Methyl anthranilate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity Study:

A reproductivetoxicity study of test chemical was performed on male and female rats. No treatment related effects on the reproductive and systemic parameters were observed during the study. Hence,No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw, when male and female wistar rats were treated with test material through oral gavage.

Link to relevant study records

Reference

Reference 1

Endpoint:

reproductive toxicity, other

Remarks:

Chronic Repeated dose 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:

experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on the available data of the 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: As mentioned below

Principles of method if other than guideline:

WoE report is based on reproductive toxicity study for read across chemicals:

GLP compliance:

not specified

Limit test:

no

Justification for study design:

not specified

Species:

rat

Strain:

other: 2.CFE 3.Wistar

Details on species / strain selection:

2. Not specified
3.The rat is the commonly used species for toxicity studies and also recommended by the regulatory guidelines specified in the study plan.

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.TEST ANIMALS
- Source: SPF colony
- Age at study initiation: (P) x wks; (F1) x wks: No data available
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: No data available
- Housing: Animals were housed 5 rat per cage
- Diet (e.g. ad libitum): Spillers' Laboratory Small Animal Diet, ad libitum
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

3.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

Route of administration:

other: 2. Oral: feed 3.Oral gavage

Vehicle:

other: 2. Spillers' Laboratory Small Animal Diet 3.corn oil

Details on exposure:

2.No data available
3.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

Details on mating procedure:

2.No data available
3.- 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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2.By using Pye 104 dual flame ionization chromatograph.
3.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.

Duration of treatment / exposure:

2.90 days
3.64 days

Frequency of treatment:

2.Daily
3.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.

Details on study schedule:

No data available

Remarks:

2.
Doses / Concentrations:
0, 300, 1200 and 3600 ppm (21, 82 and 244 mg/kg/day for the three dose levels in males and 24, 95 and 280 mg/kg/ day in females.)
Basis:
actual ingested

Remarks:

0 mg/kg bw/day: G1 (Control Group)
308 mg/kg bw/day: G2 (Low Dose Group)
556 mg/kg bw/day: G3 (Mid Dose Group)
1000 mg/kg bw/day: G4 (High Dose Group)
0 mg/kg bw/day: G1R (Recovery Control Group)
1000 mg/kg bw/day: G4R (Recovery High Dose Group)

No. of animals per sex per dose:

2.Total: 120
0 ppm : 15 male, 15 female
300 ppm : 15 male, 15 female
1200 ppm : 15 male, 15 female
3600 ppm : 15 male, 15 female

3.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

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

2.- Dose selection rationale: Because of the small amount of flavoring lost from the test diets and its apparent palatability, the material was administered in the diet at 0, 300, 1200 and 3600 ppm
3.- 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

Positive control:

2.No data available
3.No data

Parental animals: Observations and examinations:

2.Clinical sign, Body Weight, Food consumption, Water Consumption, Haematology, Clinical Chemistry and Urinalysis were observed .
3.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.

Oestrous cyclicity (parental animals):

2.No data available
3.Estrous cycle was monitored for its regularity during treatment period and in cohabitation for confirmation of pregnancy.

Sperm parameters (parental animals):

No data available

Litter observations:

2.No data available
3.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

Postmortem examinations (parental animals):

2.Reporductive organ weight, gross pathology and histopathology were exmined.
3.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.

Postmortem examinations (offspring):

2.No data available
3.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

Statistics:

2.Statistical analysis is performed by using Student's t test for the significance of Haematological examination and Absolute and relative organ weights.
3.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

Reproductive indices:

2.No data available
3.Pregnancy index/fertility index ,Mean Post-Implantation Loss (%), Post-natal Loss (%) were determined

Offspring viability indices:

2.No data available
3. Pups Survival Index (%) was determined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

2.no effects observed
3.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.

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

not specified

Mortality:

no mortality observed

Description (incidence):

2.not specified
3.No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

2.no effects observed
3.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.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

2.no effects observed
3.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

Food efficiency:

no effects observed

Description (incidence and severity):

2.no effects observed
3.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 Methyl Phenyl acetate (CAS No.:101-41-7) 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

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

not specified

Description (incidence and severity):

not specified

Ophthalmological findings:

not specified

Description (incidence and severity):

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3.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

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3.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.

