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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Toxicity to Reproduction:

No adverse effects on reproduction or development were observed at 250, 500 or 1000 mg/kg bw/day in the performed OECD 421 with rats.

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 a study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Name: N-Acetylsulphanilyl chloride
IUPAC Name: Benzenesulfonyl chloride, 4-(acetylamino)-
CAS No.: 121-60-8
Molecular Formula: C8-H8-Cl-N-O3-S
Molecular Weight: 233.67 g/mol
SMILES: CC(=O)Nc1ccc(cc1)S(=O)(=O)Cl
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a standard laboratory rodent species used for toxicity assessment and is recommended to be used in OECD 421.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 9 to 10 weeks;
- Weight at study initiation: (P) Males: 220.33 to 250.60 g; Females: 200.31 to 240.79 g;
- Fasting period before study: No data available
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, a maximum of two animals of the same sex were housed.
- Pre mating - Per cage, two animals of the same sex and group were housed.
- Cohabitation Period (mating) - Per cage, two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming the presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates, while females were housed individually.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was available ad libitum to the animals throughout the acclimatization and experimental periods.
- Water (e.g. ad libitum): Water was available ad libitum throughout the acclimatization and experimental periods. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 23.4 °C
- Humidity (%): 46 to 68%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES:
From: 16 June 2020
To: 07 September 2020
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil and formed uniform suspension at the concentration of 200 mg/mL (considering the highest dose of 1000 mg/kg body weight with a dose volume of 5 mL/kg body weight).

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle.
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): L32011001
- Purity: No data available
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear] referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No data available
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The required quantity of the test chemical was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed, and after that, the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar, and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to the required quantity with the vehicle to get the desired concentration of 50, 100, and 200 mg/mL of test item for low, mid and high dose groups, respectively.
The test formulations were maintained under stirring conditions using a magnetic stirrer to maintain homogeneity of the test chemical formulations.
The test chemical formulations were stable and homogeneous for 6 hours at room temperature and 48 hours at 2 to 8˚C with concentrations of 10.0 mg/mL and 250.0 mg/mL in corn oil. Prepared test item formulations were administered to the animals within established stability conditions.
The samples were collected in duplicates (5 mL each) from the top, middle, and bottom layers from low, mid, and high dose concentrations and in duplicates from a single layer from vehicle control.
The prepared test item formulations were stirred using magnetic stirrer during sampling.
One set of aliquots of each formulation was analyzed. The second aliquot was stored for backup purpose at established stability conditions. The second set of samples were discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is <10%.

Chromatographic Conditions:
Column : Zorbax Eclipse plus C18, 4.6 ×250 mm, 5 μm
Flow rate : 0.8 mL/min
Injection volume : 10 μL
Wavelength : 264 nm
Run time : 10 minutes
Column Oven Temperature: 25°C
Mobile Phase: Acetonitrile: Milli-Q water (80:20 %, v/v)
Diluent: Acetonitrile
Duration of treatment / exposure:
Males: 29 days
Females: 63 days
Frequency of treatment:
Once Daily
Details on study schedule:
No data available
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on available data of the substance and of read-across substances.
- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -12.09 to 14.54%; Females: -13.49 to 7.51%) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment by randomization based on the body weights. Body weight of the animals were analyzed statistically for mean body weight to rule out statistically significant differences between groups within each sex prior to dosing.
- Fasting period before blood sampling for clinical biochemistry: No Data Available
- Other: No Data Available
Positive control:
No positive control chemical was used.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: No Data Available

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Feed consumption was measured for all animals once a week during premating and weekly once for males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, the feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OTHER: No data
Oestrous cyclicity (parental animals):
Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).
Sperm parameters (parental animals):
A detailed histopathological examination was performed on the ovaries, testes, and epididymides. Particular emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals was given.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible);

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No Data Available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No Data Available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed after 29 days of treatment
- Maternal animals: All surviving females were sacrificed on lactation day 14 (i.e. after approx. 63 days of treatment)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
The F1 pups were sacrificed on PND 4 or 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as provided in Table 2 below.

GROSS NECROPSY
- The pups, which were sacrificed on PND 4 or 13 were examined for gross abnormalities with particular attention to the external reproductive genitals.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.
Statistics:
The statistical analysis was followed to the parameters in the table 3 given below.
Reproductive indices:
Mating and Fertility Indexes (both genders), Gestation Index, Parturition Index, and Pregnancy Index.
Offspring viability indices:
Litter Index and Pup Viability Index
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity in any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant changes in body weight or body weight gain with respect to day 1 of treatment in any of the groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in food intake were observed, with the exception of a significant increase in food intake during the pre-mating period in male rats treated at 1000 mg/kg bw/day. This change was considered incidental and toxicological insignificant since the increase was minimal and could not be correlated with any changes in body weight or body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant changes in serum Thyroxine (T4) hormone levels in any of the male groups. As there were no significant changes observed in serum T4 levels in adult males, the assessment was not extended to adult females (dams).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related histopathological findings at 1000 mg/kg bw/day (MF/F). A single incidence of minimal, multifocal, degeneration/atrophy of seminiferous tubules in testes was observed in high dose male without any correlating findings in epididymides of the same animal. This change was considered to be an expected spontaneous finding in rats of this age.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic lesions were observed at 0 or 1000 mg/kg bw/day.
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no irregularities observed in the oestrus cyclicity in any of the female groups during the pre-mating or mating period. No significant changes in mean oestrus cycle length were observed between the groups during the pre-mating or mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related adverse effects on the stages of spermatogenesis were observed during the microscopic examination of interstitial testicular cell structure.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 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
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
All pups showed normal behaviour during the daily inspections.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in pup weight (M/F) were observed on PND 1, PND 4, PND 7 or PND 13.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant changes in serum Thyroxine (T4) hormone levels were observed on PND 13 in any of the groups.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No significant changes were observed.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in mean pup anogenital distance (AGD) or AGD ratio were observed in any of the groups, with the exception of a significant decrease in AGD in male pups from the 1000 mg/kg dose group (mean 4.30 mm) versus the control group (mean 4.45 mm). AGD in male pups from the 1000 mg/kg dose group was within the historical control range of the test facility. Therefore, this small change in AGD at 1000 mg/kg was not considered to be an adverse effect of treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no male pups with retention of nipples/areolae on PND 13 in any of the groups.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological findings in any of the groups.
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
other: Anogenital Distance and Nipple Retention in Male Pups
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Treatment related:
no

