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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From: June 12, 2013 To: September 25, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: MAFF in Japan, 12-Nousan-No. 8147, 2-1-18, 2000
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
Lot number: 080722
Expiry: July 25, 2015

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(BrlHan:WIST@Jcl[GALAS])
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: Fuji Breeding Center, CLEA Japan, Inc. (Shizuoka, Japan)
Age at study initiation: about 13-14 weeks
Weight at study initiation: 209-246 g for females
Housing: animals were housed in suspended wire-mesh stainless steel cages (width 310 x depth 440 x height 230 mm) in groups of 4 males/cage and 5 females/cage. Aluminum cages with wire-mesh floors and fronts (width 260 mm x depth 400 mm x height 240 mm) were used for the mating (1 pair/cage). Copulated females wer ehoused individually.
Diet: ad libitum males were supplied with certified solid feed (MF; Oriental Yeast Co., Ltd., Tokyo, Japan), and females with certified pulverized feed (MF Mash; Oriental Yeast Co., Ltd., Tokyo, Japan)
Water: ad libitum local tap water (Joso-shi Water Supply, Ibaraki, Japan)
Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 2 °C
Humidity: 50 ± 20 %
Air changes: at least 10 times per hour
Photoperiod: 12 h light / 12 h dark; lights on at 7:00 a.m. and off at 7:00 p.m.

IN-LIFE DATES: From: June 25, 2013 To: October 10, 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on exposure:
The test substance was administered to the animals at approx. the same time in the morning. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance was confirmed in a previous study in which the test substance was stable for at least 15 days after preparation. For verification of stability the samples were retained at ambient temperature of the animal room for 1 day, after storing them in a sealed cylinder in a cold and dark place for 14 days.

Samples were taken from the middle layer of the graduated cylinder and verified that the test substance was presented at the target concentrations in each dosing formulation. Dosing formulations for the low- and high-dose levels were further analyzed for homogeneity of the test substance at the first preparation.

The results of homogeneity analyses demonstrated that relative standard deviations (RSD) values of mean concentrations of the test substance in dosing formulations for the low- and high-dose groups were 0.6 % and 0.4 %, respectively, indicating that the test substance was uniformly distributed in the dosing formulations.
The results of the concentration analyses showed that the test substance was detected in the samples from each dosing formulation, which was administered to animals, at a range of 103-106 % of the nominal concentrations.
Details on mating procedure:
Vaginal smears were taken from females for microscopic examination. Then, females showing proestrus or estrus vaginal smears were paired overnight with males on a 1:1 basis. The females were examined next morning for the presence of vaginal plugs and sperm in vaginal smears, and those showing vaginal plugs and/or sperm were considered to have copulated. These mating procedures were repeated for 4 days.
Duration of treatment / exposure:
Gestation day (GD) 6-19
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 pregnant rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
PREPARATION OF DOSING SOLUTION
Dosing formulations were prepared for each dose level by suspending a specified amount of the test substance (mg/10 mL/kg body weight) in an aqueous suspension of 1 % sodium carboxymethylcellulose. Dosing formulations were prepared twice at an interval of approximately 14 days.

Examinations

Maternal examinations:
CAESAREAN SECTION
- All surviving females wer euthanised by carbon dioxide inhalation on GD 20. Gravid uteri were removed and fetuses examined.

CAGE SIDE OBSERVATION
- Clinical signs and mortality: twice a day during the dosing period.
- Detailed physical examination was conducted when they were weighed or dosed.

BODY WEIGHT
- Each female was weighed on GD 0, 6, 9, 12, 15, 18 and 20.
- Adjusted body weights were calculated by subtracting the gravid uterine weight from the body weight on GD 20.

BODY WEIGHT GAINS
- Calculated by subtracting the body weight value on GD 6 from each value determined on GD 9, 12, 15, 18 and 20.
- Adjusted body weight gains were calculated by subtracting the gravid uterine weight from the body weight gain during GD 6-20.

FOOD CONSUMPTION
- The amount of food supplied and/or unconsumed was determined on the day of body weight measurement.
- Daily food consumption (g/rat/day) was calculated by dividing values of the total food consumption by the number of days.

