Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Based on the data available from different studies and applying the weight of evidence approach, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various read across chemicals
Justification for type of information:
Weight of evidence approach based on various read across chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Weight of evidence approach based on various read across chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various read across chemicals
GLP compliance:
not specified
Limit test:
no
Justification for study design:
not specified
Species:
rat
Strain:
other: 2. Wistar 3. Sprague-Dawley
Details on species / strain selection:
2. The rat is the commonly used species for toxicity studies and also recommended by the regulatory guidelines specified in the study plan.
3. No data available
Sex:
male/female
Details on test animals or test system and environmental conditions:
2. TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation.
- Weight at study initiation: Male: Minimum: 240 g Maximum: 315 g
Female: Minimum: 210 g Maximum: 260 g
- Fasting period before study: No data
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20[cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females. Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet from approved supplier was offered ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.30 to 22.70 °C
- Humidity (%): 43.90 to 67.60%
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: To: November 16, 2015 to March 26, 2016
3. TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Male 169.50 gg
Female 152.35 g
- Fasting period before study: No data available
- Housing: Animals were housed in polycarbonate cages. Three rats of same sex were housed together in each cage of size 39 cm X 28 cm X 14 cm. Paddy husk was used as bedding material.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range: 19.5 °C to 22.8 °C)
- Humidity (%):30% to 70% (actual range: 53.3% to 58.1%).
- Air changes (per hr): Ten air changes per hour of 100% fresh air that has been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
2. PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil. The test chemical was soluble in corn oil
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0.5 ml/100g body weight
- Lot/batch no. (if required): MR301015, MR161215
- Purity: No data
3. PREPARATION OF DOSING SOLUTIONS: Benzyl Propionate was diluted with Corn oil for preparation of dosing solution(s).

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data available
- Concentration in vehicle:The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg bw/day.
- Amount of vehicle (if gavage): 0.00 mg/ml/day, 26.60 mg/ml/day, 52.38 mg/ml/ day and 105.27 mg/ml/day.
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
2. - M/F ratio per cage: One male and one female (1:1)
- Length of cohabitation: Female rats were housed with same male until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data
- Further matings after two unsuccessful attempts: Yes, Re-mating of unsuccessfully paired female was done with proven male of the same group.
- After successful mating each pregnant female was caged (how): No data
- Any other deviations from standard protocol: No data
3. No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
2. yes - The analytical method was validated with respect to the following parameters.
Specificity: The specificity will be evaluated by analysing the solvent used, standard solution, and sample solution.
Linearity: The linearity was carried out by preparing and analyzing the standard solutions of at least 6 concentrations (covering the target analyte concentration i.e. 5 ppm,10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm ). A plot was drawn between the concentration and the response. The correlation coefficient, slope and intercept was calculated.
Assay accuracy and precision: Assay accuracy and precision was carried out by fortifying the standard in vehicle at two levels (covering the target analyte concentration i.e., 10 ppm & 100 ppm). Five preparations were carried out at each concentration level selected. Two controls along with the assay accuracy samples were analysed. The mean, SD, % RSD was calculated. Assay accuracy was reported as the mean % recovery whereas the precision was reported as % RSD.
Homogeneity: The homogeneity of the dose formulation prepared was determined by sampling and analyzing the formulation at top, middle and bottom layers. Sampling was done in two replicates from each layer.
Stability: The stability of the prepared dose formulation was determined by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.
3. yes - Analysis for concentration and stability of Benzyl Propionate were conducted at Subcontracted Laboratory. Test item formulation samples prepared day 1 (pre-dosing) were sent to Subcontracted Laboratory.
Duration of treatment / exposure:
2. 64 days
3. 28 days consecutively
Frequency of treatment:
2. Total days: 64
All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were further dosed till 47th day . Females were dosed during pregnancy and upto day 4 post partum.
3. Once daily
Details on study schedule:
2. No data
3. No data available
Remarks:
Study 2. G1 (Control Group) 0 mg/kg bw/day, G2 (Low Dose Group) 308 mg/kg bw/day, G3 (Mid Dose Group) 556 mg/kg bw/day, G4 (High Dose Group) 1000 mg/kg bw/day, G1R (Recovery Control Group) 0 mg/kg bw/day and G4R (Recovery High Dose Group) 1000 mg/kg bw/day.
Remarks:
study 3: 0 (vehicle), 250, 500 and 1000 mg/kg bw/day.
Basis:actual ingested
No. of animals per sex per dose:
2. Total: 124 ( 104 Test animals + 20 recovery animals)
Test animals:
0 mg/Kg bw: 13 males and 13 females
308 mg/Kg bw: 13 males and 13 females
556 mg/Kg bw: 13 males and 13 females
1000 mg/Kg bw: 13 males and 13 females

