Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

β-bromostyrene

EC number: 203-131-3 | CAS number: 103-64-0

Life Cycle description
Uses advised against

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:: developmental toxicity
Remarks:: Combined Repeated and Reproductive/Developmental Screening Test
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Justification for type of information:: Weight of evidence approach based on the data of test chemicals.

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

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:

Repeated Dose Oral Toxicity Study in combination with Reproduction / Developmental Toxicity study of test chemical in Wistar Rats

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- Source: National Institute of Biosciences
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation
- Weight at study initiation:- Fasting period before study
- Housing:Cages were cleaned at regular intervals.A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cagerotation was carried out weekly during study period except during mating and during gestation and lactation only 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.Bedding material of batch No. 8-17 (Krishana Corncob Industries, Aurangabad) was used in this study
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.40 °C
- Humidity (%): 40.50 to 64.30%
- Air changes (per hr): minimum 12 times per hour and filtered adequately.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: December 08, 2017To: April 28, 2018

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (Distilled water) 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):
- Mixing appropriate amounts with (Type of food):- Storage temperature of food:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 250, 500 and 750 mg/kg bw
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required):- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analysis by HPLC-UV

Details on mating procedure:

- M/F ratio per cage: one male and one female - Length of cohabitation: 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: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): housed individually - Any other deviations from standard protocol:

Duration of treatment / exposure:

Approx. 64 days

Frequency of treatment:

Daily

Duration of test:

Approx. 64 days

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Total: 124
0 mg/kg bw, 13 male, 13 female
250 mg/kg bw, 13 male, 13 female
500 mg/kg bw, 13 male, 13 female
750 mg/kg bw, 13 male, 13 female

Recovery group:
0 mg/kg bw, 5 male, 5 female
750 mg/kg bw, 5 male, 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels 250, 500 and 750 mg/kg body weight were selected for theMain Study based on the results of Dose Range Finding (DRF).- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. Individual body weights were considered within ±20% of the groups mean.- Rationale for selecting satellite groups: 0 and 750 mg/kg bw were selected as satellite groups- Post-exposure recovery period in satellite groups: 14 days- Section schedule rationale (if not random):

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
-Time schedule: Twice daily (morning and evening)- Cage side observations included.: Morbidity and mortality, throughout the acclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a day, preferably at the same time each day
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), on day 4 and day 13post-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- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY: No data- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weightedaverages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data- Time schedule for examinations: No data
OPHTHALMOSCOPIC EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No data
HAEMATOLOGY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters 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) and Activated Partial Thromboplastin time (aPTT) were examined.

CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters 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), Globulin (Glob), Alb/ Glb (A:G) and Bile acids were examined.URINALYSIS: No data- Time schedule for collection of urine: No data- Metabolism cages used for collection of urine: No data- Animals fasted: No data- Parameters checked in table [No.?] were examined. No dataNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Ovaries and uterine content:

Estrous cycle, Post-implantation loss and Post-natal loss were examined.

Fetal examinations:

Litter size, No. of live births, pups weight at birth and PND14 and Pups sex ratio were examined.

Statistics:

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 Dun nett’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:

Gestational length, Survival Index of pups, Pregnancy index and Pups sex ratio were examined.

Historical control data:

not specified

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. 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 increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent, 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 animals of the control and treatment groups throughout the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When treated with 500 mg/kg bw, statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 as compared to control group.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):

no effects observed

Description (incidence and severity):

No treatment related changes were observed in treated male and female rats as compared to control.

Food efficiency:

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):

At the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight.The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were consid ered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observation battery/neurobehavioral observation were comparable and no changes were r evealed i any of the animals of all the treated groups in both the sexesThe 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. However in recovery male,statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group.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. Howerver, statistically significant increase in female were noted in Distance travelled (DT),Ambulatory time (AT), and in Horizontal counts (HC)at 750 mg/kg body weight when compare to control group.The above changes observed were not dose dependent, hence considered as incidental and notattributed 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 group rats of either sex did not differ significantly when compared to respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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. 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:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No Pre and Post-implantation loss were observed in treated rats as compared to control.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No effect on Gestational length were observed in treated rats as compared to control.

