Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity: Oral

Based on the data available, the No Observed Adverse Effect Level (NOAEL) for the test chemical was considered to be 1250mg/Kg bw/day in male and female rats following oral gavage exposure for a period of 48 -66 days.

Repeated dose toxicity: inhalation

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the test chemical which is reported as mm Hg. Thus, exposure to inhalable dust, mist and vapour of the test chemical is highly unlikely. Therefore this study is considered for waiver.

Repeated dose toxicity: dermal

The acute toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Remarks:
Repeated Dose Oral Toxicity Study in combination with Reproduction / Developmental Toxicity study
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)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Animals were procured from a CPCSEA approved Vendor [National Institute of Biosciences, Pune Health
Status:Healthy young adult animals were used for the study. Females were nulliparous and non-pregnant.
Age: 12 - 13 weeks at the start of estrous cycle evaluation.
Acclimatisation: Animals were acclimatised to the test conditions for 7 days prior to test item administration.
Animal Identification: During acclimatisation period, all the animals were identified by temporary tail marking with indelible ink and cage cards. Following allocation to the study, each animal was uniquely identified by micro toe pad tattooing and colour coded cage cards labelled with study no., study type, test system, sex, dose, group, animal number, dosing start date, experimental start and completion date. Pups were identified with body marking with permanent non-toxic marker pen.
Husbandry/Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. 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]). Cage rotation was carried out weekly during study period except during mating and during gestation and lactation only for females. Pregnant females were caged individually.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. 4-17 and 817 (Krishana Corncob Industries, Aurangabad) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.
Environmental conditions: The room temperature was maintained at 19.60 to 24.70°C the relative humidity was kept between 41.80 to 66.60 %. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Air changes were about minimum 12 times per hour and filtered adequately.
Diet: A conventional laboratory pelleted diet of batch no. 040817, 041017, 040917 and 040617 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum. The copy of composition, Water Aqua guard filtered drinking water in bottles was offered ad libitum. Samples of the drinking water was subjected periodically to bacteriological tests and to chemical contaminant analysis. The latest test results are included in the raw data.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. The details of dose formulation preparation is maintained in raw data.
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): corn oil
- Concentration in vehicle: 0, 750, 1000 and 1250 mg/kg bw
- Amount of vehicle (if gavage): 2 mL/100g body weight
- Lot/batch no. (if required): A1708001 and A1703001
- 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 analyze by using HPLC-UV.
Duration of treatment / exposure:
48 days (male rats, main study)
Approx 63 days (female rats, main study)
66 days (recovery groups)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 (main study), G1-R (recovery group)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
G2
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
G3
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Remarks:
G4 (main study), G4-R (recovery group)
No. of animals per sex per dose:
13 rats per sex per dose (main study)
5 rats per sex per dose (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: - Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights were considered within ± 20% of the groups mean.
Details of the randomization was documented in the raw data. - Other:
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule:twice daily (morning and evening)
- Cage side observations checked in table [No.?] were 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 considering the peak period of anticipated effects after dosing. BODY WEIGHT: Yes - Time schedule for examinations:Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Five males and five females, randomly selected from each group, just prior to necropsy.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, Animals were fasted overnight prior to blood collection.
- How many animals:Five males and five females, randomly selected from each group,
- Parameters checked in table [No.?] were examined.: Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined. CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Five males and five females, randomly selected from each group, just prior to necropsy
.- Animals fasted: Yes, Animals were fasted overnight prior to blood collection.
- How many animals:Five males and five females, randomly selected from each group,
- Parameters checked in table [No.?] were examined.: Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb), Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN), Globulin (Glob), Alb/ Glb (A:G), Bile acids, Thyroid hormones (Total T4 and TSH), Testosterone and Estrogen were examined. URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified- Parameters checked in table [No.?] were examined.Not specified
NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations:During the last week of treatment and that of recovery groups
- Dose groups that were examined: five males and five females from control and treatment groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity were assess.
IMMUNOLOGY: Not specified
- Time schedule for examinations:Not specified
- How many animals:Not specified
- Dose groups that were examined:Not specified- Parameters checked in table [No.?] were examined.: Not specified
OTHER:number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.and Organ weight were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes 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. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.

