Registration Dossier

Endpoint:

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:

The data is from study report.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd. 4B, MN Park, Shameerpet Mandal, Turkapally Village, Medchal District, Telangana -500078
- Age at study initiation: 12 weeks
- Weight at study initiation: G1: 259.946 ± 17.79; G2: 256.054 ± 19.41; G3: 258.465 ± 18.33; G4: 259.443 ± 15.27
- Fasting period before study: No data available
- Housing: standard polysulfone rat cages (size: Length 425 mm × Width 266 mm × Height 185 mm) with stainless steel top grill.
- Bedding: Steam sterilized corn cob was used as bedding material and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets, ad libitum
- Water (e.g. ad libitum): Deep bore-well water, ad libitum in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 58 - 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light and 12-hours dark cycle

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test checmical was dissolved in Corn oil prior to dosing.

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): The test item is insoluble in water, but soluble in various oils and organic solvents. The test item was found to be miscible in corn oil and corn oil was therefore selected as vehicle.
- Concentration in vehicle: G1: 0 mg/ml; G2: 62.5 mg/ml; G3: 125 mg/ml; G4: 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): NA
- Purity: NA

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: yes
- M/F ratio per cage: 1:1
- Length of cohabitation: Two weeks
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No data available

Duration of treatment / exposure:

Two Weeks

Frequency of treatment:

Daily

Duration of test:

GD 5 to GD 19

No. of animals per sex per dose:

24 females per group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- 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 available.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: placenta, thoracic and abdominal viscera, uterine content, thyroid gland

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placenta

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

The data on maternal body weight, body weight change in interval, maternal food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, hormone analyses (T4, T3, TSH), and the weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using ANCOVA taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal-Wallis test for group comparison. Wilcoxon pairwise comparisons of the treated groups with the control group were performed, when the group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran-Armitage trend test and pair-wise comparison will be tested by Fisher’s exact test for group association.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs at at 250 and 500 mg/kg/day. At 1000 mg/kg/day, one dam had reddish discharge from vagina on GD18 which was considered to be treatment-related. No other clinical signs were observed at 1000 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

As compared to the vehicle control group, statistically and dose-dependent decreases in maternal body weight (from -5 to -32%), in maternal body weight gain (from -17 to -199%), in adjusted maternal body weight (from -8 to -14%) and in adjusted maternal body weight gain (from -61 to -133%) were observed during most recorded time points and intervals. These observed effects on body weight, body weight gain, adjusted body weight, and adjusted body weight gain at ≥250 mg/kg/day were considered treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

As compared to the vehicle control group, significant reductions in mean food consumption were noticed following treatment at 250, 500 and
1000 mg/kg/day, during GD 5 - 8, 8 - 11, 11 - 14, 14 – 17 and 17 - 20. The decreases ranged from -15 to -27% at 250 mg/kg/day, from -25 to -30% at 500 mg/kg/day, and from -41 to -53% at 1000 mg/kg/day. Net food intakes during GD 5-20 were -22%, -27%, -46% and during gestation (GD 0-20) they were -17%, -21%, -35% lower at 250, 500 and 1000 mg/kg/day, respectively, when compared to the vehicle control group. The observed effects on food intake at ≥250 mg/kg/day were considered treatment-related.

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:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in T3 or T4 levels were observed. Significant increases in TSH levels were observed at 500 and 1000 mg/kg, however these effects were not considered to be of toxicological significance since the TSH levels at 500 and 1000 mg/kg were well within the historical control range of the test facility and since no significant changes in thyroid weight or histology of the thyroid were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes in thryoid weight were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological effects were observed in the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant changes in the histology of the thyroid were observed in the study. The incidences of ectopic thymus were randomly distributed across the groups and were considered incidental background findings and not related to the test item administration.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

No significant effects were observed at 250 mg/kg/day. Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, there were only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No significant changes in pre- or post-implantation loss were observed at 250 mg/kg/day. At 500 and 1000 mg/kg/day there were significant decreases in the number of implantations by -11% and -19%, respectively; significant decreases in implantation indexes by -14 and -20%, respectively; and significant increases in % post-implantation losses by 55 and 99%, respectively.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

No significant effects were observed at 250 mg/kg/day. Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, there were only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 500 and 1000 mg/kg/day there were significant increases in % early resorptions by 50 and 98%, respectively, when compared to the control group. No other significant changes were observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed at any dose level.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant rats were 21, 24, 24, 23 at 0, 250, 500 and 1000 mg/kg /day, respectively.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Sex-combined fetal weights were significantly decreased at 250 (by -5%) and 500 mg/kg/day (by -6%) compared to the control data. Significant decreases in male fetal weight were observed at 250 (by -5%) and 500 mg/kg/day (by -8%) compared to the control data, whereas only a significant decrease in female fetal weight was observed at 250 mg/kg/day (by -4%) compared to the control data.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetuses were observed in the study.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes in sex ratio were observed.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