Recovery animals:
0 mg/Kg bw: 5 males and 5 females
1000 mg/Kg bw: 5 males and 5 females
3. Total:48
Control: 6 male, 6 female
250 mg/kg bw/day: 6 male, 6 female
500 mg/kg bw/day: 6 male, 6 female
100 0mg/kg bw/day: 6 male, 6 female
Control animals:
yes, concurrent vehicle
Details on study design:
2. - Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using validated software or the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights will be considered within ± 20% of the groups mean.
- Other: No data
3. Details on study design
- Dose selection rationale: Based on results from a preliminary 14-day study there was no chenge in the survivel, body weight, Daily clinical observations and Gross pathological examination of 1000 mg/kg/bw/day group. Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.
- Rationale for animal assignment (if not random): Animals were randomized by sex and body weight
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
Positive control:
No data
Parental animals: Observations and examinations:
2. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the acclimatization and study period
- Cage side observations checked in table [No.?] were included. Mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of animals of all groups were made once a day. Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Observations included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic
or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, During pre-mating, pregnancy and lactation, feed consumption were measured at least weekly. Feed consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT), Activated Partial Thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb) , Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN) – Calculated, Globulin (Glob) - Calculated, Alb/ Glb (A:G) – Calculated, Bile acids

URINALYSIS: Not specified
- Time schedule for collection of urine: Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION:Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

OTHER:
Functional Battery Observations: Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for five males and five females from control and treatment groups, during the last week of treatment and that of recovery groups, in the last week of recovery period.

Animals were subjected to examinations of various functional parameters which included; motor activity measurements using OPTO–VARIMEX 4, an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter; hind limb foot splay record and sensory reactivity measurements.
3. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. : Rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, At least once a week there after until

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once a day
- Dose groups that were examined: 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

HAEMATOLOGY: Yes, By using Beckman Coulter haematology analyzer.
- Time schedule for collection of blood: At termination.
- Anaesthetic used for blood collection: No data available
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked in table [No.?] were examined. : Hemoglobin, Red Blood Corpuscles, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelets, White Blood Corpuscles, Neutrophils, Lymphocytes, Eosinophils, Monocytes, Basophil and Prothrombin time were checked, Table No.H; Appendix No.VII.

CLINICAL CHEMISTRY: Yes, By using Dimension XpandPlus and Acculyte 5P.
- Time schedule for collection of blood: At termination.
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked in table [No.?] were examined. : Total Protein, Blood Urea Nitrogen, Urea Nitrogen, Alanine Aminotransferase, Aspartate Aminotransferase, Alkaline Phosphatase, Gamma Glutamyl Transferase, Glucose, Calcium, Phosphorous, Albumin, Total Bilirubin, Creatinine , Total Cholesterol, Triglycerides, Globulin Calculated Sodium, Potassium, Chloride were checked, Table No.I; Appendix No.VIII.

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. : No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes

OTHER: Viability, Behavior in Home cage, Vocalizations, Respiration, Palpebral closure, Handling Observations, Urination, Defecation, Prominence of Eye, Lacrimation, Salivation, Piloerection, Examination of Skin / Fur, Stereotype Behaviour, Rearing (Rears), Clonic and Tonic Movements and Severity of Gait were observed
Oestrous cyclicity (parental animals):
2. Estrous cycle was monitored for its regularity during treatment period and in cohabitation for confirmation of pregnancy.
3. No data available
Sperm parameters (parental animals):
2. No data
3. No data available
Litter observations:
2. STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No data
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. No data

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum. Live pups were weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No data

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No data
3. No data available
Postmortem examinations (parental animals):
2. GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.

Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.

