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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 27 July 2016 to 26 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: 40 CFR 799.9370. TSCA Prenatal developmental toxicity
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 (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dioxolane
EC Number:
211-463-5
EC Name:
1,3-dioxolane
Cas Number:
646-06-0
Molecular formula:
C3H6O2
IUPAC Name:
1,3-dioxolane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1607011300R

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Saint-Constant Québec, Canada
- Age at study initiation: approximately 6 months of age
- Weight at study initiation: 3230.0 g to 4162.0 g
- Fasting period before study: no
- Housing: individually housed in units of six to eight cages
- Diet: approximately 150 g of Certified Rabbit Chow® #5322 (PMI® Nutrition International) was available to each rabbit each day until the first day of dosing, at which time approximately 180 g to 185 g of the same certified feed was offered to each rabbit each day
- Water: ad libitum (local source and passed through a reverse osmosis membrane before use. On the day of arrival, each rabbit was provided a water supplement (Hydrogel™ water pack [Portland, ME]). During the acclimation period, the water supplement was available to each rabbit.
- Acclimation period: up to 4 days and at least 3 days prior to initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 59°F to 70°F (15°C to 21°C)
- Humidity (%): 30% to 70%.
- Air changes (per hr): minimum of 10 changes per hour of fresh air that had been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): 12-hour dark:12-hour light

IN-LIFE DATES: From: 30 July 2016 To: 25 August 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Reverse osmosis (R.O.) membrane-processed deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance dosing formulations was prepared based on Sponsor instructions at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared fresh on the date of dose administration. The dose aliquot containers were maintained at ambient conditions and blanketed with nitrogen with continuous stirring, prior to dose administration. The bottle containing the dosing formulation for each of the test agent groups was not opened until the initiation of dose administration for that group. The dosing formulation was stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 0, 4, 12, 40 and 100 mg/mL
- Amount of vehicle (if gavage): dose volume of 5mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. The method number provided by WIL Research was 419.DIW2.01.
On the first and last days of preparation, duplicate 1.0 mL samples were collected from the top, middle and bottom of the formulations for the test substance groups and from the middle of the formulations for the control substance group (Group 1). These samples were submitted for analysis following the first day of preparation. Triplicate samples were also collected in the same manner (duplicate middle samples only for Group 1), were maintained as backup samples and were retained in a refrigerator set to maintain 4°C. On days when only concentration analysis was required, the formulations were only sampled from the middle. All backup samples from the first preparation and the backup samples for Group 2 from the last preparation were shipped for analysis to investigate out of specification results. The remaining backup samples were discarded prior to issue of the final report.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. Homogeneity results were considered acceptable if the relative standard deviation (RSD) of the mean value at each sampling location was ≤ 5% for each group.
The analyzed formulations used for dose administration met the protocol-specified acceptance criteria for homogeneity (i.e., the RSD for the mean concentration was ≤ 5%), and concentration acceptability for suspension formulations, i.e., the mean analyzed concentration was 90% to 110% of the target concentration and each individual sample was within ± 15% of the target concentration. No test substance was detected in the analyzed vehicle administered to the control group (Group 1).
Details on mating procedure:
Female rabbits were naturally bred at the Supplier, by breeder male rabbits of the same source and strain, before shipment to the Testing Facility. The day mating occurred was considered to be DG 0. The rabbits were shipped to the Testing Facility after mating to arrive on DGs 3 and 4 (first shipment) and DGs 2 and 3 (second shipment). The Supplier forwarded breeding records and DG 0 body weights.
Duration of treatment / exposure:
DGs 6 through 28
Frequency of treatment:
Daily
Duration of test:
On DG 29, surviving rabbits were euthanized blind to dose group.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The inhalation route of exposure could be the likely route of human exposure. The oral route of exposure was selected to achieve significant systemic exposure to 1,3-dioxolane and to satisfy a regulatory request. Dose levels were selected by the Sponsor based on a previous dose range-finding study (Testing Facility Study No. 20096129; Sponsor’s Reference No. 1300R-OECD414-DRF).
- Rationale for animal assignment: Mated female animals were assigned to groups using a computer-based (weight-ordered) randomization procedure, using body weight assessment from DG 0.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily, once in the morning and once in the afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on the day of arrival daily after arrival at the Testing Facility, daily before dose administration during the dosing period, and on the day of scheduled euthanasia

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on DG 0 (provided by the Supplier), daily after arrival at the Testing Facility, daily during the dose period, and on the day of scheduled euthanasia

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: ovaries and uterus, cervix, esophagus, heart, kidney, liver, lung, spleen, stomach, trachea

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: placentae
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
Proportions and/or incidences were calculated to summarize categorical data, as appropriate. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics including number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. Litter values were used where appropriate. Only body weights of live fetuses were used to determine litter mean fetal body weight. Fetal body weight was analyzed using covariance analysis with live litter size as the covariate. Animal data collected during the
predose period were reported, but were not summarized or analyzed statistically.
Historical control data:
Historical control data were provided on reproductive indices, material necropsy observations, fetal gross external alterations, fetal soft tissue alterations and fetal skeletal alterations.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 200 and 500 mg/kg/day dose groups, there were increased incidences of thin body condition (2 females in each of the dose groups between DGs 14 to 29) and decreased fecal output (2 females in each of the dose groups between DGs 13 to 20). At 200 mg/kg/day, female #3070 (which aborted) was observed with thin body condition; female #3076 was observed with decreased fecal output; and female #3078 (which was observed with total resorptions) was observed with thin body condition and decreased fecal output. At 500 mg/kg/day, female #3090 (which aborted) and 3102 (which was observed with total resorptions) were observed with thin body condition and decreased fecal output. These females were also observed with a reduction in body weight gain and reduced food consumption.
All other clinical observations were considered unrelated to 1,3-dioxolane because: 1) the incidences were not dose dependent; or 2) the observations occurred in only one or two rabbits. These observations consisted of a scab on the skin; fur loss; ungroomed fur; liquid feces; red aborted material; red liquid material – tissue red; red liquid material; partially missing pinna; and pink aborted material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One unscheduled euthanasia and an early delivery occurred in the 20 mg/kg/day and 60 mg/kg/day dose groups, respectively. These unscheduled euthanasias were considered not to be test substance related because there was no additional maternal toxicity apparent in these dose groups. Female #5065 at 20 mg/kg/day had a reduction in body weight gain between DGs 20 to 21 and a reduction in food consumption between DGs 13 to 18 and 20 to 21. Female #3050 at 60 mg/kg/day had a reduction in body weight gain between DGs 25 to 28 and a reduction in food consumption between DGs 19 to 27.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reductions in body weight gain occurred at 200 mg/kg/day at all intervals in comparison with control group values, with the exception of DGs 10 to 13. In the 500 mg/kg/day dose group, there were reductions in body weight gain on DGs 6 to 10, 13 to 16, 16 to 20, 6 to 20, 24 to 29, 20 to 29 and 6 to 29 in comparison with the control group values.
Further, the decrease in body weight gain observed at 500 mg/kg/day between DGs 20 to 29 correlates with the reduced fetal body weight observed in this dose group.
Mean body weight gain was reduced in the 200 and 500 mg/kg/day dose groups (28% and 21% compared to concurrent control, respectively) over the entire dose period (DGs 6 to 29). The reductions in body weight gain correlated with reduced food consumption values observed at 200 and 500 mg/kg/day and are considered to be test substance related.
Although there were test substance related reductions in body weight gain observed in the 200 and 500 mg/kg/day dose groups, the mean body weight values were comparable throughout the dose period.
The corrected maternal body weight values (terminal body weight – gravid uterine weight) were comparable with the control group values at 500 mg/kg/day.
Body weights and body weight gains were unaffected at 20 and 60 mg/kg/day. There was a statistically significant (p≤0.01) body weight loss observed in the 20 mg/kg/day dose group between DGs 16 to 20 in comparison with the control group value. This body weight loss was considered not to be test substance related because it was not dose dependent.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reductions in food consumption occurred at 200 mg/kg/day on DGs 6 to 20, 20 to 29 and 6 to 29 (12%, 12% and 13% compared to concurrent control, respectively). In the 500 mg/kg/day dose group, mean food consumption was reduced or statistically significantly reduced beginning at the initiation of dose administration and continuing at all intervals, with exception of daily intervals from DGs 21 to 26. In the 200 mg/kg/day dose group, a statistically significant reduction (p≤ 0.01) was also observed for the DGs 20 to 21 interval. A statistically significant reduction (p≤ 0.05) was also observed for the DGs 15 to 16 interval in the 500 mg/kg/day dose group.
There were no test substance related differences in the mean food consumption values at 20 and 60 mg/kg/day as compared to the concurrent control values. There was a statistically significant (p≤0.05) reduction in food consumption observed in the 20 mg/kg/day dose group between DGs 15 to 16 in comparison with the control group value. This reduction in food consumption was considered not to be test substance related because it was not dose dependent.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although not statistically significant, mean gravid uterine weight in the 500 mg/kg/day dose group was slightly reduced at 89% of the control group value. Gravid uterine weights were unaffected at doses up to and including 200 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All does appeared normal at necropsy