There were only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was one incidence of a fetus (1/302 fetuses) with a thread like tail at 250 mg/kg/day [malformation], one small fetus (1/150 fetuses) at 500 mg/kg/day [anomaly] and two fetuses with hindlimbs turned inwards at 1000 mg/kg/day (2/3 fetuses) [malformation]. These observations at 250 and 500 mg/kg were considered incidental and not treatment-related.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg/day, there was one fetus from one dam (Rz1025) which had absent caudal vertebrae (centrum and arch absent) [malformation]. The same fetus had thread like tail during external examination [malformation]. These malformations at 250 mg/kg/day were considered to be spontaneous in nature since no such malformations were observed at 500 mg/kg/day. At 1000 mg/kg/day, one fetus from one dam (Rz1075) had tarsal bones bent inwards [malformation]. The same fetus had hind limbs turned inwards during external examination [malformation]. There were significant increases in variations of incomplete ossification of skull bones namely parietal, interparital and supraoccipital bones at 500 mg/kg/day. Delayed/incomplete ossifications are generally considered transient changes and these finding denote generalized growth delays which normally ossify late in gestation. They also do not have general predictive value for teratogenicity [Carney and Kimmel (2007)]. A significant increase in the incidene of bilateral wavy ribs (#4 to #11) [anomaly] was observed at 500 mg/kg/day as compared to vehicle control group. Wavy ribs are manifestations of delayed ossification, are transient and reversible and are not of developmental importance since they are expected to resolve during maturation (Hayasaka et al., 1985; Kast, 1994; Mitchard & Stewart, 2014). The observed variations and anomalies at 500 mg/kg/day were considered non-adverse based on the discussions above, and they were likely secondary to maternal systemic toxicity at 500 mg/kg/day.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed during visceral examination at 250 and 500 mg/kg. There was one incidence of moderate bilateral renal pelvis dilation at 250 mg/kg/day (1/145 fetuses) [anomaly]. This finding was considered spontaneous in nature and therefore not treatment-related. All fetal heads from the Wilsons-Razor blade sectioning were found to be normal (i.e. no malformations, anomalies, or variations were observed).

Details on embryotoxic / teratogenic effects:

A total of 14 litters at 500 mg/kg and only 1 litter at 1000 mg/kg were available for evaluation due to the number of complete resorptions observed at 500 and 1000 mg/kg. The results from the external, visceral, and seketal examinations performed at 1000 mg/kg were not considered to be conclusive due to low sample size.

Table for Maternal Survival, Pregnancy Status and Fetus Disposition

Parameters	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	250	500	1000
Total No. of rats found sperm positive / group		24	24	24	24
Duration of treatment		GD 5 to 19 (total 15 days)
Caesarean section (day of presumed gestation)		GD 20
Number of rats sacrificed at caesarean section		24	24	24	24
Number. of rats non-pregnant at caesarean section		3	0	0	1
Number of rats pregnant at caesarean section		21	24	24	23
Number of dams with complete resorptions Number of litters examined		0 21	0 24	10 14	22 1
Total number of fetuses		293	302	150	3
Total number of dead fetuses		0	0	0	0

Number of fetuses evaluated
a. External examination		293	302	150	3
b. Visceral examination		140	145	72	1
c. Skeletal examination		153	157	78	2

Table for Clinical Signs and Mortality

Observation Type: All Types

From Day 0 (Mating (A)) to 20 (Mating)

Female

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

Normal

Killed ‑ terminal kill

Discharge

24

0

24

0

24

0

24

1

Values = Number of Animals Affected

Table for Maternal Body Weight of Pregnant Rats

Sex: Female

Day(s) Relative to Mating

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

Body

Weight

(g)

0[a]

Mean

SD

N

%Diff

258.46

15.81

21

‑

256.05

19.41

24

‑0.93

258.47

18.33

24

0.00

259.76

15.53

23

0.51

3[a]

Mean

SD

N

%Diff

274.90

16.40

21

‑

270.79

20.34

24

‑1.49

273.00

19.47

24

‑0.69

275.44

15.62

23

0.20

5[a]

Mean

SD

N

%Diff

282.41

16.75

21

‑

278.79

21.26

24

‑1.28

281.42

21.02

24

‑0.35

283.22

16.10

23

0.29

8[a]

Mean

SD

N

%Diff

291.59

17.75

21

‑

279.67

22.14

24

‑4.09

282.03

20.26

24

‑3.28

274.09*

19.69

23

‑6.00

11[a]

Mean

SD

N

%Diff

306.63

20.97

21

‑

290.32*

22.28

24

‑5.32

291.51*

19.37

24

‑4.93

278.92*

20.20

23

‑9.04

14[a]

Mean

SD

N

%Diff

325.57

20.75

21

‑

302.34*

22.59

24

‑7.14

299.74*

19.23

24

‑7.94

279.46*

19.22

23

‑14.17

17[a]

Mean

SD

N

%Diff

356.30

27.33

21

‑

329.30*

26.60

24

‑7.58

310.98*

27.02

24

‑12.72

273.96*

22.27

23

‑23.11

20[a1]

Mean

SD

N

%Diff

400.72

31.91

21

‑

366.14*

31.33

24

‑8.63

329.87*

40.22

24

‑17.68

274.36*

21.62

23

‑31.53

[a] ‑ Anova & Dunnett: * = p < 0.05 ; [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05

