Benzyltriethylammonium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

Combined repeated dose repro-devp. Screen

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is form study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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 substance in Wistar Rats

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltriethylammonium chloride

EC Number:

200-270-1

EC Name:

Benzyltriethylammonium chloride

Cas Number:

56-37-1

Molecular formula:

C13H22N.Cl

IUPAC Name:

N-benzyl-N,N-diethylethanaminium chloride

Test material form:

solid

Details on test material:

Identification : Benzyltriethylammonium chloride
Batch number : L231511706
Appearance : Solid, white crystals
Purity : 98.81%
Manufactured date : June 2017
Expiry Date : May 2022
Storage conditions : Room Temperature (20º - 30ºC)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: National Institute of Biosciences ,Pune,India- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation- Weight at study initiation: - Fasting period before study:- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). 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 darkIN-LIFE DATES: From: December 08, 2017 To: April 28, 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled

Details on oral 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

Duration of treatment / exposure:

Approx. 64 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Total: 1240 mg/kg bw, 13 male, 13 female 250 mg/kg bw, 13 male, 13 female500 mg/kg bw, 13 male, 13 female 750 mg/kg bw, 13 male, 13 femaleRecovery 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 the Main 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):

Examinations

Observations and examinations performed and frequency:

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 13 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
-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
weighted averages 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.
-Dose groups that were examined: five males and five females from control and treatment groups.
-Battery of functions tested: Grip strength, hind limb foot splay and motor activity were examined.

OTHER: Organ weights were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.HISTOPATHOLOGY: Yes, Full histopathology was carried out on the preserved organs (ovaries, uterus, cervix with vagina, testes, epididymides, prostate, seminal vesicle with coagulating glands) of all animals and all tissues of five males and females, randomly selected from each group animals in the control and high dose groups.

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 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

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.

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:

no effects observed

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 considered 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 revealed i 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 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 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 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:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of control group and rats treated at 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. This includes in Liver: focal to multifocal minimal to mild lymphocyte infiltration (Male: G1:2/5; Female: G1/5, G4:1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), in Kidneys: focal mild tubular degeneration (Male: G1:2/5), focal mild lymphocyte infiltration (Female: G1/5); in Lungs: multifocal mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Adrenals: accessory adrenocortical tissue (Bilateral: Female: G4:1/5), in Testes: Male: multifocal mild cytoplasmic vacuolation at sertoli cell (Male: G1: 1/13), focal mild multinucleated giant cell infiltration (Male: G1: 1/13) focal mild seminiferous tubule degeneration (Male: G4: 2/13); focal minimal retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13, G1-R: 1/5); focal minimal to mild sloughing of round spermatid (Male: G1: 1/13, G2:1/13). in Epididymis: Male: multifocal mild reduced sperm count (Male: G1: 1/13).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. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). 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.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle evaluation:Estrous cycle was monitored for its regularity. Females with regular estrous cycle were allocated to control and treatment groups. In control group G1 and treatment group G2 , G3 and G4 all the females showed 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 females from G2, G3 and G4, respectively which showed precoital interval more than 5 days.Maternal and Pups ParameterThere was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. 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 was found to be 84.62, 92.31, 100.00 and 92.31 in G1, G2, G3 and G4 respectively. Pups sex ratio (Male/Female) was found to be 61/57, 72/79, 80/61 and 71/56 at birth in G1, G2, G3 and G4 respectively and 56/53, 48/51, 64/54 and 69/56 at Day 4 in G1, G2, G3 and G4 respectively. Hormonal AnalysisNo treatment related changes were noted in hormonal analysis (T4, TSH, Testosterone and Estrogen).

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

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

organ weights and organ / body weight ratios

other: Estrous cycle evaluation, Maternal and Pups Parameter and Hormonal Analysis

Remarks on result:

other: No effect observed

Target system / organ toxicity

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Any other information on results incl. tables

Mortality and Morbidity - Main Study

Sex: Male

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	250	13	NMM
G3	Mid	500	13	NMM
G4	High	750	13	NMM
G1-R	Control- Recovery	0	5	NMM
G4-R	High- Recovery	750	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	250	13	NMM
G3	Mid	500	13	NMM
G4	High	750	13	NMM
G1-R	Control -Recovery	0	5	NMM
G4-R	High- Recovery	750	5	NMM

