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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January 2021 - 23 May 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Version: June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3a,4,7,7a-tetrahydro-4,7-methanoindene
EC Number:
201-052-9
EC Name:
3a,4,7,7a-tetrahydro-4,7-methanoindene
Cas Number:
77-73-6
Molecular formula:
C10H12
IUPAC Name:
3a,4,7,7a-tetrahydro-1H-4,7-methanoindene
Test material form:
solid
Details on test material:
Supplier: Sigma Aldrich
Batch number: MKCJ2785
Expiry date: 01 February 2024
Specific details on test material used for the study:
n/a

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 dosing: 18-20 weeks old
Weight (on the first day of dosing): 2886 to 4250 g (at initiation of dosing)
Housing: individually housed in Cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. Cages were arranged on the racks according to a Latin square model.
Diet:Pellets (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Switzerland). Restricted access. On arrival, animals received approximately 25 grams pelleted diet and on subsequent days 140-160 grams was supplied.
Water: ad libitum
Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19°C
- Humidity (%): 47-55%
- Photoperiod (hrs dark / hrs light):12-hours light and 12-hours dark (except during designated procedures)
- Ventilation ten or more air changes per hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Supplier: Sigma Aldrich
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The design of test item analysis was based on ICH Harmonised Tripartite Guideline Q2 (R1): Validation of Analytical Procedures: Text and Methodology. November 2005.
A quantitative analysis of the test item in vehicle was performed by an ultra performance liquid chromatographic method with spectrophotometric detection (UPLC-UV) in order to determine the accuracy, homogeneity, stability and resuspension of formulations over a large concentration range.
Concentration results were considered acceptable if mean sample concentration results were within or equal to 90-110% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation was = 5%. Stability results were considered acceptable if the relative difference between the stored and initial taken samples was = 10%. Resuspension homogeneity was demonstrated if the coefficient of variation is = 5%.
Calibration curves were constructed using six concentration levels. For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.
The mean accuracies of the QC samples were within the criterion range of 90-110%. It demonstrated that the analytical methods were adequate for the determination of the test item in the study samples.
The formulations at 1 mg/mL and 200 mg/mL were homogeneous (i.e. coefficient of variation = 5%).
Analysis of the formulations after storage at room temperature yielded a relative difference of = 10%. The formulations were found to be stable when stored at room temperature protected from light for at least 5 hours, in a refrigerator (2-8°C) protected from light for at least 8 days, and in a freezer (= -15°C) protected from light for at least 21 days (3 weeks). Resuspension homogeneity after storage in a refrigerator for 8 days was demonstrated with a coefficient of variation of = 5%.
Results:
The concentrations analyzed in the formulation of Group 3 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the formulation of Groups 2 and 4, the mean accuracies were below the target concentration (i.e. both 88% of target). The results were accepted as they were only slightly below the target concentration.
No test item was detected in the Group 1 formulation.
The formulations of Groups 2, 3 and 4 were homogeneous (i.e. coefficient of variation = 10%).
Details on mating procedure:
Untreated females were mated at the Supplier and were at Day 1-2 post-coitum (Day 0 post-coitum is defined as the day of successful mating) when arrived at test facility.
Duration of treatment / exposure:
Day 6 to Day 28 post-coitum
Frequency of treatment:
Once daily
Duration of test:
Day 6 to Day 28 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Test System and Number of Animals:
Studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. This Study Plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Rationale for Route and Dose Levels:
The oral route of exposure was requested by the ECHA (European Chemicals Agency) in the Final Decision Letter following a Compliance Check (ECHA, 2020); they considered that this route was the most appropriate for hazard characterization.
The dose levels were selected based on the results of the Dose Range Finder, at the request of the Study Monitor and in an attempt to produce graded responses to the test item. These dose levels were considered to have a better dose spacing (i.e. 3 to 4 fold as recommended in the OECD 414 TG (2018)) than the previously used dose levels of 50, 100 and 200 mg/kg/day which were utilized in the previous dose range finding study in the rabbit. The high dose level of 200 mg/kg/day has been selected as this dose level is expected to produce some toxicity such as a reduction in body weight gain and food consumption, but not excessive lethality that would prevent meaningful evaluation, at this dose level in the previous dose range finding study in the rabbit effects were observed on body weight, bodyweight gain and food consumption, therefore, based on the magnitude of body weight loss being observed which was approximately 100 g to 150 g based on significantly reduced food consumption and/or inappetence in the first three days of dosing (Days 6 to 9 of gestation) it was considered that this would be an adequate high dose level as the previous information from the Gulati (1993) study showed that higher doses gave increased mortality at 300 mg/kg/day and 400 mg/kg/day, in 1/9 and 3/9 animals, respectively, which would be excessive and signs of systemic toxicity were also noted (body weight loss and decreased food and water consumption). A decreased body weight gain was also noted at 200 mg/kg/day on this previous study; therefore, this further justifies that the high dose level should not exceed 200 mg/kg/day, in order not to prevent meaningful evaluation.
It is also noteworthy, that there were no effects observed at 100 mg/kg/day or 25 mg/kg/day on the previous Gulati (1993) study. However, the mid-dose level of 60 mg/kg/day is expected to produce minimal to moderate toxicity and be a no observed-adverse-effect-level for maternal toxicity and developmental toxicity. The low-dose level of 20 mg/kg/day should produce no observable indications of toxicity and be a clear no-observed-effect-level for maternal and developmental toxicity.