HISTOPATHOLOGY: Yes Tissues were preserved in 10 % neutral buffered formalin (NBF) (except eyes, which was fixed using Modified Davidson’s fluid; testes and epididymis, which were fixed in Bouin’s fluid for approximately 24 hours and subsequently preserved in 10 % NBF) for subsequent histopathological examination. Organ examined. Adrenals, Pancreas, Aorta, Peyer's Patches, Bone (femur) with joint , Pituitary, Brain, (cerebrum,cerebellum,mid brain), Prostate and Seminal vesicle with coagulating glands as a whole, Cecum Rectum, Colon, Salivary glands Duodenum, Sciatic Nerve, Epididymide, Skeletal muscle, Eyes with optic nerve, Skin, Spinal Cord (cervical, mid-thoracic and lumbar), Heart, Spleen, Ileum, Sternum with marrow, Jejunum, Stomach, Kidneys, Testes, Liver, Thymus, Lungs, Thyroid with Parathyroids, Mammary glands, Trachea, Mesenteric and Mandibular lymph node, Urinary Bladder, Oesophagus, Uterus, Ovaries with oviduct, Cervix with Vagina and Gross lesion (if any), 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 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
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 period. However, statistically significant increase was noted in urine pool in group G2 in male on week 2 as compared to the G1 control group. In recovery female, statistically significant decrease was noted in rearing in G4-R on pretreatment, and increase in fecal bolus on week 1 as compared to the control recovery group G1-R. Statistically significant increase in urine pool in male G2 group on week 2 as compared to the control group G1.Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period in Main study.The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Changes in haematology in the adult rats included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. The changes in haematology were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology were not considered to be of toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in clinical chemistry were not considered to be of toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Liver: multifocal minimal to mild Neutrophil/lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female: G1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), Kidneys: focal to multifocal mild lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female: G1/5, G4:1/5); Lungs: multifocal minimal to mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Female: G1/5, G4:1/5), Heart: focal to multifocal minimal lymphocyte infiltration (Male: G1:1/5; Female: G4:1/5), Spleen: multifocal mild Extramedullary haematopoiesis (Male: G1:1/5), Adrenals: multifocal mild cytoplasmic vacuolation (male: G1:1/5), accessory adrenocortical tissue (Unilateral: Male: G1:2/5, G4:2/5; Female: G1:1/5), Testes: Male: focal mild seminiferous tubule degeneration (Male: G1: 2/13, G2:2/13, G4: 2/13, G1-R: 1/5); focal minimal to mild retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13); focal minimal to mild sloughing of round spermatid/pachytene spermatocyte (Male: G1: 1/13, G2:1/13). Lesions observed in liver, kidneys, lungs, heart, adrenals, spleen and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
other: Hormonal data
Remarks on result:
other: No effect observed
Critical effects observed:
no

Mortality and Morbidity

ex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

750

13

01/13 (FD on Day 42)

G3

Mid

1000

13

01/13 (FD on Day 21)

G4

High

1250

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

1250

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

750

13

NMM

G3

Mid

1000

13

NMM

G4

High

1250

13

NMM

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

1250

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number, FD= Found dead after dosing due to gavaging error.

Mean Body Weight (g)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

1

342.62

38.88

341.69

38.57

344.08

38.37

337.08

31.20

8

355.31

48.11

347.08

55.55

338.23

44.53

335.08

37.25

15

363.77

50.47

357.62

53.63

353.15

50.02

341.38

36.18

22

370.15

53.77

367.54

55.67

370.25

42.67

353.46

41.27

29

382.85

59.71

379.54

58.29

380.83

45.47

363.38

42.90

36

393.92

61.79

383.92

62.16

390.42

47.05

374.54

45.36

43

397.62

62.58

385.67

68.37

393.00

44.52

385.00

49.03

48

399.31

65.46

386.67

69.03

390.25

47.06

377.23

48.68

49 (Fasting)

381.23

61.15

370.17

66.41

373.83

45.13

357.62

45.10

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

1

249.00

15.23

13

245.69

13.26

13

248.31

14.01

13

247.69

10.63

13

8

255.54

17.02

13

251.69

16.84

13

251.00

13.75

13

247.00

15.71

13

15

259.54

16.76

13

254.31

13.43

13

253.69

13.91

13

252.38

14.69

13

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

Period: Gestation                                                                                                  Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

0

260.80

18.91

10

261.88

20.22

08

258.10

14.71

10

247.50

10.20

10

7

279.80

19.66

10

280.25

18.46

08

277.10

17.09

10

266.10

15.07

10

14

308.70

22.28

10

304.38

15.56

08

306.00

24.24

10

291.50

15.34

10

20

372.30

20.72

10

347.63

25.29

08

364.90

26.10

10

345.70

29.46

10

 Period: Gestation (Non pregnant)                                                                       Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. Wt.)