NECROPSY AND TISSUE PRESERVATION
Females euthanized on GD 20 were necropsied and pathological findings were recorded. One deceased female was necropsied immediately after discovery and findings were recorded. The organs and tissues were not preserved.
Ovaries and uterine content:
- Gravid uterine weight, numbers of corpora lutea and implants were determined and recorded.


The gravid uteri of females surviving to GD 20 were weighed. When no conceptus was grossly evident, the apparently non-pregnant uterus was stained with 10 % ammonium sulfide solution to detect very early resorptions. The data (clinical signs, body weights, body weight gains, food consumption, gross pathological findings, gravid uterine weights, the numbers of corpora lutea and implants, and percent pre-implantation losses) from females, which had no grossly observable
conceptus in the uteri, was excluded from statistical evaluation.
Fetal examinations:
NUMBER OF LIVE FETUSES AND PERCENT INCIDENCE OF RESOPRTIONS AND FETAL DEATHS
The numbers of live and dead fetuses were recorded with their positions in the uterus. Resorbed embryos or dead fetuses were classified into implantation sites, placental remnants, or macerated fetuses (including dead term fetuses) according to the developmental stage in which resorptions or deaths occurred; an implantation site was a metrial gland with no remnant of placenta or embryo, a placental remnant was the placenta being resorbed with little or no embryonic tissue, a macerated fetus was an embryo or fetus being resorbed or a fetus that died shortly before necropsy.

SEX RATIO, FETAL BODY WEIGHTS AND PLACENTAL WEIGHTS
Sex of each live fetus was determined, and fetal body weights and placental weights were recorded and calculated.

EXTERNAL, VISCERAL AND SKELETAL EXAMINATION
Live fetuses in a litter were individually identified by their uterine position. Then, they were examined for external and orifice abnormalities. Animals were euthanised by an intra-peritoneal injection of a pentobarbital sodium solution (Somnopentyl).

Then the fetuses were assigned serial numbers within a litter beginning at the nearest site of the right ovary in order down the right uterine horn, across cervix, and up the left horn to the nearest site of the left ovary. In odd-numbered fetuses, the thoracic and abdominal soft tissue was examined for visceral abnormalities according to the fresh visceral examination method of Stuckhardt and Poppe. The fetuses were then fixed in Bouin's solution along with the placentas. After fixation for one week or more, sections of the decapitated head were made using Wilson's razor blade sectioning technique, and the eyes, brain, nasal passages, and tongue were observed. The examined tissue of the head was preserved in Bouin's solution along with the other tissue. Even-numbered fetuses were fixed in 70 % ethanol, stained with alizarin red S and alcian blue, and cleared in 70 % glycerin for making the skeletal specimens. After examination, skeletal specimens were stored.
Statistics:
Statistical significance: α=0.05 or 0.01

Body weights, adjusted body weights, body weight gains, adjusted body weight gains, and food consumption of maternal animals, the numbers of corpora lutea, implants, and live fetuses, and the weights of gravid uteri, fetuses, and placentas: Equality of variances was first evaluated by Bartlett's test. If homogeneous, a parametric analysis of variance in one-way classifications was used to determine if any statistical differences exist among groups. If the analysis of variance would be significant, Dunnett's multiple comparison test was performed to detect any statistically significant differences between the treated groups and their corresponding controls. When Bartlett's test indicates that the variances would not be homogeneous, Kruskal-Wallis test was used for detecting any statistical differences among groups and if significant, Dunnett-type nonparametric multiple comparison test was performed to detect statistical differences between the treated groups and their corresponding controls.

