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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Bis(1,2,2,6,6-pentamethyl-4-piperidyl) sebacate and Methyl 1,2,2,6,6-pentamethyl-4-piperidyl sebacate
EC Number:
915-687-0
Cas Number:
1065336-91-5
Molecular formula:
unspecified
IUPAC Name:
Reaction mass of Bis(1,2,2,6,6-pentamethyl-4-piperidyl) sebacate and Methyl 1,2,2,6,6-pentamethyl-4-piperidyl sebacate
Details on test material:
Storage Conditions: At room temperature
Stability at higher temperatures: Stable, maximum temperature: 40°C, maximum duration: not indicated

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 185 and 284 g
- Fasting period before study:
- Housing: in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 54 to 70%.
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
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 as a solution for maximally 8 days, filled out in daily portions and stored in the refrigerator at 2-8ºC. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity/composition of the test item.
Details of the preparation and dispensing of the test item have been retained in the Study Records. Any residual volumes were discarded.

VEHICLE
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis during week 1 from all groups (concentration analysis) and from groups 2 and 4 (homogeneity). Analyses were performed using a validated analytical procedure For concentration analysis duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. For homogeneity analysis duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
Animals were received time-mated
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum
Frequency of treatment:
daily
Duration of test:
until day 21 post-coitum (necropsy)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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.

DETAILED CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily, beginning on Day 2 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 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: Yes
Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS
Animals were euthanized by an oxygen/carbon dioxide procedure on Day 21 post-coitum. No terminal body weight was recorded.
All animals 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). Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
The thyroid gland and uterus were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated. The thyroid gland was collected from all animals and preserved in 10% neutral buffered formalin. Additional tissue samples were collected to elucidate abnormal findings. Thyroid glands of all animals of Groups 1 and 4 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin and were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
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
Blood sampling:
Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected randomized at the discretion of the Study Director, between 7:00 and 09:00 a.m., from the jugular vein in the animal facility. After collection, samples were transferred to the appropriate laboratory for processing. Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for the following parameters: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube.
Litters of females surviving to 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 sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. For late resorptions a gross external examination was performed. Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were 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 this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination 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. As visceral malformations were suspected for fetus (A088-09), selected for skeletal examination, this fetus was also subjected to visceral examination. Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded. The carcasses were processed and stained with Alizarin Red S (as described below), but not examined.

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, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
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 post-implantation 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 post-implantation loss.
Indices:
Pre-implantation loss (%), Post-implantation loss (%), Viable fetuses affected/litter (%)
Historical control data:
Corrected body weight gain (gram): mean: 29, P5-P95:14.8-44.4, n=1077
Corrected body weight gain (%): mean: 13, P5-P95:7.0-20.7, n=1077
Absolute food consumption (gram), post-coitum Days 2-6: mean: 21, P5-P95: 16.3-25.8, n=1049
Total T3 (ng/dL): mean: 59.7, P5-P95:43.20-79.50, n=197
Total T4 (μg/dL): mean: 2.24, P5-P95:1.470-3.300, n=223