Changes in number of pregnant:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (actual dose received)

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

gross pathology

haematology

histopathology: non-neoplastic

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

Remarks on result:

other: No effect observed

Abnormalities:

not specified

Localisation:

not specified

Description (incidence and severity):

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No effect on pups weight at birth and PND14 were observed as compared to control.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No effect on No. of live births were observed as compared to control.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effect on Pups sex ratio were observed as compared to control.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No effect on Litter size were observed as compared to control.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

No effect on Post-natal loss were observed as compared to control.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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 died during course of study revealed various lesions among the control and treated groups viz., external examination Cannibalism (Male: G1: 1/59, G2:13/60, G3: 7/81, Female:G1 :1/59, G2:14/81, G3:1/60, G4: 1/55 ; Tail absent (Anury) G3: 1/60 and internal examination Right skin swelling G1: 1/57.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (actual dose received)

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

changes in litter size and weights

changes in postnatal survival

external malformations

Remarks on result:

other: No effect observed

Abnormalities:

not specified

Localisation:

other: not specified

Description (incidence and severity):

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)	G1(11)		G2(12)		G3(13)		G4(12)
Dose(mg/kg bwt)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
No. of Live Births	11.80	1.93	12.00	2.95	9.85	3.60	10.58	1.68
No. of alive pups at Post-natal Day 4	10.90	3.00	8.33	5.57	9.08	4.37	10.42	1.51
Post-natal Loss (%)	7.85	19.84	33.02	38.93	18.72	37.26	1.28	4.44
Fetal Survival Index at Post-natal Day 4 (%)	92.15	19.84	66.98	38.93	81.28	37.26	98.72	4.44

Mean Gestational Length and Litter size

Group(N)	G1(11)		G2(12)		G3(13)		G4(12)
Dose(mg/kg bwt)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Gestation Length	22.36	0.50	22.00	0.00	22.00	0.00	22.25	0.45
Litter size (Total No. of litter size)	10.91	3.48	12.67	2.90	10.85	2.61	10.58	1.68

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Mean Pups Weight	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0	6.34	0.57	118	6.10	0.76	151	6.24	0.86	141	6.59	0.48	127
Day 4	9.76	1.78	109	8.56	1.12	100	9.11	1.08	118	9.50	1.45	125
Day 13	24.88	3.88	87	20.70	3.00	78	22.74	2.65	86	22.05	2.90	100
Group(n)	G1(11)			G2(12)			G3(13)			G4(13)
Pups Body Weight gain (%)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0-Day 4	47.42	20.65	109	36.29	13.99	99	41.58	13.79	118	43.66	15.12	125
Day 0-Day 13	148.89	19.84	87	143.22	25.24	78	144.89	19.45	86	135.74	22.14	100
Group (Number of Litter size)	G1(118)			G2(151)			G3(141)			G4(127)
Sex Ratio at birth (Male/Female)	61/57			72/79			80/61			71/56
Sex Ratio at Day 4 (Male/Female)	56/53			48/51			64/54			69/56
Gross Observations	NAD			NAD			NAD			NAD

MeanHormonal Analysis Data (Continued)

Day: 04 (Pups)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Dose(mg/kg bwt)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	2.17	0.29	10	2.04	0.42	11	2.37	0.49	11	2.13	0.31	12
TSH (uIU/mL)	1.76	0.32	10	1.84	0.51	11	1.84	0.81	11	1.82	0.56	12

Day: 13 (Pups)

Group(n)	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Dose(mg/kg bwt)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	5.87	0.88	9	5.08	0.60	9	5.60	0.70	10	5.64	0.82	12
TSH (uIU/mL)	2.16	1.13	9	1.88	0.49	9	2.05	0.60	10	2.14	0.69	12

Absolute Organ Weight (g) Pups

Sex:Male

Group (N)	G1 (9)		G2 (8)		G3 (10)		G4 (14)
Dose (mg/Kg)	0		250		500		750
Organ	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Thyroid With Parathyroids	0.0030	0.0004	0.0037	0.0015	0.0039	0.0008	0.0036	0.0009