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

Clinical Signs and Symptoms – Main Study

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	250	13	Normal	13/13
G3	Mid	500	13	Normal	13/13
G4	High	750	13	Normal	13/13
G1-R	Control -Recovery	0	5	Normal	5/5
G4-R	High- Recovery	750	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	250	13	Normal	13/13
G3	Mid	500	13	Normal	13/13
G4	High	750	13	Normal	13/13
G1-R	Control -Recovery	0	5	Normal	5/5
G4-R	High- Recovery	750	5	Normal	5/5

Mean Body Weight (g) – Main Study

Sex: Male

Group (N)	G1 (13)		G2 (13)		G3 (13)		G4 (13)
Dose (mg/kg b. wt.)	0		250		500		750
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
1	328.62	22.46	330.46	21.52	327.92	20.07	329.08	16.28
8	343.08	26.03	348.77	26.14	350.46	24.55	350.00	20.15
14	351.62	27.09	362.92	32.04	370.00	33.60	367.46	26.28
21	364.31	26.48	380.31	33.72	385.38	39.58	379.23	30.18
28	382.77	28.60	398.77	39.39	402.62	42.57	399.00	32.01
35	397.85	31.58	407.62	37.91	412.23	44.44	407.38	32.29
40	401.15	32.82	394.92	53.26	419.54	48.74	414.69	30.05
41{fasting}	385.46	31.17	389.62	35.03	399.23	47.68	394.23	31.15

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)	G1			G2			G3			G4
Dose (mg/kg b. wt.)	0			250			500			750
Day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
1	247.54	16.31	13	245.00	11.95	13	248.23	14.22	13	247.92	14.28	13
8	251.62	16.29	13	252.00	14.93	13	254.38	16.09	13	255.77	13.40	13
14	258.15	18.38	13	259.62	17.47	13	263.62	20.39	13	265.38	16.00	13
21	267.50	14.85	2	244.00	18.38	2	274.00	12.73	2	289.00	18.38	2
28	288.00	29.70	2	276.00	./.	1	./.	./.	0	295.00	./.	1
35	309.50	28.99	2	300.00	./.	1	./.	./.	0	309.00	./.	1
42	295.00	22.63	2	319.00	./.	1	./.	./.	0	307.00	./.	1
49	282.50	34.65	2	334.00	./.	1	./.	./.	0	306.00	./.	1
56	286.50	30.41	2	328.00	./.	1	./.	./.	0	303.00	./.	1
58	280.00	29.70	2	329.00	./.	1	./.	./.	0	326.00	./.	1
59(Fasting)	272.00	28.28	2	321.00	./.	1	./.	./.	0	312.00	./.	1

Keys:N = Number of animals in group, g= gram, SD = Standard deviation./.= Not Applicable

Period: Gestation                                                                                                  Sex: Female

Group	G1			G2			G3			G4
Dose (mg/kg b. wt.)	0			250			500			750
Day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0	260.36	19.82	11	264.08	16.89	12	370.00	21.68	13	268.67	18.81	12
7	279.45	18.84	11	282.00	18.92	12	284.69	22.29	13	284.83	19.40	12
14	311.64	22.08	11	308.92	23.12	12	312.31	27.62	13	311.83	22.23	12
20	373.91	25.68	11	368.58	23.13	12	368.46	36.03	13	363.67	25.82	12

 Period: Post-Partum                                                                                                         Sex: Female

Group (N)	G1 (11)		G2 (12)		G3 (13)		G4 (12)
Dose (mg/kg b. wt.)	0		250		500		750
Day	Mean	SD	Mean	SD	Mean	SD	Mean	SD
0	298.55	28.22	297.42	17.10	306.54	26.49	294.58	21.90
4	303.09	27.13	291.25	20.91	299.38	19.49	296.58	19.46
13	313.91	33.40	297.58	21.47	307.92	23.06	304.50	21.45
14(Fasting)	285.18	25.27	272.00	16.04	282.08	19.95	272.25	18.11

Sex: Male

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/kg b. wt.)	0		750
Day	Mean	SD	Mean	SD
1	335.60	26.19	334.00	27.00
8	354.60	29.82	358.60	25.36
14	361.40	33.92	371.80	16.83
21	378.00	32.30	393.40	21.48
28	395.00	37.29	412.00	30.81
35	407.20	38.26	419.60	33.00
42	416.60	45.28	427.00	33.85
49	429.80	46.66	446.40	30.76
56	434.60	43.55	447.40	33.76
63	435.80	45.88	452.60	33.81
66	441.40	46.76	457.20	37.34
67(fasting)	421.20	45.17	436.60	34.41