Table 1. CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Mortalityb:

No. of Mortalities
(Total No. of Animals)

G1, M & 0

12

Day 1 to termination:N (12)

0 (12)

G2, M & 250

12

Day 1 to termination:N (12)

0 (12)

G3, M & 500

12

Day 1 to termination:N (12)

0 (12)

G4, M & 1000

12

Day 1 to termination:N (12)

0 (12)

M: Male; N: Normal; a: observed daily once; b: observed twice daily

Table 1. (Contd…) CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Mortalityb

No. of Mortalities
(Total No. of Animals)

G1, F & 0

12

Day 1 to termination:N (12)

0 (12)

G2, F & 250

12

Day 1 to termination:N (12)

0 (12)

G3, F & 500

12

Day 1 to termination:N (12)

0 (12)

G4, F & 1000

12

Day 1 to termination:N (12)

0 (12)

F: Female; N: Normal; a: observed daily once; b: observed twice daily

Table 2. BODY WEIGHT (g) RECORD

Group, Sex & Dose
(mg/kg body weight/day)

Body Weight (g) on Day

1

7

14

21

28

G1, M & 0

Mean

308.34

317.29

330.05

341.22

354.20

±SD

27.07

29.54

31.52

30.98

30.62

n

12

12

12

12

12

G2, M & 250

Mean

309.84

320.97

335.04

347.24

358.49

±SD

27.78

30.67

33.73

38.29

39.14

n

12

12

12

12

12

G3, M & 500

Mean

309.10

315.94

331.53

341.09

357.44

±SD

26.85

30.34

34.01

37.59

41.82

n

12

12

12

12

12

G4, M & 1000

Mean

310.77

318.10

334.03

344.30

359.74

±SD

24.54

22.14

19.72

20.07

22.00

n

12

12

12

12

12

M: Male; SD: Standard Deviation; n: Number of Animals

TABLE 2. (Contd…) BODY WEIGHT (g) RECORD

Group, Sex & Dose
(mg/kg body weight/day)

Body Weight (g) on Day

1

7

14

21#

G1, F & 0

Mean

238.06

241.92

249.61

258.80

±SD

12.86

13.25

13.50

8.01

n

12

12

12

5

G2, F & 250

Mean

239.90

244.46

251.08

253.10

±SD

12.67

14.54

13.61

7.61

n

12

12

12

7

G3, F & 500

Mean

239.99

242.97

248.77

245.89

±SD

11.44

13.34

14.54

5.02

n

12

12

12

5

G4, F & 1000

Mean

240.24

245.24

250.28

254.92

±SD

11.06

11.93

11.13

5.19

n

12

12

12

3

F: Female; SD: Standard Deviation; n: Number of Animals; #: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

Table 3. PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1

Group, Sex & Dose
(mg/kg body weight/day)

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21

1 to 28

G1, M & 0

Mean

2.86

6.99

10.66

14.90

±SD

1.33

2.26

2.37

2.21

n

12

12

12

12

G2, M & 250

Mean

3.54

8.10

12.05

15.74

±SD

1.26

4.30

7.06

8.02

n

12

12

12

12

G3, M & 500

Mean

2.15

7.14

10.17

15.40

±SD

1.83

3.11

3.81

4.96

n

12

12

12

12

G4, M & 1000

Mean

2.44

7.69

11.09

16.12

±SD

1.94

3.97

6.42

7.99

n

12

12

12

12

M: Male; SD: Standard Deviation; n: Number of Animals

Table 3. (Contd…) PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1

Group, Sex & Dose
(mg/kg body weight/day)

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21#

G1, F & 0

Mean

1.62

4.86

8.47

±SD

1.04

1.64

2.45

n

12

12

5

G2, F & 250

Mean

1.87

4.66

7.82

±SD

1.05

1.45

1.39

n

12

12

7

G3, F & 500

Mean

1.21

3.62

5.52

±SD

1.32

1.74

1.96

n

12

12

5

G4, F & 1000

Mean

2.08

4.24

9.05

±SD

1.07

3.40

5.51

n

12

12

3

F: Female; SD: Standard Deviation; n: Number of Animals; #: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

Table 4. FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex & Dose
(mg/kg body weight/day)

 

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, M & 0

Mean

21.40

22.44

±SD

0.68

1.12

n

12

12

G2, M & 250

Mean

20.88

22.97

±SD

1.47

0.70

n

12

12

G3, M & 500

Mean

20.67

23.26

±SD

1.03

0.88

n

12

12

G4, M & 1000

Mean

21.16

23.91*

±SD

0.70

0.74

n

12

12

M: Male; SD: Standard Deviation; n: Number of Animals;*: Statistically significant (P<0.05) change than the concurrent vehicle control group

