Phenethyl phenylacetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity

Based on the data available from different studies and applying the weight of evidence approach, NOAEL was considered to be 500 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

chronic toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

Reproductive toxicity study of test chemical was performed on Osborne-Mendel rats.

GLP compliance:

not specified

Limit test:

yes

Justification for study design:

No data available

Species:

rat

Strain:

Osborne-Mendel

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing:The animals were housed individually in wire cages
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS: test material soluble in corn oil.
DIET PREPARATION
- Rate of preparation of diet (frequency):Fresh diets were made and distributed weekly
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0,500 mg/kg bw/day
- Amount of vehicle (if gavage): 1ml of solution/kg daily

- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

17 weeks

Frequency of treatment:

Daily

Details on study schedule:

No data available

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Total:40
0 mg/kg bw/day: 10 male and 10 female
500.00 mg/kg bw/day: 10 male and 10female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule: every week.


BODY WEIGHT: Yes
Time schedule for examinations: every week.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes Food consumption was determined every week.

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


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

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

SACRIFICE:At the termination of the experiments the rats were sacrificed and exsanguinated.

- Gross necropsy : yes

HISTOPATHOLOGY / ORGAN WEIGHTS: The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera
were removed and the liver, kidneys, spleen, heart, and testes were weighed. These organs, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Tissues from rats dying during the experiment were examined for gross changes and were preserved if autolysis was not advanced. Organs were not weighed but abnormalities and the suspected reason for death were noted.

Postmortem examinations (offspring):

No data available

Statistics:

No data available

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No effects on reproductive organ weight

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No effects on reproductive organ was noted

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:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Remarks on result:

not measured/tested

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) for reproductive toxicity was considered to 500mg/kg/day, When male and female Osborne-Mendel rats were treated with test chemical orally for 17weeks.

Executive summary:

The reproductive toxicity of the test chemical was considered on the basis of repeated dose toxicity study.Groups of 10 male and 10 female Osbourne-Mendel rats were provided test chemical mixed in the diet at concentrations of 0, 1000, 2500 or 10,000 ppm which corresponding to an average daily intakes of 0, 50, 250, or 500 mg/kg bw per for 17 weeks. The animal’s weight, food intake and general condition were recorded every week. Haematological examinations were made at termination of the subacute studies. These examinations included white cell counts, red cell counts, haemoglobins and haematocrits. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. These organs, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Measurement of body weight and food intake recorded weekly showed no significant difference between test and control animals at any intake level. At termination, hematological examinations revealed no effects due to administration of the test chemical. At necropsy, no differences were reported in major organ weights between test and control animals. Gross examination of tissue of all animals was unremarkable and histopathological examination of six-eight animals, equally represented by gender, for the high-dose group and the control group revealed no treatment-related lesions. Hence, the highest dose tested i.e 500 mg/kg/day can be considered as the NOAEL

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Various studies have been reviewed to determine the toxicity of the test chemical to maternal and offsprings. These include in vivo experimental studies performed on rats for the test chemical. The results are mentioned below:

 

Study 1: The reproductive toxicity of the test chemical was considered on the basis of repeated dose toxicity study.Groups of 10 male and 10 female Osbourne-Mendel rats were provided test chemical mixed in the diet at concentrations of 0, 1000, 2500 or 10,000 ppm which corresponding to an average daily intakes of 0, 50, 250, or 500 mg/kg bw per for 17 weeks. The animal’s weight, food intake and general condition were recorded every week. Haematological examinations were made at termination of the subacute studies. These examinations included white cell counts, red cell counts, haemoglobins and haematocrits. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. These organs, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Measurement of body weight and food intake recorded weekly showed no significant difference between test and control animals at any intake level. At termination, hematological examinations revealed no effects due to administration of the test chemical. At necropsy, no differences were reported in major organ weights between test and control animals. Gross examination of tissue of all animals was unremarkable and histopathological examination of six-eight animals, equally represented by gender, for the high-dose group and the control group revealed no treatment-related lesions. Hence, the highest dose tested i.e 500 mg/kg/day can be considered as the NOAEL.