Table for Maternal Body Weight Gain of Pregnant Rats

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 250 mg/kg/day	G3 500 mg/kg/day	G4 1000 mg/kg/day
Absolute Weight Gain (g)	0 → 3 [a]	Mean SD N %Diff	16.44 4.88 21 -	14.74 5.73 24 ‑10.33	14.53 3.97 24 ‑11.61	15.68 4.57 23 ‑4.61
	3 → 5 [a1]	Mean SD N %Diff	7.52 3.56 21 -	8.00 3.32 24 6.38	8.43 3.84 24 12.12	7.78 3.15 23 3.47
	5 → 8 [a2]	Mean SD N %Diff	9.18 5.21 21 -	0.88* 4.37 24 ‑90.46	0.61* 5.17 24 ‑93.37	‑9.13* 9.25 23 ‑199.44
	8 → 11 [a2]	Mean SD N %Diff	15.04 7.19 21 -	10.66 4.78 24 ‑29.13	9.48* 7.51 24 ‑36.97	4.83* 10.45 23 ‑67.91
	11 → 14 [a]	Mean SD N %Diff	18.94 5.07 21 -	12.02* 5.53 24 ‑36.57	8.23* 5.85 24 ‑56.58	0.54* 7.8 23 ‑97.15
	14 → 17 [a2]	Mean SD N %Diff	30.73 9.08 21 -	26.96 8.46 24 ‑12.25	11.24* 15.43 24 ‑63.41	‑5.50* 8.91 23 ‑117.89
	17 → 20 [a2]	Mean SD N %Diff	44.42 9.78 21 -	36.83* 9.64 24 -17.09	18.90* 18.50 24 -57.46	0.41* 5.87 23 -99.09
	0 → 5 [a]	Mean SD N %Diff	23.95 5.78 21 -	22.74 5.68 24 -5.09	22.96 4.68 24 -4.16	23.46 4.63 23 -2.08
	5 → 20 [a1]	Mean SD N %Diff	118.31 21.77 21 -	87.35* 17.03 24 -26.17	48.45* 40.72 24 -59.05	8.86* 15.43 23 -107.48
	0 → 20 [a1]	Mean SD N %Diff	142.27 25.20 21 -	110.08* 18.56 24 -22.62	71.41* 40.35 24 -49.81	14.60* 17.18 23 -89.74
Adjusted Bodyweight (g)	GD20 GU WT [a]	Mean SD N %Diff	316.02 19.35 21 -	291.78* 23.20 24 -7.67	291.86* 19.67 24 -7.65	272.24* 17.79 23 -13.85
Adjusted BW Gain (g)	ADJ BWT GD5B [a]	Mean SD N %Diff	33.61 8.82 21 -	12.99* 10.08 24 -61.35	10.44* 14.59 24 -68.95	10.98* 11.40 23 -132.65

[a] ‑ Anova & Dunnett: * = p < 0.05; [a1] ‑ Anova & Dunnett(Log); [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05; [a2] ‑ Anova & Dunnett(Rank): * = p < 0.05

Table for Food Consumption of Pregnant Rats

Sex: Female

Day(s) Relative to Mating

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

Food Mean

Consumption

(g/day)

0 → 3 [a]

Mean

SD

N

%Diff

22.59

2.66

21

-

21.59

2.46

24

‑4.42

22.66

1.93

24

0.35

22.61

2.71

23

0.09

3 → 5 [a]

Mean

SD

N

%Diff

24.06

2.3

21

-

23.42

2.42

24

‑2.66

24.34

1.98

24

1.14

23.37

2.23

23

‑2.90

5 → 8 [a]

Mean

SD

N

%Diff

21.77

3.75

21

-

15.87*

2.71

24

‑27.09

15.18*

3.43

24

‑30.30

10.21*

3.59

23

‑53.11

8 → 11 [a1]

Mean

SD

N

%Diff

21.83

4.26

21

-

16.35*

2.26

24

‑25.11

16.41*

2.6

24

‑24.83

12.8*

2.62

23

‑41.39

11 → 14 [a]

Mean

SD

N

%Diff

25.96

2.87

21

-

19.35*

2.92

24

‑25.48

18.73*

2.69

24

‑27.85

14.93*

2.90

23

‑42.52

14 → 17 [a1]

Mean

SD

N

%Diff

26.83

2.57

21

-

22.00*

2.76

24

‑18.00

19.77*

4.98

24

‑26.31

13.72*

3.77

23

‑48.85

17 → 20 [a1]

Mean

SD

N

%Diff

27.73

2.42

21

-

23.67*

3.36

24

‑14.65

20.28*

4.78

24

‑26.89

15.32*

2.94

23

‑44.77

[a] ‑ Anova & Dunnett: * = p < 0.05; [a1] ‑ Anova & Dunnett(Rank): * = p < 0.05

Contd. Table for Food Consumption of Pregnant Rats

Sex: Female

Day(s) Relative to Mating (Litter: A)

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

Food Mean

Consumption

(g/day)

0 → 5 [a]

Mean

SD

N

%Diff

23.18

2.37

21

-

22.32

2.23

24

‑3.69

23.33

1.67

24

0.68

22.91

2.37

23

‑1.15

5 →20 [a1]