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Abortions were observed in one rabbit in each of the 200 and 500 mg/kg/day dose groups. These unscheduled euthanasias were considered to be test substance related because there was additional maternal toxicity observed in each of these dose groups. Female #3070 at 200 mg/kg/day was observed with thin body condition, a reduction in body weight gain and reduced food consumption values basically from the initiation of dose administration until the time the rabbit aborted. Female #3090 at 500 mg/kg/day was observed with thin body condition and decreased fecal output, a reduction in body weight gain between DGs 27 to 28 and a reduction in food consumption at each interval between DGs 6 to 13 and 14 to 24. All other female rabbits
survived until scheduled euthanasia.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There were two does in each of the 0, 200 and 500 mg/kg/day dose groups observed with litters consisting of only early resorptions (100% resorptions) at the time of Caesarean-sectioning observations. This was considered not to be test substance related because the incidence was similar in each of the groups, including the controls.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Pregnancy occurred in 21/22 (95.5%), 20/22 (90.9%), 22/22 (100%), 20/22 (90.9%) and
21/21 (100%) does in the 0, 20, 60, 200 and 500 mg/kg/day dose groups, respectively, including
those which were terminated early due to an abortion or an early delivery.
Other effects:
no effects observed
Description (incidence and severity):
All placentae appeared normal.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Average fetal body weights (total, male and female) were statistically significantly reduced (p≤0.01) at 500 mg/kg/day in comparison with the control group values (19%, 19% and 17%, respectively). This reduction in mean fetal body weights in the 500 mg/kg/day dose group correlates with the maternal toxicity observed at this dose.
Reduction in number of live offspring:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Gross external abnormalities occurred at increased incidences in the 500 mg/kg/day dose group and were considered to be test substance related.
There was a statistically significant increase (p≤ 0.01) in the litter incidence of carpal flexure at 500 mg/kg/day. Carpal flexure occurred in 12 fetuses from 7 litters within the 500 mg/kg/day dose group. Carpal flexure was generally observed in pups with decreased fetal body weights (ranging from 16.0g to 35.8g) in comparison with the mean fetal body weight for the control group (39.9g). Therefore, carpal flexure can be attributed to the reduction in fetal body weight observed at 500 mg/kg/day, which correlates with the maternal toxicity observed at this dose. The increased incidence of this finding was considered to be transient and secondary to the maternal toxicity observed at this dose. All additional external malformations were considered unrelated to 1, 3-dioxolane because the findings were not dose dependent and/or they occurred in
single litters. These malformations consisted of a small lower mandible of the jaw; short snout; anencephaly; cranial meningocele; malrotated hindlimbs; and a herniated umbilicus. No gross external abnormalities (malformations or variations) were caused by doses of the test substance as high as 200 mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal abnormalities and variations in ossification occurred at increased incidences in the 500 mg/kg/day dose group and were considered to be test substance related.
All skeletal malformations were considered unrelated to 1,3-dioxolane because the findings
were not dose dependent and/or they occurred in one or two fetuses from one or two litters
within the dose groups. These skeletal malformations abnormalities consisted of absent digits;
absent forepaw phalanges; absent metacarpals; missing frontal portion of the skull; absent
interparietal, maxillae, nasals, palatines, parietals, premaxillae; squamosals, supraoccipitals,
zygomatic arches and caudal vertebra; fused caudal vertebrae; and a hemivertebra in the lumbar
vertebrae.
Bent (angulated) hyoid ala(e) occurred in 33 fetuses from 12 litters at 500 mg/kg/day; the litter
incidence of this variation was significantly (p≤ 0.01) increased at this dose in comparison with
the control group value. The increased incidence of this finding was considered to be test
substance related because it was not within the range of Testing Facility control data collected
between 2006 and 2014. In the 500 mg/kg/day dose group, there was also a statistically
significant increase (p≤ 0.01) in the litter incidence of fused sternebrae and an increased incidence of incompletely ossified sternebrae that were considered to be test substance related
because they were dose dependent. These skeletal variations were considered to be related to the reduction in fetal body weights at this dose.
All additional skeletal variations were considered unrelated to the test substance because they
occurred at non-dose dependent incidences, they occurred in a single litter within a dose group,
and/or the fetal and litter incidences were within the ranges observed historically at the Testing
Facility. These skeletal variations consisted of small
forepaw phalanges; misshapen scapula ala and parietal; a fused or misshapen frontal; incomplete
ossification of the frontal, parietal and interparietal bones of the skull; misshapen interparietal,
mandible, supraoccipital and tympanic annulus bones of the skull; holes in the parietal bones of
the skull; presence of the suture bone; incompletely ossified or unossified pubes; nodulated
(thickened) ribs; abnormalities of the sternebrae [asymmetric, bipartite, isolated ossification site,
unossified, and misshapen (irregularly shaped)], supernumerary ribs (cervical short), and
incompletely ossified, misaligned and misshapen caudal vertebrae; unilateral ossification of the
cervical centrum; small lumbar arch; and bipartite sacral centrum.
The 500 mg/kg/day dose group had significant increases (p≤0.05) in the incidence of paired ribs
with associated significant increases and decreases (p≤0.05) in the numbers of thoracic and
lumbar vertebrae, respectively, a common variation observed at maternally toxic doses. In the
500 mg/kg/day dose group, there were also statistically significant decreases (p≤ 0.01) in the
metacarpals and forelimb phalanges in comparison with the control group values.
In the 500 mg/kg/day dose group, there were statistically significant increases (p≤0.05) in the
average number of ossified sternal centra and xiphoid in comparison with the control group
values. These differences were considered not to be test substance related because they were
slight in comparison with the concurrent control group values (i.e., 3% and 7% differences,
respectively).
There were no additional biologically important differences among the dose groups in the
average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, sacral and
caudal), manubrium, forelimbs (carpals and digits) or hindlimbs (tarsals, metatarsals, digits and
phalanges).
No skeletal fetal abnormalities (malformations or variations) were caused by doses of the test substance as high as 200 mg/kg/day.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All visceral abnormalities were considered unrelated to 1,3-dioxolane because the findings were not dose dependent and/or they occurred in single litters. These visceral abnormalities consisted of a dilated aortic arch; absent or small brain; moderate dilation of the lateral ventricles of the brain; absent or small eyes; hermaphroditism (left testis and right ovary were observed); persistent truncus arteriosus of the great vessels; defect in the ventricular septum of the heart; absent innominate artery; malpositioned intestine; narrow pulmonary trunk; retroesophageal right subclavian artery; and malpositioned origin of the right subclavian artery.
Details on embryotoxic / teratogenic effects:
Decreases in fetal body weight, carpal flexure, skeletal variations and delayed skeletal ossification observed at 500 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
Remarks on result:
other:
Remarks:
The developmental NOAEL is 200 mg/kg/day due to decreases in fetal body weight, carpal flexure, skeletal variations and delayed skeletal ossification observed at 500 mg/kg/day. The delayed ossification was considered to be related to the reduction in fetal body weight observed in the 500 mg/kg/day dose group. This change in ossification at 500 mg/kg/day was considered not to be adverse because it is a minor, reversible delay in skeletal ossification and was observed in the presence of maternal toxicity.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
external: paw
skeletal: skull
skeletal: sternum
other: reversible delay in skeletal ossification

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Test substance concentration in formulations

































 


 


 


Date of Preparation



Mean Concentration, mg/mL (% of Target)



Group 1


(0 mg/mL)



Group 2


(4 mg/mL)



Group 3


(12 mg/mL)



Group 4


(40 mg/mL)



Group 5


(100 mg/mL)



30 Jul 2016


(first preparation)



 


ND (NA)



 


3.61 (90.3)



 


11.1 (92.3)



 


37.9 (94.6)



 


94.4 (94.4)



24 Aug 2016


(last preparation)



 


ND (NA)



 


3.65 (91.1)



 


11.8 (98.4)



 


40.2 (100)



 


106 (106)



ND = No test substance chromatographic peak detected; NA = Not applicable.


