

0.00

0.00

3.65

1.69

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

6.10

4.40

10.50

6.10

4.40

10.50

0.00

0.00

0.00

2.47

10.50

100.00

±SD

2.47

2.22

1.84

2.47

2.22

1.84

0.00

0.00

0.00

3.19

1.84

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

5.70

3.60

9.30

5.70

3.60

9.30

0.00

0.00

0.00

1.79

9.30

100.00

±SD

1.77

1.07

1.25

1.77

1.07

1.25

0.00

0.00

0.00

0.88

1.25

0.00

n

10

10

10

10

10

10

10

10

10

10

10

9

Table 8.1. Summary of pup observation record during lactation period. At birth

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups at Birth

10

11

10

10

No. of Females Confirmed as
Non-Pregnant

2

1

2

2

Litter Size (No.)

116

119

120

101

No. of Dead Pups

0

0

0

0

No. of Cannibalized Pups

0

0

0

0

No. of Live Pups at Birth

116

119

120

101

No. of Live Pups with

No Abnormality Detected (No.)

116

119

120

101

Table 8.2. Summary of pup observation record during lactation period. LD1 to 4

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups on LD 4

10

11

10

10

No. of Dead Pups

during LD 1 to 4

0

0

0

0

No. of Cannibalised Pups during LD 1 to 4

0

0

0

0

No. of Live Pups during

LD 1 to 4 (No.)

116

119

120

101

No. of Live Pups with

No Abnormality Detected during LD 1 to 4

116

119

120

101

No. of Pups Sacrificed on LD 4 for blood collection

13

13

15

8

Table 8.3. Summary of pup observation record during lactation period. LD4 to 7

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups on LD 7

10

11

10

10

No. of Dead Pups

during LD 4 to 7

0

0

0

0

No. of Cannibalised Pups during

LD 4 to 7

0

0

0

0

No. of Live Pups

during LD 4 to 7

103

106

105

93

No. of Live Pups with

No Abnormality Detected during LD 4 to 7

103

106

105

93

Table 8.4. Summary of pup observation record during lactation period. LD7 to 13

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with
Live Pups on LD 13

10

11

10

10

No. of Dead Pups

during LD 7 to 13

0

0

0

0

No. of Cannibalised Pups during

LD 7 to 13

0

0

0

0

No. of Live Pups during

LD 7 to 13

103

106

105

93

No. of Live Pups with

No Abnormality Detected during

LD 7 to 13

103

106

105

93

Table 9. Summary record of mean pup weight (g) during lactation period

Group & Dose (mg/kg

body weight)

 

Mean Pup Weight (g) on LD1

 

Mean Pup Weight (g) on
LD 4

 

Mean Pup Weight (g) on
LD 7

 

Mean Pup Weight (g) on
LD 13

Male

Female

Male

Female

Male

Female

Male

Female

 

G1 & 0

Mean

6.93

6.51

11.16

10.96

15.16

14.68

25.22

24.57

±SD

0.35

0.50

0.31

0.41

0.68

0.87

0.38

0.44

n

9

10

9

10

9

10

9

10

 

G2 & 100

Mean

6.98

6.64

11.74*

11.57*

15.44

14.89

25.11

24.92

±SD

0.35

0.42

0.40

0.48

0.45

0.61

0.24

0.63

n

11

11

11

11

11

11

11

11

 

G3 & 300

Mean

6.87

6.46

11.64

11.21

15.42

14.92

25.30

24.87

±SD

0.23

0.26

0.35

0.41

0.35

0.41

0.27

0.44

n

10

10

10

10

10

10

10

10

G4 & 600

Mean

6.71

6.37

11.82*

11.49*

15.53

15.43*

25.13

24.71

±SD

0.19

0.26

0.63

0.48

0.62

0.54

0.48

0.92

n

10

10

10

10

10

10

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary of mean pup anogenital distance ratio record

Group & Dose

(mg/kg body weight)