Mean

SD

N

%Diff

24.83

2.72

21

-

19.45*

2.29

24

‑21.66

18.07*

3.00

24

‑27.20

13.39*

2.24

23

‑46.05

0 → 20 [a2]

Mean

SD

N

%Diff

24.41

2.52

21

-

20.17*

2.15

24

‑17.40

19.39*

2.37

24

‑20.58

15.77*

1.89

23

‑35.39

[a] ‑ Anova & Dunnett: * = p < 0.05

Table for Maternal Data

Sex: Female

G10

mg/kg/day

G2 250

mg/kg/day

G3 500

mg/kg/day

G4 1000

mg/kg/day

Group size

N

24

Pregnant at C/S

N

21

24

23

Gravid Uterus Weight

[a]

Mean SD

84.702

20.604

74.357

18.785

38.015 *

33.505

2.121 *

5.989

Number of

Corpora Lutea

[k]

Mean

SD

Sum

17.0

2.6

356.0

16.5

1.9

396.0

17.7

2.5

425.0

17.1

2.5

394.0

Number of

Implantation

[k]

Mean SD

Sum

15.3

2.7

321.0

15.3

2.0

368.0

13.6 *

2.5

326.0 *

12.4 *

3.2

285.0 *

Number of dams with Early Resorption

N

14

16

19

23

Number of Early Resorptions

[k]

Mean SD

Sum

1.2

1.3

26.0

2.3

2.5

56.0

6.8 *

5.9

162.0 *

12.1 *

3.5

278.0 *

% Early Resorption

/Animal

[k]

Mean SD

9.23

11.06

15.59

17.01

50.46 *

44.36

97.66 *

11.23

Number of dams with Late Resorption

N

2

7

1

Number of Late Resorptions

[k]

Mean SD

Sum

0.1

0.3

2.0

0.4

0.9

10.0

0.6

1.6

14.0

0.2

0.8

4.0

% Late Resorption

/Animal

[k]

Mean SD

0.68

2.17

2.72

5.59

4.22

12.56

1.34

6.42

Number of dams with

Resorptions

[f]

N

16

18

21

23

Total Number of Resorptions (Early + Late)

[f]

Mean SD

Sum

1.3

1.2

28.0

2.8

66.0

7.3

5.6

176.0

12.3

3.3

282.0

Pre-implantation Loss/Animal

[k]

Mean SD

1.67

1.77

35.00

1.17

1.31

28.00

4.13 *

2.64

99.00 *

4.74 *

1.94

109.00 *

% Pre-implantation

Loss

[k]

Mean

SD

9.8

10.2

6.9

7.7

22.8 *

13.7

28.4 *

12.5

Post-implantation Loss/Animal

[k]

Mean SD

1.33

1.20

28.00

2.75

2.77

66.00

7.33 *

5.60

176.00 *

12.26 *

3.25

282.00 *

% Post-implantation

Loss (%)

[k]

Mean

SD

9.9

10.7

18.3

18.7

54.7 *

41.9

99.0 *

4.8

Implantation

Index

[a1]

Mean SD

N

90.24

10.20

21

93.05

7.68

24

77.17 *

13.67

24

71.57 *

12.52

23

Note: Gross evaluation of placenta revealed no findings;

[a] - Anova & Dunnett(Rank): * = p < 0.05; [a1] - Anova & Dunnett(Log): * = p < 0.05; [k] - Kruskal-Wallis & Wilcoxon: * = p < 0.05; [f] - Cochran Armitage, Chi-Squared & Fisher's Exact

Table for Litter Data

Sex: Female			G10 mg/kg/day	G2 250 mg/kg/day	G3 500 mg/kg/day	G4 1000 mg/kg/day
Total Number of fetuses		Sum	293	302	150	3
Total Number of Dead Fetuses		Sum	0	0	0	0
Total Number of Live fetuses		Sum	293	302	150	3
Live Male fetus		Sum	150	156	75	3
% Male Fetus			51.2	51.7	50.0	100.0
Mean Fetal Weight- Male (g)	[c]	Mean SD	4.153 0.328	3.933 * 0.415	3.836 * 0.306	4.697 .
Mean AGD- Male (mm)	[c1]	Mean SD	2.67 0.16	2.62 0.13	2.64 0.15	3.00
Live Female fetus		Sum	143	146	75	0
% Female Fetus			48.8	48.3	50.0	0.0
Mean Fetal Weight- Female (g)	[c2]	Mean SD	3.898 0.335	3.728 * 0.428	3.725 0.359	. .
Mean AGD- Female (mm)	[c3]	Mean SD	0.96 0.12	1.00 0.13	0.96 0.10	. .
Mean Fetal Weight -Male+Female (g)	[c4]	Mean SD	4.027 0.318	3.827 * 0.405	3.776 * 0.302	4.697 .
Mean AGD Male + Female (mm)	[c5]	Mean SD	1.83 0.19	1.82 0.31	1.84 0.32	3.00

[c] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett;Covariate(s): Number of LiveMaleFetuses}

