Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

EC Number:

258-207-9

EC Name:

Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

Cas Number:

52829-07-9

Molecular formula:

C28H52N2O4

IUPAC Name:

bis(2,2,6,6-tetramethylpiperidin-4-yl) sebacate

Details on test material:

Test item storage: At room temperature
Stability at higher temperatures: Stable, maximum temperature: 40°C

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 4 hours at room temperature under normal laboratory light conditions has been confirmed over the concentration range 1 to 250 mg/mL (suspensions). Homogeneity of formulations was considered acceptable for the purpose of this study.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France
- Age at study initiation: 17-19 weeks
- Weight at study initiation: between 3064 and 4252 g
- Fasting period before study:
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) was provided ad libitum t
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 48 to 97%. The values that were outside the targeted range (40-70%) occurred for 15 days with a minimum or maximum of 97% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 4 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80
- Concentration in vehicle: 1.5, 5, 15 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. For the formulation of Group 2, the mean accuracy was 125%, i.e. outside the acceptance criterion of 85% to 115% of nominal concentration. However, it should be noted that the mean accuracy of the 1 mg/mL QC samples was also relatively high at 117%. The concentrations analyzed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

Details on mating procedure:

Animales were delivered time-mated

Duration of treatment / exposure:

Day 6 to 28 post-coitum

Frequency of treatment:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive.

Duration of test:

gestation day 29

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT:
Animals were individually weighed on Days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In addition, data on body weight Day 0 post-coitum (i.e. the day of mating) was provided by the Supplier (non-GLP) and included in the report. In order to monitor the health status, animal No. 44 (6 mg/kg/day) was also weighed on Day 26 post-coitum (documented in study raw data).

FOOD CONSUMPTION
Food consumption was quantitatively measured for Days 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 29

Unscheduled Deaths – F0-Generation
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%), underwent
necropsy, and specified tissues were retained.

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%) on Day 29 post-coitum. No body weight was recorded at necropsy.

Necropsy – F0-Generation
All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed.

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.

Fetal examinations:

Method of Euthanasia – F1-Generation
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube1.

Fetal Examinations (unscheduled) – F1-Generation
For normal implantations in development of females sacrificed before planned necropsy, a gross external examination was performed (if possible).

Fetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined. Late resorptions were not examined or fixed.

Visceral Examinations – F1-Generation
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations – F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) 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.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.

Indices:

Preimplantation loss (%), Postimplantation loss (%), Viable fetuses affected/litter (%)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were noted during the observation period that were considered to be related to treatment with the test item.
One animal at 6 mg/kg/day (No. 44) showed hunched posture, swelling of the genital region, piloerection, and lean appearance between post-coitum Days 24 and 28 (necropsy did not reveal any correlating findings). In absence of similar findings among other animals of this dose group or other dose groups, these findings were considered incidental and not related to treatment with the test item. Two animals at 60 mg/kg/day showed piloerection on post-coitum Day 28 (No. 67) or Days 25 and 26 (No. 85). Given the very incidental occurrence, this symptom was considered not to be related to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
Two animals (one animal at 6 mg/kg/day (No. 30) and one animal at 20 mg/kg/day (No. 48)) were sacrificed for ethical reasons on Days 1 and 11 of treatment, respectively. Both mortalities were attributed to a gavage incident, since blood was noted in or on the dosing tube immediately after dosing, followed by laboured respiration and pallor (Nos. 30 and 48) and gasping and breathing rales (Nos. 30 and 48, respectively). Necropsy showed red foci or perforations in the lungs (Nos. 30 and 48, respectively). Both females were gravid.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg/day, a minor but statistically significantly lower mean body weight gain was recorded throughout the post-coitum period. At the end of the treatment period, mean body weights were the same as the control mean. Mean body weight gain corrected for gravid uterus at this dose level appeared lower than the control mean (not statistically significant and within the historical control range; -226.5 grams (-6.2%) vs. -120.6 grams (-3.3%) in the control group, and -195.4 grams (-5.1%) and -188.4 grams (-5.0%) at 6 and 20 mg/kg/day, respectively). Body weights and body weight gain at 6 and 20 mg/kg/day remained in the same range as controls over the study period, except for a statistically significantly lower body weight gain at 6 mg/kg/day on post-coitum Day 15. Since this occurred on a single occasion and in the absence of a dose-related response, and since this change was not consistently noted as treatment progressed, this was considered not to be related to treatment with the test item. Body weights on the day of necropsy corrected for gravid uterus weight were considered not affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg/day, food consumption before and after correction for body weight was statistically significantly lower on several occasions between post-coitum Days 6 and 21. Mean over mean absolute food intake at this dose level over the post coitum period was 0.88x of the control mean. Food consumption before and after correction for body weight at 6 and 20 mg/kg/day was considered not affected by treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy showed no lesions that were considered to be related to treatment with the test item. Necropsy findings of the two animals that were sacrificed due to a gavage incident consisted of red foci or perforations in the lungs. All necropsy findings recorded for animals surviving until scheduled necropsy are occasionally seen among rabbits used in this type of study, and their incidence did not indicate a relationship to treatment with the test item.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites, pre- and postimplantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation. For one animal at 60 mg/kg/day (A084), a relatively high number of early resorptions was recorded (30.8%, exceeding the historical data range). Since the same number of early resorptions was also recorded for one control animal (A006), this was considered not to be related to treatment with the test item.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of two females in the control group, three females at 6 mg/kg/day, two females at 20 mg/kg/day and one female at 60 mg/kg/day were non-gravid. The number of non-gravid females remained within the historical control range.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects reported