References:
Gulati D.R, Grimes L. K. and Smith M. (1993). Range Finding Studies: Developmental Toxicity of Dicyclopentadiene When Administered Via Gavage In New Zeland White Rabbits. Report. Stuyd No:NTP-92-RF/DT-044

Examinations

Maternal examinations:
Mortality/Moribundity Checks:
At least twice daily throughout the study. Animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations:
At least once daily, starting on Day 6 post-coitum onwards up to the day prior to necropsy. During the dosing period, this observation were performed immediately postdose. Animals were removed from the cage for clinical sign observation. Clinical sign examination was performed according to standard procedures for the cage side observation as well as the detailed observation. Cage debris was examined to detect premature birth, if applicable.

Body Weights:
Animals were weighed on Days 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. Animals were individually weighed.

Food Consumption:
Food consumption of animals were measured daily from Day 3 post coitum onwards. Food consumption was quantitatively measured.

Water Consumption:
Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles.

Terminal Procedures:
Unscheduled Deaths: one female in the control group, three females in the 60 mg/kg/day dose group and seven females in the 2000 mg/kg/day dose group were euthanised for humane reasons by an intravenous injection of sodium pentobarbital, underwent necropsy, and specified tissues were retained.
Scheduled Euthanasia:animals surviving until scheduled euthanasia were euthanized by an intravenous injection of sodium pentobarbital on Day 29 post-coitum.

Necropsy:
All animals (including animals sacrificed before planned necropsy) 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.

Organ Weights:
The uterus was weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in poor condition or in extremis

Tissue Collection and Preservation:
Macroscopic abnormalities and placentas of live fetuses were collected from all animals and preserved in 10% buffered formalin or Davidson’s fixative and were transferred to formalin after at least 24 hours.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
-number of corpora lutea
-weight of the uterus (not for animals sacrificed before planned necropsy)
-placental weights (for live fetuses only; after weighing, placentas were fixed in formalin)
-number of implantation sites
-number and distribution of live and dead fetuses
-number and distribution of early and late resorptions
Blood sampling:
Not examined
Fetal examinations:
Fetal examinations:
For recognisable fetuses or normal implantations in development of females sacrificed before planned necropsy, a gross external examination was performed (if possible). Recognisable fetuses with abnormalities were fixed in 10% buffered formalin.
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:
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals sacrificed before planned necropsy) was determined.
Nonviable Fetus in one of the littes of the 20 mg/kg/day dose group was examined and weighed.
For late resorptions (three fetuses in the control group, three fetuses in the 20 mg/kg/day dose group and three fetuses in the 200 mg/kg/day dose group), a gross external examination was performed.

Visceral Examinations:
The sex of all fetuses was confirmed by internal examination and approximately one-half of 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. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. Abnormalities were not collected and fixed at discretion of the Study Director.
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 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:
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and processed for double staining with Alcian Blue 8GX and Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from all groups. 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:
Numerical data collected on scheduled occasions were summarised and statistically analyzed according to sex and occasion or by litter (Table 1).
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset.
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.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses were performed according to the matrix (Table 2) when possible but exclude any group with less than 3 observations.