[c2] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Female Fetuses}

[c3] - Ancova/Anova & Dunnett;{Covariate(s): Number of Live Female Fetuses}

[c4] - Ancova/Anova & Dunnett(Rank): * = p < 0.05;{Covariate(s): Number of Live Fetuses}

[c5] - Ancova/Anova & Dunnett;Covariate(s): Number of Live Fetuses}

Table for Fetal External Observations

Exam type: External

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

293

21

302

24

150

14

3

1

Limbs

Tail, Thread‑like tail – Malformation

Limb, both, Bent Inward – Malformation

General

Fetus, Small Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.3)

1(4.2)

0(0.0)

1(0.7)

1(7.1)

0(0.0)

2(66.7)

1(100.0)

0(0.0)

Fetuses N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact; [Litters N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact

Table for Fetal Visceral Observations

Exam type: Fresh Visceral Body Only

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

140

21

145

24

72

14

1

Abdomen

Kidney, both, Renal pelvis dilation, Moderate – Anomaly

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.7)

1(4.2)

0(0.0)

Fetuses N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact; [Litters N] ‑ Cochran Armitage, Chi‑Squared & Fisher's Exact

Table for Fetal Skeletal Observations

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

153

21

157

24

78

14

2

1

Hindlimbs

Tarsal bone, both, Bent – Malformation

Caudal vertebrate

14th caudal centrum, Absent – Anomaly

13th caudal centrum, Absent – Anomaly

12th caudal centrum, Absent – Anomaly

11th caudal centrum, Absent – Anomaly

10th caudal centrum, Absent – Anomaly

9th caudal centrum, Absent – Anomaly

8th caudal centrum, Absent – Malformations

7th caudal centrum, Absent – Malformations

6th caudal centrum, Absent – Malformations

5th caudal centrum, Absent – Malformations

4th caudal centrum, Absent – Malformations

3rd caudal centrum, Absent – Malformations

2nd caudal centrum, Absent – Malformations

1st caudal centrum, Absent – Malformations

6th caudal arch, Absent – Malformation

5th caudal arch, Absent – Malformation

4th caudal arch, Absent – Malformation

3rd caudal arch, Absent – Malformation

2nd caudal arch, Absent – Malformation

1st caudal arch, Absent – Malformation

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

153

21

157

24

78

14

2

1

sacral vertebrae

4th sacral centrum, Absent – Malformation

3rd sacral centrum, Absent – Malformation

2nd sacral centrum, Absent – Malformation

1st sacral centrum, Absent – Malformation

4th sacral arch, Absent – Malformation

4rd sacral arch, Absent – Malformation

4nd sacral arch, Absent – Malformation

1st sacral arch, Absent – Malformation

Lumbar vertebrae

6th lumbar centrum, Absent – Malformation

5th lumbar centrum, Hypoplastic – Anomaly

2nd lumbar centrum, Dumbbell‑shaped – Anomaly

6th lumbar arch, Displaced – Anomaly

6th lumbar arch, Incomplete ossification Anomaly

7th lumbar centrum and arch, Extra – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

153

21

157

24

78

14

2

1

thoracic vertebrae

13th thoracic centrum, Dumbbell shaped – Anomaly

13th thoracic centrum, Asymmetrical split – Anomaly

12th thoracic centrum, Unilateral Ossification –Anomaly

12th thoracic centrum, Split – Anomaly

12th thoracic centrum, Dumbbell‑shaped – Anomaly

12th thoracic centrum, Asymmetrical dumbbell shaped – Anomaly

12th thoracic centrum, Asymmetrical split – Anomaly

11th thoracic centrum, Unilateral Ossification – Anomaly

11th thoracic centrum, Dumbbell‑shaped – Anomaly

11th thoracic centrum, Asymetrical dumbbell shaped – Anomaly

10th thoracic centrum, Split – Anomaly

10th thoracic centrum, Dumbbell‑shaped – Anomaly

10th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

9th thoracic centrum, Split – Anomaly

9th thoracic centrum, Dumbbell‑shaped – Anomaly

9th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

8th thoracic centrum, Split – Anomaly

8th thoracic centrum, Dumbbell‑shaped – Anomaly

7th thoracic centrum, Split – Anomaly

7th thoracic centrum, Dumbbell‑shaped – Anomaly

6th thoracic centrum, Unilateral Ossification – Anomaly

6th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

5th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

4th thoracic centrum, Dumbbell‑shaped – Anomaly

4th thoracic centrum, Assymetrical dumbbell shaped – Anomaly

3rd thoracic centrum, Dumbbell‑shaped – Anomaly

2nd thoracic centrum, Dumbbell‑shaped – Anomaly

2nd thoracic centrum, Delayed skeletal ossification – Variation

2nd thoracic centrum, Incomplete ossification – Variation

1st thoracic centrum, Delayed skeletal ossification – Variation

1st thoracic centrum, Incomplete ossification – Variation

Cervical vertebrae

Cervical rib, Bilateral, Rudimentary – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

2(1.3)

2(9.5)

0(0.0)

2(1.3)

2(9.5)

10(6.5)

8(38.1)

2(1.3)

2(9.5)

0(0.0)

11(7.2)

9(42.9)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

23(15.0)

11(52.4)

2(1.3)

2(9.5)

0(0.0)

23(15.0)