Percent pre-implantation losses, percent incidences of resorptions and fetal deaths, and percent litter incidences of fetal malformations or variations were evaluated as follows: Kruskal-Wallis test was used for detecting any statistical differences among groups and if significant, Dunnett-type nonparametric multiple comparison test was performed to detect statistical differences between the treated groups and their corresponding controls. As for the data on the incidences of clinical and gross pathological findings in maternal animals, incidences of maternal animals having fetuses with malformations or variations, and fetal sex ratio, chi-square test was used when all expected values of control and treated groups were 5 or more, and Fisher's exact probability test was used when any expected values of control and treated groups were less than 5.
Indices:
- Percent pre-implantation losses = [(number of corpora lutea-number of implants)/number of corpora lutea] x100

- Percent resorptions and fetal deaths (percent post-implantation losses) = (total number of resorbed embryos and dead fetuses/number of implants) x100
- Sex ratio = total number of male fetuses/total number of live fetuses

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related findings in clinical observations, except for the dead female.

Incidental findings noted during the study included loose stool in 1 female in the control group (dosing period); and loss of fur in 1-3 females among the 50, 100 and 200 mg/kg bw/d groups (dosing and post-dosing periods).
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 200 mg/kg bw/d group was found dead, expectorating bloody liquid, shortly after the treatment on gestation day 19. Necropsy findings suggest that this death was treatment-related. No other deaths occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
In the 200 mg/kg group, a statistically significant decrease in maternal weight gains was seen during gestation days 6-9, during which a weight loss occurred in 6 out of 22 females. Subsequent mean values in the high-dose group were apparently comparable to those in the control group. However, this was not a clear indication of recovery, but rather being attributable to the suppression of body weight gains in the control females.

BODY WEIGHT GAINS
Body weight gains in the 50 and 100 mg/kg groups were generally comparable to or greater than those in the control group throughout the study, with the increase being statistically significant in the 100 mg/kg group during gestation days 6-15 and 6-18.
There was no significant difference in adjusted body weight gains of females between the control and any treated group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 200 mg/kg group, food consumption was moderately reduced during gestation days 6-9 when compared to the control and continued to be low until gestation days 12-15 (74-93% of control values). Statistical analysis revealed a significant decrease in food consumption by high-dose females during gestation days 6-9, 9-12 and 12-15. In the 100 mg/kg group, a slight but statistically significant decrease in food consumption was seen during gestation days 6-9 (86% of control value), although subsequent values were comparable to those in the control group.
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:
not examined
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):
Aside from uterus weight, organ weights were not collected.

There were no statistically significant differences in the numbers of corpora lutea and implants, or gravid uterine or placental weights between the control and any treated group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The dead animal in the 200 mg/kg group showed a red discolored mucosa of the ileum and lung congestion at necropsy. The red discoloration of ileum suggests that there was an intestinal hemorrhage. Despite the lung congestion, there was no evidence of esophageal and/or tracheal injury that might be attributable to gavage error.

There were no treatment-related findings observed in the necropsy of surviving females in the other dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No differences between treated and control groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No differences between treated and control groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No differences between treated and control groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No differences between treated and control groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
No differences between treated and control groups.
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No differences between treated and control groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in fetal weights of both sexes between treated and control groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All surviving females except two animals in the 200 mg/kg bw/d group had live fetuses; the total number of pregnant females examined was 24, 24, 24 and 21 in the control, 50, 100 and 200 mg/kg bw/d groups, respectively. Two females having no live fetuses did not have grossly visible conceptuses in their uteri. Subsequent examination by 10 % ammonium sulfide staining did not detect any early resorption sites in their uteri, indicating that these females were non-pregnant. The dead female in the 200 mg/kg bw/d group had some fetuses in the uterus, although not evaluated in this study.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the sex ratio between treated and control groups.
Changes in litter size and weights:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Among all live fetuses, external malformations occurred in a total of 4 fetuses. The findings included omphalocele in 1 fetus from the control group (0.28 %), local edema in 1 fetus from the 100 mg/kg bw/d group (0.32 %), and cleft palate in 2 fetuses from a single litter of 200 mg/kg bw/d group (0.79 %). There was no statistically significant difference in the litter incidences of external malformations between the control and any treated group.