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was recorded for 4/22 animals at 500 mg/kg/day (Nos. 71, 74, 82 and 83) between post-coitum Days 7 and 20. Piloerection was also recorded for 1/22 animals (No. 56) at 150 mg/kg/day between postcoitum Days 6 and 10, but given its incidental and temporary occurrence, and absence of corroborative changes in body weight or food intake, this was considered unrelated to treatment with the test item.
Other clinical signs were considered not to represent toxicity, and mainly consisted of salivation which was recorded after dosing among all animals at 150 and 500 mg/kg/day from post-coitum Day 7 onwards and among most animals at 50 mg/kg/day from post-coitum Day 8 onwards. This was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats 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:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg/day, a statistically significantly reduced mean body weight gain was recorded throughout the treatment period, with slight mean weight loss (2%) between post-coitum Days 6 and 9 (individual weight loss values ranged between 1 and 8%). Absolute mean body weights were also slightly lower than control means throughout the treatment period at this dose, but were only statistically significant on post-coitum Day 21. Mean absolute body weight at the end of the treatment period was at 0.93x of the control mean. When corrected for gravid uterus weight, mean body weight and body weight showed a more pronounced (statistically significant) change at this dose level, with absolute weight gain being 0.42x of the control mean. Both absolute and relative body weight gain corrected for gravid uterus were below the historical control data range.
At 50 and 150 mg/kg/day, mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg/day, a statistically significantly lower mean absolute and relative food consumption was recorded over post-coitum Days 6 to 9 and Days 9 to 12 post-coitum (0.59x and 0.84x control mean for absolute food intake, respectively). Absolute food consumption was also statistically significantly lower than the control mean on post-coitum Days 18-21 (0.87x).
At 50 and 150 mg/kg/day, absolute and relative food consumption of treated animals remained in the same range as controls over the treatment period.
Note: Several individual food consumption values across the dose groups over post-coitum Days 2-6 were considered unrealistically low, as these well exceeded the historical control data range and since these low values were recorded before treatment was initiated. In absence of corroborative changes in clinical appearance or body weight gain, these low values were attributed to incorrect technical conduct of these measurements.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: Values for non-gravid animals (No. 13 (Control group) and No. 49 (150 mg/kg/day) were excluded from the data tables and reported in a separate table.
At 500 mg/kg/day, mean total Triiodothyronine (T3) was statistically significantly lower than control mean (0.70x). Mean T3 values remained within the historical control data range. At 150 mg/kg/day, mean total Thyroxine (T4) appeared lower than the control mean. Since this mean was less than 20% different from the control mean (0.88x), was not statistically significant and remained within the historical control data range, this variation was considered not to be related to treatment. Serum levels of thyroid stimulating hormone (TSH) were unaffected by treatment with the test item up to 500 mg/kg/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes in thyroid gland weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item. All necropsy findings among control and treated animals were incidental, did not show a dose-related incidence trend and are occasionally seen among rats used in this type of study. Therefore, these necropsy findings were considered not to be related to treatment with the test item.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The recorded microscopic findings of the thyroid were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