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights (both sexes) were not affected by treatment with the test item. Mean combined (male and female) fetal body weights were 37.1, 38.3, 37.2 and 37.0 gram for the control, 6, 20 and 60 mg/kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered not affected by treatment with the test item. Mean sex ratios (males:females) were 47.8:52.2, 49.5:50.5, 50.3:49.7 and 52.4:47.6 for the control, 6, 20 and 60 mg/kg/day groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was not affected by treatment with the test item. Mean litter sizes were 9.1, 9.8, 10.0 and 9.0 fetuses/litter for the control, 6, 20 and 60 mg/kg/day groups, respectively.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment up to 60 mg/kg/day.
External malformations occurred in one fetus each in the control, 6 and 20 mg/kg/day groups and five fetuses from two litters at 60 mg/kg/day. Four of the affected fetuses at 60 mg/kg/day (A078-01, A078-02, A078-08 and A084-09) had carpal flexures that not occurred in the other groups. These malformations were not confirmed during skeletal examinations. Additionally, one of these fetuses, A078-02, had cleft palate that also occurred in littermate A078-04, and which was confirmed during skeletal examinations. Two of the aforementioned fetuses in litter A078 were also viscerally affected, i.e. fetus A078-01 had multiple cardiovascular defects and A078-08 had diaphragmatic hernia and malpositioned kidneys. Although external malformations occurred in five fetuses at 60 mg/kg/day, this was considered not related to treatment with the test item, as the finding “carpal and/or tarsal flexures” are the most common external malformation among historical control fetuses and none were skeletally confirmed.
Moreover, the occurrence of flexed carpal(s) was essentially confined to one litter (A078) which contained three fetuses showing this malformation (A078-01, -02 and -08). Also, since all these fetuses also had other external and/or visceral malformations as described above, it could be considered that the malformations had a maternal or hereditary origin and hence were considered not related to treatment with the test item. Other malformations in this study consisted of distended abdomen (fetus A029-06 and A056-01 at 6 and 20 mg/kg/day, respectively) and brachydactyly (control fetus A003-12). As these were recorded as single occurrences and/or in a control fetus, they were considered chance findings.
External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment with 60 mg/kg/day.
Skeletal malformations (vertebral anomalies) occurred in one control (A015-08) and four fetuses of two litters at 20 mg/kg (A045-03, -09, -10 and A059-04). These malformations occurred in absence of a dose-related incidence (i.e. absence of this finding at 60 mg/kg/day) and were essentially confined to one litter. Also, vertebral anomaly is the most common skeletal malformation among historical control foetuses. Therefore, it was considered that these malformations were not related to treatment with the test item. A caudal shift of the pelvic girdle was observed at a statistically significantly higher incidence at 60 mg/kg/day. This variation occurred at 10.2%, 20.8%, 18.3% and 38.8% in the control, 6, 20 and 60 mg/kg/day group, respectively. The incidence at the high dose marginally exceeded the historical control maximum value (38.5% per litter) and was not accompanied by a significant increase of 13th full ribs, which are frequently encountered in parallel. Therefore, the occurrence of caudal shift of pelvic girdle was considered not to be related to treatment.
A statistically significantly higher incidence of unossified hyoid bodies was recorded at 60 mg/kg/day. Mean incidences per litter of this variation were 0.0%, 0.4% (one fetus, i.e. A026-07), 0.0% and 1.9% (four fetuses, i.e. A075-03, A076-06, A078-02 and A088-06) in the control, 6, 20 and 60 mg/kg/day group, respectively. Three of these fetuses at 60 mg/kg/day (A075-03, A076-06 and A088-06) had a body weight that was notably lower than the mean litter weight and mean group litter weight. However, mean fetal weights were considered not affected by treatment with the test item. Also, the mean incidence of unossified metacarpals and/or metatarsals and unossified sternebra nos. 5 and/or 6 (the two main ossification parameters) appeared higher or lower than the control mean, respectively, instead of a consistent increase that would be expected in case of skeletal ossification delay. Therefore, it was considered that unossified hyoid bodies were not related to treatment with the test item.
All other skeletal variations that occurred were not considered treatment related as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 60 mg/kg/day.
Visceral malformations occurred in one fetus of the control and 20 mg/kg/day group, five fetuses of three litters at 6 mg/kg/day and in three fetuses of two litters at 60 mg/kg/day. At 60 mg/kg/day, one fetus (A084-05) had one large lung cyst, one fetus (A078-01) had
multiple cardiovascular defects and one fetus (A078-08) had diaphragmatic hernia and malpositioned kidneys. One fetus at 20 mg/kg/day (A059-07) had two cysts associated with the colon. At 6 mg/kg/day, three fetuses (A026-01, -07 and A033-12) had one or more
cardiovascular abnormalities and two fetuses (A037-02 and -12) had internal hydrocephaly. One control fetus (A003-10) had a malpositioned testis. Based on the group distribution, single occurrence and/or occurrence in a control fetus, these malformations were considered not to be related to treatment with the test item.
All variations noted were considered unrelated to treatment with the test item as they occurred in the absence of a dose-related trend, occurred infrequently and/or at frequencies that were within the range of historical control data.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