Sex: Female

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/kg b. wt.)	0		750
Day	Mean	SD	Mean	SD
1	246.60	22.73	248.60	20.13
8	255.20	19.20	257.60	23.44
14	263.20	19.68	269.60	19.63
21	270.40	20.32	276.40	22.50
28	275.60	19.93	280.00	27.96
35	279.80	20.27	278.80	25.94
42	287.00	22.44	283.80	22.70
49	292.60	25.86	287.60	23.80
56	287.80	25.97	288.00	25.56
63	291.20	26.33	287.00	23.79
66	291.00	25.66	287.40	23.04
67(fasting)	275.40	26.02	272.80	25.12

Sensory Reactivity Measurements – Main Study

Sex:Male

Group (N)		G1 (5)	G2 (5)	G3 (5)	G4 (5)
Dose (mg/kg b. wt.)		0	250	500	750
Parameter	Observation	No. of Animals Showing Observation
Approach response	Fast	5	5	5	5
Touch response	Normal	5	5	5	5
Click response	Normal	5	5	5	5
Pupil response	Normal	5	5	5	5
Air righting reflex	Normal	5	5	5	5
Tail pinch response	Flinch	5	5	5	5

 Sex:Female

Group (N)		G1 (5)	G2 (5)	G3 (5)	G4 (5)
Dose (mg/kg b. wt.)		0	250	500	750
Parameter	Observation	No. of Animals Showing Observation
Approach response	Fast	5	5	5	5
Touch response	Normal	5	5	5	5
Click response	Normal	5	5	5	5
Pupil response	Normal	5	5	5	5
Air righting reflex	Normal	5	5	5	5
Tail pinch response	Flinch	5	5	5	5

Group (N)		G1R M (5)	G1R F (5)	G4R M (5)	G4R M (5)
Dose (mg/kg b. wt.)		0	0	750	750
Parameter	Observation	No. of Animals Showing Observation
Approach response	Fast	5	5	5	5
Touch response	Normal	5	5	5	5
Click response	Normal	5	5	5	5
Pupil response	Normal	5	5	5	5
Air righting reflex	Normal	5	5	5	5
Tail pinch response	Flinch	5	5	5	5

Mean Foot Splay Record – Main Study

Sex:Male

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose           (mg/kg b. wt.)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
FS (mm)	84.00	5.69	95.93	7.19	105.93	6.55	84.53	10.58

 Sex: Female

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose        (mg/kg b. wt.)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
FS (mm)	86.47	10.88	71.13	8.40	83.73	9.11	79.47	10.30

 

Group (N)	G1R M (5)		G4R M (5)		G1R F (5)		G4R F (5)
Dose        (mg/kg b. wt.)	0		0		750		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
FS (mm)	102.33	5.59	108.53	7.55	79.13	3.04	63.27↓	1.07

 Keys:FS= Foot splay,N = Number of animals in group, R= Recovery, SD = Standard deviation, mm= milimeter,F=Female, M=Male

Mean Grip Strength Record – Main Study

Sex: Male

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose             (mg/kg b. wt.)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Fore limb (g)	817.27	59.78	790.60	87.55	804.73	102.15	770.47	89.81
Hind limb(g)	514.47	48.43	531.13	65.31	517.33	54.63	557.53	60.26

 Sex: Female

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose             (mg/kg b. wt.)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Fore limb (g)	740.33	81.50	750.40	93.95	834.87	69.47	768.27	64.34
Hind limb(g)	533.80	60.97	561.27	49.31	561.27	49.31	515.67	42.92

                                                                                                                                   

Group (N)	G1R M (5)		G1R F (5)		G4R M (5)		G4R F (5)
Dose             (mg/kg b. wt.)	0		0		750		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Fore limb (g)	779.40	95.68	813.27	64.72	749.33	53.31	797.60	75.71
Hind limb(g)	519.73	31.85	540.87	51.12	574.40↑	35.94	527.93	62.16