AGD Ratio

Mean Male AGD Ratio

Mean Female AGD Ratio

G1 & 0

Mean

2.10

1.18

±SD

0.09

0.06

n

9

10

G2 & 100

Mean

2.04

1.12

±SD

0.07

0.05

n

11

11

G3 & 300

Mean

1.98*

1.10*

±SD

0.05

0.04

n

10

10

G4 & 600

Mean

1.95*

1.10*

±SD

0.04

0.06

n

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group & Dose
(mg/kg body weight)

No. of Nipples/Areolae on
Lactation Day 13

G1 & 0

Mean

0.00

±SD

0.00

n

10

G2 & 100

Mean

0.00

±SD

0.00

n

11

G3 & 300

Mean

0.00

±SD

0.00

n

10

G4 & 600

Mean

0.00

±SD

0.00

n

10

n: Number of dams

Table 12. Summary of uteri observation record

Group & Dose
(mg/kg body

weight)

No. of

Corpora lutea

No. of Implantations

Implantation Index (%)

Pre-Implantation Loss

(%)

Post-Implantation Loss

(%)

Pre-natal Loss (No.)

Post-natal Loss

(At birth to

LD 13)

(%)

Post-natal Loss

(At birth to

LD 13)

(No.)

No. of Early Resorptions

No. of Late Resorptions

G1 & 0

Mean

11.60

11.60

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

3.31

3.31

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

10.82

10.82

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.52

2.52

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

12.00

12.00

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.31

2.31

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

10.10

10.10

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.02

2.02

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

Conclusions:
The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. 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 (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 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 of 50 days with a 14 days recovery. 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 corn oil was established before initiation of the treatment. 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 group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.

There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no clinical signs and no external abnormalities noted in pups from all the tested dose groups. No treatment related changes in mean pup weight and anogenital distance noted at all the tested dose groups. The gross pathology conducted for the pups at all the tested dose groups revealed no changes. No occurrences of nipples/areolae in male pups from all the dose groups observed on PND 13.There were no treatment related changes noted in serum thyroxine hormone (T4) levels.

The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for parental reproduction was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). Finally, the No Observed Adverse Effect Level (NOAEL) for parental and reproductive toxicity was determined to be 600 mg/kg bw/day as no adverse effects were found on general toxicity, fertility or development of offspring at any dose level tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (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 were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of 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.
Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: No




Duration of treatment / exposure:
Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.
Frequency of treatment:
Once a day
Duration of test:
Main group Males: 13 February - 21 march 2019
Main group Females: 13 February - 18 April 2019
Recovery Animals: 13 February - 03 April 2019
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
G3 - Mid dose
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose + G4R - High dose Recovery Group
No. of animals per sex per dose:
Main Group: 12
Recovery Group: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) 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 main group 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 (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 14 (dams)
- Organs examined: see table 1

OTHER:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Ovaries and uterus were examined for histopathology.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No data
- Head examinations: No data
Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.
Indices:
See table 2 below.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.

There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.

There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and 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):
The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. 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):
Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. 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 tested dose group recovery group animals of either sex conducted at termination.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is 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):
There 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, statistically significant decrease in absolute seminal vesicles with coagulating glands at group G3 males; statistically significant decrease in absolute heart weight at group G4R females were noted when compared with concurrent vehicle control group. These changes are considered as incidental and not treatment related as the observations do not occur in dose dependant manner, are not found in both sexes and also no gross or histopathological changes were noted in these organs at this dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during necropsy at any of the main and recovery group animals of either sex.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males the assessment of T4 in blood samples from the dams was not performed.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions ocurred in any dose group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No pre or post-implantation losses were observed during the study.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No resorptions were detected at any dose group during the study.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No resorptions were detected at any dose group during the study.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead pups were counted at any dose group during the study.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
There were no changes observed in the gestation length at groups G2 and G3 when compared with vehicle control group. However, a treatment related statistically significant reduction in mean gestation length at group G4 was noted when compared with vehicle control group. This observation is considered as treatment related due to observed clinical signs during the treatment period at this dose level.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the number of dams between the treated groups and the vehicle control group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There were no changes observed in the copulatory interval and number of conceiving days at groups G2 and G3 when compared with vehicle control group. However, an increased copulatory interval was noted at group G4 when compared with vehicle control group. This change is considered as treatment related due to occurrence of treatment related clinical signs and reduced mean body weights at this dose group during the treatment period.