BODY WEIGHTS (GRAM) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	6 mg/kg bw	20 mg/kg bw	60 mg/kg bw
DAY 0	MEAN	3525	3599	3640	3587
	ST.DEV.	303.3	316.2	298.5	236.8
	N	20	19	20	21
DAY 3	MEAN	3463	3589	3574	3539
	ST.DEV.	322.7	308.4	279.4	235.1
	N	20	19	20	21
DAY 6	MEAN	3514	3673	3649	3623
	ST.DEV.	336	320.7	272.7	237.6
	N	20	19	20	21
DAY 9	MEAN	3602	3724	3712	3618
	ST.DEV.	357.2	329	286.4	238
	N	20	18	20	21
DAY 12	MEAN	3655	3782	3762	3658
	ST.DEV.	345.2	309.6	292.5	239.5
	N	20	18	20	21
DAY 15	MEAN	3726	3819	3819	3703
	ST.DEV.	343.3	297.2	287.6	222.4
	N	20	18	20	21
DAY 18	MEAN	3755	3868	3853	3747
	ST.DEV.	364.3	299	293.8	240.3
	N	20	18	19	21
DAY 21	MEAN	3791	3900	3887	3763
	ST.DEV.	358.9	308.2	275.5	238.8
	N	20	18	19	21
DAY 24	MEAN	3815	3910	3916	3801
	ST.DEV.	352.8	357.9	263.2	259.8
	N	20	18	19	21
DAY 27	MEAN	3834	3942	3942	3845
	ST.DEV.	343	337.3	258.6	240.1
	N	20	18	19	21
DAY 29	MEAN	3867	3987	3980	3867
	ST.DEV.	364.7	320.9	259	252.3
	N	20	18	19	21