Cleft palates occurred in two high-dose fetuses from one single litter. These fetuses weighed 2048 mg and 1906 mg while their normal littermates weighed 2231-3560 mg, and were recognized as the two smallest fetuses in this litter. Hence, the affected fetuses were much smaller than their littermates and weighed less than 60 % of their groupmates (average high-dose fetal weight 3571 mg).

From this notable weight and size decrement it is likely that the process of palatal closure was disrupted perhaps by the accidental delay in fetal development and could not be caught up in these two fetuses after passing the critical window. Hence these cleft palates are considered to be a manifestation of a very distinct developmental delay rather than as a specific malformation. It is regarded to be highly unlikely that these cleft palates were caused by a specific teratogenic potential of the test substance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS
Skeletal malformations were found in 4 and 1 fetuses from the control and 100 mg/kg bw/d groups, respectively. In the control group, 1 fetus exhibited multiple malformations in cervical and thoracic skeleton (including fused cervical centrum cartilage, fused sternebra and 1st rib cartilage not fused to sternum) together with fused phalanx. Another fetus in the same group had fused sternebra and fused rib cartilage, while the remaining 2 fetuses exhibited 1st rib cartilage not fused to sternum. In the 100 mg/kg bw/d group, the affected fetus had fused cervical centrum cartilage accompanied by fused cervical arch. Mean litter incidence of each skeletal malformation in the control and 100 mg/kg group ranged from 0.00 % to 1.89 % and from 0.00 % to 0.60 %, respectively. Mean litter incidence of fetus having any skeletal malformations in these two groups were 2.48 % and 0.60 %, respectively. Statistical analysis also showed significantly lower litter incidences of 1st rib cartilage not fused to sternum and fetus having any skeletal malformations, for the 50, 100 and/or 200 m/kg bw/d groups, when compared to the control: however, these are thought to be toxicologically meaningless fluctuations.

VARIATIONS
There were also many skeletal variations found in all groups including controls. Although these skeletal variations consisted exclusively of those commonly observed in term rat fetuses such as cervical rib, discontinuous rib cartilage, and 27 presacral vertebrae, a few findings listed below showed increased litter incidences in the 200 mg/kg bw/d fetuses.

94.36 % of fetuses in the 200 mg/kg bw/d group had one or more skeletal variations, one of which was supernumerary rib in almost all cases. In addition, a significantly increased incidence was noted for zygomatic bone fused with maxilla in the 200 mg/kg bw/d group, although the incidence was much lower than that of the supernumerary rib.

No other statistically significant difference was found in the litter incidence of any skeletal variation between the control and treated group.

There was no statistically significant difference between the control and any treated group in the incidence for either females having fetuses with malformations or those having fetuses with variations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS
There were no visceral malformations in all fetuses examined (half of the live fetuses obtained) except one in the 100 mg/kg bw/d group. The fetus that had local edema further exhibited the following visceral malformations: microphthalmia, small nasal cavity and complex cardiovascular malformations consisting of right-sided aortic arch, overriding aorta, narrowed pulmonary trunk and narrowed left subclavian, with the mean litter incidences of 0.60 %.

VARIATIONS
Visceral variations were found in in all groups including the control; these included left umbilical artery (13.79 %-18.50 %), dilated renal pelvis (0.00 %-0.68 %) and thymic remnant in the neck (0.00 %-0.69 %). Statistical analysis revealed no significant difference in either the litter incidences of visceral malformations or those of visceral variations between the control and any treated group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: slightly increased incidence of skeletal variations

Fetal abnormalities

open allclose all
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
variation: increased incidence of supernumerary rib at maternal toxic dose of 200 mg/kg bw/d
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
Description (incidence and severity):
variation: increased incidence of zygomatic bone fused with maxilla at maternal toxic dose of 200 mg/kg bw/d

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Maternal food consumption

Dose level [mg/kg]

0

50

100

200

Day 0 - 6

SD

17.4

1.5

17.7

1.4

17.3

1.6

17.9

2.1

Day 6 - 9

SD

18.7

2.3

18.0

1.6

16.1**

2.5

13.8**

2.3

Day 9-12

SD

19.2

3.5

19.2

2.0

18.4

1.7

16.7**

2.4

Day 12-15

SD

19.4

3.0

19.6

1.7

18.9

1.8

18.1*

2.1

Day 15-18

SD

21.5

1.7

21.7

1.5

21.6

1.5

21.5

2.7

Day 18-20

SD

21.7

2.1

21.8

1.9

21.0

2.0

19.8

4.0

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Body weight [g]