Table 4. (Contd…) FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex & Dose
(mg/kg body weight/day)

 

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, F & 0

Mean

16.44

17.73

±SD

1.24

1.35

n

12

12

G2, F & 250

Mean

15.55

16.26

±SD

1.10

1.36

n

12

12

G3, F & 500

Mean

15.26

16.89

±SD

1.09

0.70

n

12

12

G4, F & 1000

Mean

15.97

17.38

±SD

1.33

1.08

n

12

12

F: Female; SD: Standard Deviation; n: Number of Animals

Table 5. VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Determination of Oestrus Cyclicity during Pre-Mating Treatment Period

Group, Sex & Dose
(mg/kg body weight/day)

Total No. of

Females Evaluated

No. of Females with Complete Regular Oestrus Cycle

No. of Females with at least one Irregular Oestrus Cycle

Average Length of

Oestrus Cycle (Days)

G1, F & 0

12

n

12

0

Mean

4.96

%

100.0

0.0

±SD

0.14

n

12

G2, F & 250

12

n

12

0

Mean

5.00

%

100.0

0.0

±SD

0.00

n

12

G3, F & 500

12

n

12

0

Mean

5.00

%

100.0

0.0

±SD

0.00

n

12

G4, F & 1000

12

n

12

0

Mean

4.92

%

100.0

0.0

±SD

0.19

n

12

F: Female; SD: Standard Deviation; n: Number of Animals

Table 6. GESTATION BODY WEIGHT (g)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Gestation Day (GD)

0

7

14

20

G1, F & 0

Mean

255.89

265.36

287.90

350.03

±SD

14.75

14.26

12.93

15.45

n

11

11

11

11

G2, F & 250

Mean

259.51

272.15

296.43

355.73

±SD

14.04

13.24

10.69

10.17

n

10

10

10

10

G3, F & 500

Mean

255.30

265.51

289.00

353.63

±SD

12.00

11.63

14.22

20.46

n

11

11

11

11

G4, F & 1000

Mean

255.13

265.39

291.32

349.51

±SD

10.99

12.46

14.26

14.57

n

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table 7. PERCENT CHANGE IN BODY WEIGHT (%) DURING GESTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Gestation Day (GD)

0 to 7

7 to 14

14 to 20

G1, F & 0

Mean

3.73

8.55

21.60

±SD

1.11

1.96

2.39

n

11

11

11

G2, F & 250

Mean

4.91

9.00

20.07

±SD

1.54

2.52

3.36

n

10

10

10

G3, F & 500

Mean

4.02

8.83

22.34

±SD

1.32

1.65

3.09

n

11

11

11

G4, F & 1000

Mean

4.01

9.77

20.12

±SD

1.22

1.56

5.45

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams;Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table 8. FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

 

Feed Consumption (g/animal/day) during Gestation Day (GD)

0 to 7

7 to 14

14 to 20

G1, F & 0

Mean

18.83

20.07

22.26

±SD

1.36

1.07

0.87

n

11

11

11

G2, F & 250

Mean

17.78

19.30

22.09

±SD

0.79

0.75

1.24

n

10

10

10

G3, F & 500

Mean

18.69

19.91

22.77

±SD

1.43

1.58

1.16

n

11

11

11

G4, F & 1000

Mean

19.08

21.06

22.55

±SD

1.07

1.12

2.13

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams;Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis

Table 9. LACTATION BODY WEIGHT (g)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Lactation Day (LD)

1

4

7

13

G1, F & 0

Mean

266.00

272.73

287.58

307.40

±SD

15.39

15.24

13.98

17.20

n

11

11

11

11

G2, F & 250

Mean

271.21

278.63

294.88

315.58

±SD

10.75

10.27

9.75

11.90

n

10

10

10

10

G3, F & 500

Mean

270.12

276.19

292.81

313.45

±SD

18.66

18.87

20.60

17.26

n

11

11

11

11

G4, F & 1000

Mean

269.63

273.55

288.30

308.16

±SD

10.61

11.45

11.16

13.53

n

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams

Table 10. PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1, F & 0

Mean

2.55

5.50

6.88

±SD

1.39

1.81

2.62

n

11

11

11

G2, F & 250

Mean

2.75

5.86

7.02

±SD

1.21

1.90

2.25

n

10

10

10

G3, F & 500

Mean

2.26

6.02

7.18

±SD

1.27

2.10

3.18

n

11

11

11

G4, F & 1000

Mean

1.45

5.42

6.90

±SD

1.36

2.07

2.75

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams

Table 11. FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

 

Feed Consumption (g/animal/day) during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1, F & 0

Mean

25.76

31.06

34.70

±SD

0.97

2.24

1.17

n

11

11

11

G2, F & 250

Mean

27.07

32.23

36.14

±SD

1.25

2.76

1.11

n

10

10

10

G3, F & 500

Mean

25.95

31.39

35.59

±SD

1.42

2.86

1.59

n

11

11

11

G4, F & 1000

Mean

26.12

31.92

35.55

±SD

1.93

2.89

1.45

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams

Table 12. SUMMARY OF DELIVERYDATA (AT BIRTH) PER LITTER

Group, Sex

& Dose
(mg/kg body

weight/day)

Litter Delivered (No.)

Total Litter

Size (No.)