 

Study 2: Combined repeated dose repro-developmental toxicity study was conducted to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The study was performed as per OECD 422 Guidelines.The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control), 308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups. Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed in any of the groups throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the respective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of both sexes did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of test chemical at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical. Based on the findings of repeated dose oral toxicity study in combination with reproduction/ developmental toxicity of test chemical in Wistar rats with 14 days recovery, where in 0, 308, 556 and 1000 mg/kg body weight, doses were tested; No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw, when male and female wistar rats were treated with test chemical orally.

 

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

 

Based on the data available from different studies and applying the weight of evidence approach, NOAEL was considered to be 500 mg/kg/day for reproductive toxicity, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

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:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.

Details on mating procedure:

- 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

Duration of treatment / exposure:

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.

Frequency of treatment:

Daily

Duration of test:

64 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1 (Control Group)

Dose / conc.:

308 mg/kg bw/day

Remarks:

G2 (Low Dose Group)

Dose / conc.:

556 mg/kg bw/day

Remarks:

G3 (Mid Dose Group)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4 (High Dose Group)

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1R (Control Recovery Group)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4R (Recovery High Dose Group)

No. of animals per sex per dose:

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 vehicle

Details on study design:

- 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

Maternal examinations:

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.

Ovaries and uterine content:

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:

Fetal examinations:

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

Statistics:

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

Indices:

Pregnancy index/fertility index was determined

Historical control data:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

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):

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):

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

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):

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):

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.

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):

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.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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 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):

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:

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):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

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

Abnormalities:

not specified

Localisation:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

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.

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):

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)

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

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

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

 Table 1 Mortality and Morbidity

Sex: Male

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	308	13	NMM
G3	Mid	556	13	NMM
G4	High	1000	13	NMM
G1-R	Control- Recovery	0	5	NMM
G4-R	High- Recovery	1000	5	NMM

 

Sex: Female

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	308	13	NMM
G3	Mid	556	13	NMM
G4	High	1000	13	NMM
G1-R	Control -Recovery	0	5	NMM
G4-R	High- Recovery	1000	5	NMM

Keys:NMM = No mortality and morbidity observed, No.= Num

Table 2 Clinical Signs and Symptoms

Sex: Male

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Clinical Sign	Incidences During Study period
G1	Control	0	13	Normal	13/13
G2	Low	308	13	Normal	13/13
G3	Mid	556	13	Normal	13/13
G4	High	1000	13	Normal	13/13
G1-R	Control -Recovery	0	5	Normal	5/5
G4-R	High- Recovery	1000	5	Normal	5/5

 

   Sex: Female

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Clinical Sign	Incidences During Study period
G1	Control	0	13	Normal	13/13
G2	Low	308	13	Normal	13/13
G3	Mid	556	13	Normal	13/13
G4	High	1000	13	Normal	13/13
G1-R	Control -Recovery	0	5	Normal	5/5
G4-R	High- Recovery	1000	5	Normal	5/5

Key:No.= Number

Table 3 Summary of Detailed Clinical Examinations

Week:Pre-Treatment                                                                                    Sex:Male

Group (N)		G1 (13)	G2 (13)	G3 (13)	G4 (13)	G1-R (5)	G4-R (5)
Dose (mg/kg body weight)		0	308	556	1000	0	1000
Parameter	Observation	No. of Animals Showing Observation
Posture	Curled up, often asleep	1	3	2	3	1	1
	Sitting B	0	2	2	2	0	0
	Sitting C	1	2	1	1	2	1
	Sitting A	6	2	2	3	1	2
	Rearing	5	4	6	4	1	1
Convulsions	Absent	13	13	13	13	5	5
Ease of removing from the cage	Very easy	11	11	12	10	4	5
	Easy	2	2	1	3	1	0
	Moderately difficult	0	0	0	0	0	0
Handling reactivity	Easy	13	13	13	13	5	5
	Moderately easy	0	0	0	0	0	0
Palpebral closure	Eyelids wide open	13	13	13	13	5	5
Lacrimation	None (No lacrimation)	13	13	13	13	5	5
Eye Examination	Absent	13	13	13	13	5	5
Piloerection	Absent	13	13	13	13	5	5
Skin Examination	Absent	13	13	13	13	5	5
Salivation	None	13	13	13	13	5	5
Gait	Normal	13	13	13	13	5	5
Mobility	Normal	13	13	13	13	5	5
Arousal	Normal	13	13	13	13	5	5
Respiration	Normal	13	13	13	13	5	5
Tonic Movement	Absent	13	13	13	13	5	5
Clonic Movement	Absent	13	13	13	13	5	5
Stereotypy	Absent	13	13	13	13	5	5
Bizzare Behaviour	Absent	13	13	13	13	5	5
Number of Rears	Mean	11.5	10.2	7.5↓	7.2↓	7.2	6.4
Vocalization Count	Mean	0.0	0.0	0.0	0.0	0.0	0.0
No. of urine pools	Mean	1.2	0.9	3.5↑	4.2↑	3.6	3.8
No. of faecal bolus	Mean	1.2	0.5	3.8↑	1.8	2.4	1.4