BODY WEIGHT GAIN (%) SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4
		Control	6 mg/kg bw	20 mg/kg bw	60 mg/kg bw
DAY 6	MEAN	0	0	0	0
	ST.DEV.	0	0	0	0
	N	20	19	20	21
DAY 9	MEAN	2	1	2	0 **
	ST.DEV.	2.2	1.5	1.6	1.7
	N	20	18	20	21
DAY 12	MEAN	4	3	3	1 **
	ST.DEV.	2.6	1.8	2.2	1.5
	N	20	18	20	21
DAY 15	MEAN	6	4 *	5	2 **
	ST.DEV.	2.8	2.8	3	2.5
	N	20	18	20	21
DAY 18	MEAN	7	5	6	3 **
	ST.DEV.	3	2.4	3.8	2.8
	N	20	18	19	21
DAY 21	MEAN	8	6	7	4 **
	ST.DEV.	3.7	2.7	3.8	3
	N	20	18	19	21
DAY 24	MEAN	9	6	8	5 **
	ST.DEV.	3.4	3.5	3.9	2.9
	N	20	18	19	21
DAY 27	MEAN	9	7	8	6 *
	ST.DEV.	3.6	3.3	4.3	3.4
	N	20	18	19	21
DAY 29	MEAN	10	8	9	7 *
	ST.DEV.	4.1	3.7	4.3	3.7
	N	20	18	19	21

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

DOSE GROUP	1		2		3		4
	NO.	%	NO.	%	NO.	%	NO.	%
FEMALES ON STUDY	22		22		22		22
FEMALES THAT ABORTED OR DELIVERED	0	0	0	0	0	0	0	0
FEMALES THAT DIED	0	0	0	0	0	0	0	0
FEMALES THAT ABORTED	0	0	0	0	0	0	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	0	0	0	0	0	0
FEMALES THAT WERE EUTHANIZED	0	0	1	4.5	1	4.5	0	0
NONGRAVID	0	0	0	0	0	0	0	0
GRAVID	0	0	1	100	1	100	0	0
FEMALES EXAMINED AT SCHEDULED NECROPSY	22	100	21	95.5	21	95.5	22	100
NONGRAVID	2	9.1	3	14.3	2	9.5	1	4.5
GRAVID	20	90.9	18	85.7	19	90.5	21	95.5
WITH RESORPTIONS ONLY	0	0	0	0	0	0	0	0
WITH VIABLE FETUSES	20	100	18	100	19	100	21	100
TOTAL FEMALES GRAVID	20	90.9	19	86.4	20	90.9	21	95.5

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

Group		Males	Females	Viable Fetuses	Dead Fetuses	Early Resorptions	Late resorptions
1	TOTAL	87	94	181	0	8	4
	MEAN	4.4	4.7	9.1	0	0.4	0.2
	S.D.	1.87	1.84	1.96	0	0.94	0.52
2	TOTAL	91	86	177	0	4	2
	MEAN	5.1	4.8	9.8	0	0.2	0.1
	S.D.	2.29	1.26	2.96	0	0.43	0.32
3	TOTAL	95	95	190	0	7	3
	MEAN	5	5	10	0	0.4	0.2
	S.D.	1.6	1.76	1.7	0	0.68	0.37
4	TOTAL	98	92	190	0	9	3
	MEAN	4.7	4.4	9	0	0.4	0.1
	S.D.	1.43	2.01	1.99	0	0.93	0.36

None significantly different from control group

Group		Post Implantation Loss	Implantation Sites	Corpora Lutera	Pre-Implantation Loss	Fetal weights (g)	No. of gravid females
1	TOTAL	12	193	206	13	NA	20
	MEAN	0.6	9.7	10.3	0.7	37.1
	S.D.	1.39	2.28	2.58	0.75	4.52
2	TOTAL	6	183	192	9	NA	18
	MEAN	0.3	10.2	10.7	0.5	38.3
	S.D.	0.49	3.15	3.25	0.71	4.25
3	TOTAL	10	200	205	5	NA	19
	MEAN	0.5	10.5	10.8	0.3	37.2
	S.D.	0.84	1.84	2.02	0.56	3.68
4	TOTAL	12	202	219	17	NA	21
	MEAN	0.6	9.6	10.4	0.8	37
	S.D.	0.93	2.11	1.83	1.36	5.66