Live Pups (No.)

Dead Pups (No.)

Cannibalized Pups (No.)

Sex Ratio

(m/f)

at Birth

Live Birth

Index (%)

Male

Female

Total

Male

Female

Total

Undetermined

Male

Female

Total

G1, F & 0

Mean

10.09

5.27

4.73

10.00

0.09

0.00

0.09

0.00

0.00

0.00

0.00

1.21

99.09

±SD

1.97

1.90

1.27

2.00

0.30

0.00

0.30

0.00

0.00

0.00

0.00

0.53

3.02

n

11

11

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

10.80

5.30

5.40

10.70

0.10

0.00

0.10

0.00

0.00

0.00

0.00

1.28

99.00

±SD

1.69

1.25

2.12

1.77

0.32

0.00

0.32

0.00

0.00

0.00

0.00

1.05

3.16

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 500

Mean

9.82

4.91

4.91

9.82

0.00

0.00

0.00

0.00

0.00

0.00

0.00

1.15

100.00

±SD

1.17

1.22

1.58

1.17

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.57

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

10.33

5.92

4.42

10.33

0.00

0.00

0.00

0.00

0.00

0.00

0.00

1.52

100.00

±SD

1.44

1.51

1.51

1.44

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.64

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: male/female

Table 13. LITTER OBSERVATION DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body

weight/day)

During Lactation Day 1 to 4

Sex Ratio

(M/F) on

LD 4

Pup Survival

Index (%)

during

LD 1 to 4

Live Pups (No.)

Dead Pups (No.)

Cannibalized Pups (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

5.27

4.73

10.00

0.00

0.00

0.00

0.00

0.00

0.00

1.21

100.00

±SD

1.90

1.27

2.00

0.00

0.00

0.00

0.00

0.00

0.00

0.53

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

5.30

5.40

10.70

0.00

0.00

0.00

0.00

0.00

0.00

1.28

100.00

±SD

1.25

2.12

1.77

0.00

0.00

0.00

0.00

0.00

0.00

1.05

0.00

n

10

10

10

10

10

10

10

10

10

10

10

G3, F & 500

Mean

4.91

4.91

9.82

0.00

0.00

0.00

0.00

0.00

0.00

1.15

100.00

±SD

1.22

1.58

1.17

0.00

0.00

0.00

0.00

0.00

0.00

0.57

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

5.92

4.42

10.33

0.00

0.00

0.00

0.00

0.00

0.00

1.52

100.00

±SD

1.51

1.51

1.44

0.00

0.00

0.00

0.00

0.00

0.00

0.64

0.00

n

12

12

12

12

12

12

12

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; M/F: Male/Female; LD: Lactation Day

Table 13. (Contd...) LITTER OBSERVATION DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body weight/day)

Pups Sacrificed for

blood collection (No.)

Live Pups (No.) on LD 4

after blood collection (No.)

During LD 5 to 7

Sex Ratio (M/F) on

LD 7

Pup Survival Index (%) during

LD 5 to 7

Live Pups (No.)

Dead Pups (No.)

Cannibalized (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

0.00

0.73

0.73

5.27

4.00

9.27

5.27

4.00

9.27

0.00

0.00

0.00

0.00

0.00

0.00

1.62

100.00

±SD

0.00

1.01

1.01

1.90

1.48

1.19

1.90

1.48

1.19

0.00

0.00

0.00

0.00

0.00

0.00

1.07

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

0.00

0.90

0.90

5.30

4.50

9.80

5.30

4.50

9.80

0.00

0.00

0.00

0.00

0.00

0.00

1.41

100.00

±SD

0.00

0.88

0.88

1.25

1.35

1.03

1.25

1.35

1.03

0.00

0.00

0.00

0.00

0.00

0.00

0.99

0.00

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 500

Mean

0.00

0.36

0.36

4.91

4.55

9.45

4.91

4.55

9.45

0.00

0.00

0.00

0.00

0.00

0.00

1.24

100.00

±SD

0.00

0.50

0.50

1.22

1.37

0.82

1.22

1.37

0.82

0.00

0.00

0.00

0.00

0.00

0.00

0.63

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

0.00

0.58

0.58

5.92

3.83

9.75

5.92

3.83

9.75

0.00

0.00

0.00

0.00

0.00

0.00

1.83

100.00

±SD

0.00

0.79

0.79

1.51

1.34

0.87

1.51

1.34

0.87

0.00

0.00

0.00

0.00

0.00

0.00

1.06

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; M/F: Male/Female

Table 13. (Contd...) LITTER OBSERVATION DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body

weight/day)

During LD 8 to 13

Sex Ratio

(M/F)

on LD 13

Pup Survival

Index (%)

during

LD 8 to 13

Live Pups (No.)

Dead Pups (No.)