Sex:Female

Group (N)	G1 (9)		G2 (9)		G3 (10)		G4 (14)
Dose (mg/Kg)	0		250		500		750
Organ	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Thyroid With Parathyroids	0.0038	0.0004	0.0035	0.0010	0.0043	0.0008	0.0040	0.0010

Gross Pathology Observations (Pups)

Sex: Male

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	44	40	51	55
Organ & Lesion
External Observations
No Abnormality Detected	44	39	51	55
Cannibalism	./.	1	./.	./.
Internal Observations
No Abnormality Detected	44	40	51	55

Sex: Female

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	51	65	62	47
Organ & Lesion
External Observations
No Abnormality Detected	50	65	61	47
Cannibalism	./.	./.	1	./.
Right skin swelling	1	./.	./.	./.
Tail absent (Anury)	./.	./.	1	./.
Internal Observations
No Abnormality Detected	50	65	62	47
Right pus swollen joint	1	./.	./.	./.

Gross Pathology Observations (Pups)

Sex: Male

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	44	40	51	55
Organ & Lesion
External Observations
No Abnormality Detected	44	39	51	55
Cannibalism	./.	1	./.	./.
Internal Observations
No Abnormality Detected	44	40	51	55

Sex: Female

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	51	65	62	47
Organ & Lesion
External Observations
No Abnormality Detected	50	65	61	47
Cannibalism	./.	./.	1	./.
Right skin swelling	1	./.	./.	./.
Tail absent (Anury)	./.	./.	1	./.
Internal Observations
No Abnormality Detected	50	65	62	47
Right pus swollen joint	1	./.	./.	./.

Microscopic Observations (Pups)

Sex: Female

Group	G1	G2	G3	G4	G1R	G4R
Dose (mg/kg)	0	250	500	750	0	750
Total Number of Animals Observed	21	-	-	21	-	-
Organ & Lesion
No Abnormality Detected	21	X	X	21	X	X

Conclusions:

NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Executive summary:

In a experimental study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test). Wistar male and female rat treated with test chemical in the concentration of 0, 250,500 and 750 mg/kg bw orally by gavage for more than 63 days. No mortality or morbidity was observed inany animals of the control and treatment groups throughout the study period. No apparent treatment relatedclinical signs were observed in any of the animals throughout the treatment. 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 increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. Statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 at 500 mg/kg bw as compared to control group. 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.

No treatment related changes in food consumption were observed in treated male and female rats as compared to control. Similarly, at the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance. At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight. The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response. The functional observation battery/neurobehavioral observation was comparable and no changes were revealed in any of the animals of all the treated groups in both the sexes. 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. However in recovery male, statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R). when compare to control recovery group. Motor activity measurements were comparable and no changeswere revealed in any of the animals from all treated groups of both the sexes as compare to control group. However, statistically significant increase in female were noted in Distance travelled (DT), Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group. The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. In addition, 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 respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous. 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. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Microscopic examination of control group and rats treated with 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs.

Lesions observed in liver, kidneys, lungs, adrenals and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship.Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. No effects on regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 female from G2, G3 and G4, respectively which showed precoital interval more than 5 days. There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. No developmental effect were observed such as Gestational length, Litter size, No. of live births, Post-implantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index and Pups sex ratio (Male/Female) respectively of treated rats as compared to control. No treatment related changes were noted in hormonal analysis (T4, TSH, Testosterone and Estrogen). Therefore, NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Cross-reference 2

Reason / purpose for cross-reference:: read-across source

Reference

Endpoint:: developmental toxicity
Type of information:: experimental study
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
Description (incidence and severity):: 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 the 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.
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 the 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.
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
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.
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
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 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.