Following organs from randomly selected 5 male and 5 female rats were collected and preserved : Adrenals, Aorta, Bone (femur) with joint, Brain (cerebrum, cerebellum, mid brain), Cecum, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mammary glands, Mesenteric and Mandibular lymph node, Oesophagus, Ovaries with oviduct, Pancreas, Peyer's Patches, Pituitary, Prostate and Seminal vesicle with coagulating glands, Rectum, Salivary glands, Sciatic Nerve, Skeletal muscle, Skin, Spleen, Spinal Cord (cervical, mid-thoracic and lumbar), Sternum with marrow, Stomach, Testes, Thymus, Thyroid with Parathyroids, Trachea, Urinary Bladder, Uterus, Cervix with Vagina

Testes, epididymides, prostate and seminal vesicle with coagulating glands of all male rats and ovaries, uterus and cervix with vagina of all female rats were collected and preserved. Thyroid gland of one male and one female pup from each litter was collected and preserved. Collected Organs/tissues were fixed and preserved in 10 % Neutral buffered formalin. Testes and epididymis were initially fixed in Bouin’s fluid for approximately 24 hr, then 4 changes were given in 70% alcohol and transferred to 10 % neutral buffered formalin for preservation. Eyes were initially fixed in Modified Davidson‘s fluid for approximately 24 hr and then transferred to 10 % neutral buffered formalin for preservation.

Organ Weight: Weighing of brain, adrenals, ovaries with oviduct, testes, epididymides, heart, liver, kidneys, thymus and spleen was performed for randomly selected 5 male and 5 female rats. Testes and epididymides of all male rats were weighed. Before weighing adherent tissue/fat from organs were trimmed off and were kept in normal saline.

HISTOPATHOLOGY: Yes, All the preserved organs (Testes, epididymides, prostate and seminal vesicle with coagulating glands, ovaries, uterus and cervix with vagina) of all the rats, all the preserved tissues of randomly selected five male and five female rats of groups G1 and G4 and preserved thyroid of one male and one female pup of each litter were subjected to histopathological examination. All the tissues were trimmed, processed, embedded in paraffin wax. Sections were cut at a thickness of 3-5 micron and stained with hematoxylin and eosin stain. Processed tissues were subjected to histopathological examination. The prepared slides were examined under microscope by the Pathologist to note histopathological lesions, if any in different organs. Special attention was paid to observe effect of test item on reproductive system and spermatogenesis. The observed abnormalities were described according to morphological character, distribution, severity.
3. SACRIFICE: male and female animals sacrificed on day 29,
GROSS PATHOLOGY: Yes
Gross necropsy was conducted. All the animals of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day group were sacrificed and gross lesions were noted.

HISTOPATHOLOGY: Yes
Control and treated at the highest dose level of 1000 mg/kg were subjected to sacrifice.

Organs examined: Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder and Uterus of 1000 mg/kg/bw/day group.
Postmortem examinations (offspring):
2. SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age. No data
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Gross pathology- Pups: Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. No data
3. No data available
Statistics:
2. Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks
3. Data were analysed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.

Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:
* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01).
Reproductive indices:
2. Pregnancy index/fertility index ,Mean Post-Implantation Loss (%), Post-natal Loss (%) were determined
3. No data available
Offspring viability indices:
2. Pups Survival Index (%) was determined
3. No data available
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.
Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight).
The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.
3. no effects observed - No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days before commencement of treatment: Home cage observations in rats from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of rats did not reveal any abnormality from all treated groups and control group. Open field observation of rats did not reveal any abnormality from all treated groups and control group. During study period: At the end of dosing period: Week 1, Home cage observations in animals from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of animals did not reveal any abnormality from all treated groups and control group. Open field observation of animals did not reveal any abnormality from all treated groups and control group. Week 2: Home cage observations in animals from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of animals did not reveal any abnormality from all treated groups and control group. Open field observation of animals did not reveal any abnormality from all treated groups and control group. Week 3: Home cage observations in animals from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of animals did not reveal any abnormality from all treated groups and control group.Open field observation of animals did not reveal any abnormality from all treated groups and control group. Week 4: Home cage observations in animals from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of animals did not reveal any abnormality from all treated groups and control group. Open field observation of animals did not reveal any abnormality from all treated groups and control group.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
2. no mortality observed - No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period
3. no mortality observed - No mortality was observed in any of the traeted groups of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight).
Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
These changes observed were inconsistent, hence not considered as effect of the test item administration.
3. no effects observed - Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration
3. no effects observed - Animals from control and all treated dose groups exhibited normal feed consumption at the end of the dosing period of 28 days
Food efficiency:
no effects observed
Description (incidence and severity):
2. no effects observed - Formulations were found to be homogeneous and stable upto 6 hour in vehicle corn oil. The mean active ingredient content at 61.6, 111.2 and 200 mg/ml concentration of test chemical was 61.770, 110.321 and 200.007 mg/ml on day 1; 62.045, 110.902 and 198.199 mg/ml on day 21 and 60.726, 111.912 and 201.231 mg/ml on day 40, respectively
3. not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed
for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R.
The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables
3. effects observed, treatment-related - Male and Female - Haematological investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.
Male : Hb : Increased values were obtained for animals from 500 mg/kg (p≤0.01) and 1000 mg/kg (p≤0.05) dose groups,
MCHC : Increased values were obtained for animals from 500 mg/kg dose group (p≤0.01) and
Total WBC : Increased values were obtained for animals from 1000 mg/kg dose group (p≤0.05).
Female : Platelets : Increased values were obtained for animals from 1000 mg/kg dose group (p≤0.05).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistical ly significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.
The above changes were inconsistent, not dose dependent hence considered as incidental in nature.
3. effects observed, treatment-related - Male and Female - Biochemical investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.
Male :Sodium : Elevated levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.01) and
Chloride : Elevated levels were observed in animals from 250 mg/kg dose group (p≤0.05).
Female : Calcium : Elevated levels were observed in animals from 250 mg/kg dose group (p≤0.01),
Bilirubin : Elevated levels were observed in animals from 500 mg/kg dose group (p≤0.05),
Sodium : Elevated levels were observed in animals from 250 mg/kg (p≤0.01), 500 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups,
Alkaline Phosphatase : Decreased levels were observed in animals from 250 mg/kg dose group (p≤0.05),
Potassium : Decreased levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.05) and
Chloride : Decreased levels were observed in animals from 1000 mg/kg dose group (p≤0.05).
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to the repective control group G1-R. The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-R in female as compared to G1-R. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.
3. no effects observed - Sensory Reactivity Observations: All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.
Grip Strength: Grip strength values observed in male and female animals for control and different dose groups were comparable.
Motor Activity: Higher values were observed in male animals from 500 mg/kg dose group for first interval (p≤0.05). Lower values were observed in female animals from 500 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance.
Immunological findings:
not specified
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):
2. effects observed, non-treatment-related - Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5); Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5); Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5); Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5); Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5); Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5); Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild
cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5); Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5); Adrenal s: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5); Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13); Seminal Vesicles: multifocal mild neutrophilic /lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13); Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13). Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.
From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs.
3. no effects observed - No treatment related histopathological changes were evident in male and female rats from control and high dose groups. Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the Test Item.
3. not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
2. no effects observed - No test item related changes in estrous cyclicity and precoital interval were observed. In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G3, 3 females and in group G4, 4 females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. Precoital Interval was calculated, all females showed precoital interval less than 5 days, except 1, 1 and 4 females from G1, G3 and G4, respectively which showed precoital interval more than 5 days.
3. not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
2. effects observed, treatment-related - Pregnancy index was observed to be 92.31, 84.62, 84.62 and 61.54 in G1, G2, G3 and G4 respectively. Marked decrease in Pregnancy index / Fertility index in G4 (1000 mg/kg body weight) was considered to be treatment related.
3. not specified
Dose descriptor:
NOAEL
Remarks:
2
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive performance
Dose descriptor:
NOAEL
Remarks:
3
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No effects on reproductive organ was observed
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
2. mortality observed, non-treatment-related - There was no statistically significant difference between the control (G1) and treatment groups for mortality, no. of live births and survival index
3. not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
2. no effects observed - There was no statistically significant difference between the control (G1) and treatment groups for pups weight at birth and PND4 and weight gain at PND4
3. not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - Pups died during course of study revealed various lesions among the control and treated groups viz., external examination emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54); Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54); Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and internal examination absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18); blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18); reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18); reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18); paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18); congested intestine (Female: G1: 1/56, G3: 1/54); autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18)
3. not specified
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.
3. not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
2. effects observed, non-treatment-related - Pups sex ratio (Male/Female) was observed to be 55/57, 44/30, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.
3. not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Remarks:
2
Generation:
F1
Effect level:
556 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility index/pregnancy index was decreased at dose level of 1000 mg/Kg bw in dams which was correlated to the viability of offsprings
Remarks on result:
other: No developmental toxic effects observed
Remarks on result:
not determinable
Remarks:
3
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
Based on the data available from different studies and applying the weight of evidence approach, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.
Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical. The studies are as mentioned below:

 

Combined repeated dose repro-developmental toxicity study was conducted to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The study was performed as per OECD 422 Guidelines.The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control), 308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups. Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed in any of the groups throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the respective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of both sexes did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of test chemical at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical. Based on the findings of repeated dose oral toxicity study in combination with reproduction/ developmental toxicity of test chemical in Wistar rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw, when male and female wistar rats were treated with test chemical orally.