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 up to 500 mg/kg/day. Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.2 and 5.1 gram for the control, 50, 150 and 500 mg/kg/day groups, respectively. The statistically significantly higher mean fetal body weights of males (and combined for both sexes) at 50 mg/kg/day occurred in the absence of a dose-related trend and only marginally exceeded the historical control data range. As such, this variation was considered not to be related to treatment with the test item.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group. Mean litter sizes were 11.6 fetuses/litter in the control group and 11.0 fetuses/litter at 50, 150 and 500 mg/kg/day
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment with the test item up to 500 mg/kg/day. Mean sex ratios (males:females) were 43.5:56.5, 52.4:47.6, 43.2:56.8 and 50.8:49.2 for the control, 50, 150 and 500 mg/kg/day groups, respectively. The statistically significant alteration in sex ratio at 50 mg/kg/day occurred in the absence of a dose-related trend and was slight in magnitude. As such, this variation was considered not to be related to treatment with the test item.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Fetal ano-genital distance of both sexes (before and after correction for body weight) was unaffected by treatment with the test item up to 500 mg/kg/day.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological examination were 243 (21), 243 (22), 231 (21) and 241 (22) in the control, 50, 150 and 500 mg/kg/day groups, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Moreover, as during eviscerating of the fetuses prior to skeletal staining, a prominent visceral malformation was noticed for one fetus (A088-09) at 500 mg/kg/day that was selected for skeletal examination, this fetus was also subjected to a visceral examination.
There were no treatment-related effects on external morphology following treatment up to 500 mg/kg/day.
The only two external malformations in this study were observed in the control group. Fetus A009-07 had an omphalocele and the late resorption in litter A020 had general anasarca. Since both malformations occurred in the control group, these were considered to be spontaneous in origin. There were no external variations in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 500 mg/kg/day.
Skeletal malformations in test item treated groups were confined to sternoschisis in one fetus at 500 mg/kg/day (A088-09) and bent scapula and humerus in one fetus at 50 mg/kg/day (A023-07). The group distribution and single occurrence of these two malformations did not suggest a relationship to treatment with the test item. Moreover, the control fetus with omphalocele externally (A009-07) also had sternoschis (in addition to a vertebral anomaly) and both malformations were previously observed in historical controls.
No treatment-related skeletal variations were recorded. Skeletal variations occurred at an incidence of 79.5%, 82.2%, 77.3% and 74.4% per litter in the control, 50, 150 and 500 mg/kg/day groups, respectively. The incidence of 14th full ribs in the control group (12.6%) was close to the historical control maximum value of 13.1% per litter. The statistically significantly lower incidence of this skeletal variation at 50 and 500 mg/kg/day (2.7% and 1.9% per litter at 50 and 500 mg/kg/day, respectively, and 6.0% at 150 mg/kg/day) was considered to have resulted from this relatively high control incidence. Moreover, since there was no apparent dose-related trend in incidence of 14th full ribs, the statistically significant changes in this skeletal variation were considered not to represent a test itemrelated effect. The statistically non-significant higher incidence of 7th cervical ossification sites at 500 mg/kg/day (11.3% per litter, i.e. 11 fetuses in 7 litters) was higher than the control mean (5 fetuses in 5 litters) and exceeded the historical control maximum value (mean: 3.8% per litter, and maximum value of 9.9% per litter). However, statistical significance was not achieved, the mean was only slightly above the upper historical control limit, and a more generalized/advanced ossification process is expected in case of a test item-related effect.
Therefore, the presence of 7th cervical ossification sites at 500 mg/kg/day was considered not to be related to treatment with the test item. Moreover, an extra cervical ossification site in the costal cartilage region will disappear postnatally by incorporation in the transverse process. All other variations occurred in the absence of a dose-related incidence trend, in control fetuses only and/or at frequencies that were within the range of available historical control data. Therefore, they were considered not related to treatment with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 500 mg/kg/day.
Two viscerally malformed fetuses were observed. One fetus at 500 mg/kg/day (A088-09) had a narrow pulmonary trunk, and one fetus at 150 mg/kg/day (A050-08) had situs inversus. Due to the single occurrence and absence of any clear dose-related incidence trend, both were considered chance findings. Only one visceral variation (small supernumerary liver lobes) was observed in this study in two fetuses at 50 mg/kg/day and one fetus at 500 mg/kg/day. Given the low incidence and absence of any clear dose-related incidence trend, this was considered not to be related to treatment with the test item.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects reported

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

BODY WEIGHTS (GRAM) SUMMARY FEMALES

    Group 1 Group 2 Group 3 Group 4
    Control 50 mg/kg bw 150 mg/kg bw 500 mg/kg bw
DAY 0 MEAN 213 213 215 214
  ST.DEV. 19.9 20.4 20.6 23.8
  N 21 22 21 22
DAY 3 MEAN 228 231 229 228
  ST.DEV. 18.2 20.2 19.6 24.9
  N 21 22 21 22
DAY 6 MEAN 235 238 236 223
  ST.DEV. 16.7 20.9 20.0 22.2
  N 21 22 21 22
DAY 9 MEAN 251 254 251 239
  ST.DEV. 18.0 21.7 20.3 25.3
  N 21 22 21 22
DAY 12 MEAN 267 269 264 253
  ST.DEV. 19.5 23.2 20.7 26.2
  N 21 22 21 22
DAY 15 MEAN 299 299 296 282
  ST.DEV. 22.6 26.6 23.9 29.4
  N 21 22 21 22
DAY 18 MEAN 338 337 332 316*
  ST.DEV. 27.5 29.5 26.5 37.3
  N 21 22 21 22
DAY 21 MEAN 3791 3900 3887 3763
  ST.DEV. 358.9 308.2 275.5 238.8
  N 20 18 19 21