Urinalysis findings:

not specified

Description (incidence and severity):

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3.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.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.no effects observed
3.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.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3.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.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

2.not specified
3.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.

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

2.not specified
3.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

2.not specified
3.Pregnancy index was found 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.

2.Mortality: No data available

Clinical signs:No signs of ill health were observed in treated rat during the study as compared to control.

Body weight: No effect was observed on body weight and weight gain of treated rat as compared to control.

Food consumption: No effect was observed on food consumption of treated rat as compared to control.

Test substance intake:Average intakes of were 21, 82 and 244 mg/kg/day for the three dose levels in males and 24, 95 and 280 mg/kg/ day in females.

Organ weights: When male and female rats treated with 244 and 280 mg/kg bw, Absolute and relative kidney weight was significantly increased in male and relative weight in female as compared to control.
When male and female rats treated with 82 and 95 mg/kg bw, Absolute and relative kidney weight was significantly increased in male and relative kidney, adrenals and ovaries weight in female as compared to control.
No effect on reproductive oragn weight of female gonads were observed at 288 mg/kg bw.

Gross pathology: No gross abnormalities were observed in Gonads of treated male and female rat as compared to control.

Histopathology: No histopathological changes were observed in Gonads of treated male and female rat as compared to control.

other findings:
Water consumption: No effect was observed on water consumption of treated rat as compared to control.

Haematology:
At 6 week,
When treated with 244 and 280 mg/kg bw, in male rat significant decrease were observed in hemoglobin, RBC and WBC (particularly the lymphocytes) and in female significant decrease hemoglobin were observed.

When treated with 82 and 96 mg/kg bw, in male rat significant decrease were observed in WBC (particularly the lymphocytes) and in female hemoglobin level as compared to control.

At autopsy,
No significant differences between treated and control groups were observed.

Clinical chemistry:No significant differences were observed in treated rat as compared to control.

Urinanalysis: No significant differences were observed in urine analyses of treated rat as compared to control.
3.

Dose descriptor:

NOAEL

Remarks:

2

Effect level:

244 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effect on reporductive organweight, gorss pathology and histopathology

Remarks on result:

other: not specified

Dose descriptor:

NOAEL

Remarks:

2

Effect level:

288 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effect on reporductive organ weight, gorss pathology and histopathology

Remarks on result:

other: No effect on reporductive organ weight

Dose descriptor:

NOAEL

Remarks:

3

Effect level:

556 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Significant changes in the pregnancy index were found at 1000 mg/Kg bw.

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: Not specified

Organ:

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.not specified
3.There was no statistically significant difference between the control (G1) and treatment groups for mortality, no. of live births and survival index

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2.not specified
3.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

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.not specified
3.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)

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3. Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

2.not specified
3. Pups sex ratio (Male/Female) was found to be 55/57, 44/30, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

other: not specified

Remarks:

2

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: Not specified

Dose descriptor:

NOAEL

Remarks:

3.

Generation:

F1

Effect level:

556 mg/kg bw/day

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

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw, when male and female wistar rats were treated with test material through oral gavage.

Executive summary:

Reproductive Toxicity Study:

The reproductive toxicity study of the test chemical is as follows:

In a Chronic repeated dose toxicity study, CFE male and female rat were treated with test material in the concentration of 0, 300, 1200 and 3600 ppm orally in diet equivalant to average intakes of 21, 82 and 244 mg/kg/day for males and 24, 95 and 280 mg/kg/ day for females.No signs of ill health, change in body weight,food and water consumption, Clinical chemistry and Urinanalysis were observed in treated male and female rat during the study as compared to control. At 6 week, significant decrease was observed in hemoglobin, RBC and WBC (particularly the lymphocytes) of male and female rat when treated with 82 and 244 mg/kg bw and 82 and 96 mg/kg bw treated rats. Similarly, Absolute and relative kidney weight was significantly increased in male rat and absolute and relative kidney weight , relative adrenals and ovaries weight in female when treated with 82 and 244 mg/kg bw and 82 and 96 mg/kg bw treated rats as compared to control. As no effect on reproductive oragn weight of female gonads were observed in 288 mg/kg bw, the observed change in relative organ weight of female gonads were considered to be non significant. In addition,No gross abnormalities and histopathological changes were observed in Gonads of treated male and female rat as compared to control.Therefore, NOAEL was considered to be 244 mg/kg body weight/day for male and 288 mg/kg bw for female when CFE male and female rat treated with test material orally in feed for 90 days.

In another study, Combined repeated dose repro-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test material in Wistar rats. 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 any of the groups of animals 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 repective 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 item 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 either sex 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 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. 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. Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material 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) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

Thus, based on the above data, the test chemical did not show any reproductive toxicity, and thus is not likely to classify as a 'Reproductive Toxicant' according to CLP criteria of classification.

Reference 2

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and developmental immunotoxicity (Cohorts 1A, 1B with extension, and 3)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

556 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is Klimish rating 2

Additional information

Reproductive Toxicity Study:

The reproductive toxicity study of the test chemical is as follows:

In a Chronic repeated dose toxicity study, CFE male and female rat were treated with test material in the concentration of 0, 300, 1200 and 3600 ppm orally in diet equivalant to average intakes of 21, 82 and 244 mg/kg/day for males and 24, 95 and 280 mg/kg/ day for females.No signs of ill health, change in body weight,food and water consumption, Clinical chemistry and Urinanalysis were observed in treated male and female rat during the study as compared to control. At 6 week, significant decrease was observed in hemoglobin, RBC and WBC (particularly the lymphocytes) of male and female rat when treated with 82 and 244 mg/kg bw and 82 and 96 mg/kg bw treated rats. Similarly, Absolute and relative kidney weight was significantly increased in male rat and absolute and relative kidney weight , relative adrenals and ovaries weight in female when treated with 82 and 244 mg/kg bw and 82 and 96 mg/kg bw treated rats as compared to control. As no effect on reproductive oragn weight of female gonads were observed in 288 mg/kg bw, the observed change in relative organ weight of female gonads were considered to be non significant. In addition,No gross abnormalities and histopathological changes were observed in Gonads of treated male and female rat as compared to control.Therefore, NOAEL was considered to be 244 mg/kg body weight/day for male and 288 mg/kg bw for female when CFE male and female rat treated with test material orally in feed for 90 days.

In another study, Combined repeated dose repro-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test material in Wistar rats. 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 any of the groups of animals 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 repective 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 item 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 either sex 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 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. 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. Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material 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) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

Thus, based on the above data, the test chemical did not show any reproductive toxicity, and thus is not likely to classify as a 'Reproductive Toxicant' according to CLP criteria of classification.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

A Developmental study of test chemical was performed on male and female rats. No treatment related effects on the reproductive and systemic parameters were observed during the study. Hence, No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw when male and female wistar rats were treated with test material orally.

Link to relevant study records

Reference

Endpoint:

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:

guideline study

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar 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: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

The objective of this study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test material in Wistar rats.

GLP compliance:

yes

Limit test:

no

Species:

other: 2. rat 3.mouse

Strain:

other: 2. Wistar 3.CD-1

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 was offered ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: A conventional laboratory pelleted diet was offered. Aqua guard filtered drinking water in bottles was offered.