Haematology

Sex: Male                                

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose (mg/kg)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
RBC (X 10^6/µl)	9.13	0.63	8.83	0.17	9.30	0.35	9.01	0.35
HCT (%)	43.50	2.60	43.46	1.37	43.42	1.41	43.04	1.53
MCV(µm^3)	47.76	2.51	49.20	0.99	46.72	1.51	47.74	1.26
HGB (g/dl)	14.82	0.79	14.80	0.42	14.72	0.40	14.64	0.46
MCH (pg)	16.28	0.79	16.78	0.24	15.86	0.60	16.26	0.32
MCHC( g/dl)	34.08	0.33	34.08	0.31	33.98	0.36	34.08	0.26
PLT (X 10^3/µl)	525.20	66.38	464.40	29.86	480.20	21.32	603.40	105.57
WBC (X 10^3/µl)	16.58	5.60	15.52	2.27	16.92	3.04	19.40	3.26
Neutrophil (%)	16.20	2.49	15.80	2.39	19.60	1.34	17.80	2.28
Lymphocyte (%)	82.80	3.27	83.20	1.92	79.40	0.89	81.00	2.12
Monocyte (%)	0.00	0.00	0.00	0.00	0.20	0.45	0.20	0.45
Eosinophil (%)	1.00	1.00	1.00	1.00	0.80	0.84	1.00	0.71
Basophil (%)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Prothrombin time(Sec)	16.49	8.14	21.56	1.01	18.68	5.10	16.61	8.29
aPTT (Sec)	34.53	2.52	27.92	11.14	31.49	4.82	26.74	11.76

Sex: Female

Group (N)	G1 (5)		G2 (5)		G3 (5)		G4 (5)
Dose (mg/kg)	0		250		500		750
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
RBC (X 10^6/µl)	7.24	0.41	6.88	0.42	6.67	0.57	7.03	0.35
HCT (%)	38.56	1.11	36.96	1.63	35.88	3.75	36.86	1.41
MCV(µm^3)	53.34	1.83	53.76	1.72	53.70	1.30	52.44	0.76
HGB (g/dl)	13.36	0.39	12.92	0.65	12.62	1.28	12.88	0.51
MCH (pg)	18.46	0.67	18.80	0.41	18.92	0.32	18.34	0.35
MCHC( g/dl)	34.62	0.41	34.96	0.38	35.22	0.42	34.98	0.18
PLT (X 10^3/µl)	378.20	96.51	377.00	51.50	400.60	88.86	412.40	103.75
WBC (X 10^3/µl)	13.40	3.04	12.98	2.65	10.08	3.18	11.76	5.01
Neutrophil (%)	22.00	3.24	25.00	1.87	20.60	1.52	22.40	2.88
Lymphocyte (%)	76.40	3.78	74.00	2.35	78.80	2.28	77.00	2.45
Monocyte (%)	0.40	0.55	0.20	0.45	0.00	0.00	0.40	0.89
Eosinophil (%)	1.20	0.84	0.80	0.45	0.60	0.89	0.20	0.45
Basophil (%)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Prothrombin time(Sec)	20.69	8.40	22.21	2.96	22.84	2.32	21.28	8.94
aPTT (Sec)	25.86	5.42	21.07	8.68	21.19	9.32	22.56	2.00

Sex: Male

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/kg)	0		750
Parameter	Mean	SD	Mean	SD
RBC (X 10^6/µl)	9.13	0.44	8.87	0.28
HCT (%)	43.84	1.25	42.30	1.55
MCV(µm^3)	48.06	1.82	47.70	1.62
HGB (g/dl)	14.76	0.25	14.30	0.46
MCH (pg)	16.18	0.66	16.14	0.33
MCHC( g/dl)	33.72	0.43	33.86	0.52
PLT (X 10^3/µl)	488.00	76.41	533.60	40.18
WBC (X 10^3/µl)	14.80	3.45	16.14	4.41
Neutrophil (%)	19.00	2.55	16.40	1.67
Lymphocyte (%)	81.00	2.55	82.80	1.30
Monocyte (%)	0.00	0.00	0.00	0.00
Eosinophil (%)	0.00	0.00	0.80	0.84
Basophil (%)	0.00	0.00	0.00	0.00
Prothrombin time(Sec.)	23.57	0.86	22.18	2.01
aPTT (Sec.)	32.13	4.72	31.91	10.09