 

In supporting with the above result, another reproductive toxicity study of the given test chemical was considered on the basis of repeated dose 28-day oral toxicity study performed as per OECD 407 Guidelines. The male and female Sprague-Dawley rats were administered with test chemical in dose concentration 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day by oral gavage route. Corn oil used as vehicle. Analysis for concentration and stability of test chemical were conducted at Subcontracted Laboratory. The animals of uniform body weight were selected. The individual body weight of the animals did not exceed ± 20% of group mean body weight. The group means body weights of all the groups were approximately equal. A total of 48 animals (24 males + 24 females) were selected and randomly distributed into four groups with 6 animals/sex/group and 3/sex/cage. The doses were selected based on the results of the Dose Range Finding study,  Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment, every day, for a period of 28 days. The test and/or control item was administered by oral gavage route, using a 18 gauge ball–tipped intubation needle fitted onto a gauge syringe of appropriate size. Doses were calculated using recent body weights, 10 ml per kg body weight is considered the volume which could be administered to a rat. All the animals were observed for viability twice daily. Body weight was recorded on the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29. The quantity of feed consumed by control and different treatment groups was recorded weekly until scheduled sacrifice and the feed consumption per animal was calculated for each group. All animals were examined for clinical signs such as skin and fur changes, eye and mucous membrane changes, respiratory, circulatory and general changes were recorded once daily. In home cage, rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure. After completion of 28 days study period, all surviving study rats were sacrificed on day 29. Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Lungs, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together. All the rats survived through the dosing period of 28 days and were sacrificed and gross lesions were noted. From each rat, samples or the whole of the tissue were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin. Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination. Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder, Uterus. All the animals from control and all the treated dose groups survived throughout the dosing period of 28 days. Animals from control and different dose groups exhibited normal body weight gain and normal feed consumption throughout the dosing period of 28 days. No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days. Home cage observations in rats from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of rats did not reveal any abnormality from all treated groups and control group. Open field observation of rats did not reveal any abnormality from all treated groups and control group. All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4. Grip strength values observed in male and female animals for control and different dose groups were comparable. Higher values for motor activity were observed in male animals from 500 mg/kg dose group for first interval (p≤0.05). Lower values were observed in female animals from 500 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance. In comparison with controls organ weight data of female animals sacrificed on day 29, revealed increased relative weight of liver (p≤0.05), ovaries (p≤0.01) and lungs (p≤0.05) of animals from 1000 mg/kg dose group. Although significant changes in organ weights were observed in female animals from high dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance. Gross pathological examination in male and female animals from control and different treatment groups did not reveal any abnormality. Hematological analysis performed on 29th day revealed statistically significant increase in the values of Hb of male rats dosed at 500 mg/kg and 1000 mg/kg, MCHC of male rats dosed at 500 mg/kg, Total WBC of male rats dosed at 1000 mg/kg and Plateles of female rats dosed at 1000 mg/kg. The increase in the values of different parameters was marginal and within the normal laboratory limits. Clinical biochemistry analysis results, when compared between the test and control groups revealed below observations. Statistically significant increase of Sodium in male rats dosed at 250 mg/kg and 1000 mg/kg of test item. Statistically significant increase of Chloride levels in male rats dosed at 250 mg/kg of test item. Statistically significant increase of Calcium levels in female rats dosed at 250 mg/kg of test item. Statistically significant increase of Bilirubin levels in female rats dosed at 500 mg/kg of test item. Statistically significant increase of Sodium in female rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of test item. Statistically significant decrease of Alkaline Phosphatase levels in female rats dosed at 250 mg/kg of test item. Statistically significant decrease of Potassium levels in female rats dosed at 250 mg/kg and 1000 mg/kg of test item. Statistically significant decrease of Chloride levels in female rats dosed at 1000 mg/kg of test item. Although there was an increase/decrease in the values of various biochemical parameters as mentioned above, the deviations were marginal and within the range of normal laboratory limits. No treatment related histopathological changes were evident in male and female rats from control and high dose groups. Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings. Hence, No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw on the basis of no effects on reproductive organ, when male and female Sprague-Dawley rats were treated with the given test chemical orally for 28 days.

 

Based on the data available from different studiesand applying the weight of evidence approach, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Klimisch Rating 2
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical. The studies are as mentioned below:

 

Combined repeated dose repro-developmental toxicity study was conducted to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The study was performed as per OECD 422 Guidelines.The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control), 308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups. Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed in any of the groups throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the respective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of both sexes did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of test chemical at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical. Based on the findings of repeated dose oral toxicity study in combination with reproduction/ developmental toxicity of test chemical in Wistar rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw, when male and female wistar rats were treated with test chemical orally.