Dose level [mg/kg]

0

50

100

200

Day 0

SD

229

11

229

12

229

11

228

11

Day 6

SD

250

10

249

12

249

11

248

13

Day 9

SD

260

12

258

11

258

11

250*

12

Day 12

SD

270

16

273

13

271

10

267

15

Day 15

SD

284

15

287

12

288

11

281

14

Day 18

SD

318

15

321

15

324

11

314

19

Day 20

SD

348

16

351

19

354

14

342

23

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Body weight gain [g]

Dose level [mg/kg]

0

50

100

200

Day 6-9

SD

9

4

9

3

8

5

2**

6

Day 6-12

SD

20

11

23

4

22

6

19

7

Day 6-15

SD

33

11

38

4

39*

7

33

8

Day 6-18

SD

68

9

72

6

75*

9

66

14

Day 6-20

SD

98

10

102

11

105

11

93

20

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Mean gravid uterus weights and net body weight change of pregnant rats
administered the test substance during Days 6 to 19 of gestation

Dose level [mg/kg bw/d]

0

50

100

200

Gravid uterus (g)

73 ± 9

72 ± 9

76 ± 6

71 ± 15

Carcass (g)

275±12

279±16

278±12

271±17

Net weight change from
Day 6 (g)

25±7

29±8

29±9

22±12

* p< 0.05

Caesarean section data

Dose level [mg/kg bw/d]

Unit of measure

0

50

100

200

Rats tested

N

24

24

24

24

Pregnant

N (%)

24

24

24

22

Dams with viable fetuses

N

24

24

24

21

Rats pregnant and caesarean-sectioned on day 20 of gestation

N

24

24

24

21

Corpora Lutea

Mean ± S.D.

15±1.5

14.6±1.1

15.0±1.0

14.8±1.1

Implantations

Mean ± S.D.

13.9±1.9

13.7±1.0

14.3±0.9

13.2±2.4

% pre-implantation losses

Mean

7.7

6.1

4.6

10.6

Litter Size

Mean ± S.D.

13.3±2.0

13.0±1.7

13.7±1.0

12.5±2.8

Live Fetuses

Mean ± S.D.

13.3±2.0

13.0±1.7

13.7±1.0

12.5±2.8

Resorptions and fetal deaths

Mean ± S.D.

0.7±4.8

0.7±5.4

0.5±3.8

0.7±5.2

Macerated fetus

Mean

0.0

0.0

0.1

0.0

Placental remnants

Mean

0.1

0.1

0.1

0.1

Implantation Sites

Mean

0.5

0.6

0.4

0.6

Dams with resorptions

N (%)

9 (37.5)

10 (41.7)

8 (33.3)

4 (19.0)

Dams with all conceptuses resorbed

N (%)

0

0

0

0

Litter Observations

% Live male fetuses/litter

Mean ± S.D.

0.519

0.497

0.505

0.529

Live Fetal body weights (mg/litter)

Male

Mean ± S.D.

3594±231

3653±223

3589±212

3571±324

Female

Mean ± S.D.

3414±210

3430±181

3420±181

3448±302

Placental weight (mg)

Mean ± S.D.