There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: parental general (systemic) toxicity
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
changes in pregnancy duration
other: Changes in the copulatory interval and number of conceiving days
Remarks on result:
other: Increased copulatory interval and gestation length observed at high dose tested (600 mg/kg bw/day).
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal general and developmental toxicity
Remarks on result:
other: No adverse effects were found at any dose tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in mean pup weight of either sex during lactation period at all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean male and female pup weight on lactation day 4 at groups G2 and G4; statistically significant increase in mean female pup weight on LD 7 at group G4 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.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the live birth index (Number of pups born alive / Total number of pups born X 100) between the treated groups and the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the sex ratio of pups between the treated groups and the vehicle control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the litter size (mean number of pups delivered per dam) between the treated groups and the vehicle control group.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in pup survival from lactation day 1 to 13 between the treated groups and the vehicle control group.
External malformations:
no effects observed
Description (incidence and severity):
No external abnormalities were detected in any of the pups sacrificed.
Skeletal malformations:
not specified
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral abnormalities were detected in any of the pups sacrificed.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum Thyroxine (T4) Levels: There were no effects found in serum Thyroxine (T4) hormone levels noted at lactation day 13 pups. As no effects were noted in lactation day 13 pups the assessment of T4 in blood samples from the day 4 pups was not performed.
Anogenital Distance of Pups on Lactation Day 4: There were no treatment related changes observed in mean pup ano-genital distance ratio of either sex during measured on LD 4 at all the tested dose groups when compared with vehicle control group. However, statistically significant decrease in mean male and female mean pup ano-genital distance ratio at groups G3 and G4 was noted when compared with vehicle control. These changes cannot be considered as treatment related effects as the obtained values are within historical control range and also no treatment related effects noted in mean pup weight on the day of measurement of ano-genital distance.
Nipple Retention in Male Pups on Lactation Day 13: There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: No treatment related effects were observed at the highest dose tested (600 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: No adverse effects were observed at any dose level tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 4. Summary of vaginal smear examination for determination of oestrus cyclicity

Group & Dose
(mg/kg body weight/day)

Total No. of Females

No. of Females with Regular Oestrus Cyclicity during

Pre-mating and Mating

No. of Females confirmed with pregnancy

 

No. of Females with Regular Oestrus Cyclicity on Lactation Day 14

No. of Females with Irregular Oestrus Cyclicity during

Pre-mating, Mating and on Lactation day 14

G1 & 0

12

12

10

10

0

G2 & 100

12

12

11

11

0

G3 & 300

12

12

10

10

0

G4 & 600

12

12

10

10

0

Table 5. Summary of cohabitation record

Group & Dose
(mg/kg body weight)

Copulatory Interval

(Days)

 

Conceiving Days

(1 to 5)

Conceiving Days

(6 to 14)

 

G1 & 0

Mean

8.17

n

5

7

±SD

6.37

n

12

%

41.7

58.3

 

G2 & 100

Mean

8.50

n

4

8

±SD

5.96

n

12

%

33.3

66.7

 

G3 & 300

Mean

8.58

n

5

7

±SD

6.14

n

12

%

41.7

58.3

 

G4 & 600

Mean

10.42

n

3

9

±SD

5.81

n

12

%

25.0

75.0

Table 6. Summary of gestation length and delivery data record

Group & Dose
(mg/kg body weight)

Gestation Length (Days)

Litter Size

(No.)

Live Pups (No.)

Dead Pups (No.)

Sex Ratio (M/F) at Birth

Live Birth Index
(%)

Total (No.)

Male

(No.)