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

BODY WEIGHT GAIN (%) SUMMARY FEMALES

    Group 1 Group 2 Group 3 Group 4
    Control 50 mg/kg bw 150 mg/kg bw 500 mg/kg bw
DAY 6 MEAN 0 0 0 0
  ST.DEV. 0.0 0.0 0.0 0.0
  N 21 22 21 22
DAY 9 MEAN 3 3 3 -2**
  ST.DEV. 2.3 1.5 1.8 2.8
  N 21 22 21 22
DAY 12 MEAN 10 10 9 5**
  ST.DEV. 2.1 1.8 2.4 3.3
  N 21 22 21 22
DAY 15 MEAN 17 17 15 11**
  ST.DEV. 2.7 2.4 2.5 4.1
  N 21 22 21 22
DAY 18 MEAN 31 30 29 24**
  ST.DEV. 4.0 2.5 4.1 5.3
  N 21 22 21 22
DAY 21 MEAN 49 46 45 38**
  ST.DEV. 6.1 5.1 6.9 7.8
  N 21 22 21 22

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

CLINICAL BIOCHEMISTRY SUMMARY FEMALES pregnant

    Group 1 Group 2 Group 3 Group 4
    Control 50 mg/kg bw 150 mg/kg bw 500 mg/kg bw
TSH MEAN 0.300 0.287 0.286 0.247
uIU/mL ST.DEV 0.176 0.178 0.251 0.172
  N 21 22 21 22
TotalT3 MEAN 62.0 55.3 48.5 43.2**
ng/dL ST.DEV 19.7 17.0 18.1 18.4
  N 21 20 21 22
TotalT4 MEAN 2.08 2.25 2.25 1.83
ug/dL ST.DEV 0.44 0.58 0.65 0.56
  N 21 22 21 22

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

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES pregnant

    Group 1 Group 2 Group 3 Group 4
    Control 50 mg/kg bw 150 mg/kg bw 500 mg/kg bw
BODYW. MEAN 338 337 332 316*
(GRAM) ST.DEV 28 30 26 37
  N 21 22 21 22
THYROIDS MEAN 0.017 0.017 0.018 0.015
(GRAM) ST.DEV 0.004 0.003 0.003 0.004
  N 21 22 21 22

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

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY FEMALES pregnant

    Group 1 Group 2 Group 3 Group 4
    Control 50 mg/kg bw 150 mg/kg bw 500 mg/kg bw
BODYW. MEAN 338 337 332 316*
(GRAM) ST.DEV 28 30 26 37
  N 21 22 21 22
THYROIDS MEAN 0.005 0.005 0.005 0.005
(%) ST.DEV 0.001 0.001 0.001 0.001
  N 21 22 21 22

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 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 EXAMINED AT SCHEDULED NECROPSY 22 100 22 100 22 100 22 100
NONGRAVID 1 4.5 0 0 1 4.5 0 0
GRAVID 21 95.5 22 100 21 95.5 22 100
WITH RESORPTIONS ONLY 0 0 0 0 0 0 0 0
WITH VIABLE FETUSES 21 100 22 100 21 100 22 100
TOTAL FEMALES GRAVID 21 95.5 22 100 21 95.5 22 100

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

Group   Males Females Viable Fetuses Dead Fetuses Early Resorptions Late resorptions
1 TOTAL 108 135 243 0 6 3
  MEAN 5.1 6.4 11.6 0 0.3 0.1
  S.D. 2.06 1.86 2.38 0 0.56 0.36
2 TOTAL 127 116 243 0 15 0
  MEAN 5.8 5.3 11 0 0.7 0
  S.D. 2.27 2.33 3.03 0 0.84 0
3 TOTAL 101 130 231 0 9 1
  MEAN 4.8 6.2 11 0 0.4 0
  S.D. 1.86 1.72 2.3 0 0.6 0.22
4 TOTAL 123 118 241 0 13 0
  MEAN 5.6 5.4 11 0 0.6 0
  S.D. 2.17 1.89 2.5 0 0.85 0