Parametric/Non-Parametric
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Non-Parametric
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test.
ANCOVA
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.
Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Indices:
n/a
Historical control data:
Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Females with premature deaths showed hunched posture, piloerection and/or a thin appearance (Table 1). One female at 200 mg/kg/day, which was sacrificed in extremis on Day 13 post-coitum, was noted with limited usage of the hindlegs during the morning check. Examination by a veterinarian confirmed paralysis of both the hindlegs. This finding was correlated with a dislocation of lumbar vertebra no. 7 and accumulation of dark red fluid in the same area at necropsy. No explanation was found for the dislocated vertebra, however given the convulsions observed for another female at 200 mg/kg/day, this female may have had convulsions as well that could have caused the dislocation of the vertebra. As such, a possible relationship with treatment with the test item could not be excluded.
At 60 and 200 mg/kg/day, decreased feces output was observed for most females at a higher incidence compared to control animals and with a higher incidence with increasing dose.
At 200 mg/kg/day, erected fur and/or a thin appearance were regularly noted for 6/15 surviving animals between Days 9 and 21 post-coitum.
One animal in the 200 mg/kg/day dose group was in a poor condition on Day 20 post-coitum and was examined by a veterinarian. The animal showed convulsions, slight tremors, an abnormal stance, small pupils and hyperemia of the right nictitans with a fixed stare. Additionally, the animal showed abnormal chewing, an increased heart rate, panting and had a pale appearance. These clinical signs were of short duration; the next morning, no abnormalities were noted for this animal.
At 20 mg/kg/day, no test item-related clinical observations were noted. Decreased feces output was observed for most females. This finding was considered to be unrelated to treatment with the test item because it also occurred in control animals at a comparable incidence.
Other clinical observations recorded 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. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of 11 females did not survive until scheduled necropsy: one control female, three females at 60 mg/kg/day and seven females at 200 mg/kg/day. Of these premature deaths, one female at 60 mg/kg/day and five females at 200 mg/kg/day were considered to be related to treatment with the test item. All of these females showed strongly reduced or no food intake for at least 6 consecutive days, along with body weight loss (12%) and a decreased feces output. Additionally, one female at 200 mg/kg/day was sacrificed in extremis on Day 13 post-coitum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg/day, a reduced body weight gain compared to controls was observed between Days 6 and 15 post coitum, followed by a slight body weight loss between Days 18-21 post coitum and increased body weight gain thereafter (Table 2 and 3). Overall body weight gain between Days 6 and 29 post-coitum was 90% of control.
At 200 mg/kg/day, body weight losses were noted mainly between Days 6 and 9 post-coitum. Mean body weight gain remained lower than controls up to Day 15 post-coitum, after which mean body weight gain was similar or even higher compared to control animals. Overall body weight gain between Days 6 and 29 post-coitum was 61% of control.
At 20 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 control over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 60 and 200 mg/kg/day, a (slightly) lower food consumption was noted, mainly between Days 6 and 18 post-coitum (not always statistically significant) (Table 4). In subsequent intervals, food consumption was similar to controls. Mean food consumption over Days 6 to 29 post-coitum was 90% and 70% of control, respectively (reaching no statistical significance at 60 mg/kg/day).
At 20 mg/kg/day, mean food consumption was similar to the control level over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
All females, including the preterm sacrificed animals, were found to be pregnant, except for one female in the 20 mg/kg/day dose group , one female in the 60 mg/kg/day dose group and one female in the 200 mg/kg/day dose group.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre- and post implantation loss in the control and test groups were considered in the range of normal biological variation (Table 5).
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Description (incidence and severity):
There were 2 females of the 60 and 200 mg/kg/day groups each with early resorptions only (Table 5). These 4 females were all preterm sacrifices: one female had an early abortion on Day 20, and three females were sacrificed for welfare reasons due to severe body weight loss and low food consumption. At the incidence observed, a relationship with the test item could not be excluded.
Dead fetuses:
not specified
Description (incidence and severity):
The total number of litters with viable fetuses available for evaluation was 21, 21, 18 and 15 in the control, 20, 60 and 200 mg/kg/day groups, respectively (Table 5).
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites in the control and test groups were considered in the range of normal biological variation (Table 5).
Other effects:
not examined