0

750

1000

1250

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

0

270.67

11.59

3

255.20

16.02

5

253.67

5.03

3

267.00

12.12

3

7

281.67

13.58

3

269.00

22.89

5

273.00

7.21

3

284.00

12.49

3

14

287.67

10.69

3

282.60

23.45

5

277.33

15.63

3

288.00

10.54

3

20

287.33

12.66

3

278.80

29.56

5

271.33

11.93

3

282.33

14.29

3

27

283.00

11.53

3

273.00

23.13

5

268.33

14.57

3

281.33

14.05

3

28

./.

./.

-

243.00

./.

1

./.

./.

./.

./.

./.

./.

29

287.00

8.49

2

270.00

57.98

2

275.00

./.

1

./.

./.

./.

30

272.50

4.95

2

288.50

4.95

2

260.50

2.12

2

291.00

./.

1

31

./.

./.

-

274.00

./.

1

247.00

./.

1

275.67

7.09

3

32

272.00

./.

1

272.50

13.44

2

258.00

./.

1

257.00

8.49

2

33

261.00

./.

1

258.00

11.31

2

239.00

./.

1

./.

./.

./.

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

Period: Post-Partum                                                                                                         Sex: Female

Group (N)

G1 (10)

G2 (8)

G3 (10)

G4 (10)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0

300.50

23.88

300.13

19.72

300.80

28.32

285.30

21.07

4

296.10

22.48

296.88

23.19

291.80

26.25

281.70

22.17

13

303.10

17.79

291.25

23.00

294.10

27.20

281.00

22.41

14(Fasting)

276.30

18.68

272.13

19.43

270.50

26.91

255.50

17.84

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

Note: Three female was non-pregnant in Group G1, G3, G4 and five females were non-pregnant in G2 group.

Mean Body Weight

Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1250

Day

Mean

SD

Mean

SD

1

348.00

45.43

344.80

45.15

8

357.60

53.02

341.40

51.21

15

362.40

57.13

355.20

54.20

22

376.80

63.36

345.00

63.65

29

385.80

69.32

360.00

65.83

36

388.20

71.38

370.00

73.75

43

389.40

76.07

377.00

82.97

50

396.60

79.96

383.40

85.84

57

392.80

80.53

387.00

80.74

64

398.60

85.97

400.20

86.04

66

395.00

77.78

397.40

84.00

67(fasting)

378.8

77.81

378.40

78.41

Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1250

Day

Mean

SD

Mean

SD

1

252.00

15.02

252.20

16.65

8

257.40

16.64

255.80

19.77

15

263.80

23.56

257.60

18.80

22

265.60

20.33

258.00

19.79

29

272.20

23.40

264.00

21.27

36

275.20

28.01

268.60

23.33

43

277.20

28.74

271.80

21.29

50

278.00

28.35

266.00

24.53

57

277.60

28.88

269.60

20.72

64

282.20

33.37

276.20

20.14

66

275.40

29.78

270.20

20.15

67(fasting)

265.40

31.34

258.40

18.71

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

Mean Feed Consumption (g/day/animal)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(1-8)

54.80

8.82

53.93

10.59

49.92

8.83

49.33

8.63

(8-15)

52.64

8.74

53.28

9.50

53.90

9.02

52.06

7.93

(15-29)

51.10

8.56

53.16

8.66

55.60

7.29

52.07

7.15

(29-36)

52.46

9.45

53.65

8.30

58.17

8.45

53.31

7.93

(36-43)

51.79

9.45

56.11

11.46

56.00

7.66

52.87

8.41

(43-48)

13.06

1.87

14.04

2.19

14.28

1.40

13.43

2.84

 

Period: Pre-mating                                                                                                     Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(1-8)

14.51

1.14

13.36

1.48

13.03

0.96

12.28

2.07

(8-14)

14.47

1.14

14.22

0.77

14.20

0.88

14.89

0.75

Period: Gestation                                                                                                  Sex: Female

Group

G1

Dose

(mg/kg b. wt.)

0

Day

Mean

SD

N

(0-7)

16.95

2.15

13

(7-14)

19.34

2.30

13

(14-20)

21.03

4.77

13

(20-22)

13.29

4.21

7

(20-23)

13.75

4.55

3

(20-27)

11.96

4.61

3

(20-29)

3.60

0.71

2

(20-32)

6.62

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G2

Dose

(mg/kg b. wt.)