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.Details on test animal
TEST ANIMALS
- Source: Charles River Breeding Laboratory Inc. (Kingston, N.Y.).
- Age at study initiation: (P) x wks; (F1) x wks: F0: 11 weeks
F1: 70 ± 10 days
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: F0: male- 34.5 ± 0.58 to 35.6 ± 0.76g , Female: 26.3 ± 0.66 to 27.6 ± 0.60g

F1: male- 1.65 ± 0.014 to 1.60 ± 0.023g, female: 1.53 ± 0.016 to 1.60 ± 0.014g

- Fasting period before study: No data available
- Housing: Animals were housed 2 per Polycarbonate shoebox type cages (5" x 11" x 7") with stainless steel wire bar lids, one of these animals will be punched in the left ear for identification purposes. Cages will be uniformly placed on stainless steel racks (66" x 60" x 30"). Cages will be rotated (relative placement) at least once a week. Bedded on Ab-Sorb-Dri , Approximately 100g of bedding will be used per cage.
- Diet (e.g. ad libitum): Purina certified Rodent Chow animal diet #5002, ad libitum.
- Water (e.g. ad libitum): Water, ad libitum.
- Acclimation period: F0: 1 week , F1: five weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25° C
- Humidity (%):20 to 70%
- Air changes (per hr): 10 or more air changes per hour of HEPA-filtered air.
- Photoperiod (hrs dark / hrs light): 14 hour light cycle

IN-LIFE DATES: From: 4/20/82
To: 8/25/82

Route of administration:

oral: gavage

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.Details on exposure

PREPARATION OF DOSING SOLUTIONS: 0.1, 0.25 and 0.5 g/kg test chemical were mixed with corn oil directly as Methyl salicylate is completely miscible with corn oil.

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): A preliminary stability study in feed showed losses of ~ 7% of the chemical after 1 day of storage at -20°C. Because of these losses, all subsequent chemical/vehicle studies were performed in a corn oil vehicle. Water was not used because the chemical is only slightly soluble (< 1 mg/ml) in water.
- Concentration in vehicle: 0.1, 0.25 and 0.5 g/kg/day (0, 100, 250 and 500 mg/kg/day)
- Amount of vehicle (if gavage): 30 mg/ml
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2.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.not specified

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.
- 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.- M/F ratio per cage: 1: 1 ratio
- Length of cohabitation: 100 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy No data available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: Litters produced during the cohabitation period (day 7 to day 107) will be humanely sacrificed. Litters produced during the period day 107 to day 127 will be allowed to remain with their mothers

Duration of treatment / exposure:

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.
3.127 days

Frequency of treatment:

Daily

Duration of test:

2. 64 days
3. 127 days

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: 200
0 mg/kg/day: 40 male, 40 female
100 mg/kg/day: 20 male, 20 female
250 mg/kg/day: 20 male, 20 female
500 mg/kg/day: 20 male, 20 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. Not specified

Maternal 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.Survival, clinical sign, Body weight were observed .

Ovaries and uterine content:

2.The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:

3.No data availavble

Fetal examinations:

2.- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes:
- Head examinations: No data

3.Number and percent of live pups per litter and Mean body weight of live offspring, Proportion of pups born alive and sex of pups born alive were observed

Statistics:

2.Raw data was analysed using statistical software “Sigma Plot 11.0”. 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.Statistical analysis wre performed by using Pairwise comparisons for Fertility oT Pairs During Continuous Breedins, Litters per Fertile Pair and Litter Size (Live offsprins) per Fertile Pair by using Chise square Pairwise comparisons .

Indices:

2.Pregnancy index/fertility index was determined
3.Fertility, No. Fertile/ No. Cohabited and litters per pair were observed.

Historical control data:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

2.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 treatment related sign of toxicity were observed in treated rats as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2.No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.
3.Whe treated with 100 and 250 mg/kg/day, 2 male and 2 female were died as compared to control.
Whe treated with 500 mg/kg/day, 4 rats were died as compared to control.

The cause of death varied from case to case but it was neither chemical nor dose related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

2.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 effect was observed body weight of treated rats as compared to control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

2.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.not specified

Food efficiency:

no effects observed

Description (incidence and severity):

2.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.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.not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.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 statistically 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.
3.not specified

The above changes were inconsistent, not dose dependent hence considered as incidental in nature.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

2.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 respective 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.not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

2.At the end of treatment and recovery period, absolute and relative weight of organs of treated rats of either sex did not differ significantly except a significant increase in relative wieght of Adrenal of G4-R male group when compared to the respective control group rats.
3.not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.External Findings: External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance.