470±46

454±33

463±34

476±48

Incidences of external malformations

Dose level [mg/kg]

Measure

0

50

100

200

Litters Evaluated

N

24

24

24

21

Fetuses Evaluated

N

318

312

329

263

Litters with external malformations

N

1

0

1

1

Fetuses with external malformations

N

1

0

1

2

Omphalocele

Number of affected litters

N (%)

1 (4 %)

0

0

0

Number of affected fetuses

N

1

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.28±1.36

0

0

0

Local edema

Number of affected litters

N (%)

0

0

1 (4 %)

0

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

1

0

Cleft palate

Number of affected litters

N (%)

0

0

0

1

Number of affected fetuses

N

0

0

0

2

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0

0.79±3.64

Incidence of visceral malformation

Dose level [mg/kg]

Measure

0

50

100

200

Litters Evaluated

N

24

24

24

21

Fetuses Evaluated

N

165

150

169

135

Litters with visceral malformations

N

0

0

1

0

Fetuses with visceral malformations

N

0

0

1

0

Right-sided aortic arch

Number of affected litters

N (%)

0 (0%)

0 (0%)

1 (4 %)

0 (0 %)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Overriding aorta

Number of affected litters

N (%)

0 (0 %)

0 (0 %)

1 (4%)

0 (0 %)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Narrowed pulmonary trunk

Number of affected litters

N (%)

0 (0 %)

0 (0 %)

1 (4%)

0 (0 %)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Narrowed left subclavian

Number of affected litters

N (%)

0 (0 %)

0 (0 %)

1 (4%)

0 (0 %)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Small nasal cavity

Number of affected litters

N (%)

0 (0 %)

0 (0 %)

1 (4%)

0 (0%)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Microphthalmia

Number of affected litters

N (%)

0 (0%)

0 (0%)

1 (4%)

0 (0%)

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.6±2.92

0

Incidence of visceral variations

Dose level [mg/kg]

Measure

0

50

100

200

Litters Evaluated

N

24

24

24

21

Fetuses Evaluated

N

165

162

169

135

Litters with variations

N

17

16

17

15

Fetuses with variations

N

28

25

31

19

Left umbilical artery

Number of affected litters

N (%)

17 (71 %)

16 (67 %)

17 (71 %)

14 (67 %)

Number of affected fetuses

N

28

25

31

18

Mean incidence of affected fetuses in litters ± SD

(%)

16.75 ± 13.59

15.58 ±14.89

18.50 ±14.64

13.79 ±12.68

Dilated renal pelvis

Number of affected litters

N (%)

1 (4%)

0

0

1 (5%)

Number of affected fetuses

N

1

0

0

1

Mean incidence of affected fetuses in litters ± SD

(%)

0.52 ±2.55

0.00 ±0.00

0.00 ±0.00

0.68 ±3.12

Thymic remnant in the neck

Number of affected litters

N (%)

1 (4%)

1 (4%)

0

0

Number of affected fetuses

N

1

1

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.69 ±3.40

0.60 ±2.92

0.00 ±0.00

0.00 ±0.00

Incidence of skeletal malformations

Dose level [mg/kg]

Measure

0

50

100

200

Litters Evaluated

N

24

24

24

21

Fetuses Evaluated

N

153

150

160

128

Litters with skeletal malformations

N

4

0

1

0

Fetuses with skeletal malformations

N

4

0

1

0

Fusion between occipital and first cervical centrum

Number of affected litters

N (%)

1

0

0

0

Number of affected fetuses

N

1

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.69±3.40

0

0

0

Fused cervical centrum cartilage

Number of affected litters

N (%)

1

0

1

0

Number of affected fetuses

N

1

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.69±3.40

0

0.60±2.92

0

Fused cervical arch

Number of affected litters

N (%)

0

0

1

0

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.60±2.92

0

Split cartilage of ventral arch

Number of affected litters

N (%)

1

0

0

0

Number of affected fetuses

N

1

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.69±3.40

0

0

0

Fused sternebra

Number of affected litters

N (%)

2

0

0

0

Number of affected fetuses

N

2

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

1.29±4.38

0

0

0

Rib cartilage not fused to sternum (1st)

Number of affected litters

N (%)

3

0

0

0

Number of affected fetuses

N

3

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

1.89±5.11

0

0

0

Fused rib cartilage

Number of affected litters

N (%)

1

0

0

0

Number of affected fetuses

N

1

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.60±2.92

0

0

0

Fused phalanx

Number of affected litters

N (%)

1

0

0

0

Number of affected fetuses

N

1

0

0

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.69±3.40

0

0

0

Mean % litter incidences of major skeletal variations

Skeletal findings

Dose levels (mg/kg/day)