750

Day

Mean

SD

N

(0-7)

18.55

2.86

13

(7-14)

20.00

3.83

13

(14-20)

17.41

4.42

13

(20-22)

15.67

7.07

2

(20-23)

14.63

2.71

4

(20-26)

15.50

0.51

2

(20-27)

13.85

1.23

5

(27-28)

1.44

./.

1

(27-29)

4.30

./.

1

(27-30)

5.09

./.

1

(27-32)

7.27

1.47

2

Period: Gestation                                                                                                  Sex: Female

Group

G3

Dose

(mg/kg b. wt.)

1000

Day

Mean

SD

N

(0-7)

18.11

2.84

13

(7-14)

21.29

3.79

13

(14-20)

19.29

5.61

13

(20-22)

15.22

3.80

9

(20-23)

8.00

./.

1

(20-27)

12.92

0.52

3

(27-29)

2.50

./.

1

(27-30)

4.73

./.

1

(20-32)

6.62

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G4

Dose

(mg/kg b. wt.)

1250

Day

Mean

SD

N

(0-7)

17.46

2.42

13

(7-14)

18.98

2.23

13

(14-20)

17.13

4.16

13

(20-22)

14.50

3.32

8

(20-23)

16.88

0.88

1

(20-27)

15.00

0.78

1

(27-30)

4.91

./.

1

(20-31)

5.75

0.59

2

Period: Lactation                                                                                                        Sex: Female

Group (N)

G1 (10)

G2 (8)

G3 (10)

G4 (10)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(0-4)

25.30

7.97

21.63

3.85

26.03

5.93

27.18

6.81

(4-13)

37.77

11.39

32.35

9.45

38.77

5.23

35.64

13.22

Sex:Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1250

Day

Mean

SD

Mean

SD

(1-8)

58.30

14.02

54.40

15.79

(8-15)

55.37

15.34

54.21

17.07

(15-22)

55.11

14.70

56.56

17.29

(22-29)

53.70

12.85

53.39

16.52

(29-36)

54.27

12.85

55.43

16.16

(36-43)

53.02

11.68

55.81

17.64

(43-50)

54.33

12.26

57.21

18.08

(50-57)

56.02

10.88

56.58

15.03

(57-64)

62.60

13.10

61.77

16.18

(64-66)

46.33

14.07

48.37

5.24

 

 Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1250

Day

Mean

SD

Mean

SD

(1-8)

50.71

10.71

49.92

11.94

(8-15)

50.45

10.67

51.39

11.87

(15-22)

49.33

9.43

50.88

11.68

(22-29)

48.80

10.08

49.60

12.49

(29-36)

50.68

10.25

50.38

11.58

(36-43)

49.25

11.06

50.24

11.18

(43-50)

48.88

10.07

52.80

14.13

(50-57)

52.19

10.64

52.45

11.68

(57-64)

55.42

10.42

54.92

10.32

(64-66)

41.48

10.64

41.65

10.89

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

Sensory Reactivity Measurements

Sex:Male

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose (mg/kg b. wt.)

0

750

1000

1250

Parameter

Observation

No. of Animals Showing Observation

Approach response

Fast

5

5

5

5

Touch response

Normal

5

5

5

5

Click response

Normal

5

5

5

5

Pupil response

Normal

5

5

5

5

Air righting reflex

Normal

5

5

5

5

Tail pinch response

Flinch

5

5

5

5

 

Sex:Female

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose (mg/kg b. wt.)

0

750

1000

1250

Parameter

Observation

No. of Animals Showing Observation

Approach response

Fast

5

5

5

5

Touch response

Normal

5

5

5

5

Click response

Normal

5

5

5

5

Pupil response

Normal

5

5

5

5

Air righting reflex

Normal

5

5

5

5

Tail pinch response

Flinch

5

5

5

5

Mean Foot Splay Record

Sex:Male

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose           (mg/kg b. wt.)

0

750

1000

1250

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

FS (mm)

86.67

14.15

82.53

10.20

96.20

19.58

96.80

11.25

 

Sex: Female

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose        (mg/kg b. wt.)

0

750

1000

1250

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

FS (mm)

99.40

10.65

99.67

10.29

103.47

7.85

103.80

9.02

Mean Grip Strength Record

Sex: Male

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose             (mg/kg b. wt.)

0

750

1000

1250

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Fore limb (g)

758.00

68.93

749.53

50.02

823.60↑

80.15

743.40

76.87

Hind limb(g)

485.87

48.47

502.80

66.48

519.00

38.47

511.53

52.06

 Sex: Female

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose             (mg/kg b. wt.)