Female

(No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

22.20

11.60

11.60

4.50

7.10

0.00

0.00

0.00

0.83

100.00

±SD

0.42

3.31

3.31

2.76

2.64

0.00

0.00

0.00

0.91

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

22.73

10.82

10.82

6.09

4.73

0.00

0.00

0.00

1.55

100.00

±SD

0.65

2.52

2.52

3.18

1.68

0.00

0.00

0.00

1.10

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

22.40

12.00

12.00

6.10

5.90

0.00

0.00

0.00

1.33

100.00

±SD

0.52

2.31

2.31

2.47

2.33

0.00

0.00

0.00

1.06

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

23.40*

10.10

10.10

5.70

4.40

0.00

0.00

0.00

1.47

100.00

±SD

0.70

2.02

2.02

1.77

1.35

0.00

0.00

0.00

0.83

0.00

n

10

10

10

10

10

10

10

10

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7.1. Summary of litter observation record during lactation period. LD1 to 4

Group & Dose
(mg/kg body weight)

No. of Live Pups At Birth

During LD 1 to 4

Sex Ratio (M/F) at

LD 4

No. of Survived Pups during

LD 1 to 4

Pup Survival

Index
(%)

LD1 to 4

Live Pups (No.)

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

11.60

11.60

4.50

7.10

0.00

0.00

0.00

0.83

11.60

100.00

±SD

3.31

3.31

2.76

2.64

0.00

0.00

0.00

0.91

3.31

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

10.82

10.82

6.09

4.73

0.00

0.00

0.00

1.55

10.82

100.00

±SD

2.52

2.52

3.18

1.68

0.00

0.00

0.00

1.10

2.52

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

12.00

12.00

6.10

5.90

0.00

0.00

0.00

1.33

12.00

100.00

±SD

2.31

2.31

2.47

2.33

0.00

0.00

0.00

1.06

2.31

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

10.10

10.10

5.70

4.40

0.00

0.00

0.00

1.47

10.10

100.00

±SD

2.02

2.02

1.77

1.35

0.00

0.00

0.00

0.83

2.02

0.00

n

10

10

10

10

10

10

10

10

10

10

Table 7.2. Summary of litter observation record during lactation period. LD4 to 7

Group & Dose
(mg/kg body weight/day)

Live Pups (No.) on LD 4

 

 

 

 

 

 

 

 

 

Pups Sacrificed for Blood Collection on LD 4 (No.)

 

 

 

 

 

 

 

 

 

Live Pups (No.) on LD 4 after Sacrificed for Blood Collection

During LD 4 to 7

Sex Ratio (M/F) at LD 7

No. of Survived Pups during

LD 4 to 7

Pup Survival Index
(%) during

LD 4 to 7

Live Pups (No.)

Dead Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0  

Mean

4.50

7.10

11.60

0.00

1.30

1.30

4.50

5.80

10.30

4.50

5.80

10.30

0.00

0.00

0.00

1.57

10.30

100.00

±SD

2.76

2.64

3.31

0.00

0.95

0.95

2.76

2.39

2.54

2.76

2.39

2.54

0.00

0.00

0.00

2.70

2.54

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

6.09

4.73

10.82

0.00

1.18

1.18

6.09

3.55

9.64

6.09

3.55

9.64

0.00

0.00

0.00

3.31

9.64

100.00

±SD

3.18

1.68

2.52

0.00

0.98

0.98

3.18

2.16

1.69

3.18

2.16

1.69

0.00

0.00

0.00

3.65

1.69

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G3 & 300 

Mean

6.10

5.90

12.00

0.00

1.50

1.50

6.10

4.40

10.50

6.10

4.40

10.50

0.00

0.00

0.00

2.47

10.50

100.00

±SD

2.47

2.33

2.31

0.00

0.71

0.71

2.47

2.22

1.84

2.47

2.22

1.84

0.00

0.00

0.00

3.19

1.84

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

5.70

4.40

10.10

0.00

0.80

0.80

5.70

3.60

9.30

5.70

3.60

9.30

0.00

0.00

0.00

1.79

9.30

100.00

±SD

1.77

1.35

2.02

0.00

0.92

0.92

1.77

1.07

1.25

1.77

1.07

1.25

0.00

0.00

0.00

0.88

1.25

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 7.3. Summary of litter observation record during lactation period. LD7 to 13

Group & Dose
(mg/kg body weight)

Live Pups (No.) on LD 7

During LD 7 to 13

Sex Ratio

(M/F) at

LD 7

No. of Survived

Pups during LD 7 to 13

Pup Survival Index
(%) during

LD 7 to 13

Live Pups (No.)