Sex: Female

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/kg)	0		750
Parameter	Mean	SD	Mean	SD
RBC (X 10^6/µl)	8.31	0.53	8.21	0.34
HCT (%)	41.20	1.92	41.10	2.01
MCV(µm^3)	49.60	1.18	50.04	0.90
HGB (g/dl)	14.32	0.68	14.36	0.55
MCH (pg)	17.24	0.36	17.52	0.19
MCHC( g/dl)	34.78	0.08	35.00	0.41
PLT (X 10^3/µl)	406.40	66.24	412.20	51.91
WBC (X 10^3/µl)	9.06	1.30	7.04↓	1.22
Neutrophil (%)	15.60	2.70	15.60	2.61
Lymphocyte (%)	83.60	2.88	83.80	2.95
Monocyte (%)	0.20	0.45	0.40	0.55
Eosinophil (%)	0.60	0.89	0.20	0.45
Basophil (%)	0.00	0.00	0.00	0.00
Prothrombin time(Sec)	24.67	5.68	21.88	1.37
aPTT (Sec)	27.99	2.32	24.53	11.51

Clinical Chemistry

Sex: Male

Group (N)	G1 (5)		G2 (5)
Dose (mg/Kg)	0		250
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	89.08	39.28	78.18	4.56
Cholesterol (mg/dl)	51.06	8.08	51.06	7.36
Triglyceride (mg/dl)	97.74	19.29	66.66	5.90
ALT (U/l)	61.72	9.52	66.46	9.37
AST (U/l)	164.90	41.12	142.92	29.56
Calcium (mg/dl)	9.57	0.23	9.66	0.15
Albumin (g/dl)	3.81	0.17	3.75	0.10
Total Protein (g/dl)	6.84	0.23	6.73	0.29
Creatinine (mg/dl)	0.56	0.07	0.54	0.02
Phosphorus (mg/dl)	6.45	0.27	6.37	0.40
Urea (mg/dl)	43.66	6.05	41.30	2.21
Bile Acid (µmol/l)	32.82	13.75	23.42	10.19
BUN (mg/dl)	20.41	2.82	19.29	1.03
Glob (g/dl)	3.03	0.14	2.98	0.25
A/G – Calculated	1.26	0.08	1.27	0.11
Na (mmol/l)	143.40	2.70	143.80	2.49
K (mmol/l)	5.80	1.12	5.54	0.78

Sex: Male

Group (N)	G3 (5)		G4 (5)
Dose (mg/Kg)	500		750
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	87.12	15.08	77.62	22.56
Cholesterol (mg/dl)	43.48	5.71	49.14	9.08
Triglyceride (mg/dl)	55.50↓	19.30	70.58	28.18
ALT (U/l)	64.06	4.52	65.62	8.66
AST (U/l)	162.70	28.35	153.66	14.61
Calcium (mg/dl)	9.61	0.40	9.69	0.10
Albumin (g/dl)	3.68	0.23	3.76	0.14
Total Protein (g/dl)	6.82	0.14	6.73	0.37
Creatinine (mg/dl)	0.56	0.03	0.51	0.02
Phosphorus (mg/dl)	6.71	0.92	6.85	0.46
Urea (mg/dl)	44.32	2.21	42.80	4.10
Bile Acid (µmol/l)	18.68	11.41	16.36	11.75
BUN (mg/dl)	20.71	1.03	19.99	1.91
Glob (g/dl)	3.14	0.22	2.98	0.30
A/G – Calculated	1.18	0.14	1.27	0.12
Na (mmol/l)	147.00	5.15	143.40	0.89
K (mmol/l)	5.34	0.98	5.90	1.40

Sex: Female

Group (N)	G1 (5)		G2 (5)
Dose (mg/Kg)	0		250
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	73.10	11.87	67.12	22.06
Cholesterol (mg/dl)	89.80	18.11	79.28	14.01
Triglyceride (mg/dl)	175.82	123.99	126.86	74.90
ALT (U/l)	105.44	28.70	107.94	33.75
AST (U/l)	191.28	50.28	182.72	14.33
Calcium (mg/dl)	9.82	0.30	9.42	0.21
Albumin (g/dl)	3.79	0.22	3.63	0.14
Total Protein (g/dl)	7.36	0.30	6.88	0.50
Creatinine (mg/dl)	0.58	0.05	0.58	0.13
Phosphorus (mg/dl)	6.66	1.61	7.35	1.76
Urea (mg/dl)	57.22	8.33	62.26	19.53
Bile Acid (µmol/l)	38.48	23.45	45.80	32.59
BUN (mg/dl)	26.75	3.89	29.09	9.13
Glob (g/dl)	3.57	0.16	3.25	0.39
A/G – Calculated	1.06	0.07	1.13	0.12
Na (mmol/l)	145.40	4.77	141.40	5.68
K (mmol/l)	5.28	0.61	5.90	0.48