Keys:N = Number of animals in group, No. = Number,↓ = Statistically Significant Decrease (atp < 0.05),↑=Statistically Significant Increase (atp < 0.05)

 

Table 3 Summary of Detailed Clinical Examinations Continued

Week:Pre-Treatment                                                                                    Sex:Female

Group (N)		G1 (13)	G2 (13)	G3 (13)	G4 (13)	G1-R (5)	G4-R (5)
Dose (mg/kg body weight)		0	308	556	1000	0	1000
Parameter	Observation	No. of Animals Showing Observation
Posture	Curled up, often asleep	3	4	2	2	1	1
	Sitting B	2	1	4	2	1	1
	Sitting C	2	0	1	1	1	0
	Sitting A	2	2	3	1	1	1
	Rearing	4	6	3	7	1	2
Convulsions	Absent	13	13	13	13	5	5
Ease of removing from the cage	Very easy	10	12	12	11	4	4
	Easy	3	1	1	2	1	1
	Moderately difficult	0	0	0	0	0	0
Handling reactivity	Easy	13	13	13	13	5	5
	Moderately easy	0	0	0	0	0	0
Palpebral closure	Eyelids wide open	13	13	13	13	5	5
Lacrimation	None (No lacrimation)	13	13	13	13	5	5
Eye Examination	Absent	13	13	13	13	5	5
Piloerection	Absent	13	13	13	13	5	5
Skin Examination	Absent	13	13	13	13	5	5
Salivation	None	13	13	13	13	5	5
Gait	Normal	13	13	13	13	5	5
Mobility	Normal	13	13	13	13	5	5
Arousal	Normal	13	13	13	13	5	5
Respiration	Normal	13	13	13	13	5	5
Tonic Movement	Absent	13	13	13	13	5	5
Clonic Movement	Absent	13	13	13	13	5	5
Stereotypy	Absent	13	13	13	13	5	5
Bizzare Behaviour	Absent	13	13	13	13	5	5
Number of Rears	Mean	6.2	10.0↑	11.2↑	10.5↑	10.4	11.2
Vocalization Count	Mean	0.0	0.0	0.0	0.0	0.0	0.0
No. of urine pools	Mean	1.0	1.3	1.2	1.1	1.2	1.8
No. of faecal bolus	Mean	0.9	0.8	1.2	1.2	1.2	1.0

Keys:N = Number of animals in group, No.= Number,↑= Statistically Significant Increase (atp < 0.05)

Table 3 Summary of Detailed Clinical Examinations Continued

Week:1^st                                                                                                                   Sex:Male