Data source

Referenceopen all close all

Reference 1

Reference Type:: study report
Title:: Unnamed
Year:: 2018
Report date:: 2018

Reference 2

Reference Type:: study report
Title:: Unnamed
Year:: 2016
Report date:: 2016

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: other: OECD 422 (Combined Repeated and Reproductive/Developmenta Toxicity Screening Test)

Principles of method if other than guideline:: According to OECD 422 (Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test)
GLP compliance:: yes
Limit test:: no

Test material

Test material information

Test material form:: not specified
Details on test material:: - Name of test material: β-bromostyrene
- Molecular formula: C8H7Br
- Molecular weight: 183.0473 g/mole
- Substance type: Organic
- Physical state: Yellow clear liquid
- Impurities: 0.4%
- Purity: 99.6%

Test animals

Species:: other: Study 2: rat; Study 3: rat
Strain:: other: Study 2: Wistar; Study 3: Wistar
Details on test animals or test system and environmental conditions:: Study 2:
- Source: National Institute of Biosciences
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation
- Weight at study initiation:- Fasting period before study
- Housing:Cages were cleaned at regular intervals.A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cagerotation was carried out weekly during study period except during mating and during gestation and lactation only 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.Bedding material of batch No. 8-17 (Krishana Corncob Industries, Aurangabad) was used in this study
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.40 °C
- Humidity (%): 40.50 to 64.30%
- Air changes (per hr): minimum 12 times per hour and filtered adequately.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: December 08, 2017 To: April 28, 2018

Study 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 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

Administration / exposure

Route of administration:: other: Study 2: oral: gavage; Study 3: oral: gavage
Vehicle:: other: Study 2: water; Study 3: corn oil
Details on exposure:: Study 2:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (Distilled water) 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):
- Mixing appropriate amounts with (Type of food):- Storage temperature of food:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 250, 500 and 750 mg/kg bw
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required):
- Purity:

Study 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
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Study 2: Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analysis by HPLC-UV.

Study 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.
Details on mating procedure:: Study 2:
- M/F ratio per cage: one male and one female
- Length of cohabitation: 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: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): housed individually
- Any other deviations from standard protocol: N/A

Study 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.
- 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:: Study 2: Approx. 64 days
Study 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.
Frequency of treatment:: Study 2: Daily
Study 3: Daily
Duration of test:: Study 2: Approx. 64 days
Study 3: 64 days

Doses / concentrations

Remarks:: Study 2: 0, 250, 500 and 750 mg/kg bw
Study 3: 0, 308, 556 and 1000 mg/kg bw

No. of animals per sex per dose:: Study 2:
Total: 124 animals
0 mg/kg bw, 13 male, 13 female
250 mg/kg bw, 13 male, 13 female
500 mg/kg bw, 13 male, 13 female
750 mg/kg bw, 13 male, 13 female
Recovery group:
0 mg/kg bw, 5 male, 5 female
750 mg/kg bw, 5 male, 5 female

Study 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 vehicle
Details on study design:: Study 2:
- Dose selection rationale: The dose levels 250, 500 and 750 mg/kg body weight were selected for theMain Study based on the results of Dose Range Finding (DRF).- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. Individual body weights were considered within ±20% of the groups mean.- Rationale for selecting satellite groups: 0 and 750 mg/kg bw were selected as satellite groups- Post-exposure recovery period in satellite groups: 14 days- Section schedule rationale (if not random): N/A

Study 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

Examinations

Maternal examinations:: Study 2: CAGE SIDE OBSERVATIONS: Yes
-Time schedule: Twice daily (morning and evening)- Cage side observations included.: Morbidity and mortality, throughout the acclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a day, preferably at the same time each day
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), on day 4 and day 13post-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- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY: No data- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weightedaverages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data- Time schedule for examinations: No data
OPHTHALMOSCOPIC EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No data
HAEMATOLOGY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters 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) and Activated Partial Thromboplastin time (aPTT) were examined.

CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters 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), Globulin (Glob), Alb/ Glb (A:G) and Bile acids were examined.URINALYSIS: No data- Time schedule for collection of urine: No data- Metabolism cages used for collection of urine: No data- Animals fasted: No data- Parameters checked in table [No.?] were examined. No data NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Study 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.
Ovaries and uterine content:: Study 2: Estrous cycle, Post-implantation loss and Post-natal loss were examined.
Study 3: 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: N/A
Fetal examinations:: Study 2: Litter size, No. of live births, pups weight at birth and PND14 and Pups sex ratio were examined.
Study 3: - External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes:
- Head examinations: No data
Statistics:: Study 2: Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dun nett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal- Wallis, ANOVA on ranks.