Internal Findings: Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.
3.not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.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);

Adrenals: 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 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.
3.not specified

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.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

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

2.not specified

Changes in number of pregnant:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

2.Pregnancy index was found 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.Reproductive performance:
No effect on Fertility Index were observed on treated rats as compared to control.
Significant decrase in litters per pair, proportion of pups born alive and sex of pups born alive when treated with 500 mg/kg/day as compared to control.

Dose descriptor:

NOAEL

Remarks:

2.

Effect level:

556 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: decrease in pregancy index was observed in 1000mg/kg bw dose group

Dose descriptor:

NOAEL

Remarks:

3

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Localisation:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

2.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.Significant decrase in live pup weight and adjusted live pup weight was observed in 500 mg/kg/day treated rats as compared to control.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

2.Pups sex ratio (Male/Female) was found to be 55/57, 33/40, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.
3. Not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

2.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

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

2.

Effect level:

556 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

Remarks on result:

other: No developmental toxic effects was observed

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Not specified

Remarks on result:

other: Not specified

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg bw when male and female wistar rats were treated with test material orally.

Executive summary:

Developmental Toxicity Study:

The developmental toxicity study of the test chemical is as follows:

Combined repeated dose and reproductive-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of the test material in Wistar rats. 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 any of the groups of animals 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 repective 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 item 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 either sex 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 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. 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.Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material 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) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

In a developmental toxicity study, CD-1 a1bino male and female mice were treated wtih test chemical in the concentration of 0, 100, 250 and 500 mg/kg/day by oral gavage. 2 male and 2 female were died in 100 and 250 mg/kg/day dose group and 4 rats were died in 500 mg/kg/day dose group as compared to control. The cause of death varied from case to case but it was neither chemical nor dose related. No treatment related sign of toxicity and change in body weight were observed ion treated rats as compared to control. No effect on Fertility Index were observed but Significant decrease in litters per pair, proportion of pups born alive and sex of pups born alive when treated with 500 mg/kg/day as compared to control. In addition, significant decrease in number of live pups, live pup weight and adjusted live pup weight was observed in 500 mg/kg/day treated rats as compared to control. Therefore, NOAEL was considered to be 250 mg/kg/dy for F0 and F1 generation when CD-1 a1bino male and female mice were treated with test chemical for 127 days.

Thus, based on the above data, the test chemical did not show any developmental toxicity, and thus is not likely to classify as a 'Developmental Toxicant' according to CLP criteria of classification.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

556 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is Klimish rating 2

Additional information

Developmental Toxicity Study:

The developmental toxicity study of the test chemical is as follows:

Combined repeated dose and reproductive-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of the test material in Wistar rats. 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 any of the groups of animals 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 repective 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 item 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 either sex 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 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. 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.Based on the findings of Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity of test material 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) is considered to be 556 mg/kg bw. When male and female wistar rats were treated with test material orally.

In a developmental toxicity study, CD-1 a1bino male and female mice were treated wtih test chemical in the concentration of 0, 100, 250 and 500 mg/kg/day by oral gavage. 2 male and 2 female were died in 100 and 250 mg/kg/day dose group and 4 rats were died in 500 mg/kg/day dose group as compared to control. The cause of death varied from case to case but it was neither chemical nor dose related. No treatment related sign of toxicity and change in body weight were observed ion treated rats as compared to control. No effect on Fertility Index were observed but Significant decrease in litters per pair, proportion of pups born alive and sex of pups born alive when treated with 500 mg/kg/day as compared to control. In addition, significant decrease in number of live pups, live pup weight and adjusted live pup weight was observed in 500 mg/kg/day treated rats as compared to control. Therefore, NOAEL was considered to be 250 mg/kg/dy for F0 and F1 generation when CD-1 a1bino male and female mice were treated with test chemical for 127 days.

Thus, based on the above data, the test chemical did not show any developmental toxicity, and thus is not likely to classify as a 'Developmental Toxicant' according to CLP criteria of classification.

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation, thetest chemicalis not likely to classify as reproductive and Developmental toxicant.