Group   Post Implantation Loss Implantation Sites Corpora Lutera Pre-Implantation Loss Fetal weights (g) No. of gravid females
1 TOTAL 9 252 262 10 NA 21
  MEAN 0.4 12 12.5 0.5 5.2  
  S.D. 0.75 2 1.94 0.68 0.3  
2 TOTAL 15 258 283 25 NA 22
  MEAN 0.7 11.7 12.9 1.1 5.4*  
  S.D. 0.84 2.75 2.03 2.05 0.34  
3 TOTAL 10 241 268 27 NA 21
  MEAN 0.5 11.5 12.8 1.3 5.2  
  S.D. 0.6 2.16 1.84 1.71 0.23  
4 TOTAL 13 254 284 30 NA 22
  MEAN 0.6 11.5 12.9 1.4 5.1  
  S.D. 0.85 2.74 2.04 1.73 0.26  

* = Significantly different from the control group at 0.05

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

Group 1 2 3 4
CORPORA LUTEA        
MEAN 12.5 12.9 12.8 12.9
S.D. 1.94 2.03 1.84 2.04
N 21 22 21 22
IMPLANTATION SITES        
MEAN        
S.D. 12 11.7 11.5 11.5
N 2 2.75 2.16 2.74
VIABLE FETUSES (%) 21 22 21 22
MEAN        
S.D.        
N 95.7 92.8 95.6 95.4
DEAD FETUSES (%) 9.58 9.72 5.4 6.4
MEAN 21 22 21 22
S.D.        
N 0 0 0 0
EARLY RESORPTIONS (%) 0 0 0 0
MEAN 21 22 21 22
S.D.        
N        
LATE RESORPTIONS (%) 2.8 7.2 3.8 4.6
MEAN 6.69 9.72 5.15 6.4
S.D. 21 22 21 22
N        
TOTAL RESORPTIONS (%)        
MEAN 4.3 7.2 4.4 4.6
S.D. 9.58 9.72 5.4 6.4
N 21 22 21 22
PRE-IMPLANTATION LOSS (%)        
MEAN 3.8 8.7 9.7 10.9
S.D. 5.5 16.68 12.39 14.44
N 21 22 21 22
POST-IMPLANTATION LOSS (%)        
MEAN 4.3 7.2 4.4 4.6
S.D. 9.58 9.72 5.4 6.4
N 21 22 21 22
MALES (%)        
MEAN 43.5 52.4* 43.2 50.8
S.D. 14.2 15.31 12.35 15.73
N 21 22 21 22
FEMALES (%)        
MEAN 56.5 47.6* 56.8 49.2
S.D. 14.2 15.31 12.35 15.73
N 21 22 21 22
MALE FETAL WEIGHTS (g)        
MEAN 5.3 5.6* 5.3 5.2
S.D. 0.39 0.28 0.28 0.26
N 21 22 21 22
FEMALE FETAL WEIGHTS (g)        
MEAN 5 5.3 5.1 4.9
S.D. 0.25 0.39 0.22 0.27
N 21 22 21 21
COMBINED FETAL WEIGHTS (g)        
MEAN 5.2 5.4* 5.2 5.1
S.D. 0.3 0.34 0.23 0.26
N 21 22 21 22
MALE AGD (MM)        
MEAN 3.03 2.98 3.08 3.09
S.D. 0.391 0.502 0.42 0.354
N 21 22 21 22
FEMALE AGD (MM)        
MEAN 1.68 1.65 1.72 1.7
S.D. 0.156 0.205 0.2 0.207
N 21 22 21 21
MALE AGD corrected by Body Weight        
MEAN 1.74 1.68 1.76 1.79
S.D. 0.211 0.288 0.238 0.207
N 21 22 21 22
FEMALE AGD corrected by Body Weight        
MEAN 0.98 0.95 1 1
S.D. 0.087 0.118 0.121 0.13
N 21 22 21 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