Study 3: 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:: Study 2: Gestational length, Survival Index of pups, Pregnancy index and Pups sex ratio were examined.
Study 3: Pregnancy index/fertility index was determined.
Historical control data:: No Data Available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Study 2: No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. 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 increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent, hence considered as incidental and not attributed to the effect of test item administration.

Study 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.
Mortality:: no mortality observed
Description (incidence):: Study 2: No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period.

Study 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):: Study 2: When treated with 500 mg/kg bw, statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 as compared to control group.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.

Study 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):: no effects observed
Description (incidence and severity):: Study 2: No treatment related changes were observed in treated male and female rats as compared to control.

Study 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):: Study 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 the 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.
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):: Study 2: At the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.

Study 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):: Study 2: At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight.The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.

Study 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 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):: no effects observed
Description (incidence and severity):: Study 2: The functional observation battery/neurobehavioral observation were comparable and no changes were r evealed i any of the animals of all the treated groups in both the sexesThe 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. However in recovery male,statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group.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. Howerver, statistically significant increase in female were noted in Distance travelled (DT),Ambulatory time (AT), and in Horizontal counts (HC)at 750 mg/kg body weight when compare to control group.The above changes observed were not dose dependent, hence considered as incidental and notattributed to the effect of test item administration.

Study 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 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):: Study 2: 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 respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous.

Study 3: 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):: Study 2: 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. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.

Study 3: 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):: Study 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);
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 the 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.
Histopathological findings: neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: Study 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.
Other effects:: not specified

Maternal developmental toxicity

Number of abortions:: not specified
Pre- and post-implantation loss:: no effects observed
Description (incidence and severity):: Study 2: No Pre and Post-implantation loss were observed in treated rats as compared to control.
Total litter losses by resorption:: not specified
Early or late resorptions:: not specified
Dead fetuses:: not specified
Changes in pregnancy duration:: no effects observed
Description (incidence and severity):: Study 2: No effect on Gestational length were observed in treated rats as compared to control.
Changes in number of pregnant:: not specified
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Study 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.

Effect levels (maternal animals)

Dose descriptor:: NOAEL
Effect level:: 750 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: behaviour (functional findings); body weight and weight gain; clinical biochemistry; clinical signs; effects on pregnancy duration; food consumption and compound intake; gross pathology; haematology; mortality; organ weights and organ / body weight ratios; pre and post implantation loss
Remarks on result:: other: Not Specified

Maternal abnormalities

Abnormalities:: not specified

Results (fetuses)

Fetal body weight changes:: no effects observed
Description (incidence and severity):: Study 2: No effect on pups weight at birth and PND14 were observed as compared to control.
Study 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.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: Study 2: No effect on No. of live births were observed as compared to control.
Study 3: no effects observed
Changes in sex ratio:: no effects observed
Description (incidence and severity):: Study 2: No effect on Pups sex ratio were observed as compared to control. '
Study 3: 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:: no effects observed
Description (incidence and severity):: Study 2: No effect on Litter size were observed as compared to control.
Changes in postnatal survival:: no effects observed
Description (incidence and severity):: Study 2: No effect on Post-natal loss were observed as compared to control.
External malformations:: effects observed, non-treatment-related
Description (incidence and severity):: Study 2: 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 died during course of study revealed various lesions among the control and treated groups viz., external examination Cannibalism (Male: G1: 1/59, G2:13/60, G3: 7/81, Female:G1 :1/59, G2:14/81, G3:1/60, G4: 1/55 ; Tail absent (Anury) G3: 1/60 and internal examination Right skin swelling G1: 1/57.