Cannibalized (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

5.27

4.00

9.27

0.00

0.00

0.00

0.00

0.00

0.00

1.62

100.00

±SD

1.90

1.48

1.19

0.00

0.00

0.00

0.00

0.00

0.00

1.07

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

5.30

4.50

9.80

0.00

0.00

0.00

0.00

0.00

0.00

1.41

100.00

±SD

1.25

1.35

1.03

0.00

0.00

0.00

0.00

0.00

0.00

0.99

0.00

n

10

10

10

10

10

10

10

10

10

10

10

G3, F & 500

Mean

4.91

4.55

9.45

0.00

0.00

0.00

0.00

0.00

0.00

1.24

100.00

±SD

1.22

1.37

0.82

0.00

0.00

0.00

0.00

0.00

0.00

0.63

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

5.92

3.83

9.75

0.00

0.00

0.00

0.00

0.00

0.00

1.83

100.00

±SD

1.51

1.34

0.87

0.00

0.00

0.00

0.00

0.00

0.00

1.06

0.00

n

12

12

12

12

12

12

12

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; M/F: Male/Female

Table 14. REPRODUCTIVE PERFORMANCE

Group, Sex & Dose

(mg/kg body weight/day)

No. of Males with Evidence of Mating
(Total No. of Males used for Mating)

Male Mating Index (%)

No. of Males Capable of Impregnating a Female
(No. of males used for Mating)

Male Fertility Index (%)

G1, M & 0

12 (12)

100.0

11 (12)

91.7

G2, M & 250

12 (12)

100.0

10 (12)

83.3

G3, M & 500

12 (12)

100.0

11 (12)

91.7

G4, M & 1000

12 (12)

100.0

12 (12)

100.0

M: Male

Table 14. (Contd…) REPRODUCTIVE PERFORMANCE

Group, Sex & Dose                               (mg/kg body weight/day)

 

Copulatory interval / Pre-coital interval (Mean Time to Mating) / Cohabitation Record

 

Gestation Length / Duration of Pregnancy (Days)

Pre-coital Interval

Conceiving Days

(1 to 5)

Conceiving Days

(5 to More)

G1, F & 0

Mean

6.83

n

5

7

22.09

±SD

3.79

%

41.7

58.3

0.30

n

12

11

G2, F & 250

Mean

7.75

n

3

9

22.50

±SD

3.14

%

25

75

0.71

n

12

10

G3, F & 500

Mean

6.50

n

6

6

22.45

±SD

3.87

%

50

50

0.52

n

12

11

G4, F & 1000

Mean

5.33

n

7

5

22.42

±SD

3.85

%

58.3

41.7

0.51

n

12

12

F: Female; SD: Standard Deviation; n: Number of Females confirmed with mating (in case ofPre-Coital Interval) /Number of Females confirmed with pregnancy (in case ofgestation length)

Table 14. (Contd…)REPRODUCTIVE PERFORMANCE

Group, Sex & Dose

(mg/kg body weight/day)

No. of Females with Evidence of Mating (No. of Females used for Mating)

Female Mating Index (%)

 

 

 

 

 

 

 

 

No. of Females Confirmed as Fertile
(No. of Females used for Mating)

Female Fertility

Index (%)

G1, F & 0

12 (12)

100.0

11 (12)

91.7

G2, F & 250

12 (12)

100.0

10 (12)

83.3

G3, F & 500

12 (12)

100.0

11 (12)

91.7

G4, F & 1000

12 (12)

100.0

12 (12)

100.0

F: Female

Table 14. (Contd…)REPRODUCTIVE PERFORMANCE

Group, Sex

& Dose

(mg/kg body weight/day)

Pregnancy Index (%)

 

 

 

 

 

 

 

 

Gestation Index (%)

 

Parturition Index (%)

No. of Pregnant Females

No. of Females Confirmed with Mating

Female Fecundity or Pregnancy Index (%)

Females with Live Born Pups at Parturition

No. of Females with Evidence of Pregnancy

Gestation Index (%)

No. of females littered

No. of Females with Evidence of Pregnancy

Parturition Index (%)

G1, F & 0

11

12

91.7

11

11

100.0

11

11

100.0

G2, F & 250

10

12

83.3

10

10

100.0

10

10

100.0

G3, F & 500

11

12

91.7

11

11

100.0

11

11

100.0

G4, F & 1000

12

12

100.0

12

12

100.0

12

12

100.0

F: Female

Table 14. (Contd…)REPRODUCTIVE PERFORMANCE

Group, Sex

& Dose

(mg/kg body weight/day)

Post-implantation Loss (%)

 

Postnatal Loss (%)

No. of Implantations

No. of

Viable Pups

Post-implantation Loss (No.)

Post-implantation Loss (%)

Total No. of Deaths/

Cannibalized during Lactation Period

Postnatal Loss

(%)

G1, F & 0

Mean

10.36

10.00

0.36

3.76

0.00

0.00

±SD

1.75

2.00

0.67

7.22

0.00

0.00

n

11

11

11

11

11

11

G2, F & 250

Mean

11.60

10.70

0.90

7.83

0.00

0.00

±SD

1.17

1.77

1.29

11.08

0.00

0.00

n

10

10

10

10

10

10

G3, F & 500

Mean

10.36

9.82

0.55

5.14

0.00

0.00

±SD

1.12

1.17

0.82

7.55

0.00

0.00

n

11

11

11

11

11

11

G4, F & 1000

Mean

10.83

10.33

0.50

4.62

0.00

0.00

±SD

1.19

1.44

0.90

8.43

0.00

0.00

n

12

12

12

12

12

12

F: Female; n: Number of Dams

Table 15. ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Testes

Epididymis

Prostate (P) + Seminal vesicles

with coagulating glands (SC)