None significantly different from control group

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY (% PER LITTER)

Group	1	2	3	4
CORPORA LUTEA
MEAN	10.3	10.7	10.8	10.4
S.D.	2.58	3.25	2.02	1.83
N	20	18	19	21
IMPLANTATION SITES
MEAN	9.7	10.2	10.5	9.6
S.D.	2.28	3.15	1.84	2.11
N	20	18	19	21
VIABLE FETUSES (%)
MEAN	95	97.1	95.4	94.6
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
DEAD FETUSES (%)
MEAN	0	0	0	0
S.D.	0	0	0	0
N	20	18	19	21
EARLY RESORPTIONS (%)
MEAN	3.5	2	3.2	4
S.D.	7.62	4.13	5.86	7.71
N	20	18	19	21
LATE RESORPTIONS (%)
MEAN	1.5	0.9	1.4	1.4
S.D.	4.03	2.59	3.37	3.59
N	20	18	19	21
TOTAL RESORPTIONS (%)
MEAN	5	2.9	4.6	5.4
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
PRE-IMPLANTATION LOSS (%)
MEAN	5.7	4.7	2.1	7.6
S.D.	6.52	6.36	4.44	12.04
N	20	18	19	21
POST-IMPLANTATION LOSS (%)
MEAN	5	2.9	4.6	5.4
S.D.	10.97	4.46	7.21	7.76
N	20	18	19	21
MALES (%)
MEAN	47.8	49.5	50.3	52.4
S.D.	15.09	13.1	15.38	16.13
N	20	18	19	21
FEMALES (%)
MEAN	52.2	50.5	49.7	47.6
S.D.	15.09	13.1	15.38	16.13
N	20	18	19	21
MALE FETAL WEIGHTS (g)
MEAN	37.6	39.5	38.3	37.6
S.D.	4.87	4.61	4.65	5.31
N	20	18	19	21
FEMALE FETAL WEIGHTS (g)
MEAN	37	37.2	35.4	36.9
S.D.	4.66	4.65	3.89	5.98
N	20	18	19	21
COMBINED FETAL WEIGHTS (g)
MEAN	37.1	38.3	37.2	37
S.D.	4.52	4.25	3.68	5.66
N	20	18	19	21

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA AND IMPLANTATION SITES COMPARED USING DUNNETT'S TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

	FETUSES				LITTERS
Dose Group	1	2	3	4	1	2	3	4
NUMBER EXAMINED EXTERNALLY	181	177	190	190	20	18	19	21
ABDOMEN- DISTENDED	0	1	1	0	0	1	1	0
BRACHYDACTYLY	1	0	0	0	1	0	0	0
CARPAL AND/OR TARSAL FLEXURE	0	0	0	4	0	0	0	2
CLEFT PALATE	0	0	0	2	0	0	0	1
NUMBER EXAMINED VISCERALLY	181	177	190	190	20	18	19	21
AORTIC ARCH- DILATED	0	2	0	0	0	1	0	0
PULMONARY TRUNK- NARROW	0	1	0	0	0	1	0	0
ATRIAL SEPTUM DEFECT	0	1	0	0	0	1	0	0
ATRIOVENTRICULAR VALVE- ABSENT	0	1	0	1	0	1	0	1
TESTIS- MALPOSITIONED	1	0	0	0	1	0	0	0
TETRALOGY OF FALLOT	0	1	0	0	0	1	0	0
AORTIC ARCH- NARROW	0	0	0	1	0	0	0	1
PULMONARY TRUNK- DILATED	0	0	0	1	0	0	0	1
VENTRICULAR SEPTUM DEFECT	0	0	0	1	0	0	0	1
ATRIUM- SMALL	0	0	0	1	0	0	0	1
DIAPHRAGMATIC HERNIA	0	0	0	1	0	0	0	1
KIDNEY(S)- MALPOSITIONED	0	0	0	1	0	0	0	1
HYDROCEPHALY- INTERNAL	0	2	0	0	0	1	0	0
VISCERA- CYST(S)	0	0	1	0	0	0	1	0
LUNG- CYST	0	0	0	1	0	0	0	1
NUMBER EXAMINED SKELETALLY	181	177	190	190	20	18	19	21
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	1	0	4	0	1	0	2	0
TOTAL NUMBER WITH MALFORMATIONS
EXTERNAL	1	1	1	5	1	1	1	2
SOFT TISSUE	1	5	1	3	1	3	1	2
SKELETAL	1	0	4	0	1	0	2	0
COMBINED	3	6	6	6	2	4	3	2