Group (N)		G1 (13)	G2 (13)	G3 (13)	G4 (13)	G1-R (5)	G4-R (5)
Dose (mg/kg body weight)		0	308	556	1000	0	1000
Parameter	Observation	No. of Animals Showing Observation
Posture	Curled up, often asleep	3	3	6	5	2	2
	Sitting B	8	8	6	7	3	2
	Sitting C	1	2	1	1	0	1
	Sitting A	1	0	0	0	0	0
	Rearing	0	0	0	0	0	0
Convulsions	Absent	13	13	13	13	5	5
Ease of removing from the cage	Very easy	11	12	13	10	5	5
	Easy	2	0	0	3	0	0
	Moderately difficult	0	1	0	0	0	0
Handling reactivity	Easy	13	12	12	13	5	5
	Moderately easy	0	1	1	0	0	0
Palpebral closure	Eyelids wide open	13	13	13	13	5	5
Lacrimation	None (No lacrimation)	13	13	13	13	5	5
Eye Examination	Absent	13	13	13	13	5	5
Piloerection	Absent	13	13	13	13	5	5
Skin Examination	Absent	13	13	13	13	5	5
Salivation	None	13	13	13	13	5	5
Gait	Normal	13	13	13	13	5	5
Mobility	Normal	13	13	13	13	5	5
Arousal	Normal	13	13	13	13	5	5
Respiration	Normal	13	13	13	13	5	5
Tonic Movement	Absent	13	13	13	13	5	5
Clonic Movement	Absent	13	13	13	13	5	5
Stereotypy	Absent	13	13	13	13	5	5
Bizzare Behaviour	Absent	13	13	13	13	5	5
Number of Rears	Mean	5.7	6.3	5.5	7.3	6.0	6.2
Vocalization Count	Mean	0.0	0.1	0.0	0.0	0.0	0.0
No. of urine pools	Mean	2.9	2.8	3.2	2.7	3.0	4.6
No. of faecal bolus	Mean	1.5	1.4	2.4	1.8	1.4	1.6

Keys:N = Number of animals in group, No.= Number

 Table 3 Summary of Detailed Clinical Examinations Continued

Week:1^st                                                                                                                Sex:Female

Group (N)		G1 (13)	G2 (13)	G3 (13)	G4 (13)	G1-R (5)	G4-R (5)
Dose (mg/kg body weight)		0	308	556	1000	0	1000
Parameter	Observation	No. of Animals Showing Observation
Posture	Curled up, often asleep	0	5	8	6	0	1
	Sitting B	5	5	4	5	3	2
	Sitting C	4	2	1	1	2	2
	Sitting A	4	1	0	1	0	0
	Rearing	0	0	0	0	0	0
Convulsions	Absent	13	13	13	13	5	5
Ease of removing from the cage	Very easy	11	13	11	13	5	4
	Easy	2	0	2	0	0	1
	Moderately difficult	0	0	0	0	0	0
Handling reactivity	Easy	11	13	10	13	5	4
	Moderately easy	2	0	3	0	0	1
Palpebral closure	Eyelids wide open	13	13	13	13	5	5
Lacrimation	None (No lacrimation)	13	13	13	13	5	5
Eye Examination	Absent	13	13	13	13	5	5
Piloerection	Absent	13	13	13	13	5	5
Skin Examination	Absent	13	13	13	13	5	5
Salivation	None	13	13	13	13	5	5
Gait	Normal	13	13	13	13	5	5
Mobility	Normal	13	13	13	13	5	5
Arousal	Normal	13	13	13	13	5	5
Respiration	Normal	13	13	13	13	5	5
Tonic Movement	Absent	13	13	13	13	5	5
Clonic Movement	Absent	13	13	13	13	5	5
Stereotypy	Absent	13	13	13	13	5	5
Bizzare Behaviour	Absent	13	13	13	13	5	5
Number of Rears	Mean	10.5	9.9	10.2	10.4	10.2	10.2
Vocalization Count	Mean	0.0	0.0	0.0	0.0	0.0	0.0
No. of urine pools	Mean	2.0	1.2	1.5	1.3	2.2	1.4
No. of faecal bolus	Mean	1.1	1.2	1.1	1.4	1.6	1.2