Thyroid along with parathyroid#

G1, M & 0

Mean

3.2035

1.3530

2.6717

0.0242

±SD

0.3542

0.0968

0.2587

0.0046

n

12

12

12

12

G2, M & 250

Mean

3.2802

1.3835

2.8051

0.0237

±SD

0.2207

0.1027

0.5185

0.0043

n

12

12

12

12

G3, M & 500

Mean

3.2392

1.3028

2.9552

0.0233

±SD

0.4203

0.1243

0.5374

0.0040

n

12

12

12

12

G4, M & 1000

Mean

3.1350

1.3175

2.7737

0.0247

±SD

0.2501

0.0992

0.4644

0.0046

n

12

12

12

12

M: Male; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation

Table 15. (Contd…) ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Ovaries

Thyroid along with parathyroid#

G1, F & 0

Mean

0.1194

0.0206

±SD

0.0247

0.0023

n@

11

11

G2, F & 250

Mean

0.1346

0.0222

±SD

0.0316

0.0020

n@

10

10

G3, F & 500

Mean

0.1209

0.0208

±SD

0.0192

0.0019

n@

11

11

G4, F & 1000

Mean

0.1189

0.0217

±SD

0.0122

0.0027

n

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; #: Weighed post fixation; @: the data of non-pregnant females one from G1, G3, and two from G2 were excluded from mean calculations and statistical analysis.

Table 16. TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Terminal Body Weight (g)

Testes

Epididymis

Prostate (P) + Seminal vesicles with coagulating glands (SC)

Thyroid along with parathyroid

G1, M & 0

Mean

335.21

0.9598

0.4055

0.8034

0.0072

±SD

28.97

0.1121

0.0350

0.1113

0.0012

n

12

12

12

12

12

G2, M & 250

Mean

338.95

0.9751

0.4111

0.8310

0.0070

±SD

36.37

0.0930

0.0401

0.1418

0.0008

n

12

12

12

12

12

G3, M & 500

Mean

337.71

0.9765

0.3906

0.8837

0.0069

±SD

40.42

0.1944

0.0586

0.1789

0.0006

n

12

12

12

12

12

G4, M & 1000

Mean

334.56

0.9406

0.3950

0.8272

0.0073

±SD

22.99

0.0917

0.0349

0.1092

0.0010

n

12

12

12

12

12

M: Male; SD: Standard Deviation; n: Number of Animals

Table 16. (Contd…) SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Terminal Body Weight (g)

Ovaries

Thyroid along with parathyroid

G1, F & 0

Mean

297.21

0.0402

0.0069

±SD

17.41

0.0081

0.0005

n

11

11

11

G2, F & 250

Mean

305.00

0.0440

0.0073

±SD

10.46

0.0098

0.0006

n

10

10

10

G3, F & 500

Mean

303.50

0.0399

0.0069

±SD

17.07

0.0063

0.0005

n

11

11

11

G4, F & 1000

Mean

299.36

0.0398

0.0072

±SD

14.82

0.0038

0.0008

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams

Table 17. SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - MALES

Group, Sex & Dose

(mg/kg body weight/day)

Serum T4 Levels (ng/mL)

G1, M & 0

Mean

76.973

±SD

21.316

n

11

G2, M & 250

Mean

72.150

±SD

14.877

n

12

G3, M & 500

Mean

79.823

±SD

13.505

n

12

G4, M & 1000

Mean

89.738

±SD

25.060

n

9

M: Male;SD: Standard Deviation; n: Number of Animals

Table 18. PUP OBSERVATIONS DURING POSTNATAL PERIOD

Group, Sex & Dose                  (mg/kg body weight/day)

At Birth (PND 1)

PND

5 to 7

Pups Sacrificed for Blood Collection on PND 4 (No.)*

PND

4 to 7$

PND

8 to 13$

G1, F & 0

No. of Dams / Litters#

11

11

4

11

11

No. of Live Pups

110

110

8

102

102

Pup Observation/

No. of Pups observed

N/110

N/110

-

N/102

N/102

G2, F & 250

No. of Dams / Litters#

10

10

6

10

10

No. of Live Pups

107

107

9

98

98

Pup Observation/

No. of Pups observed

N/107

N/107

-

N/98

N/98

G3, F & 500

No. of Dams / Litters#

11

11

4

11

11

No. of Live Pups

108

108

4

104

104

Pup Observation/

No. of Pups observed

N/108

N/108

-

N/104

N/104

G4, F & 1000

No. of Dams / Litters#

12

12

5

12

12

No. of Live Pups

124

124

7

117

117

Pup Observation/

No. of Pups observed

N/124

N/124

-

N/117

N/117

F: Female; N: Normal; PND: Postnatal Day;#: confirmed with live pups*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table 19. MEAN PUP WEIGHT (g) PER LITTER DURING LACTATION PERIOD

Group, Sex & Dose                  (mg/kg body weight/day)

 

PND 1

 

PND 4

 

PND 7

 

PND 13

Mean Pup Weight (g)

Mean Pup Weight (g)

Mean Pup Weight (g)

Mean Pup Weight (g)

Male

Female

Male

Female

Male

Female

Male

Female

G1, F & 0

Mean

6.54

5.58

10.91

10.07

14.49

13.33

25.03

23.85

±SD

0.34

0.20

0.27

0.30

0.57

0.60

0.46

0.43

n

11

11

11

11

11

11

11

11

G2, F & 250

Mean

6.54

5.73

10.85

10.02

14.27

13.45

24.78

23.71

±SD

0.41

0.15

0.17

0.30

0.37

0.93

0.44

0.38

n

10

10

10

10

10

10

10

10

G3, F & 500

Mean

6.50

5.62

10.86

9.96

14.58

13.57

25.02

23.98

±SD

0.34

0.27

0.40

0.35

0.64

0.71

0.29

0.47

n

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

6.40

5.63

10.76

9.92

14.34

13.47

25.00

24.00

±SD

0.38

0.26

0.36

0.40

0.49

0.95

0.47

0.33

n

12

12

12

12

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day

Table 20. MEAN PUP ANOGENITAL DISTANCE (AGD) MEASUREMENT (mm) AND ANOGENITAL DISTANCE (AGD) RATIO PER LITTER ON POSTNATAL DAY 4