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

Dose Group		1	2	3	4
NUMBER OF LITTERS EXAMINED EXTERNALLY		20	18	19	21
ABDOMEN- DISTENDED	MEAN	0	0.5	0.4	0
	S.D.	0	1.96	1.91	0
BRACHYDACTYLY	MEAN	0.5	0	0	0
	S.D.	2.24	0	0	0
CARPAL AND/OR TARSAL FLEXURE	MEAN	0	0	0	2.3
	S.D.	0	0	0	8.42
CLEFT PALATE	MEAN	0	0	0	1.2
	S.D.	0	0	0	5.46

None significantly different from control group

NUMBER OF LITTERS EXAMINED VISCERALLY		20	18	19	21
AORTIC ARCH- DILATED	MEAN	0	0.8	0	0
	S.D.	0	3.37	0	0
PULMONARY TRUNK- NARROW	MEAN	0	0.4	0	0
	S.D.	0	1.68	0	0
ATRIAL SEPTUM DEFECT	MEAN	0	0.4	0	0
	S.D.	0	1.68	0	0
ATRIOVENTRICULAR VALVE- ABSENT	MEAN	0	0.4	0	0.6
	S.D.	0	1.68	0	2.73
TESTIS- MALPOSITIONED	MEAN	0.5	0	0	0
	S.D.	2.24	0	0	0
TETRALOGY OF FALLOT	MEAN	0	0.4	0	0
	S.D.	0	1.81	0	0
AORTIC ARCH- NARROW	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
PULMONARY TRUNK- DILATED	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
VENTRICULAR SEPTUM DEFECT	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
ATRIUM- SMALL	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
DIAPHRAGMATIC HERNIA	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
KIDNEY(S)- MALPOSITIONED	MEAN	0	0	0	0.6
	S.D.	0	0	0	2.73
HYDROCEPHALY- INTERNAL	MEAN	0	1	0	0
	S.D.	0	4.29	0	0
VISCERA- CYST(S)	MEAN	0	0	0.6	0
	S.D.	0	0	2.55	0
LUNG- CYST	MEAN	0	0	0	0.5
	S.D.	0	0	2.42

None significantly different from control group

NUMBER OF LITTERS EXAMINED SKELETALLY		20	1819	19	21
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY	MEAN	0.6	0	1.9	0
	S.D.	2.48	0	6.15	0

None significantly different from control group

NUMBER OF LITTERS EXAMINED		20	18	19	21
PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS	MEAN	0.5	0.5	0.4	2.9
	S.D.	2.24	1.96	1.91	11.06
PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS	MEAN	0.5	2.2	0.6	1.7
	S.D.	2.24	5.45	2.55	5.86
PERCENT PER LITTER WITH SKELETAL MALFORMATIONS	MEAN	0.6	0	1.9	0
	S.D.	2.48	0	6.15	0

None significantly different from control group

Applicant's summary and conclusion

Executive summary:

The objectives of this study were to determine the potential of the test item to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. Based on the results of the dose range finder, groups of 22 females per dose group were exposed to the test article at dose levels of 0, 6, 20, and 60 mg/kg body weight per day. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80 were prepared accurately. Homogeneity of

formulations was considered acceptable for the purpose of this study. No adverse maternal effects were recorded up to a dose level of 60 mg/kg/day. There were no test item-related effects in fetal external, visceral or skeletal morphology or other developmental parameters. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test item was established as being at least 60 mg/kg/day. Based on the Dose Range Finding Study, a slightly higher dose level of 100 mg/kg/day was not tolerated.