 Sex: Female

Group (N)	G3 (5)		G4 (5)
Dose (mg/Kg)	500		750
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	82.56	16.70	79.64	20.37
Cholesterol (mg/dl)	72.90	13.52	68.16	9.14
Triglyceride (mg/dl)	83.08	21.23	83.44	33.81
ALT (U/l)	111.04	16.66	95.36	18.63
AST (U/l)	154.20	13.02	172.38	29.09
Calcium (mg/dl)	9.58	0.25	9.19↓	0.18
Albumin (g/dl)	3.75	0.27	3.59	0.15
Total Protein (g/dl)	7.53	0.60	7.02	0.13
Creatinine (mg/dl)	0.59	0.09	0.56	0.06
Phosphorus (mg/dl)	6.41	0.83	6.15	0.68
Urea (mg/dl)	53.10	1.52	55.38	5.27
Bile Acid (µmol/l)	51.58	35.66	30.80	19.84
BUN (mg/dl)	24.82	0.72	26.08	2.12
Glob (g/dl)	3.77	0.37	3.44	0.26
A/G – Calculated	1.00	0.06	1.05	0.12
Na (mmol/l)	154.40	16.52	143.80	5.76
K (mmol/l)	6.00	0.58	5.88	0.80

Sex: Male

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/Kg)	0		750
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	80.00	13.41	92.76	17.10
Cholesterol (mg/dl)	46.96	10.04	56.96	13.25
Triglyceride (mg/dl)	65.06	19.79	94.38	46.41
ALT (U/l)	66.08	10.33	62.48	8.72
AST (U/l)	190.52	34.85	152.62	29.67
Calcium (mg/dl)	9.51	0.28	9.87	0.38
Albumin (g/dl)	3.76	0.27	4.00	0.19
Total Protein (g/dl)	7.52	0.32	7.59	0.45
Creatinine (mg/dl)	0.50	0.01	0.48	0.02
Phosphorus (mg/dl)	5.48	0.19	5.25	0.87
Urea (mg/dl)	45.94	3.54	42.90	5.86
Bile Acid (µmol/l)	17.82	8.65	22.26	6.26
BUN (mg/dl)	21.68	1.84	20.05	2.75
Glob (g/dl)	3.76	0.12	3.59	0.27
A/G – Calculated	1.00	0.07	1.12↑	0.04
Na (mmol/l)	139.80	1.92	141.20	1.10
K (mmol/l)	5.32	0.26	5.20	0.60

Sex: Female

Group (N)	G1-R (5)		G4-R (5)
Dose (mg/Kg)	0		750
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	84.86	13.63	87.94	18.69
Cholesterol (mg/dl)	60.94	5.49	68.84	4.02
Triglyceride (mg/dl)	49.46	15.45	39.72	10.62
ALT (U/l)	64.78	6.10	47.80↓	4.27
AST (U/l)	146.56	26.43	120.84	33.19
Calcium (mg/dl)	9.91	0.20	10.13	0.50
Albumin (g/dl)	4.08	0.15	4.13	0.21
Total Protein (g/dl)	8.08	0.37	7.62	0.39
Creatinine (mg/dl)	0.55	0.08	0.54	0.04
Phosphorus (mg/dl)	4.17	0.53	4.58	0.34
Urea (mg/dl)	43.90	4.60	41.98	4.24
Bile Acid (µmol/l)	20.40	8.59	15.48	6.28
BUN (mg/dl)	20.51	2.14	19.61	1.98
Glob (g/dl)	3.99	0.41	3.49↓	0.23
A/G – Calculated	1.03	0.12	1.18↑	0.07
Na (mmol/l)	140.00	2.55	142.00	2.00
K (mmol/l)	4.82	0.33	4.72	0.20

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction / Developmental Toxicity study, Wistar male and female rat treated with test substance 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 in any 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.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. At the end of treatment period a 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 a statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight and in 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 the treatment-free recovery period a 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 a statistical significantly 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. The functional observation battery/neurobehavioral observation were 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 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 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. Estrous cycle was monitored for its regularity. Females with regular estrous cycle were allocated to control and treatment groups. Therefore, NOAEL for the test chemical was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test substance orally by gavage for more the 63 days.