Dead Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0

Mean

4.50

5.80

10.30

4.50

5.80

10.30

0.00

0.00

0.00

1.57

10.30

100.00

±SD

2.76

2.39

2.54

2.76

2.39

2.54

0.00

0.00

0.00

2.70

2.54

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

6.09

3.55

9.64

6.09

3.55

9.64

0.00

0.00

0.00

3.31

9.64

100.00

±SD

3.18

2.16

1.69

3.18

2.16

1.69

0.00

0.00

0.00

3.65

1.69

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

6.10

4.40

10.50

6.10

4.40

10.50

0.00

0.00

0.00

2.47

10.50

100.00

±SD

2.47

2.22

1.84

2.47

2.22

1.84

0.00

0.00

0.00

3.19

1.84

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

5.70

3.60

9.30

5.70

3.60

9.30

0.00

0.00

0.00

1.79

9.30

100.00

±SD

1.77

1.07

1.25

1.77

1.07

1.25

0.00

0.00

0.00

0.88

1.25

0.00

n

10

10

10

10

10

10

10

10

10

10

10

9

Table 8.1. Summary of pup observation record during lactation period. At birth

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups at Birth

10

11

10

10

No. of Females Confirmed as
Non-Pregnant

2

1

2

2

Litter Size (No.)

116

119

120

101

No. of Dead Pups

0

0

0

0

No. of Cannibalized Pups

0

0

0

0

No. of Live Pups at Birth

116

119

120

101

No. of Live Pups with

No Abnormality Detected (No.)

116

119

120

101

Table 8.2. Summary of pup observation record during lactation period. LD1 to 4

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups on LD 4

10

11

10

10

No. of Dead Pups

during LD 1 to 4

0

0

0

0

No. of Cannibalised Pups during LD 1 to 4

0

0

0

0

No. of Live Pups during

LD 1 to 4 (No.)

116

119

120

101

No. of Live Pups with

No Abnormality Detected during LD 1 to 4

116

119

120

101

No. of Pups Sacrificed on LD 4 for blood collection

13

13

15

8

Table 8.3. Summary of pup observation record during lactation period. LD4 to 7

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with

Live Pups on LD 7

10

11

10

10

No. of Dead Pups

during LD 4 to 7

0

0

0

0

No. of Cannibalised Pups during

LD 4 to 7

0

0

0

0

No. of Live Pups

during LD 4 to 7

103

106

105

93

No. of Live Pups with

No Abnormality Detected during LD 4 to 7

103

106

105

93

Table 8.4. Summary of pup observation record during lactation period. LD7 to 13

Parameter ↓

Group & Dose (mg/kg body weight)

G1 & 0

G2 & 100

G3 & 300

G4 & 600

No. of Females with
Live Pups on LD 13

10

11

10

10

No. of Dead Pups

during LD 7 to 13

0

0

0

0

No. of Cannibalised Pups during

LD 7 to 13

0

0

0

0

No. of Live Pups during

LD 7 to 13

103

106

105

93

No. of Live Pups with

No Abnormality Detected during

LD 7 to 13

103

106

105

93

Table 9. Summary record of mean pup weight (g) during lactation period

Group & Dose (mg/kg

body weight)

 

Mean Pup Weight (g) on LD1

 

Mean Pup Weight (g) on
LD 4

 

Mean Pup Weight (g) on
LD 7

 

Mean Pup Weight (g) on
LD 13

Male

Female

Male

Female

Male

Female

Male

Female

 