Study 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)
Skeletal malformations:: not specified
Visceral malformations:: not specified
Other effects:: not specified

Effect levels (fetuses)

Dose descriptor:: NOAEL
Effect level:: 750 mg/kg bw/day (actual dose received)
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; changes in litter size and weights; changes in postnatal survival; external malformations
Remarks on result:: other: Not Specified

Fetal abnormalities

Abnormalities:: not specified

Overall developmental toxicity

Developmental effects observed:: not specified
Treatment related:: not specified

Any other information on results incl. tables

Study 2:

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)	G1(11)		G2(12)		G3(13)		G4(12)
Dose(mg/kg bwt)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
No. of Live Births	11.80	1.93	12.00	2.95	9.85	3.60	10.58	1.68
No. of alive pups at Post-natal Day 4	10.90	3.00	8.33	5.57	9.08	4.37	10.42	1.51
Post-natal Loss (%)	7.85	19.84	33.02	38.93	18.72	37.26	1.28	4.44
Fetal Survival Index at Post-natal Day 4 (%)	92.15	19.84	66.98	38.93	81.28	37.26	98.72	4.44

Mean Gestational Length and Litter size

Group(N)	G1(11)		G2(12)		G3(13)		G4(12)
Dose(mg/kg bwt)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Gestation Length	22.36	0.50	22.00	0.00	22.00	0.00	22.25	0.45
Litter size (Total No. of litter size)	10.91	3.48	12.67	2.90	10.85	2.61	10.58	1.68

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Mean Pups Weight	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0	6.34	0.57	118	6.10	0.76	151	6.24	0.86	141	6.59	0.48	127
Day 4	9.76	1.78	109	8.56	1.12	100	9.11	1.08	118	9.50	1.45	125
Day 13	24.88	3.88	87	20.70	3.00	78	22.74	2.65	86	22.05	2.90	100
Group(n)	G1(11)			G2(12)			G3(13)			G4(13)
Pups Body Weight gain (%)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0-Day 4	47.42	20.65	109	36.29	13.99	99	41.58	13.79	118	43.66	15.12	125
Day 0-Day 13	148.89	19.84	87	143.22	25.24	78	144.89	19.45	86	135.74	22.14	100
Group (Number of Litter size)	G1(118)			G2(151)			G3(141)			G4(127)
Sex Ratio at birth (Male/Female)	61/57			72/79			80/61			71/56
Sex Ratio at Day 4 (Male/Female)	56/53			48/51			64/54			69/56
Gross Observations	NAD			NAD			NAD			NAD

MeanHormonal Analysis Data (Continued)

Day: 04 (Pups)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Dose(mg/kg bwt)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	2.17	0.29	10	2.04	0.42	11	2.37	0.49	11	2.13	0.31	12
TSH (uIU/mL)	1.76	0.32	10	1.84	0.51	11	1.84	0.81	11	1.82	0.56	12

Day: 13 (Pups)

Group(n)	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
Dose(mg/kg bwt)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	5.87	0.88	9	5.08	0.60	9	5.60	0.70	10	5.64	0.82	12
TSH (uIU/mL)	2.16	1.13	9	1.88	0.49	9	2.05	0.60	10	2.14	0.69	12

Absolute Organ Weight (g) Pups

Sex:Male

Group (N)	G1 (9)		G2 (8)		G3 (10)		G4 (14)
Dose (mg/Kg)	0		250		500		750
Organ	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Thyroid With Parathyroids	0.0030	0.0004	0.0037	0.0015	0.0039	0.0008	0.0036	0.0009

Sex:Female

Group (N)	G1 (9)		G2 (9)		G3 (10)		G4 (14)
Dose (mg/Kg)	0		250		500		750
Organ	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Thyroid With Parathyroids	0.0038	0.0004	0.0035	0.0010	0.0043	0.0008	0.0040	0.0010

Gross Pathology Observations (Pups)

Sex: Male

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	44	40	51	55
Organ & Lesion
External Observations
No Abnormality Detected	44	39	51	55
Cannibalism	./.	1	./.	./.
Internal Observations
No Abnormality Detected	44	40	51	55

Sex: Female

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	51	65	62	47
Organ & Lesion
External Observations
No Abnormality Detected	50	65	61	47
Cannibalism	./.	./.	1	./.
Right skin swelling	1	./.	./.	./.
Tail absent (Anury)	./.	./.	1	./.
Internal Observations
No Abnormality Detected	50	65	62	47
Right pus swollen joint	1	./.	./.	./.