Table 2: Mortality





















































































































































































Dose Level (mg/kg/day)



20 ( Group 2 )



60 ( Group 3 )



200 ( Group 4 )



500 ( Group 5 )



Rabbit Number



5065



3050



3070



3090



Doses Administered



16



22



21



22



Mode of Death



UE



UE – Early Delivery



UE - Abortion



UE - Abortion



Day of Death



DG 21



DG 28



DG 27



DG 28



Clinical Observations



 



 



 



 



Thin body condition



-



-



DGs 16 to 26



DGs 16 to 28



Decreased fecal output



-



-



-



DGs 16 to 20



Ungroomed fur



-



-



-



DGs 19 to 22



Red liquid material: red tissue



DG 21



-



-



-



Red liquid material



-



-



-



DG 28



Aborted material



-



DG 28



-



-



Bodyweights



 



 



 



 



Body weight change (grams)



-7.6% (-281.8g)


DGs 20 to 21



-3.5% (-139.4g)


DGs 25 to 28



-16.6% (-573.3g)


DGs 7 to 26



-4.6% (-178.7g)


DGs 27 to 28



Food Consumption



 



 



 



 



 


Cumulative food consumption



Reduced at each interval


between DGs 13 to 18 and


20 to 21



Reduced at each interval between DGs 19 to 27



Reduced at all intervals after


the initiation of dose administration



Reduced at each interval


between DGs 6 to 13 and


14 to 24



Necropsy Observations



 



 



 



 



All tissues appear normal



X



X



X



X



  Uterine Contents                                                                                                                                                                                                                                                                     



 



 



 



 



Implantation sites



9



11



11



11



Viable conceptuses



-



-



-



-



Live fetuses



8



10



-



-



Dead fetuses



-



1



11



11



Early resorptions



-



-



-



-



Late resorptions



1



-



-



-



X = Procedure conducted, - = Finding not present; DG = Day of presumed gestation; NA = Not applicable; UE = Unscheduled euthanasia


 


Table 3: Animal accountability






































































 



0


mg/kg/day



20


mg/kg/day



60


mg/kg/day



200


mg/kg/day



500


mg/kg/day



Initial group size



22



22



22



22



22



No. of rabbits dosed



22



22



22



22



21



No. of early deliveries/abortions during the study



0



0



1



1



1



No. of other intercurrent deaths



0



1



0



0



0



No. of rabbits not pregnant



1



2



0



2



0



No. of rabbits with total resorption



2



0



0



2



2



No. of rabbits with live fetuses in utero at term



19



19



21



17



18



 


Table 4: Maternal clinical observations




































































































































































































































































 



Day numbers relative to mating date



0 mg/kg/day



20 mg/kg/day



60 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Thin



Number of Observations



-



10



-



23



29



Number of animals



-



2



-



2



2



Days from – to



-



18   29



-



14   26



14   29



Skin, Scab



Number of Observations



-



-



1



-



-



Number of animals



-



-



1



-



-



Days from – to



-



-



6   6



-



-



Fur, Loss



Number of Observations



15



6



11



15



12



Number of animals



3



1



1



3



2



Days from – to



22   29



24   29



19   29



23   29



21   29



Fur, Ungroomed



Number of Observations



-



-



11



-



4



Number of animals



-



-



1



-



1



Days from – to



-



-



19   29



-



19   22



Feces, Liquid



Number of Observations



-



-



1



-



-



Number of animals



-



-



1



-



-



Days from – to



-



-



20   20



-



-



Feces, Output decreased



Number of Observations



-



6



 



8



11



Number of animals



-



2



 



2



2



Days from – to



-



17   22



 



13   18



13   20



Material, Liquid, Red



Number of Observations



2



2



 



-



1



Number of animals



1



2



 



-



1



Days from – to



15   16



16   21



 



-



28   28



Pinna Partly Missing



Number of Observations



-



-



-



24



-



Number of animals



-



-



-



1



-



Days from – to



-



-



-



6   29



-



Material, Aborted tissue, Pink



Number of Observations



-



-



1



-



-



Number of animals



-



-



1



-



-



Days from – to



-



-



28   28



-



-



 


Table 5: Maternal body weights

























































































































































































































































Day



Group /


Sex



 



6



7



8



9



10



11



12



13



 


1F



 


Mean



 


3653 .58



 


3695 .24



 


3719 .47



 


3766 .08



 


3786 .09



 


3805 .76



 


3822 .49



 


3839 .09



 



SD



230 .53



233 .11



240 .00



245 .38



234 .07



241 .34



233 .31



236 .73



 



N



19



19



19



19



19



19



19



19



2F



Mean



3558.82



3619.12



3629.99



3657.20



3705.10



3721.01



3741.36



3757.23



 



SD



173.85



181.69



178.63



175.64



184.38



197.81



191.98



196.45



 



N



19



19



19



19



19



19



19



19



 



%Diff G1



-2 59



-2 06



-2 41



-2 89



-2 14



-2 23



-2 12



-2 13



3F



Mean



3659.47



3687.01



3698.23



3728.42



3756.88



3775.76



3798.74



3821.37



 



SD



182.17



214.70



221.64



225.03



201.70



193.19



179.84



181.86



 



N



21



21



21



21



21



21



21



21



 



%Diff G1



0.16



-0.22



-0.57



-1.00



-0.77



-0.79



-0.62



-0.46



4F



Mean



3653.28



3641.72



3674.05



3700.25



3708.61



3743.46



3764.11



3786.57



 



SD



205.19



181.87



163.72



175.87



186.66



193.48



179.03



187.19



 



N



17



17



17



17



17



17



17



17



 



%Diff G1



-0.01



-1.45



-1.22



-1.75



-2.05



-1.64



-1.53



-1.37



5F



Mean



3694.40



3705.29



3730.50



3748.59



3761.81



3762.71



3813.09



3827.48



 



SD



167.72



198.81



201.78



190.05



198.46



177.52



190.07



209.69



 



N



18



18



18



18



18



18



18



18



 



%Diff G1



1.12



0.27



0.30



-0.46



-0.64



-1.13



-0.25



-0.30



 


Table 5 (cont): Maternal body weights

























































































































































































































































Day



Group /


Sex



 



14



15



16



17



18



19



20



21



1F



Mean



3870.12



3901.73



3908.47



3909.33



3924.14



3934.40



3970.76



3961.01



 



SD



231.28



236.16



239.02



228.84



232.17



227.61



235.44



231.44



 



N



19



19



19



19



19



19



19



19



2F



Mean



3790.18



3794.27



3809.99



3804.39



3811.68



3803.07



3800.45



3807.49



 



SD



199.72



193.37



201.82



196.08



191.83



199.95



209.57



197.91



 



N



19



19



19



19



18



19



19



19



 



%Diff G1



-2.07



-2.75



-2.52



-2.68



-2.87



-3.34



-4.29



-3.88



3F



Mean



3860.90



3887.44



3898.08



3902.47



3911.68



3923.61



3924.72



3941.54



 



SD



196.10



214.27



210.86



215.31



205.25



212.35



207.09



200.49



 



N



21



21



21



21



21



21



21



21



 