11(52.4)

0(0.0)

5(3.3)

5(23.8)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

0(0.0)

1(0.7)

1(4.8)

0(0.0)

3(1.9)

3(12.5)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

9(5.7)

9(37.5)

0(0.0)

13(8.3)

11(45.8)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

13(8.3)

9(37.5)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

12(7.6)*

9(37.5)*

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)*

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(1.3)

1(7.1)

4(5.1)

3(21.4)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

4(5.1)

4(28.6)

1(1.3)

1(7.1)

0(0.0)

6(7.7)

3(21.4)

1(1.3)

1(7.1)

0(0.0)

2(2.6)*

2(14.3)*

0(0.0)

3(3.8)

3(21.4)

0(0.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

153

21

157

24

78

14

2

1

ribs

14th rib, Right, Rudimentary - Anomaly

14th rib, Right, Accessory – Anomaly

14th rib, Left, Rudimentary – Anomaly

14th rib, Left, Accessory – Anomaly

14th rib, Bilateral, Rudimentary – Anomaly

14th rib, Bilateral, Accessory – Anomaly

14th rib, Bilateral, Extra – Anomaly

13th rib, Right, Wavy Rib, Moderate – Anomaly

13th rib, Left, Wavy Rib, Moderate – Anomaly

13th rib, Bilateral, Wavy Rib, Moderate – Anomaly

12th rib, Left, Wavy Rib, Moderate – Anomaly

12th rib, Right, Wavy Rib, Moderate – Anomaly

12th rib, Bilateral, Wavy Rib, Moderate – Anomaly

11th rib, Left, Wavy Rib, Slight – Anomaly

11th rib, Right, Wavy Rib, Slight – Anomaly

11th rib, Right, Wavy Rib, Moderate – Anomaly

11th rib, Bilateral, Wavy Rib, Slight – Anomaly

11th rib, Bilateral, Wavy Rib, Moderate – Anomaly

10th rib, Right, Wavy Rib, Slight – Anomaly

10th rib, Right, Wavy Rib, Moderate – Anomaly

10th rib, Left, Wavy Rib, Slight – Anomaly

10th rib, Bilateral, Wavy Rib, Slight – Anomaly

10th rib, Bilateral, Wavy Rib, Moderate – Anomaly

9th rib, Left, Wavy Rib, Slight – Anomaly

9th rib, Right, Wavy Rib, Slight – Anomaly

9th rib, Right, Wavy Rib, Moderate – Anomaly

9th rib, Bilateral, Wavy Rib, Slight – Anomaly

9th rib, Bilateral, Wavy Rib, Moderate – Anomaly

8th rib, Left, Wavy Rib, Slight – Anomaly

8th rib, Right, Wavy Rib, Slight – Anomaly

8th rib, Right, Wavy Rib, Moderate – Anomaly

8th rib, Bilateral, Wavy Rib, Slight – Anomaly

8th rib, Bilateral, Wavy Rib, Moderate – Anomaly

7th rib, Right, Wavy Rib, Slight – Anomaly

7th rib, Right, Wavy Rib, Moderate – Anomaly

7th rib, Left, Wavy Rib, Slight – Anomaly

7th rib, Bilateral, Wavy Rib, Slight – Anomaly

7th rib, Bilateral, Wavy Rib, Moderate – Anomaly

6th rib, Left, Wavy Rib, Slight – Anomaly

6th rib, Right, Wavy Rib, Slight – Anomaly

6th rib, Right, Wavy Rib, Moderate – Anomaly

6th rib, Bilateral, Wavy Rib, Slight – Anomaly

6th rib, Bilateral, Wavy Rib, Moderate – Anomaly

5th rib, Right, Wavy Rib, Slight – Anomaly

5th rib, Right, Wavy Rib, Moderate – Anomaly

5th rib, Left, Wavy Rib, Slight – Anomaly

5th rib, Bilateral, Wavy Rib, Slight – Anomaly

5th rib, Bilateral, Wavy Rib, Moderate – Anomaly

4th rib, Right, Wavy Rib, Slight – Anomaly

4th rib, Right, Wavy Rib, Moderate – Anomaly

4th rib, Left, Wavy Rib, Slight – Anomaly

4th rib, Bilateral, Wavy Rib, Slight – Anomaly

4th rib, Bilateral, Wavy Rib, Moderate – Anomaly

3rd rib, Bilateral, Wavy Rib, Moderate – Anomaly

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

12(7.8)

8(38.1)

2(1.3)

2(9.5)

14(9.2)

11(52.4)

0(0.0)

12(7.8)

9(42.9)

2(1.3)

2(9.5)

0(0.0)

7(4.5)

6(25.0)

1(0.6)

1(4.2)

12(7.6)

9(37.5)

4(2.5)

3(12.5)

17(10.8)

12(50.0)

0(0.0)

1(0.6)

1(4.2)

2(1.3)

2(8.3)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

3(1.9)

3(12.5)

4(2.5)

3(12.5)

0(0.0)

1(0.6)

1(4.2)

7(4.5)*

7(29.2)*

0(0.0)

3(1.9)

2(8.3)

2(1.3)

2(8.3)