G1 & 0

Mean

6.93

6.51

11.16

10.96

15.16

14.68

25.22

24.57

±SD

0.35

0.50

0.31

0.41

0.68

0.87

0.38

0.44

n

9

10

9

10

9

10

9

10

 

G2 & 100

Mean

6.98

6.64

11.74*

11.57*

15.44

14.89

25.11

24.92

±SD

0.35

0.42

0.40

0.48

0.45

0.61

0.24

0.63

n

11

11

11

11

11

11

11

11

 

G3 & 300

Mean

6.87

6.46

11.64

11.21

15.42

14.92

25.30

24.87

±SD

0.23

0.26

0.35

0.41

0.35

0.41

0.27

0.44

n

10

10

10

10

10

10

10

10

G4 & 600

Mean

6.71

6.37

11.82*

11.49*

15.53

15.43*

25.13

24.71

±SD

0.19

0.26

0.63

0.48

0.62

0.54

0.48

0.92

n

10

10

10

10

10

10

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary of mean pup anogenital distance ratio record

Group & Dose

(mg/kg body weight)

AGD Ratio

Mean Male AGD Ratio

Mean Female AGD Ratio

G1 & 0

Mean

2.10

1.18

±SD

0.09

0.06

n

9

10

G2 & 100

Mean

2.04

1.12

±SD

0.07

0.05

n

11

11

G3 & 300

Mean

1.98*

1.10*

±SD

0.05

0.04

n

10

10

G4 & 600

Mean

1.95*

1.10*

±SD

0.04

0.06

n

10

10

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group & Dose
(mg/kg body weight)

No. of Nipples/Areolae on
Lactation Day 13

G1 & 0

Mean

0.00

±SD

0.00

n

10

G2 & 100

Mean

0.00

±SD

0.00

n

11

G3 & 300

Mean

0.00

±SD

0.00

n

10

G4 & 600

Mean

0.00

±SD

0.00

n

10

n: Number of dams

Table 12. Summary of uteri observation record

Group & Dose
(mg/kg body

weight)

No. of

Corpora lutea

No. of Implantations

Implantation Index (%)

Pre-Implantation Loss

(%)

Post-Implantation Loss

(%)

Pre-natal Loss (No.)

Post-natal Loss

(At birth to

LD 13)

(%)

Post-natal Loss

(At birth to

LD 13)

(No.)

No. of Early Resorptions

No. of Late Resorptions

G1 & 0

Mean

11.60

11.60

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

3.31

3.31

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

G2 & 100

Mean

10.82

10.82

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.52

2.52

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

12.00

12.00

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.31

2.31

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

G4 & 600

Mean

10.10

10.10

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.02

2.02

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

10

10

10

10

10

10

10

10

10

10

Applicant's summary and conclusion

Conclusions:
The NOAEL of the test substance for parental and developmental toxicity in rats by oral route was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.


Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. 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 (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 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 of 50 days with a 14 days recovery. 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 corn oil was established before initiation of the treatment. 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 group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.

There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no clinical signs and no external abnormalities noted in pups from all the tested dose groups. No treatment related changes in mean pup weight and anogenital distance noted at all the tested dose groups. The gross pathology conducted for the pups at all the tested dose groups revealed no changes. No occurrences of nipples/areolae in male pups from all the dose groups observed on PND 13.There were no treatment related changes noted in serum thyroxine hormone (T4) levels.

The No Observed Effect Level (NOEL) for parental general toxicity was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day. The No Observed Effect Level (NOEL) for maternal developmental toxicity was established at 300 mg/kg bw/day based on the increased copulatory interval and gestation length observed at the high dose tested (600 mg/kg bw/day). The No Observed Effect Level (NOEL) for developmental toxicity was established at 600 mg/kg bw/day based no effects found in offspring at any dose tested.

Finally, the No Observed Adverse Effect Level (NOAEL) for parental and developmental toxicity was determined to be 600 mg/kg bw/day as no adverse effects were found on maternal or developmental toxicity at any dose level tested.