%Diff G1



-0.24



-0.37



-0.27



-0.18



-0.32



-0.27



-1.16



-0.49



4F



Mean



3818.16



3830.09



3845.67



3850.26



3863.05



3875.31



3876.00



3887.87



 



SD



188.56



200.23



181.51



181.24



199.52



222.01



209.42



218.02



 



N



17



17



17



17



17



17



17



17



 



%Diff G1



-1.34



-1.84



-1.61



-1.51



-1.56



-1.50



-2.39



-1.85



5F



Mean



3847.66



3867.89



3882.69



3893.92



3904.70



3919.44



3932.02



3956.38



 



SD



239.00



250.81



257.60



252.94



240.51



238.59



225.90



229.34



 



N



18



18



18



18



18



18



18



18



 



%Diff G1



-0.58



-0.87



-0.66



-0.39



-0.50



-0.38



-0.98



-0.12



 


Table 5 (cont): Maternal body weights

























































































































































































































































Day



Group /


Sex



 



22



23



24



25



26



27



28



29



 


1F



 


Mean



3984.00



3994.59



4003.01



4014.23



4020.17



4025.56



4036.27



4080.64



 



SD



229.83



237.89



232.28



233.88



227.26



218.41



213.77



219.49



 



N



19



19



19



19



19



19



19



19



2F



Mean



3812.13



3827.63



3849.01



3862.47



3892.46



3893.33



3910.30



3947.47



 



SD



214.46



223.49



227.27



223.31



207.15



223.85



234.30



248.08



 



N



19



19



19



19



19



19



19



19



 



%Diff G1



-4.31



-4.18



-3.85



-3.78



-3.18



-3.28



-3.12



-3.26



3F



Mean



3953.52



3980.08



3983.10



3978.50



3991.57



3992.79



4003.25



4041.90



 



SD



205.85



209.47



208.42



206.68



217.15



234.28



230.62



214.73



 



N



21



21



21



21



21



21



21



21



 



%Diff G1



-0 .77



-0 .36



-0 .50



-0 .89



-0 .71



-0 .81



-0 .82



-0 .95



4F



Mean



3888.53



3890.97



3901.65



3907.43



3924.05



3941.74



3938.88



3960.04



 



SD



220.69



202.51



211.06



206.58



208.41



208.94



203.62



197.38



 



N



17



17



17



17



17



17



17



17



 



%Diff G1



-2.40



-2.59



-2.53



-2.66



-2.39



-2.08



-2.41



-2.96



5F



Mean



3973.82



3991.04



4013.51



4015.06



4020.30



4024.65



4021.90



4032.56



 



SD



218.22



211.94



218.98



210.98



230.61



225.34



213.75



204.42



 



N



18



18



18



18



18



18



18



18



 



%Diff G1



-0.26



-0.09



0.26



0.02



0.00



-0.02



-0.36



-1.18



 


Table 6: Maternal body weight gains















































































































































































































Group/


Sex



 



Day



Change


6-10



Change


10-13



Change


13-16



Change


16-20



Change


6-20



Change


20-24



Change


24-29



Change


20-29



Change


6-29



1F



Mean



132.51



53.00



69.37



62.29



317.18



32.24



77.63



109.87



427.05



SD



83.69



42.55



47.10



57.42



143.55



80.78



90.69



135.56



122.36



N



19



19



19



19



19



19



19



19



19



2F



Mean



146.28



52.13



52.76



-9.54a



241.64



48.56



98.46



147.02



388.65



SD



85.14



86.22



50.43



57.32



115.59



60.50



63.96



96.44



158.63



N



19



19



19



19



19



19



19



19



19



3F



Mean



97.41



64.49



76.71



26.64



265.26



58.38



58.80



117.17



382.43



SD



100.98



103.00



61.56



61.42



104.99



46.76



71.77



88.97



143.46



N



21



21



21



21



21



21



21



21



21



4F



Mean



55.34



77.96



59.10



30.33



222.72



25.65



58.39



84.04



306.76



SD



173.68



124.26



77.60



62.12



228.21



71.73



81.56



117.63



219.42



N



17



17



17



17



17



17



17



17



17



5F



Mean



67.41



65.67



55.22



49.33



237.62



81.48



19.05



100.53



338.16



SD



97.68



122.29



117.15



81.34



128.91



49.63



66.81



88.59



148.65



N



18



18



18



18



18



18



18



18



18



a: significantly different from control group 1 p≤0.01 (Dunnett)


Table 7: Percent body weight gain relative to Group 1 (Control)









































 



20 mg/kg/day



60 mg/kg/day



200 mg/kg/day



500 mg/kg/day



DGs 6 to 10



+10%



-26%



-58%



-49%



DGs 6 to 20



-24%



-16%



-30%



-25%



DGs 20 to 29



+34%



+7%



-24%



-9%



DGs 6 to 29



-9%



-10%



-28%



-21%



 


Table 8: Gravid uterine weights and corrected maternal body weights










































































































































Group/


Sex



 



Gravid uterus weight



Day 29 (DTW)



Change from Day 6-29



 



g



g



g



1F



Mean



556.69



3523.95



-129.64



SD



128.00



232.16



126.47



N



19



19



19



2F



Mean



541.32



3406.15



-152.66



SD



158.13



280.81



211.65



N



19



19



19



%Diff G1



-2.76



-3.34



17.76



3F



Mean



528.16



3513.73



-145.73



SD



160.81



271.57



190.31



N



21



21



21



%Diff G1



-5.12



-0.29



12.42



4F



Mean



528.03



3432.01



-221.26



SD



131.06



229.08



283.55



N



17



17



17



%Diff G1



-5.15



-2.61



70.68



5F



Mean



498.01



3534.55



-159.85



SD



100.55



207.09



133.77



N



18



18



18



%Diff G1



-10.54



0.30



23.31



 


Table 9: Maternal gross pathology observations














































































































































































































































































 



Female



 



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



Number of animals:



22



22



22



22



21



Number of completed animals:



22



22



22



22



21



Cervix



Submitted



1



2



0



2



0



No visible lesions



1



2



-



2



-



Esophagus



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Gallbladder



Submitted



1



1



0



0



0



No visible lesions



1



0



-



-



-



Focus; white, multifocal



0



1



-



-



-



Heart



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Kidney



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Liver



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Lung



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Ovary



Submitted



1



2



0



2



0



No visible lesions



1



2



-



2



-



Spleen



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Stomach



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Trachea



Submitted



0



1



1



1



1



No visible lesions



-



1



1



1



1



Uterus



Submitted



1



2



0



2



0



No visible lesions



1



2



-



2



-



Table 10: Maternal performance


































































































Day(s): 6 to 29 Relative to Mating



Sex: Female



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



No Pregnant



N-ve


N+ve


%



1


21


95.5



2


20


90.9



0


22


100.0



2


20


90.9



0


21


100.0



Females with live fetuses



 



19



20



22



17



18



Females with all dead or resorbed



 



2



0



0



3



3



Females euthanized preterminally



N+ve



0



1



1



1



1



Pregnant/Early PM



N+ve



0



1



1



1



1



Not pregnant/Early PM



N+ve



0



0



0



0



0



Females aborted



N+ve



0



0



0



1



1



Females delivered



N+ve



0



0



1



0



0



Placenta exam Normal



N-ve


N+ve



0


19



0


20



0


22



0


18



0


19



 


Table 11: Ovarian and uterine findings




















































































































































































































































































































































































Group/


Sex



 