Group & Dose

(mg/kg body weight/day)

 

Mean Male Pup
AGD Measurement (mm)

Mean Female Pup
AGD Measurement (mm)

 

Mean Male Pup
AGD Ratio

Mean Female Pup
AGD Ratio

G1 & 0

Mean

4.45

2.48

2.01

1.15

±SD

0.16

0.08

0.08

0.04

n

11

11

11

11

G2 & 250

Mean

4.34

2.44

1.96

1.13

±SD

0.15

0.14

0.06

0.06

n

10

10

10

10

G3 & 500

Mean

4.38

2.40

1.98

1.12

±SD

0.15

0.11

0.08

0.06

n

11

11

11

11

G4 & 1000

Mean

4.30*

2.37

1.95

1.10

±SD

0.09

0.11

0.03

0.05

n

12

12

12

12

F: Female; SD: Standard Deviation; n: Number of Litters; AGD: Anogenital Distance

*: Statistically significant (P<0.05) change than the concurrent vehicle control group

Table 21. MALE PUP NIPPLE/AREOLAE RETENTION (no.) RECORD PER LITTER

Group & Dose
(mg/kg body weight/day)

Mean No. of Pups with Retention of Nipples/

Areolae on Postnatal Day 13

G1 & 0

Mean

0.00

±SD

0.00

n

11

G2 & 250

Mean

0.00

±SD

0.00

n

10

G3 & 500

Mean

0.00

±SD

0.00

n

11

G4 & 1000

Mean

0.00

±SD

0.00

n

12

SD: Standard Deviation; n: Number of Litters

Table 22. SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY 13 PUPS (PND)

 Group & Dose

(mg/kg body weight/day)

Serum T4 Levels (ng/mL)

G1 & 0

Mean

82.863

±SD

18.754

n

11

G2 & 250

Mean

81.388

±SD

16.661

n

10

G3 & 500

Mean

88.978

±SD

15.694

n

11

G4 & 1000

Mean

80.389

±SD

14.195

n

12

SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day

Conclusions:
The study-derived NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.
Executive summary:

The study was performed according to OECD 421 (2016) and according to GLP. The substance was given to 12 rats per sex per dose level at 0 (corn oil), 250, 500 and 1000 mg/kg bw/day. Male rats were treated before mating and during mating for a total of 29 days. Female rats were treated before mating, during mating, during pregnancy, and up to day 13 of lactation for a total of approx. 63 days. Results (parental generation): All animals survived to planned death and there were no clinical signs of toxicity. No significant changes in body weight or body weight gain with respect to day 1 of treatment were observed. No significant changes in food intake were observed, with the exception of a significant increase in food intake during the pre-mating period in male rats treated at 1000 mg/kg bw/day. This change was considered incidental and toxicological insignificant since the increase was minimal and could not be correlated with any changes in body weight or body weight gain. There were no significant changes in serum Thyroxine (T4) hormone levels in any of the male groups. As there were no significant changes observed in serum T4 levels in adult males, the assessment was not extended to adult females (dams). There were no significant changes in absolute or relative weight of testes, epididymis, prostate with seminal vesicle and coagulation gland, ovaries, or thyroid with parathyroidinin any of the groups.There were no gross pathological findings in any of the groups. There were no treatment-related histopathological findings at 1000 mg/kg bw/day (MF/F). A single incidence of minimal, multifocal, degeneration/atrophy of seminiferous tubules in testes was observed in high dose male without any correlating findings in epididymides of the same animal. This change was considered to be an expected spontaneous finding in rats of this age. There were no irregularities observed in the oestrus cyclicity in any of the female groups during the pre-mating or mating period. No significant changes in mean oestrus cycle length were observed between the groups during the pre-mating or mating period. No treatment related adverse effects on the stages of spermatogenesis were observed during the microscopic examination of interstitial testicular cell structure. No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed. Results (F1 generation): All pups showed normal behaviour during the daily inspections. No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period. No significant changes in pup weight (M/F) were observed on PND 1, PND 4, PND 7 or PND 13. No significant changes in serum Thyroxine (T4) hormone levels were observed on PND 13 in any of the groups. No significant changes in mean pup anogenital distance (AGD) or AGD ratio were observed in any of the groups, with the exception of a significant decrease in AGD in male pups from the 1000 mg/kg dose group (mean 4.30 mm) versus the control group (mean 4.45 mm). AGD in male pups from the 1000 mg/kg dose group was within the historical control range of the test facility. Therefore, this small change in AGD at 1000 mg/kg was not considered to be an adverse effect of treatment. There were no male pups with retention of nipples/areolae on PND 13 in any of the groups. No significant changes in sex ratio were observed. There were no gross pathological findings in any of the groups. Conclusion: NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from a Klimisch 1 database and provides a robust study summary.
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:

The study was performed according to OECD 421 (2016) and according to GLP. The substance was given to 12 rats per sex per dose level at 0 (corn oil), 250, 500 and 1000 mg/kg bw/day. Male rats were treated before mating and during mating for a total of 29 days. Female rats were treated before mating, during mating, during pregnancy, and up to day 13 of lactation for a total of approx. 63 days.Results (parental generation): All animals survived to planned death and there were no clinical signs of toxicity. No significant changes in body weight or body weight gain with respect to day 1 of treatment were observed. No significant changes in food intake were observed, with the exception of a significant increase in food intake during the pre-mating period in male rats treated at 1000 mg/kg bw/day. This change was considered incidental and toxicological insignificant since the increase was minimal and could not be correlated with any changes in body weight or body weight gain. There were no significant changes in serum Thyroxine (T4) hormone levels in any of the male groups. As there were no significant changes observed in serum T4 levels in adult males, the assessment was not extended to adult females (dams). There were no significant changes in absolute or relative weight of testes, epididymis, prostate with seminal vesicle and coagulation gland, ovaries, or thyroid with parathyroidinin any of the groups.There were no gross pathological findings in any of the groups. There were no treatment-related histopathological findings at 1000 mg/kg bw/day (MF/F). A single incidence of minimal, multifocal, degeneration/atrophy of seminiferous tubules in testes was observed in high dose male without any correlating findings in epididymides of the same animal. This change was considered to be an expected spontaneous finding in rats of this age. There were no irregularities observed in the oestrus cyclicity in any of the female groups during the pre-mating or mating period. No significant changes in mean oestrus cycle length were observed between the groups during the pre-mating or mating period. No treatment related adverse effects on the stages of spermatogenesis were observed during the microscopic examination of interstitial testicular cell structure. No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed.Results (F1 generation):All pups showed normal behaviour during the daily inspections. No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period. No significant changes in pup weight (M/F) were observed on PND 1, PND 4, PND 7 or PND 13. No significant changes in serum Thyroxine (T4) hormone levels were observed on PND 13 in any of the groups. No significant changes in mean pup anogenital distance (AGD) or AGD ratio were observed in any of the groups, with the exception of a significant decrease in AGD in male pups from the 1000 mg/kg dose group (mean 4.30 mm) versus the control group (mean 4.45 mm). AGD in male pups from the 1000 mg/kg dose group was within the historical control range of the test facility. Therefore, this small change in AGD at 1000 mg/kg was not considered to be an adverse effect of treatment. There were no male pups with retention of nipples/areolae on PND 13 in any of the groups. No significant changes in sex ratio were observed. There were no gross pathological findings in any of the groups.Conclusion:NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Developmental Toxicity:

No adverse effects on reproduction or development were observed at 250, 500 or 1000 mg/kg bw/day in the performed OECD 421 with rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from a Klimisch 1 database and provides a robust study summary.
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:

The study was performed according to OECD 421 (2016) and according to GLP. The substance was given to 12 rats per sex per dose level at 0 (corn oil), 250, 500 and 1000 mg/kg bw/day. Male rats were treated before mating and during mating for a total of 29 days. Female rats were treated before mating, during mating, during pregnancy, and up to day 13 of lactation for a total of approx. 63 days.Results (parental generation): All animals survived to planned death and there were no clinical signs of toxicity. No significant changes in body weight or body weight gain with respect to day 1 of treatment were observed. No significant changes in food intake were observed, with the exception of a significant increase in food intake during the pre-mating period in male rats treated at 1000 mg/kg bw/day. This change was considered incidental and toxicological insignificant since the increase was minimal and could not be correlated with any changes in body weight or body weight gain. There were no significant changes in serum Thyroxine (T4) hormone levels in any of the male groups. As there were no significant changes observed in serum T4 levels in adult males, the assessment was not extended to adult females (dams). There were no significant changes in absolute or relative weight of testes, epididymis, prostate with seminal vesicle and coagulation gland, ovaries, or thyroid with parathyroidinin any of the groups.There were no gross pathological findings in any of the groups. There were no treatment-related histopathological findings at 1000 mg/kg bw/day (MF/F). A single incidence of minimal, multifocal, degeneration/atrophy of seminiferous tubules in testes was observed in high dose male without any correlating findings in epididymides of the same animal. This change was considered to be an expected spontaneous finding in rats of this age. There were no irregularities observed in the oestrus cyclicity in any of the female groups during the pre-mating or mating period. No significant changes in mean oestrus cycle length were observed between the groups during the pre-mating or mating period. No treatment related adverse effects on the stages of spermatogenesis were observed during the microscopic examination of interstitial testicular cell structure. No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed.Results (F1 generation):All pups showed normal behaviour during the daily inspections. No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period. No significant changes in pup weight (M/F) were observed on PND 1, PND 4, PND 7 or PND 13. No significant changes in serum Thyroxine (T4) hormone levels were observed on PND 13 in any of the groups. No significant changes in mean pup anogenital distance (AGD) or AGD ratio were observed in any of the groups, with the exception of a significant decrease in AGD in male pups from the 1000 mg/kg dose group (mean 4.30 mm) versus the control group (mean 4.45 mm). AGD in male pups from the 1000 mg/kg dose group was within the historical control range of the test facility. Therefore, this small change in AGD at 1000 mg/kg was not considered to be an adverse effect of treatment. There were no male pups with retention of nipples/areolae on PND 13 in any of the groups. No significant changes in sex ratio were observed. There were no gross pathological findings in any of the groups.Conclusion:NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.

Justification for classification or non-classification

No adverse effects on reproduction or development were observed at 250, 500 or 1000 mg/kg bw/day in the performed OECD 421 with rats.

The chemical is therefore regarded to be classified as Not Classified for toxicity to reproduction (category 1A, 1B or 2) as per CLP (Regulation (EC) No 1272/2008).

Additional information