 

In supporting with the above result, another reproductive toxicity study of the given test chemical was considered on the basis of repeated dose 28-day oral toxicity study performed as per OECD 407 Guidelines. The male and female Sprague-Dawley rats were administered with test chemical in dose concentration 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day by oral gavage route. Corn oil used as vehicle. Analysis for concentration and stability of test chemical were conducted at Subcontracted Laboratory. The animals of uniform body weight were selected. The individual body weight of the animals did not exceed ± 20% of group mean body weight. The group means body weights of all the groups were approximately equal. A total of 48 animals (24 males + 24 females) were selected and randomly distributed into four groups with 6 animals/sex/group and 3/sex/cage. The doses were selected based on the results of the Dose Range Finding study,  Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment, every day, for a period of 28 days. The test and/or control item was administered by oral gavage route, using a 18 gauge ball–tipped intubation needle fitted onto a gauge syringe of appropriate size. Doses were calculated using recent body weights, 10 ml per kg body weight is considered the volume which could be administered to a rat. All the animals were observed for viability twice daily. Body weight was recorded on the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29. The quantity of feed consumed by control and different treatment groups was recorded weekly until scheduled sacrifice and the feed consumption per animal was calculated for each group. All animals were examined for clinical signs such as skin and fur changes, eye and mucous membrane changes, respiratory, circulatory and general changes were recorded once daily. In home cage, rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure. After completion of 28 days study period, all surviving study rats were sacrificed on day 29. Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Lungs, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together. All the rats survived through the dosing period of 28 days and were sacrificed and gross lesions were noted. From each rat, samples or the whole of the tissue were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin. Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination. Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder, Uterus. All the animals from control and all the treated dose groups survived throughout the dosing period of 28 days. Animals from control and different dose groups exhibited normal body weight gain and normal feed consumption throughout the dosing period of 28 days. No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days. Home cage observations in rats from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of rats did not reveal any abnormality from all treated groups and control group. Open field observation of rats did not reveal any abnormality from all treated groups and control group. All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4. Grip strength values observed in male and female animals for control and different dose groups were comparable. Higher values for motor activity were observed in male animals from 500 mg/kg dose group for first interval (p≤0.05). Lower values were observed in female animals from 500 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance. In comparison with controls organ weight data of female animals sacrificed on day 29, revealed increased relative weight of liver (p≤0.05), ovaries (p≤0.01) and lungs (p≤0.05) of animals from 1000 mg/kg dose group. Although significant changes in organ weights were observed in female animals from high dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance. Gross pathological examination in male and female animals from control and different treatment groups did not reveal any abnormality. Hematological analysis performed on 29th day revealed statistically significant increase in the values of Hb of male rats dosed at 500 mg/kg and 1000 mg/kg, MCHC of male rats dosed at 500 mg/kg, Total WBC of male rats dosed at 1000 mg/kg and Plateles of female rats dosed at 1000 mg/kg. The increase in the values of different parameters was marginal and within the normal laboratory limits. Clinical biochemistry analysis results, when compared between the test and control groups revealed below observations. Statistically significant increase of Sodium in male rats dosed at 250 mg/kg and 1000 mg/kg of test item. Statistically significant increase of Chloride levels in male rats dosed at 250 mg/kg of test item. Statistically significant increase of Calcium levels in female rats dosed at 250 mg/kg of test item. Statistically significant increase of Bilirubin levels in female rats dosed at 500 mg/kg of test item. Statistically significant increase of Sodium in female rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of test item. Statistically significant decrease of Alkaline Phosphatase levels in female rats dosed at 250 mg/kg of test item. Statistically significant decrease of Potassium levels in female rats dosed at 250 mg/kg and 1000 mg/kg of test item. Statistically significant decrease of Chloride levels in female rats dosed at 1000 mg/kg of test item. Although there was an increase/decrease in the values of various biochemical parameters as mentioned above, the deviations were marginal and within the range of normal laboratory limits. No treatment related histopathological changes were evident in male and female rats from control and high dose groups. Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings. Hence, No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw on the basis of no effects on reproductive organ, when male and female Sprague-Dawley rats were treated with the given test chemical orally for 28 days.

 

Based on the data available from different studiesand applying the weight of evidence approach, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the data available from different studiesand applying the weight of evidence approach, NOAEL was considered to be 1000 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Additional information

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