Benzyltriethylammonium chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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 from 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 Benzyltriethylammonium Chloride (CAS No.: 56-37-1) in Wistar Rats

GLP compliance:

yes

Limit test:

no

Justification for study design:

not specified

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

Details on species / strain selection:

not specified

Sex:

male/female

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]). .Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier,was used as bedding material. 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

Administration / exposure

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. Atthe 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:

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: - After successful mating each pregnant female was caged (how): housed individually - Any other deviations from standard protocol:

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

Details on study schedule:

not specified

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: 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):

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily (morning and evening)- Cage side observations : Morbidity and mortality, throughout theacclimatization 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/kgbody weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gaindata: 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 exam

Oestrous cyclicity (parental animals):

Estrous cycle were monitored daily from beginning of the treatment period until evidence of mating. When taking vaginal smear care was taken to avoid disturbance to vaginal mucosa.

Sperm parameters (parental animals):

not specified

Litter observations:

Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), body weight and ano-genital distance (AGD) were examined.

Postmortem examinations (parental animals):

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 andgross 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.

Postmortem examinations (offspring):

presence of gross abnormalities 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, FunctionalObservational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

Reproductive indices:

Pregnancy Index (%), Post-natal Loss (%), Gestation Length were examined.

Offspring viability indices:

Fetal Survival Index at Post-natal Day 4 (%) were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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,

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 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

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 theselesions 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:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There 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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: No effect observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

other:

Remarks on result:

other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

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	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

Mean Body Weight (g)

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

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

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
(1-8)	4.37	2.19	5.50	2.43	6.87	3.52	6.34	2.59
(1-15)	7.01	4.39	9.72	4.29	12.70↑	4.92	11.61	4.42
(1-28)	10.87	3.42	15.02	5.90	17.30↑	6.28	15.13	4.96
(1-29)	16.54	5.36	20.54	6.86	22.54	7.07	21.13	5.48
(1-35)	21.12	6.19	23.28	7.21	25.45	7.17	23.72	6.20
(1-40)	22.11	6.39	19.45	17.33	27.61	8.21	25.98	5.72
(1-41)	17.33	5.86	17.77	7.65	21.41	8.18	19.74	6.12

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
(1-8)	1.66	1.31	13	2.83	2.41	13	2.48	2.86	13	3.20	1.19	13
(1-15)	4.28	2.24	13	5.90	3.15	13	6.10	2.68	13	7.05	2.45	13
(1-21)	9.59	2.27	2	6.02	5.39	2	9.57	2.61	2	10.53	0.44	2
(1-28)	17.89	8.07	2	17.95	./.	1	./.	./.	1	18.47	./.	1
(1-35)	26.71	7.48	2	28.21	./.	1	./.	./.	1	24.10	./.	1
(1-42)	20.81	5.07	2	36.32	./.	1	./.	./.	1	23.29	./.	1
(1-49)	15.60	10.18	2	42.74	./.	1	./.	./.	1	22.89	./.	1
(1-56)	17.27	8.38	2	40.17	./.	1	./.	./.	1	21.69	./.	1
(1-59)	14.61	8.19	2	40.60	./.	1	./.	./.	1	30.92	./.	1

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
(0-7)	7.43	3.09	11	6.78	2.19	12	5.85	2.40	13	6.08	3.55	12
(0-14)	19.85	5.33	11	16.97	4.42	12	16.04	3.47	13	16.17	5.54	12
(0-20)	43.90	8.24	11	39.63	3.82	12	36.88	6.74	13	35.58	8.39	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
(0-4)	-4.32	4.81	-8.47	4.07	-7.73	5.39	-7.34	6.33
(0-13)	5.29	7.73	0.04	4.14	0.72	6.21	3.61	7.07
(0-14)	-4.32	4.81	-8.47	4.07	-7.73	5.39	-7.34	6.33

Summary of Days of Conception and Pregnancy Index (%)

Group(N)	G1(13)	G2(13)	G3(13)	G4(13)
Dose (mg/kg b.wt.)	0	250	500	750
No. of females showed evidence of copulation	11	12	13	12
No. of females concieved between Days 1-5 of cohabitation	11	11	11	11
No. of females concieved after Day 5 of cohabitation	0	1	2	1
Females achieved pregnancy	11	12	13	12
Pregnancy Index (%)	84.62	92.31	100.00	92.31

Key:N= number of dams in a group, No. = Number

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

Hormonal Analysis Data

Sex: Male (Termination)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	4.62	0.79	13	4.34	0.96	13	4.63	0.75	13	4.18	0.73	13
TSH (uIU/mL)	3.48	2.05	13	4.44	2.52	13	3.31	1.59	13	3.97	1.62	13
Testosterone (ng/dL)	71.85	77.91	13	115.22	96.43	13	99.50	118.60	13	99.75	112.05	13

                  Sex: Female (Day 4)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	3.54	0.56	11	3.27	0.63	12	3.60	0.74	13	3.46	0.48	12
TSH (uIU/mL)	3.10	1.31	11	3.38	1.58	12	2.69	1.41	13	3.21	1.67	12

                                                                                        Sex: Female (Day 13)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	3.48	0.60	11	3.28	0.69	12	3.30	0.50	13	3.04	0.31	12
TSH (uIU/mL)	2.45	2.09	11	3.25	1.84	12	3.23	2.71	13	2.21	1.32	12
E2 (pg/mL)	33.13	11.94	11	41.09	18.51	12	29.94	15.08	13	25.25	8.02	12

Sex: Female (Non-Pregnenat)

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	5.6	3.39	2	3.90	./.	1	./.	./.	0	3.90	./.	1
TSH (uIU/mL)	5.45	1.15	2	2.62	./.	1	./.	./.	0	2.50	./.	1
E2 (pg/mL)	36.4	2.50	2	56.37	./.	1	./.	./.	0	39.28	./.	1

 

Sex: Male (Termination)

 

Group	G1R			G4R
Dose(mg/kg bwt)	0			750
	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	2.98	0.63	5	4.76	2.18	5
TSH (uIU/mL)	5.03	1.73	5	3.91	1.65	5
Testosterone (ng/dL)	54.67	37.03	5	160.34	183.40	5

 

Sex: Female (Termination)

 

Group	G1-R			G4-R
Dose(mg/kg bwt)	0			750
	Mean	SD	N	Mean	SD	N
T4 (ug/dL)	2.82	0.11	5	2.72	0.43	5
TSH (uIU/mL)	4.69	4.74	5	2.82	2.74	5
Testosterone (ng/dL)	45.64	33.01	5	24.04	21.81	5

Day: 04 (Pups)

 

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			750
	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
	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

Applicant's summary and conclusion

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).The 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 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. 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,

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. 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 reproductive parameters were observed such as 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. Gestational length, Litter size, No. of live births, Postimplantation 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.

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