Gross Pathology Observations (Pups)

Sex: Male

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	44	40	51	55
Organ & Lesion
External Observations
No Abnormality Detected	44	39	51	55
Cannibalism	./.	1	./.	./.
Internal Observations
No Abnormality Detected	44	40	51	55

Sex: Female

Group	G1	G2	G3	G4
Dose (mg/kg)	0	250	500	750
Total No. of Pups observed	51	65	62	47
Organ & Lesion
External Observations
No Abnormality Detected	50	65	61	47
Cannibalism	./.	./.	1	./.
Right skin swelling	1	./.	./.	./.
Tail absent (Anury)	./.	./.	1	./.
Internal Observations
No Abnormality Detected	50	65	62	47
Right pus swollen joint	1	./.	./.	./.

Microscopic Observations (Pups)

Sex: Female

Group	G1	G2	G3	G4	G1R	G4R
Dose (mg/kg)	0	250	500	750	0	750
Total Number of Animals Observed	21	-	-	21	-	-
Organ & Lesion
No Abnormality Detected	21	X	X	21	X	X

Study 3:

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
Handling reactivity	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
Handling reactivity	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
Handling reactivity	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
Handling reactivity	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
Handling reactivity	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)

Applicant's summary and conclusion

Conclusions:

Study 2: NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Study 3: 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 summaries of the data of the developmental toxicity studies are as follows:

Study 2:

In a experimental study conducted , where Wistar male and female rat treated with test chemical in the concentration of 0, 250, 500 and 750 mg/kg bw orally by gavage for more than 63 days. No mortality or morbidity was observed inany animals of the control and treatment groups throughout the study period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. 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 increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. Statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 at 500 mg/kg bw as compared to control group. 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. No treatment related changes in food consumption were observed in treated male and female rats as compared to control. Similarly, at the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance. At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight. The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response. The functional observation battery/neurobehavioral observation was comparable and no changes were revealed in any of the animals of all the treated groups in both the sexes. 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. However in recovery male, statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R). when compare to control recovery group. Motor activity measurements were comparable and no changeswere revealed in any of the animals from all treated groups of both the sexes as compare to control group. However, statistically significant increase in female were noted in Distance travelled (DT), Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group. The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. In addition, 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 respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous. 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. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Microscopic examination of control group and rats treated with 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. Lesions observed in liver, kidneys, lungs, adrenals and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship.Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. No effects on regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 female from G2, G3 and G4, respectively which showed precoital interval more than 5 days. There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. No developmental effect were observed such as Gestational length, Litter size, No. of live births, Post-implantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index and Pups sex ratio (Male/Female) respectively of treated rats as compared to control. No treatment related changes were noted in hormonal analysis (T4, TSH, Testosterone and Estrogen). Therefore, NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Study 3:

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.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

β-bromostyrene

Developmental toxicity / teratogenicity

Administrative data

Cross-referenceopen allclose all

Cross-reference 1

Reference

Mean Gestational Length and Litter size

Gross Pathology Observations (Pups)

Gross Pathology Observations (Pups)

Cross-reference 2

Reference

Table 1 Mortality and Morbidity

Table 2 Clinical Signs and Symptoms

Table 3 Summary of Detailed Clinical Examinations

Table 4 Mean Body Weight (g)

Table 4 Mean Body Weight (g) Continued

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

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

Data source

Referenceopen allclose all

Reference 1

Reference 2

Materials and methods

Test guideline

Test material

Test material information

Constituent 1

Test animals

Administration / exposure

Doses / concentrations

Examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Maternal developmental toxicity

Effect levels (maternal animals)

Maternal abnormalities

Results (fetuses)

Effect levels (fetuses)

Fetal abnormalities

Overall developmental toxicity

Any other information on results incl. tables

Mean Gestational Length and Litter size

Gross Pathology Observations (Pups)

Gross Pathology Observations (Pups)

Table 1 Mortality and Morbidity

Table 2 Clinical Signs and Symptoms

Table 3 Summary of Detailed Clinical Examinations

Table 4 Mean Body Weight (g)

Table 4 Mean Body Weight (g) Continued

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

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

Applicant's summary and conclusion

Cross-referenceopen all close all

Referenceopen all close all