 

0

50

100

200

Fetuses with one or more variations

62.64

74.82

81.29

94.36**

Supernumerary rib

51.35

67.26

72.82

89.95**

Zygomatic bone fused with maxilla

3.87

0.69

5.30

13.14**

** significant different from control at p ≤ 0.01

Incidences of skeletal malformations

Dose level [mg/kg]

Measure

0

50

100

200

Litters Evaluated

N

24

24

24

21

Fetuses Evaluated

N

153

150

160

128

Litters with skeletal variations

N

23

24

24

21

Fetuses with skeletal variations

N

95

112

132

120

Zygomatic bone fused with maxilla

Number of affected litters

N (%)

3

1

6

11

Number of affected fetuses

N

6

1

8

17

Mean incidence of affected fetuses in litters ± SD

(%)

3.87±11.72

0.69±3.40

5.30±10.09

13.14±18.83**

Cervical rib

Number of affected litters

N (%)

0

2

3

3

Number of affected fetuses

N

0

2

3

3

Mean incidence of affected fetuses in litters ± SD

(%)

0

1.19±4.03

1.98±5.38

2.34±6.00

Bipartite ossification of cervical centrum

Number of affected litters

N (%)

0

1

0

1

Number of affected fetuses

N

0

1

0

1

Mean incidence of affected fetuses in litters ± SD

(%)

0

0.60±2.92

0

0.60±2.73

Bipartite ossification of sternebra

Number of affected litters

N (%)

0

0

1

0

Number of affected fetuses

N

0

0

1

0

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

0.69±3.40

0

Misaligned sternebra

Number of affected litters

N (%)

1

1

2

0

Number of affected fetuses

N

1

1

2

0

Mean incidence of affected fetuses in litters ± SD

(%)

0.60±2.92

0.69±3.40

1.29±4.38

0

Discontinuous rib cartilage (false rib)

Number of affected litters

N (%)

21

38

16

19

Number of affected fetuses

N

48

19

39

38

Mean incidence of affected fetuses in litters ± SD

(%)

31.98±19.27

25.81±23.85

23.81±22.02

31.09±18.30

Branched rib cartilage (false rib)

Number of affected litters

N (%)

3

6

1

3

Number of affected fetuses

N

4

3

1

4

Mean incidence of affected fetuses in litters ± SD

(%)

3.51±11.05

4.58±13.18

0.60±2.92

2.72±7.31

Supernumerary rib

Number of affected litters

N (%)

22

24

23

21

Number of affected fetuses

N

79

101

119

114

Mean incidence of affected fetuses in litters ± SD

(%)

51.35±33.31

67.26±31.91

72.82±30.42

89.95±15.18**

Dumbbell ossification of thoracic centrum

Number of affected litters

N (%)

0

0

3 (12.5%)

1 (4.8%)

Number of affected fetuses

N

0

0

3

1

Mean incidence of affected fetuses in litters ± SD

(%)

0

0

1.89±5.11

0.79±3.64

Bipartite ossification of thoracic centrum

Number of affected litters

N (%)

0

1 (4.1%)

0

2 (8.3%)

Number of affected fetuses

N

0

1

0

2

Mean incidence of affected fetuses in litters ± SD

(%)

0

0.60±2.92

0

1.36±4.30

27 presacral vertebrae

Number of affected litters

N (%)

6 (25%)

12 (50%)

7 (29%)

2 (9.5%)

Number of affected fetuses

N

9

17

14

5

Mean incidence of affected fetuses in litters ± SD

(%)

6.13±12.46

11.67±16.11

8.63±16.45

3.40±10.98

Lumbosacral transitional vertebra

Number of affected litters

N (%)

9 (37.5%)

10 (41.7%)

9 (37.5%)

12 (57.1%)

Number of affected fetuses

N

15

13

12

18

Mean incidence of affected fetuses in litters ± SD

(%)

9.70±14.49

8.61±12.02

7.44±10.86

13.87±13.82

Applicant's summary and conclusion