0

750

1000

1250

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Fore limb (g)

759.00

52.84

711.60

82.65

723.13

44.97

752.60

52.89

Hind limb(g)

500.13

36.19

526.67

62.71

525.67

64.58

518.93

51.85

Mean Motor Activity Record

Sex:Male

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose

(mg/kg b. wt.)

0

750

1000

1250

Parameter↓

Mean

SD

Mean

SD

Mean

SD

Mean

SD

DT(cm)

1546.60

431.24

1531.60

696.95

1753.60

478.07

1456.80

798.05

RT(sec)

213.40

83.79

221.60

117.98

174.40

65.96

239.60

101.98

ST (sec)

124.40

9.24

106.40

34.07

128.60

19.32

118.60

12.22

AT(sec)

262.20

79.78

272.00

99.21

297.00

66.55

241.80

104.44

BSM

88.60

3.78

78.80

21.74

89.40

9.81

84.00

13.84

HC

1328.60

355.89

1268.20

516.23

1504.00

310.06

1232.60

590.69

AC

885.80

246.84

844.20

373.21

992.60

250.47

823.60

462.65

HB

0.40

0.55

1.40

2.07

0.80

0.45

1.00

0.71

CR

23.40

7.40

22.80

10.18

23.20

7.60

21.00

9.59

CCR

23.60

8.02

21.60

10.71

24.60

3.91

17.20

7.60

Sex: Female

Group (N)

G1 (5)

G2 (5)

G3 (5)

G4 (5)

Dose

(mg/kg b.wt.)

0

750

1000

1250

Parameter↓

Mean

SD

Mean

SD

Mean

SD

Mean

SD

DT(cm)

986.00

675.81

955.20

793.46

1825.20

1602.61

1155.60

620.76

RT (sec)

328.60

159.43

346.00

154.97

260.20

168.97

290.80

159.72

ST (sec)

122.20

79.47

108.00

57.37

110.60

56.39

130.00

73.75

AT(sec)

149.20

92.23

146.00

109.95

229.20

141.97

179.20

90.59

BSM

65.20

35.56

61.40

30.44

67.80

29.84

76.40

41.00

HC

937.20

621.38

944.60

700.99

1530.60

1259.24

1089.80

630.68

AC

549.80

380.47

562.00

473.70

1066.20

996.01

617.60

362.50

HB

2.20

3.27

3.80

4.32

2.20

1.79

1.40

1.67

CR

12.20

7.09

11.00

7.91

22.20

15.97

16.00

9.62

CCR

11.00

5.15

13.80

10.26

20.80

16.18

15.80

9.18

Keys:DT=Distance travelled (cm), RT=Resting time (sec), ST=Stereotypic time (sec), AT=Ambulatory time (sec), BSM=Burst of stereotypic movements,HC= Horizontal counts, AC=Ambulatory count, HB=Horizontal break, CR=Clockwise rotation and CCR=Counter clockwise rotation and N = Number of animals in the group, SD= Standard Deviation, cm= centimeter, sec= seconds,


 

MeanHormonal Analysis Data

 Sex: Male

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

750

1000

1250

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.33

0.55

13

4.24

2.66

13

4.28

2.67

13

3.62

0.66

13

TSH (uIU/mL)

2.16

1.47

13

4.55

4.01

13

2.69

3.22

13

2.38

1.65

13

Testosterone (ng/dL)

185.98

147.96

13

188.00

192.69

13

148.34

187.15

13

169.54

140.02

13

 

Sex: Female

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

750

1000

1250

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.66

0.56

10

3.81

1.02

8

3.35

0.44

10

3.75

0.58

10

TSH (uIU/mL)

3.45

1.05

10

3.49

2.52

8

2.21

0.93

10

3.26

1.03

10

E2 (pg/mL)

 

24.26

17.29

10

45.51

31.42

8

23.88

6.77

10

31.09

16.46

10

 Sex: Female (Non-Pregnenat)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

750

1000

1250

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.47

0.15

3

3.24

 

0.43

5

3.57

 

0.40

3

3.07

0.35

3

TSH (uIU/mL)

1.63

0.21

3

2.22

1.28

5

1.81

0.88

3

0.93

0.27

3

E2 (pg/mL)

 

38.06

5.98

3

34.73

26.87

5

19.28

0.83

2

34.69

14.33

3

Sex: Male

 

Group(n)