Keys:N = Number of animals in group, No.= Number

Table 3 Summary of Detailed Clinical Examinations Continued

Week:7^th                                                                                           

Sex		Male		Female
Group (N)		G1-R (5)	G4-R (5)	G1-R (5)	G4-R (5)
Dose (mg/kg body weight)		0	1000	0	1000
Parameter	Observation	No. of Animals Showing Observation
Posture	Curled up, often asleep	2	3	2	3
	Sitting B	1	1	2	1
	Sitting C	1	0	1	0
	Sitting A	1	1	0	1
	Rearing	0	0	0	0
Convulsions	Absent	5	5	5	5
Ease of removing from the cage	Very easy	4	3	4	2
	Easy	1	2	1	3
	Moderately difficult	0	0	0	0
Handling reactivity	Easy	5	4	3	3
	Moderately easy	0	1	2	2
Palpebral closure	Eyelids wide open	5	5	5	5
Lacrimation	None (No lacrimation)	5	5	5	5
Eye Examination	Absent	5	5	5	5
Piloerection	Absent	5	5	5	5
Skin Examination	Absent	5	5	5	5
Salivation	None	5	5	5	5
Gait	Normal	5	5	5	5
Mobility	Normal	5	5	5	5
Arousal	Normal	5	5	5	5
Respiration	Normal	5	5	5	5
Tonic Movement	Absent	5	5	5	5
Clonic Movement	Absent	5	5	5	5
Stereotypy	Absent	5	5	5	5
Bizzare Behaviour	Absent	5	5	5	5
Number of Rears	Mean	4.2	5.6	9.6	11.2
Vocalization Count	Mean	0.0	0.0	0.0	0.0
No. of urine pools	Mean	2.0	0.6	2.0	3.2
No. of faecal bolus	Mean	2.8	1.2	1.2	1.4

Keys:N = Number of animals in group, No.= Number

Table 4 Mean Body Weight (g)

Sex: Male

Group (N)	G1 (13)		G2 (13)		G3 (13)		G4 (13)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 1	247.38	11.20	262.38	20.32	261.62	18.86	261.92	17.03
Day 8	282.31	9.33	294.92	19.40	289.31	20.12	278.69	19.98
Day 14	315.00	13.00	324.38	21.35	316.15	20.45	299.15	21.37
Day 21	333.15	15.35	339.46	25.50	336.23	22.65	316.23	18.79
Day 28	350.08	18.06	362.62	30.37	358.54	27.49	334.15	20.60
Day 30	355.77	18.46	368.00	30.44	364.54	27.22	331.62↓	19.88
Day 37	370.77	22.36	381.92	30.46	381.77	31.58	351.62	24.13
Day 44	393.31	26.08	407.69	34.24	402.23	34.23	368.23	26.45
Day 46	397.23	24.79	414.77	34.07	405.38	34.10	370.92	26.71
Day 47 (Fasting)	372.00	25.57	391.08	33.83	383.38	33.84	350.15	26.15

 

Period: Pre-mating                                                                                                     Sex: Female

Group (N)	G1 (13)		G2 (13)		G3 (13)		G4 (13)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 1	226.62	6.50	231.54	7.34	230.15	6.73	228.77	7.90
Day 8	229.85	8.37	233.46	7.85	233.54	9.00	227.92	7.39
Day 14	232.77	8.32	236.92	8.23	237.00	9.65	232.62	8.01

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05). 

Table 4 Mean Body Weight (g) Continued

Period: Gestation                                                                                                   Sex: Female

Group (N)	G1 (12)		G2 (11)		G3 (11)		G4 (8)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 0	232.83	10.14	236.36	9.15	237.64	8.02	231.13	6.98
Day 7	245.25	18.20	249.91	8.83	251.36	9.75	237.13	8.56
Day 14	268.58	26.15	268.55	7.10	275.36	15.50	248.50	9.89
Day 20	302.00	37.56	302.45	13.13	310.64	27.21	264.50↓	11.74

Period: Post-Partum                                                                                              Sex: Female

Group (N)	G1 (12)		G2 (11)		G3 (11)		G4 (8)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 0/1	240.83	25.04	254.36	26.47	252.27	19.08	228.63	15.00
Day 4	242.58	24.39	251.27	26.88	256.27	22.95	225.50	15.12
Day 5 (Fasting)	226.83	20.94	236.18	24.64	240.00	20.97	214.25	13.44

Keys:N= Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically   Significant Decrease (atp<0.05)

Table 6 Mean Feed Consumption (g/day/animal)