No. of CL Left



No. of CL Right



No. of Corpora Lutea



Number of Implants



Pre-implantation loss %



Live Male Fetuses %



Live Male Fetuses



Live Female Fetuses



No. of Live Fetuses



No. of Dead Fetuses



Total Fetuses



No. of Resorptions



No. of Early Resorptions



No. of Late Resorptions



Post Implantation Loss %



1F



Mean



6.0



5.1



11.1



10.5



6.77



48.32



4.3



4.7



9.0



0.0



10.0



1.4



1.3



0.1



11.69



SD



1.5



1.6



2.3



3.0



15.06



12.77



2.3



2.6



4.1



0.0



3.0



3.7



3.7



0.3



29.63



N



21



21



21



21



21



19



21



21



21



21



19



21



21



21



21



2F



Mean



5.7



5.5



11.3



10.5



7.29



46.77



4.7



5.4



10.1



0.0



10.1



0.4



0.1



0.4



3.90



SD



2.2



1.7



2.1



2.7



10.60



13.69



2.0



2.1



2.6



0.0



2.6



0.8



0.2



0.8



7.20



N



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



%Diff G1



-5.1



8.5



1.1



0.5



7.80



-3.20



9.3



13.9



11.7



-



1.1



-70.5



-96.1



286.8



-66.64



3F



Mean



5.6



5.2



10.9



10.4



6.69



46.77



4.4



5.2



9.7



0.0



9.7



0.7



0.6



0.1



10.50



SD



1.5



1.8



1.9



2.9



16.61



19.68



1.9



2.3



3.4



0.0



3.4



1.1



1.0



0.5



17.87



N



21



21



21



21



21



21



21



21



21



21



21



21



21



21



21



%Diff G1



-7.1



2.8



-2.6



-0.9



-1.20



-3.21



2.2



11.1



6.8



-



-3.3



-50.0



-57.1



50.0



-10.18



4F



Mean



5.7



5.5



11.3



10.6



7.02



50.17



4.5



4.4



8.9



0.1



10.1



1.6



1.3



0.3



18.21



SD



2.5



1.9



2.6



3.5



18.59



12.27



2.5



2.5



4.5



0.5



3.6



2.7



2.6



0.6



31.38



N



19



19



19



19



19



17



19



19



19



19



17



19



19



19



19



%Diff G1



-5.1



8.5



1.1



1.0



3.76



3.83



3.2



-6.2



-1.7



-



0.6



10.5



-5.3



231.6



55.73



5F



Mean



6.0



5.2



11.2



10.9



3.00



34.99



3.2



5.5



8.7



0.1



9.7



2.1



1.5



0.6



19.70



SD



1.9



1.9



2.3



2.6



9.79



22.45



2.6



3.1



3.6



0.2



2.2



3.1



3.1



1.0



29.40



N



20



20



20



20



20



18



20



20



20



20



18



20



20



20



20



%Diff G1



-0.8



1.1



0.1



3.6



-55.67



-27.59



-27.3



16.7



-3.8



-



-2.8



47.0



12.5



530.0



68.47



 


Table 12: Mean fetal body weights




































































































































Group/


Sex



 



Mean Fetal Weight (M)



Mean Fetal Weight (F)



Mean Fetal Weight (both)



1F



Mean



40.475



39.372



39.914



SD



5.529



4.840



4.941



N



19



19



19



2F



Mean



40.340



38.047



38.928



SD



4.193



4.528



3.873



N



19



19



19



%Diff G1



-0.334



-3.366



-2.471



3F



Mean



40.419



38.466



39.953



SD



5.894



6.339



6.448



N



20



20



21



%Diff G1



-0.139



-2.303



0.098



4F



Mean



38.247



37.169



37.581



SD



6.437



6.448



6.064



N



17



17



17



%Diff G1



-5.505



-5.596



-5.845



5F



Mean



32.759a



32.563a



32.272a



SD



6.236



5.493



5.472



N



16



18



18



%Diff G1



-19.065



-17.295



-19.147



a: significantly different from control group 1 p≤0.01 (Dunnett)


Table 13: Fetal abnormalities by finding







































































































































































































































































































































































































































































































































































































































































































































 



 



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



 



Number of fetuses examined:


Number of fetuses evaluated:


Number of litters examined:


Number of litters evaluated:



190


190


19


19



192


192


19


19



203


203


21


21



169


171


17


17



174


175


18


18



Face



Jaw, lower (Mandible), Small-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Snout, Short-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Head/neck



Head/neck, Anencephaly-malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Head/neck, Cranial meningocele-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Limb



Hindlimb, Malrotated-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Paw



Forepaw, Hyperflexion-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



12 (6.9)


7 (38.9)**



Trunk



Umbilicus, Herniated-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Aortic arch



Aortic arch, Dilated-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Brain



Brain, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Brain, Small-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Lateral ventricle, Dilated, Moderate-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.0)


2 (9.5)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Eye



Eye, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Eye, Small-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Gonad



Gonad, Hermaphroditism-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Great vessels



Truncus arteriosus, Persistent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



1 (0.5)


1 (4.8)



1 (0.6)


1 (5.9)



0 (0.0)


0 (0.0)



Heart



Ventricular septum, Defect-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



2 (1.0)


2 (10.5)



1 (0.5)


1 (4.8)



1 (0.6)


1 (5.9)



0 (0.0)


0 (0.0)



Innominate artery



Innominate artery, Absent-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Intestine



Intestine, Malpositioned-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Pulmonary trunk



Pulmonary trunk, Narrow-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Subclavian artery



Subclavian artery, Retroesophageal-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Subclavian artery origin, Malpositioned-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Forelimb



Digits, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



2 (1.1)


2 (11.1)



Forepaw phalanges, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



2 (1.1)


2 (11.1)



Forepaw phalanges, Small-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Metacarpal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



2 (1.1)


2 (11.1)



Pelvic girdle



Pubis, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



2 (1.1)


2 (10.5)



1 (0.5)


1 (5.3)



2 (1.0)


2 (9.5)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Pubis, Unossified-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Rib



Rib, Nodulated-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



3 (1.8)


2 (11.8)



2 (1.1)


2 (11.1)



Scapula



Scapula ala, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Skull



Frontal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Frontal, Fused-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Frontal, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



3 (1.5)


2 (9.5)



0 (0.0)


0 (0.0)



14 (8.0)


2 (11.1)



Frontal, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Hyoid ala, Bent-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



33 (19.0)


12 (66.7)**



Interparietal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Interparietal, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Interparietal Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Mandible, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Maxilla, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Nasal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Palatine, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Parietal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Parietal, Hole-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



3 (1.5)


3 (14.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Parietal, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.0)


1 (4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Parietal, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1(4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Premaxilla, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Squamosal, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Supraoccipital, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1(4.8)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Supraoccipital, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Suture bone, Present-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1(4.8)



0 (0.0)


0 (0.0)



3 (1.7)


3 (16.7)



Tympanic annulus, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Zygomatic arche, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Sternebra, Asymmetric-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



1 (0.6)


1 (5.9)



2 (1.1)


1 (5.6)



Sternebra, Bipartite ossification-Variation



Fetuses N (%)


Litters N (%)



5 (2.6)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Sternebra, Fused-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



3 (1.6)


2 (10.5)



3 (1.5)


2 (9.5)



5 (3.0)


3 (17.6)



9 (5.2)


6 (33.3)**



Sternebra, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



2 (1.1)


2 (10.5)



2 (1.0)


2 (10.5)



1 (0.5)


1 (4.8)



2 (1.2)


1 (5.9)



8 (4.6)


4 (22.2)



Sternebra, Isolated ossification site-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.2)


2 (11.8)



1 (0.6)


1 (5.6)



Sternebra, Misshapen-Variation



Fetuses N (%)


Litters N (%)



1 (0.5)


1 (5.3)



2 (1.0)


1 (5.3)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



4 (2.3)


2 (11.1)



Sternebra, Unossified-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Supernumerary rib



Cervical, Short-Variation



Fetuses N (%)


Litters N (%)



1 (0.5)


1 (5.3)



2 (1.0)


1 (5.3)



2 (1.0)


1 (4.8)



3 (1.8)


2 (11.8)



3 (1.7)


3 (16.7)



Vertebra



Caudal vertebra, Absent-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



Caudal vertebra, Fused-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Caudal vertebra, Incomplete ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



3 (1.5)


2 (9.5)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Causal vertebra, Misaligned-Variation



Fetuses N (%)


Litters N (%)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



1 (0.5)


1 (4.8)



0 (0.0)


0 (0.0)



3 (1.7)


2 (11.1)



Caudal vertebra, Misshapen-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



1 (0.5)


1 (5.3)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



Cervical centrum, Unilateral ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



2 (1.1)