FETAL WEIGHTS AND (CORRECTED) AGD COMPARED USING DUNNETT'S TEST

* = Significantly different from the control group at 0.05

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

  FETUSES       LITTERS      
Dose Group 1 2 3 4 1 2 3 4
NUMBER EXAMINED EXTERNALLY 243 243 231 241 21 22 21 22
TRUNK- OMPHALOCELE 1 0 0 0 1 0 0 0
NUMBER EXAMINED VISCERALLY 121 123 114 121 21 22 21 22
SITUS INVERSUS 0 0 1 0 0 0 1 0
PULMONARY TRUNK- NARROW 0 0 0 1 0 0 0 1
NUMBER EXAMINED SKELETALLY 122 120 117 121 21 22 21 22
BENT LIMB BONES 0 1 0 0 0 1 0 0
STERNOSCHISIS 1 0 0 1 1 0 0 1
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY 1 0 0 0 1 0 0 0
TOTAL NUMBER WITH MALFORMATIONS                
EXTERNAL : 1 0 0 0 1 0 0 0
SOFT TISSUE : 0 0 1 1 0 0 1 1
SKELETAL : 1 1 0 1 1 1 0 1
COMBINED : 1 1 1 1 1 1 1 1

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

Dose Group   1 2 3 4
NUMBER OF LITTERS EXAMINED EXTERNALLY   21 22 21 22
TRUNK- OMPHALOCELE MEAN MEAN 0.5 0 0 0
  S.D. 2.18 0 0 0

None significantly different from control group

NUMBER OF LITTERS EXAMINED VISCERALLY   21 22 21 22
SITUS INVERSUS MEAN 0 0 1 0
  S.D. 0 0 4.36 0
PULMONARY TRUNK- NARROW MEAN 0 0 0 0.6
  S.D. 0 0 0 3.05

None significantly different from control group

NUMBER OF LITTERS EXAMINED SKELETALLY   20 18 19 21
BENT LIMB BONES MEAN 0 0.6 0 0
  S.D. 0 3.05 0 0
STERNOSCHISIS MEAN 1 0 0 0.9
  S.D. 4.36 0 0 4.26
VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY MEAN 1 0 0 0
  S.D. 4.36 0 0 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 0 0
  S.D. 2.18 0 0 0
PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS MEAN 0 0 1 0.6
  S.D. 0 0 4.36 3.05
PERCENT PER LITTER WITH SKELETAL MALFORMATIONS MEAN 1 0.6 0 0.9
  S.D. 4.36 3.05 0 4.26
TOTAL PERCENT PER LITTER WITH MALFORMATIONS MEAN 0.5 0.6 1 0.6
  S.D. 2.18 3.05 4.36 3.05

None significantly different from control group

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) were established:
- Maternal NOAEL: 150 mg/kg/day (based on reduced weight gain).
- Developmental NOAEL: at least 500 mg/kg/day.
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 Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 50, 150 or 500 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4 and TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland). 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, ano-genital distance, external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Maternal findings

Adverse effects on body weight were recorded at 500 mg/kg/day, at which absolute weight gain corrected for gravid uterus weight was less than half of the control mean (and below the historical control data range). This magnitude of change in weight gain was considered to represent an adverse effect. Over post-coitum Days 6-9, slight mean weight loss (2%) and lower food intake (0.59x control mean) was recorded, which showed a partial recovery as treatment progressed, although food intake was again reduced over post-coitum Days 18-21 (0.87x control mean). This was accompanied by piloerection for a few animals at 500 mg/kg/day between post-coitum Days 7 and 20. A non-adverse but statistically significant lower mean total T3 was recorded at 500 mg/kg/day (0.70x of control means, respectively). Since the mean remained within the historical control data range, and no treatment-related changes in thyroid histopathology or thyroid weight was recorded, this lower mean total T3 was considered not adverse.

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Fetal Examinations

There were no test item-related effects in external, visceral or skeletal morphology. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights and anogenital distance).

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Levels (NOAEL) were established:

Maternal NOAEL: 150 mg/kg/day (based on reduced weight gain).

Developmental NOAEL: at least 500 mg/kg/day.