1,3-dioxolane

Developmental toxicity / teratogenicity

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9370. TSCA Prenatal developmental toxicity

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 5

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.