9(5.7)*

6(25.0)*

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

6(3.8)*

4(16.7)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

8(5.1)*

5(20.8)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

7(4.5)*

5(20.8)

0(0.0)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

0(0.0)

1(0.6)

1(4.2)

6(3.8)*

4(16.7)

1(0.6)

1(4.2)

1(0.6)

1(4.2)

2(1.3)

2(8.3)

4(2.5)

4(16.7)

0(0.0)

1(0.6)

1(4.2)

0(0.0)

3(3.8)

3(21.4)

0(0.0)

5(6.4)

4(28.6)

0(0.0)

12(15.4)

7(50.0)

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

3(3.8)

3(21.4)

2(2.6)

2(14.3)

3(3.8)*

3(21.4)

4(5.1)*

3(21.4)

10(12.8)*

8(57.1)*

1(1.3)

1(7.1)

6(7.7)*

6(42.9)*

5(6.4)*

4(28.6)*

10(12.8)*

8(57.1)*

3(3.8)*

3(21.4)

2(2.6)

2(14.3)

4(5.1)*

4(28.6)*

3(3.8)*

3(21.4)

1(1.3)

1(7.1)

11(14.1)*

9(64.3)*

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

4(5.1)*

4(28.6)*

3(3.8)*

3(21.4)*

11(14.1)*

9(64.3)

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

2(2.6)

2(14.3)

10(12.8)*

8(57.1)*

5(6.4)*

5(35.7)*

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

4(5.1)*

4(28.6)*

1(1.3)

1(7.1)

8(10.3)*

7(50.0)*

1(1.3)

1(7.1)

4(5.1)

4(28.6)

1(1.3)

1(7.1)

6(7.7)*

5(35.7)*

2(2.6)

2(14.3)

1(1.3)

1(7.1)

4(5.1)*

4(28.6)*

1(1.3)

1(7.1)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

3(3.8)*

3(21.4)*

1(1.3)

1(7.1)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

Contd.

Exam type: Skeletal-Entire

Number of Live Fetuses Examined

Number of Litters Evaluated

G1

0

mg/kg/day

G2

250

mg/kg/day

G3

500

mg/kg/day

G4

1000

mg/kg/day

153

21

157

24

78

14

2

1

sternebrae

6th sternebra, Hypoplastic – Anomaly

6th sternebra, Delayed skeletal ossification – Variation

6th sternebra, Incomplete ossification – Variation

5th sternebra, Hypoplastic – Anomaly

5th sternebra, Asymmetrical ossification – Anomaly

5th sternebra, Split – Anomaly

4th sternebra, Asymmetrical ossification – Anomaly

4th sternebra, Incomplete ossification – Variation

4th sternebra, Split – Anomaly

3rd sternebra, Asymmetrical ossification – Anomaly

3rd sternebra, Incomplete ossification – Variation

2nd sternebra, Hypoplastic – Anomaly

2nd sternebra, Split – Anomaly

1st sternebra, Incomplete ossification – Variation

Skull

Supraoccipital, Incomplete ossification – Variation

Parietal, Incomplete ossification – Variation

Interparietal, Incomplete ossification – Variation

Hyoid, Incomplete ossification – Variation

Frontal, Incomplete ossification – Variation

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

Fetuses N(%)

Litters N(%)

0(0.0)

7(4.6)

6(28.6)

43(28.1)

18(85.7)

19(12.4)

11(52.4)

1(0.7)

1(4.8)

2(1.3)

2(9.5)

1(0.7)

1(4.8)

13(8.5)

9(42.9)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

7(4.6)

7(33.3)

2(1.3)

2(9.5)

1(0.7)

1(4.8)

1(0.7)

1(4.8)

0(0.0)

2(1.3)

2(9.5)

2(1.3)

2(9.5)

0(0.0)

1(0.6)

1(4.2)

15(9.6)

6(25.0)

50(31.8)

19(79.2)

21(13.4)

13(54.2)

0(0.0)

2(1.3)

2(8.3)

0(0.0)

6(3.8)

4(16.7)

0(0.0)

1(0.6)

1(4.2)

6(3.8)

5(20.8)

1(0.6)

1(4.2)

0(0.0)

20(12.7)*

13(54.2)*

35(22.3)*

15(62.5)*

41(26.1)*

16(66.7)*

0(0.0)

2(2.6)

2(14.3)

34(43.6)

14(100.0)

10(12.8)

8(57.1)

0(0.0)

2(2.6)

2(14.3)

1(1.3)

1(7.1)

0(0.0)

1(1.3)

1(7.1)

0(0.0)

5(6.4)

3(21.4)

0(0.0)

1(1.3)

1(7.1)

25(32.1)*

10(71.4)*

31(39.7)*

12(85.7)*

34(43.6)*

12(85.7)*

7(9.0)*

3(21.4)

2(2.6)

1(7.1)

0(0.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

1(50.0)

1(100.0)

0(0.0)

[Fetuses N] Cochran Armitage, Chi Squared & Fisher's Exact; [Litters N] Cochran Armitage, Chi Squared & Fisher's Exact

Conclusions:

NOAEL for maternal toxicity could not be established due to significant decreases in body weight, body weight gain, and food intake at ≥250mg/kg/day. NOAEL for maternal developmental toxicity was considered at 250 mg/kg/day. Significant changes in uterine weight, early resorptions, postimplantation loss, and implantation index were observed at ≥500 mg/kg/day and this dose level was therefore considered to be LOAEL for maternal developmental toxicity. NOAEL for fetal developmental toxicity could not be established due to significant decreases in fetal weights at ≥250mg/kg/day. NOAEL for teratogenicity was considered at 500 mg/kg/day since fetal external, visceral and skeletal examinations did not reveal any signs of teratogenicity at 250 and 500 mg/kg/day. The observed effects on fetal developmental toxicity (i.e. the small changes in fetal body weight) were considered to be secondary to maternal systemic toxicity whereas the observed effects on maternal developmental toxicity were not considered to be entirely due to maternal systemic toxicity.

Executive summary:

The substance was given by oral gavage to 24 pregnant wistar rats at 0, 250, 500 and 1000 mg/kg bw/day from GD5 to GD19. The study was performed according to OECD 414 (adopted in 2018) and GLP. Results (adults): There were no unscheduled deaths, clinical signs, or gross pathological findings at 250 and 500 mg/kg/day. At 1000 mg/kg/day, one dam (Rz1075) had reddish discharge from the vagina on GD18 which was attributed to test item. No other clinical signs were observed at 1000 mg/kg/day, and there were no unschedulded deaths nor any gross pathological findings at 1000 mg/kg/day. As compared to the vehicle control group, statistically and dose-dependent decreases in maternal body weight (from -5 to -32%), in maternal body weight gain (from -17 to -199%), in adjusted maternal body weight (from -8 to -14%) and in adjusted maternal body weight gain (from -61 to -133%) were observed in all dosed groups. These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.As compared to vehicle control group, significant and dose-dependent decreases in mean food consumption (from -15 to -53%) were observed in all dosed groups during different periods of gestation.These effects at 250, 500 and 1000 mg/kg/day were attributed to the test item.No significant changes in T3 or T4 levels were observed.Significant increases in TSH levels were observed at 500 and 1000 mg/kg, however these effects were not considered to be of toxicological significance since the TSH levels at 500 and 1000 mg/kg were well within the historical control range of the test facility and since no significant changes in thyroid weight or histology of the thyroid were observed.No significant effects were observed at 250 mg/kg/day. Ten dams of 24 pregnant rats treated at 500 mg/kg/day and 22 dams of 23 pregnant rats treated at 1000 mg/kg/day had complete resorptions (i.e. only implantation sites with no fetuses). Hence, therewere only 14 litters at 500 mg/kg/day and 1 litter at 1000 mg/kg/day available for evaluation. Significant changes in uterine weight, number of implantations, implantation index, early resorptions, and post-implantation losses were observed at 500 and 1000 mg/kg/day. The effects observed at 500 and 1000 mg/kg/day were attributed to the test item. Gross evaluations of the placenta revealed no remarkable findings.Results (foetus): The total number of fetuses were 293, 302, 150, and 3 at 0, 250, 500 and 1000 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in sex ratio or AGD were observed. Significant decreases in male fetal weight and in sex-combined fetal weight were observed at 250 and 500 mg/kg/day and these effects were attributed to the test item. External, visceral and skeletal examinations revealed no teratogenic effects attributed to the test item at 250 and 500 mg/kg/day. There were 14 litters at 500 mg/kg/day and only 1 litter at 1000 mg/kg/day available for evaluation. Due to low sample size at 1000 mg/kg/day, the data from fetal external, visceral and skeletal evaluations were considered insufficient for evaluation of teratological effects. Conclusions: NOAEL for maternal toxicity could not be established due to significant decreases in body weight, body weight gain, and food intake at ≥250mg/kg/day. NOAEL for maternal developmental toxicity was considered at 250 mg/kg/day. Significant changes in uterine weight, early resorptions, postimplantation loss, and implantation index were observed at ≥500mg/kg/dayand this dose level was therefore considered to be LOAEL for maternal developmental toxicity. NOAEL for fetal developmental toxicity could not be established due to significant decreases in fetal weights at ≥250mg/kg/day. NOAEL for teratogenicity was considered at 500 mg/kg/day since fetal external, visceral and skeletal examinations did not reveal any signs of teratogenicity at 250 and 500 mg/kg/day.

The observed effects on fetal developmental toxicity (i.e. the small changes in fetal body weight) were considered to be secondary to maternal systemic toxicity whereas the observed effects on maternal developmental toxicity were not considered to be entirely due to maternal systemic toxicity.

Registration Dossier

Registration Dossier

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Diss Factsheets

3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Developmental toxicity / teratogenicity

Administrative data

Data source

Reference

Materials and methods

Test guideline

Test material

Constituent 1

Test animals

Administration / exposure

Doses / concentrationsopen allclose all

Examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Maternal developmental toxicity

Effect levels (maternal animals)

Maternal abnormalities

Results (fetuses)

Effect levels (fetuses)

Fetal abnormalities

Overall developmental toxicity

Any other information on results incl. tables

Applicant's summary and conclusion

Doses / concentrationsopen all close all