1 (5.6)



Lumbar arch, Small-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



0 (0.0)


0 (0.0)



Lumbar vertebra, Hemivertebra-Malformation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.9)



0 (0.0)


0 (0.0)



Sacral centrum, Bipartite ossification-Variation



Fetuses N (%)


Litters N (%)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



0 (0.0)


0 (0.0)



1 (0.6)


1 (5.6)



** p≤0.01


Table 14: Fetal findings by classification - External









































































Exam Type: External



 



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



Number of fetuses examined:


Number of fetuses evaluated:


Number of litters examined:


Number of litters evaluated:



190


190


19


19



192


192


19


19



203


203


21


21



169


171


17


17



174


175


18


18



Malformation



Number of Fetuses



0



1



2



1



12



Group % of Fetuses



0.0



0.5



1.0



0.6



6.9



Number of Litters (f)



0



1



2



1



7**



All classifications



Number of Fetuses



0



1



2



1



12



Group % of Fetuses



0.0



0.5



1.0



0.6



6.9



Number of Litters (f)



0



1



2



1



7**



**: p≤0.01 (Fisher’s Exact)


Table 15: Fetal findings by classification – Fresh visceral


































































































Exam Type: Fresh visceral



 



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



Number of fetuses examined:


Number of fetuses evaluated:


Number of litters examined:


Number of litters evaluated:



190


190


19


19



192


192


19


19



203


203


21


21



169


171


17


17



174


175


18


18



Variation



Number of Fetuses



0



0



2



0



2



Group % of Fetuses



0.0



0.0



1.0



0.0



1.1



Number of Litters (f)



0



0



2



0



1



Malformation



Number of Fetuses



0



3



2



1



5



Group % of Fetuses



0.0



1.6



1.0



0.6



2.9



Number of Litters (f)



0



2



2



1



2



All classifications



Number of Fetuses



0



3



4



1



5



Group % of Fetuses



0.0



1.6



2.0



0.6



2.9



Number of Litters (f)



0



2



3



1



2



 


Table 16: Fetal findings by classification – Skeletal


































































































Exam Type: Skeletal



 



0 mg/kg/day


Group 1



20 mg/kg/day


Group 2



60 mg/kg/day


Group 3



200 mg/kg/day


Group 4



500 mg/kg/day


Group 5



Number of fetuses examined:


Number of fetuses evaluated:


Number of litters examined:


Number of litters evaluated:



190


190


19


19



192


192


19


19



203


203


21


21



169


171


17


17



174


175


18


18



Variation



Number of Fetuses



11



9



12



14



70



Group % of Fetuses



5.8



4.7



5.9



8.3



40.2



Number of Litters (f)



7



6



6



8



15**



Malformation



Number of Fetuses



0



1



1



2



4



Group % of Fetuses



0.0



0.5



0.5



1.2



2.3



Number of Litters (f)



0



1



1



1



2



All classifications



Number of Fetuses



11



9



12



15



70



Group % of Fetuses



5.8



4.7



5.9



8.9



40.2



Number of Litters (f)



7



6



6



8



15**



**: p≤0.01 (Fisher’s Exact)


Table 17: Ossification of site data
















































































































































































































































































































































































































































Group/


Sex



 



Hyoid



Cervical Vertebrae



Thoracic Vertebrae



Lumbar Vertebrae



Sacral Vertebrae



Caudal Vertebra



Ribs, Paired



Manubrium



Sternal Centra



Xiphoid



Carpals



Metacarpals



Forelimb Digits



Forelimb Phalanges



Tarsals



Metatarsals



Hindlimb Digits



Hindlimb Phalanges



1F



Mean



1.06



7.00



12.44



6.57



4.00



15.47



12.37



1.00



3.86



0.93



0.00



4.98



5.00



13.96



1.99



4.00



4.00



11.99



SD



0.27



0.00



0.30



0.30



0.00



0.31



0.28



0.00



0.14



0.11



0.00



0.05



0.00



0.10



0.02



0.00



0.00



0.02



N



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



2F



Mean



1.00



7.00



12.43



6.58



4.00



15.44



12.39



1.00



3.73



0.93



0.00



4.98



5.00



13.95



1.99



4.00



4.00



11.99



SD



0.00



0.00



0.32



0.33



0.00



0.25



0.31



0.00



0.26



0.12



0.00



0.05



0.00



0.07



0.02



0.00



0.00



0.02



N



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



19



%Diff G1



-5.94



0.00



-0.08



0.16



0.00



-0.24



0.13



0.00



-3.41



0.00



-



0.00



0.00



-0.04



0.00



0.00



0.00



0.00



3F



Mean



1.04



7.00



12.53



6.48



4.00



15.49



12.50



1.00



3.88



0.94



0.00



4.96



5.00



13.97



1.99



4.00



4.00



12.00



SD



0.20



0.00



0.32



0.33



0.00



0.38



0.34



0.00



0.14



0.11



0.00



0.08



0.00



0.07



0.04



0.00



0.00



0.02



N



21



21



21



21



21



21



21



21



21



21



21



21



21



21



21



21



21



21



%Diff G1



-1.91



0.00



0.74



-1.40



0.00



0.11



0.98



0.00



0.60



0.70



-



-0.44



0.00



0.06



-0.21



0.00



0.00



0.00



4F



Mean



0.99



7.00



12.41



6.59



4.00



15.25



12.35



1.04



3.93



0.99



0.00



4.85



5.00



13.91



1.99



4.00



4.00



11.99



SD



0.05



0.00



0.34



0.34



0.00



0.43



0.33



0.17



0.12



0.02



0.00



0.22



0.00



0.13



0.03



0.00



0.00



0.02



N



17



17



17



17



17



17



17



17



17



17



17



17



17



17



17



17



17



17



%Diff G1



-7.05



0.00



0.39



0.39



0.00



-1.43



-0.22



4.12



1.85



6.71



-



-2.65



0.00



-0.33



-0.33



0.00



0.00



-0.01



5F



Mean



0.99



7.00



12.73a



6.28a



4.00



15.36



12.68a



1.00



3.97a



1.00a



0.00



4.41b



5.00



12.84b



1.98



4.00



4.00



11.94



SD



0.02



0.00



0.26



0.27



0.00



0.31



0.28



0.00



0.12



0.00



0.00



0.39



0.00



0.78



0.05



0.00



0.00



0.10



N



18



18



18



18



18



18



18



18



18



18



18



18



18



18



18



18



18



18



%Diff G1



-6.46



0.00



2.38



-4.42



0.00



-0.73



2.50



0.00



2.96



7.34



-



-11.40



0.00



-8.02



-0.85



0.00



0.00



-0.42



a: p≤0.05 (Dunnett)


b: p≤0.01 (Dunnett)


Table 18: Historical control data – Reproductive indices









































Period:



January 2006 – July 2014



Number of studies:



19



Number of rabbits



Tested:



273



Pregnant:



262



Found dead:



10



Aborted:



10



Delivered prematurely:



1



Number of rabbits pregnant at caesarean-sectioning:



241



 






























































































 



Mean or %



Range/Study mean or %



% pregnant



95.2



(80.0-100)



Average # corpora lutea



10.7



(9.2-13.0)



Average # implantations



10.0



(8.5-12.8)



Average litter size



Average # live fetuses



9.4



(8.3-12.0)



Average # dead fetuses



0.0



(0.0-0.2)



Average # resorptions



0.6



(0.0-2.5)



Average # early resorptions



0.3



(0.0-1.0)



Average # late resorptions



0.3



(0-2.0)



Average % does with any resorptions



37.3



(0.0-76.9)



Average % does with all conceptuses resorbed



0.0



-



Average % does with one or more live fetuses



100.0



-



Average sex ratio (% males/litter)



48.6



(32.0-58.9)



Average fetal body weight (g)



39.70



(35.66-44.38)



Average for males (g)



37.57



(35.94-42.67)



Average for females (g)



36.69



(34.12-41.22)



Average % dead or resorbed conceptuses/litter



6.3



(0.0-18.0)



 


Table 19: Historical control data – Maternal necropsy observations






































Period:



2006-2014



Total no. studies



19



Total no. does:



273



No. pregnant:



262



No. died:



14



No. aborted:



13



No. delivered prematurely:



1



No. does with 100% resorptions:



0



 




































































































































































 



Range/Study



N



%



N



%



Gross lesions



Esophagus



Small amount of red gelatinous material present



1



0.37



0-1



(0-5.0)



Heart



Pale



2



0.73



0-1



(0-5.0)



Lungs



All lobes, spongy



1



0.37



0-1



(0-5.0)



Right bronchi, perforation



1



0.37



0-1



(0-5.0)



Stomach



Trichobezoar



1



0.37



0-1



(0-5.0)



Pup tissue present



1



0.37



0-1



(0-5.0)



Cardiac region, mucosal surface, numerous black ulcerations



1



0.37



0-1



(0-5.0)



Gallbladder



Numerous white areas or distended tan areas



3



1.10



0-2



(0-7.5)



Liver



All lobes, pale



4



1.47



0-1



(0-5.0)



Caudate and right lateral lobes, white area



1



0.37



0-1



(0-5.0)



Median lobe, adjacent to gallbladder raised dark area



1



0.37



0-1



(0-5.0)



Spleen



Misshapen



1



0.37



0-1



(0-5.0)



Two accessory spleens



1



0.37



0-1



(0-5.0)



Kidneys



Right, misshapen and low set



1



0.37



0-1



(0-5.0)



Adrenal glands



Left, misshapened round



1



0.37



0-1



(0-5.0)



Left, absent



1



0.37



0-1



(0-5.0)



Uterus



Right horn, reduced to a ligament and ended in blind sac



1



0.37



0-1



(0-5.0)



Ovaries



Parovarian cysts



7



2.56



0.3



(0-8.75)



Adipose



All or abdominal, friable



10



3.66



0-8



(0-16.6)



 


Table 20: Historical control data – Fetal gross external alterations






















Period:



2006-2014



No. of studies:



18



No. litters examined:



241



No. live fetuses examined (Day 29):



2279



 















































































































































































 



Range/Study



N



%



N



%



Alteration



Head



Meningocele



L



2



0.83



0-1



(0-5.6)



F



2



0.09



0-1



(0-0.6)



Domed



L



1



0.41



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Eye(s)



Lids open



L



1



0.41



0-1



(0-5.6)



F



1



0.04



0-1



(0-0.6)



Bulge depressed



L



2



0.83



0-1



(0-5.9)



F



2



0.09



0-1



(0-0.6)



Palate



Cleft



L



1



0.41



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Body



Acrania



L



1



0.41



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Abdominal distension



L



1



0.41



0-1



(0-4.2)



F



1



0.04



0-1



(0-0.5)



Forelimb(s) and/or hindlimb(s)



Limb(s): Flexed



L



4



1.66



0-1



(0-6.7)



F



4



0.18



0-1



(0-0.8)



Limb(s): Rotated



L



1



0.41



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Tail



Short



L



1



0.41



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



L: Litter incidence


F: Fetal incidence


Table 21: Historical control data – Fetal soft tissue alterations






















Period:



2006-2014



No. of studies:



14



No. litters examined:



221



No. live fetuses examined (Day 29):



2079



 




























































































































































































































































 



Range/Study



N



N



N



N



Alteration



Eye(s)



Corneal opacity



L



1



0.45



0-1



(0-5.6)



F



1



0.05



0-1



(0-0.6)



Microphthalmia



L



2



0.90



0-1



(0-5.9)



F



2



0.10



0-1



(0-0.6)



Brain



Ventricles, slight dilation



L



1



0.45



0-1



(0-5.0)



F



1



0.05



0-1



(0-1.0)



Ventricles, extreme dilation



L



1



0.45



0-1



(0-5.0)



F



1



0.05



0-1



(0-1.0)



Lungs



One or more lobes, partial or complete agenesis



L



1



0.45



0-1



(0-5.6)



F



1



0.05



0-1



(0-0.6)



Small



L



1



0.45



0-1



(0-5.9)



F



1



0.05



0-1



(0-0.6)



Heart



Interventricular septal defect



L



3



1.36



0-1



(0-5.9)



F



3



0.14



0-1



(0-0.6)



Vessels



Persistent truncus ateriosis



L



1



0.45



0-1



(0-20.0)



F



1



0.05



0-1



(0-1.7)



Distended aorta



L



1



0.45



0-1



(0-5.0)



F



1



0.05



0-1



(0-0.5)



Constricted pulmonary



L



1



0.45



0-1



(0-5.0)



F



1



0.05



0-1



(0-0.5)



Spleen



Small



L



1



0.45



0-1



(0-5.9)



F



1



0.05



0-1



(0-0.6)



Kidney(s)



Low set



L



2



0.90



0-1



(0-5.9)



F



2



0.10



0-1



(0-0.6)



Small



L



1



0.45



0-1



(0-5.9)



F



1



0.05



0-1



(0-0.6)



Dilated, moderate



L



1



0.45



0-1



(0-5.9)



F



1



0.05



0-1



(0-0.6)



Abdominal cavity



Red cloudy fluid



L



1



0.45



0-1



(0-4.2)



F



1



0.05



0-1



(0-0.5)



L: Litter incidence


F: Fetal incidence


 


Table 22: Fetal skeletal alterations






















Period:



2006-2014



No. of studies:



15



No. litters examined:



269



No. live fetuses examined (Day 29):



2476



 































































































































































































































































































































































































































































































































































































































































































































 



Range/Study



N



N



N



N



Alterations



Skull



Frontals: interfrontals present



L



3



1.12



0-1



(0-5.6)



F



3



0.12



0-1



(0-0.6)



Frontals: Contains a hole



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Frontals: Incompletely ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Frontals: Large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Nasals: Internasals



L



9



3.35



0-2



(0-11.8)



F



10



0.40



0-3



(0-1.7)



Nasals: Intranasals



L



4



1.49



0-2



(0-11.8)



F



4



0.16



0-2



(0-1.3)



Nasals: Displaced suture



L



18



6.69



0-6



(0-31.6)



F



19



0.77



0-6



(0-3.3)



Eye socket: Small



L



1



0.37



0-1



(0-5.6)



F



1



0.04



0-1



(0-0.6)



Skull: Incompletely or not ossified



L



3



1.12



0-1



(0-6.7)



F



3



0.12



0-1



(0-0.8)



Palate: Incompletely



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Parietal: Contains a hole



L



3



1.12



0-2



(0-10.0)



F



3



0.12



0-2



(0-1.0)



Parietal: Large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Interparietal: Incompletely ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.9)



Interparietal: Large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Interparietal: Small



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Fontanelle: Anterior, large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Fontanelle: Posterior, large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Supraoccipital: Large



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Supraoccipital: Incompletely ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Hyoid



Ala(e), angulated



L



4



1.49



0-2



(0-15.4)



F



7



0.28



0-4



(0-3.7)



Body, not ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.05)



Vertebrae



Cervical: Arch, misaligned



L



1



0.37



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Thoracic: Hemivertebra



L



2



0.74



0-1



(0-5.6)



F



2



0.08



0-1



(0-0.6)



Thoracic: Centra, fused



L



1



0.37



0-1



(0-4.2)



F



1



0.04



0-1



(0-0.5)



Caudal: One or more misaligned



L



15



5.58



0-3



(0-15.8)



F



16



0.65



0-3



(0-1.6)



Caudal: Fused



L



1



0.37



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Caudal: Small



L



1



0.37



0-1



(0-5.3)



F



1



0.04



0-1



(0-0.5)



Caudal: 12 present



L



1



0.37



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Ribs



Two or more, fused



L



1



0.37



0-1



(0-5.6)



F



1



0.04



0-1



(0-0.6)



One or more, split



L



1



0.37



0-1



(0-5.6)



F



1



0.04



0-1



(0-0.6)



One or more, thickened



L



8



2.97



0-3



(0-20.0)



F



8



0.32



0-3



(0-2.4)



Flat



L



1



0.37



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Short



L



1



0.37



0-1



(0-5.6)



