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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction:
NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test material orally by gavage for more the 63 days. Thus, comparing this value with the criteria of CLP regulation test chemical not likely to classify as reproductive toxicant.

Link to relevant study records
Reference
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
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
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
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
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):
Positive control:
not specified
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.
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: 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.
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
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
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.
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
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
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
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

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
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is of K1 reliability from study report
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction:

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.



Based on the experimental studies available for the test chemical were reviewed to determine the developmental toxicity, NOAEL for test chemical was considered to be 750mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive toxicant.


Effects on developmental toxicity

Description of key information

Developmental toxicity/ teratogenicity:

Based on the experimental studies available for the test chemical were reviewed to determine the developmental toxicity, NOAELfor test chemical was considered to be 750mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulationtest chemical is not likely to classify as reproductive and developmental toxicant.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
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 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:
no
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:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): 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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects observed
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

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

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is of K1 reliability from study report
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity/ teratogenicity:

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.

Based on the various studies available for the test chemical were reviewed to determine the developmental toxicity, NOAELfor test chemical was considered to be 1000 mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria ofCLP regulationtest chemicalisnot likely to classify as reproductive and developmental toxicant.

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation Test chemical not likely to classify as reproductive and developmental toxicant.

Additional information