Sex: Male

Group (N)	G1 (13)		G2 (13)		G3 (13)		G4 (13)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 1-8	23.01	1.29	22.97	1.66	22.33	1.67	20.99	1.93
Day 8-14	24.39	1.69	23.48	0.90	22.81	1.55	22.52	1.10
Day 30-37	23.71	1.20	23.19	1.22	23.50	1.64	23.52	1.38
Day 37-44	29.33	3.03	28.84	4.00	28.07	4.45	26.89	4.34
Day 44-46	26.63	1.27	25.45	1.73	25.18	0.98	25.15	1.92

 

 

 

Period: Pre-mating                                                                                                      Sex: Female

Group (N)	G1 (13)		G2 (13)		G3 (13)		G4 (13)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 1-8	12.76	1.21	13.08	0.41	13.15	0.85	11.80	0.68
Day 8-14	11.23	1.04	11.10	0.67	11.80	0.48	10.71	0.96

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,

Table 6 Mean Feed Consumption (g/day/animal) Continued

Period: Gestation                                                                                                      Sex: Female

Group (N)	G1 (12)		G2 (11)		G3 (11)		G4 (8)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 0-7	15.93	3.64	16.18	2.29	16.16	3.06	13.55	1.59
Day 7-14	17.96	3.53	18.26	1.76	18.05	2.50	15.29	1.97
Day 14-20	19.46	4.85	17.44	1.98	17.23	3.35	13.71↓	1.35

 

Period: Gestation and Post-Partum                                                                        Sex: Female

Group (N)	G1 (12)		G2 (11)		G3 (11)		G4 (8)
Dose (mg/kg b. wt.)	0		308		556		1000
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Day 20 -Day 5 PP	18.40	5.22	18.82	4.69	18.25	5.56	13.12	2.06

Keys:g= gram, SD = Standard deviation, N = Number of animals in group, PP= Post Partum↓= Statistically Significant Decrease (atp<0.05)

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:

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 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

556 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Study 1: 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 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.

 

Study 2: Reproductive and development toxicity study of test chemical was performed on mated female SpragueDawley rats according to OECD Test Guideline 414.The test material73.5% & 26.5% biphenyl mixed in corn oil at volume of 5 ml/kg were administered in dose concentration 0,50, 200, 500 mg/kg/day on gestation days 6-15by oral gavage route..96 rats were divided as 24 rats /dose group .Animals was checked daily for clinical signs. Food consumption and body weight were recorded; the dams were sacrificed and subjected to a macroscopic examination. Approximately 1/2 of the fetuses in each litter were processed for soft-tissue evaluations while the other half for skeletal evaluations. Statistical evaluation of equality of means was made by the appropriate one-way analysis of variance technique (ANOVA) for parametric procedures and Kruskal-Wallis test for nonparametric procedures were used after applying Bartlett's test for determination of equal variance. Statistical tests for trend, using either standard regression techniques (parametric cases) or Jonckheere's test in nonparametric cases. Levels of statistical significance used were either p<0.05 or p<0.01. Two deaths occurred at 500 mg/kg. Statistically reduced maternal weight gain and food consumption were observed at 200 and 500 mg/kg/d. Excessive alopecia, salivation and/or anogenital staining was observed but no pattern of treatment relationship could be determined. No effects observed on fetal resorptions, fetal viability, postimplantation loss or total implantations. Mean litter weights in treated and control groups were similar. No significant increases were observed in incidence of malformations or variations at any treatment level.As no indications of any influence on reproductive parameters or damage to reproductive organs were found and No developmental toxic effects were seen up to the highest dose of 500 mg/kg bw. Hencethe dose-level of 500 mg/kg/day was considered to be the NOAEL for embryo-foetal toxicity and reproductive toxicity .When femaleSprague Dawley rats were treated with test chemical orally.

Justification for classification or non-classification

Based on the data available from different studies and applying the weight of evidence approach, NOAEL was considered to be 500 mg/kg/day for reproductive toxicity and 556 mg/kg/day for developmental toxicitya, when rodents were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation, the test chemical is not likely to classify as reproductive toxicant.