F



1



0.04



0-1



(0-0.6)



T13, Supernumerary



L



19



7.06



0-19



(0-95.0)



F



57



2.30



0-57



(0-28.9)



T13, Short



L



15



5.58



0-15



(0-75.0)



F



30



1.21



0-30



(0-15.2)



Sternebrae



Two or more, fused



L



5



1.86



0-2



(0-25.0)



F



7



0.28



0-3



(0-4.1)



One or more, asymmetric



L



3



1.12



0-1



(0-5.6)



F



3



0.12



0-1



(0-0.6)



One or more, incompletely or not ossified



L



15



5.58



0-1



(0-50.0)



F



23



0.93



0-3



(0-7.6)



Manubrium



Irregularly shaped



L



1



0.37



0-1



(0-6.7)



F



1



0.04



0-1



(0-0.8)



Pelvis



Pubis, incompletely ossified



L



4



1.49



0-2



(0-10.0)



F



4



0.16



0-2



(0-1.0)



Pubis, not ossified



L



2



0.74



0-2



(0-10.0)



F



3



0.12



0-3



(0-1.5)



Xiphoid



Not ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Fused



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



Forelimb



Metacarpal, not ossified



L



5



1.86



0-5



(0-25.0)



F



6



0.24



0-6



(0-3.0)



Phalanx, not ossified



L



1



0.37



0-1



(0-5.0)



F



1



0.04



0-1



(0-0.5)



L: Litter incidence


F: Fetal incidence


 

Applicant's summary and conclusion

Conclusions:
The maternal no-observable-adverse effect level (NOAEL) for 1,3-dioxolane is 60 mg/kg/day,
based on mortality, clinical observations (thin body condition and decreased fecal output),
reductions in body weight gain and reductions in food consumption observed at 200 and
500 mg/kg/day during the study. The developmental NOAEL is 200 mg/kg/day due to decreases
in fetal body weight, carpal flexure, skeletal variations and delayed skeletal ossification observed
at 500 mg/kg/day. The delayed ossification was considered to be related to the reduction in fetal
body weight observed in the 500 mg/kg/day dose group. This change in ossification at 500
mg/kg/day was considered not to be adverse because it is a minor, reversible delay in skeletal
ossification and was observed in the presence of maternal toxicity. Reductions in gravid uterine
weights were also observed in the 500 mg/kg/day dose group. The test substance, 1,3-dioxolane, is not a selective developmental toxicant because fetal effects only occurred at the 500 mg/kg/day dose that was toxic to the does.
Executive summary:

The objectives of this study were to detect adverse effects of 1,3-dioxolane on pregnant New Zealand White [Crl:KBL(NZW)] rabbits and development of the embryo and fetus consequent to exposure of the female from implantation to the day prior to termination. This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C to D of the reproductive process.


The following parameters and endpoints were evaluated in this study: viability, clinical signs, maternal body weights, maternal body weight changes, maternal food consumption, ovarian and uterine examinations, gravid uterine weights, fetal examinations (body weight, sex, external, visceral, and skeletal) and gross necropsy findings.


One unscheduled euthanasia and an early delivery occurred in the 20 mg/kg/day and 60 mg/kg/day dose groups, respectively. These unscheduled euthanasias were considered not to be test substance related because there was no additional maternal toxicity apparent in these dose groups.


Abortions were also observed in one rabbit in each of the 200 and 500 mg/kg/day dose groups. These unscheduled euthanasias were considered to be test substance related because there was additional maternal toxicity observed in each of these dose groups, such as thin body condition, decreased fecal output, reduction in body weight gain and reduced food consumption. All other female rabbits survived until scheduled euthanasia.


In the 200 and 500 mg/kg/day dose groups, there were increased incidences of thin body condition (2 females in each of the dose groups between DGs 14 and 29) and decreased fecal output (2 females in each of the dose groups between DGs 13 and 20). At 200 mg/kg/day, female #3070 (which aborted) was observed with thin body condition; female #3076 was observed with decreased fecal output; and female #3078 (which was observed with total resorptions) was observed with thin body condition and decreased fecal output. At 500 mg/kg/day, female #3090 (which aborted) and 3102 (which was observed with total resorptions) were observed with thin body condition and decreased fecal output. These females were also observed with a reduction in body weight gain and reduced food consumption.


Reductions in body weight gain occurred in the 200 mg/kg/day dose group at all intervals in comparison with the control group values, with the exception of DGs 10 to 13. In the 500 mg/kg/day dose group, there were reductions in body weight gain on DGs 6 to 10, 13 to 16, 16 to 20, 6 to 20, 24 to 29, 20 to 29 and 6 to 29 in comparison with the control group values.


Mean body weight gain was reduced in the 200 and 500 mg/kg/day dose groups (28% and 21% compared to concurrent control, respectively) over the entire dose period (DGs 6 to 29). The reductions in body weight gain correlated with reduced food consumption values observed at 200 and 500 mg/kg/day and are considered to be test substance related.


Although not statistically significant, mean gravid uterine weight in the 500 mg/kg/day dose group was slightly reduced at 89% of the control group value. The corrected maternal body weight values were comparable with the control group values at 500 mg/kg/day.


Reductions in food consumption occurred at 200 mg/kg/day on DGs 6 to 20, 20 to 29 and 6 to 29 (12%, 12% and 13% compared to concurrent control, respectively). In the 500 mg/kg/day dose group, mean food consumption were reduced beginning at the initiation of dose administration and continuing at all intervals, with exception of daily intervals from DGs 21 to 26. A statistically significant reduction was observed for the DGs 20 to 21 interval fot the 200 mg/kg/day dose group and the DGs 15 to 16 interval in the 500 mg/kg/day dose group.


There were no necropsy findings observed in the gestating female rabbits.



Average fetal body weights (total, male and female) were statistically significantly reduced at 500 mg/kg/day (19%, 19% and 17% lower than controls, respectively). This reduction in mean fetal body weights in the 500 mg/kg/day dose group correlates with the maternal toxicity observed at this dose.


Gross external and skeletal abnormalities and variations in ossification occurred at increased incidences in the 500 mg/kg/day dose group and were considered to be test substance related. These abnormalities included significantly increased numbers of litters with fetuses with carpal flexure at gross examination. Carpal flexure was generally observed in pups with decreased fetal body weights (ranging from 16.0g to 35.8g) in comparison with the mean fetal body weight for the control group (39.9g). Therefore, carpal flexure can be attributed to the reduction in fetal body weight observed at the 500 mg/kg/day dose, which correlates with the maternal toxicity observed at this dose. There was also an increase in the numbers of litters with fetuses with variations in skeletal development. Most common of these skeletal variations were bent hyoid ala, fused sternebrae, and incompletely ossified sternebrae and were likely associated with the reduced fetal weights at this dose. Statistically significant increases in the number of paired ribs
with associated significant increases and decreases in the numbers of thoracic and lumbar vertebrae occurred at 500 mg/kg/day, as well as also statistically significant reductions in the numbers of ossified metacarpals and forelimb phalanges. No gross external, soft tissue or skeletal fetal abnormalities (malformations or variations) were caused by doses of the test substance as high as 200 mg/kg/day.


In conclusion, the maternal no-observable-adverse effect level (NOAEL) for 1,3-dioxolane is 60 mg/kg/day, based on mortality, clinical observations (thin body condition and decreased fecal output), reductions in body weight gain and reductions in food consumption observed at 200 and 500 mg/kg/day during the study. The developmental NOAEL is 200 mg/kg/day due to decreases in fetal body weight, carpal flexure, skeletal variations and delayed skeletal ossification observed at 500 mg/kg/day. The delayed ossification was considered to be related to the reduction in fetal body weight observed in the 500 mg/kg/day dose group. This change in ossification at 500 mg/kg/day was considered not to be adverse because it is a minor, reversible delay in skeletal ossification and was observed in the presence of maternal toxicity. Reductions in gravid uterine weights were also observed in the 500 mg/kg/day dose group. The test substance, 1,3-dioxolane, is not a selective developmental toxicant because fetal effects only occurred at the 500 mg/kg/day dose that was toxic to the does.