G1R

G4R

Dose(mg/kg bwt)

0

1250

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.86

0.79

5

3.40

0.85

5

TSH (uIU/mL)

3.04

2.08

5

1.21

1.16

5

Testosterone (ng/dL)

20.95

10.16

5

90.59

84.78

5

 

 

 

Sex: Female

 

Group(n)

G1R

G4R

Dose(mg/kg bwt)

0

1250

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.62

 

0.31

5

2.58

0.54

5

TSH (uIU/mL)

0.86

0.42

5

2.43

0.83

5

Testosterone (ng/dL)

20.69

7.96

4

21.31

7.49

5
Conclusions:
NOAEL for the test chemical was established at 1250 mg/kg bw/day in male and female rats after oral gavage treatment for a period of 48 to 66 days  
Executive summary:

The chemical was given to 13 rats per sex per dose level at 0, 750, 1000 and 1250 mg/kg bw/day by oral gave. Male rats were treated two weeks before mating and thereafter for a total of 48 dosing days. Female rats were treated two weeks before mating, during mating, during gestation and during lactation, for a total of approx. 63 dosing days. Recovery groups of male and female rats (5/sex/dose) were treated at 0 or 1250 mg/kg bw/day for 66 days in total. Animals in the recovery groups were allowed to recover for two weeks after the final dose was given. No morbidity was observed during the study period. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg). Clinical findings were sporadic and of no biological significance. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical. Food consumption was unaffected by treatment. Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical. Oestrous cyclicity was unaffected by treatment. All female rats showed evidence of copulation after the cohabitation/mating period. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births, pup survival, pup weight or sex ratio. Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical. Microscopic examination of the pups’ thyroid and parathyroid glands in the 0 and 1250 mg/kg treatment groups revealed no abnormalities. Changes in haematology in the adult rats included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in haematology and clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology and clinical chemistry were not considered to be of toxicological importance. Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females). There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides. All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg. Microscopic examination revealed no treatment-related effects, that is, the incidences and types of lesions observed at 1250 mg/kg were comparable to that of the concurrent control groups. Ophthalmological examination was not performed, however, detailed clinical examinations and microscopic examination of the eyes with optic nerve (at 0 and 1250 mg/kg) did not reveal any abnormalities. NOAEL for the test chemical was established at 1250 mg/kg bw/day in male and female rats after oral gavage treatment for a period of 48 to 66 days  

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is Klimicsh 1 and from expeimental study report

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: Oral

Data available from experimental study for the test chemicals was reviewed to determine the toxic nature of test chemical. The study is mentioned below:

The chemical was given to 13 rats per sex per dose level at 0, 750, 1000 and 1250 mg/kg bw/day by oral gave. Male rats were treated two weeks before mating and thereafter for a total of 48 dosing days. Female rats were treated two weeks before mating, during mating, during gestation and during lactation, for a total of approx. 63 dosing days. Recovery groups of male and female rats (5/sex/dose) were treated at 0 or 1250 mg/kg bw/day for 66 days in total. Animals in the recovery groups were allowed to recover for two weeks after the final dose was given. No morbidity was observed during the study period. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg). Clinical findings were sporadic and of no biological significance. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical. Food consumption was unaffected by treatment. Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical. Oestrous cyclicity was unaffected by treatment. All female rats showed evidence of copulation after the cohabitation/mating period. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births, pup survival, pup weight or sex ratio. Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical. Microscopic examination of the pups’ thyroid and parathyroid glands in the 0 and 1250 mg/kg treatment groups revealed no abnormalities. Changes in haematology included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in haematology and clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology and clinical chemistry were not considered to be of toxicological importance. Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females). There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides. All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg. Microscopic examination revealed no treatment-related effects, that is, the incidences and types of lesions observed at 1250 mg/kg were comparable to that of the concurrent control groups. Ophthalmological examination was not performed, however, detailed clinical examinations and microscopic examination of the eyes with optic nerve (at 0 and 1250 mg/kg) did not reveal any abnormalities. NOAEL for the test chemical was established at 1250 mg/kg bw/day in male and female rats after oral gavage treatment for a period of 48 to 66 days  

Repeated dose toxicity: inhalation

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the test chemical which is reported as mm Hg. Thus, exposure to inhalable dust, mist and vapour of the test chemical is highly unlikely. Therefore this study is considered for waiver.

Repeated dose toxicity: dermal

The acute toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

The chemical is regarded to be classified as Not Classified for STOT RE by the oral route.