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: clinical effects
Description (incidence and severity):
hunched posture, piloerection and/or a thin appearance, convulsions, slight tremors, an abnormal stance, small pupils and hyperemia of the right nictitans with a fixed stare, abnormal chewing, an increased heart rate, panting and had a pale appearance, reduced food intake and body weight loss.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, male fetal weights were considered affected by treatment with the test item (Table 5). Male fetal weights appeared to be more affected than female fetal weights, as mean values were, respectively, 11 and 3% lower than concurrent control weights (not statistically significant). The mean combined fetal weight was 7% lower than concurrent controls.
The fetal weights (male, female and combined) were comparable to the controls for the 20 and 60 mg/kg/day dose groups and remained within the historical control range of the Test Facility.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was considered unaffected by treatment with the test item up to 200 mg/kg/day (Table 5).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group (Table 5).
Mean litter sizes were 9.0, 8.3, 9.2 and 9.5 live fetuses/litter for the control, 20, 60 and 200 mg/kg/day groups, respectively.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, one fetus of the control, 20 mg/kg/day and 200 mg/kg/day dose groups each presented with external malformations (Table 7). The fetus in the control group appeared with multiple external malformations including hyperflexion of both forepaws, ectrodactyly of the left forepaw, a misshapen right forepaw and lastly omphalocele. The (dead) fetus from the low dose group (20 mg/kg/day) presented with a distended abdomen, and the fetus from the high dose group (200 mg/kg/day) had a hyperflexion of the left forepaw and gastroschisis.
Additionally, one fetus from the mid-dose group (60 mg/kg/day) that did not survive until scheduled necropsy had an omphalocele.
Due to a lack of a dose-related response and/or comparable observations in the control group, the recorded external malformations were considered chance findings and not related to the test-item.
No external variations were observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were observed in 4, 3, 2 and 1fetuses of the control, 20, 60 and 200 mg/kg/day groups, respectively (Table 6) and 7.
Skeletal malformations occurred in the forepaw, ribs, sternebra and vertebra. All occurred in single animals and/or in absence of a dose-related response and were therefore considered to be chance findings.
Higher incidences of unossified fore- and hindpaw phalanges (variations) were noted in fetuses of the 200 mg/kg/day group. Mean litter incidences were 8 and 25%, respectively, vs 1 and 10% in the concurrent group (not statistically significant).
All other skeletal variations occurred in the absence of a dose-related incidence trend and/or infrequently. Therefore, they were considered not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations were observed in 3, 2, 2 and 2 fetuses of the control, 20, 60 and 200 mg/kg/day groups, respectively (Table 6 and 7).
Visceral malformations occurred in the aortic arch, heart, lungs, diaphragm, ductus arteriosus, pulmonary trunk and intestines. All occurred in single animals and/or in absence of a dose related response and were therefore considered to be chance findings.
All visceral variations occurred in absence of a dose-related response. Therefore, they were considered not test item-related.
Other effects:
no effects observed
Description (incidence and severity):
Placental weight of live fetuses was considered not to be affected by treatment with the test item.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: resorptions

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day
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:
presumably yes

Any other information on results incl. tables

Table 1 Test Item-Related Premature Decedents

Dose Level

Female No.

Day of Sacrifice

Findings

60 mg/kg/day

56

Day 23 PC

-  Extremely low FC on 7 consecutive days

-  BW loss of 5% between Days 12 and 21 PC

-  Decreased feces output from Day 13 PC onwards, enlarged eyeballs on Days 22 and 23 PC

-  No macroscopic findings at necropsy

-  Gravid with 14 live fetuses

200 mg/kg/day

75

Day 14 PC

-  Extremely low/absent FC on 7 consecutive days

-  BW loss of 12% between Days 6 and 14 PC

-  Decreased feces output from Day 8 PC onwards, thin appearance on Days 12 and 14 PC, and erected fur and hunched posture on Day 14 PC

-  No macroscopic findings at necropsy

-  Gravid with 12 normal implantations

76

Day 14 PC

-  Extremely low/absent FC on 8 consecutive days

-  BW loss of 11% between Days 6 and 14 PC

-  Decreased feces output from Day 8 PC onwards, thin appearance on Days 12 and 14 PC, and erected fur and hunched posture on Day 14 PC

-  Enlarged uterus at necropsy (with no microscopic correlate)

-  Non-gravid

81

Day 14 PC

-  Extremely low FC on 8 consecutive days

-  BW loss of 9% between Days 6 and 14 PC

-  Decreased feces output from Day 7 PC onwards, thin appearance and erected fur on Days 12 and 13 PC, and hunched posture on Day 13 PC

-  No relevant macroscopic findings at necropsy

-  Gravid with 9 normal implantations

83

Day 13 PC

-  Extremely low/absent FC on 7 consecutive days

-  BW loss of 9% between Days 6 and 13 PC

-  Decreased feces output from Day 8 PC onwards, erected fur and hunched posture on Day 13 PC

-  No relevant macroscopic findings at necropsy

-  Gravid with 12 early resorptions

87

Day 18 PC

-  Extremely low/absent FC on 9 consecutive days

-  BW loss of 11% between Days 6 and 18 PC

-  Decreased feces output from Day 7 PC onwards, erected fur from Day 12 PC onwards, and thin appearance on Day 18 PC

-  No macroscopic findings at necropsy

-  Gravid with 10 normal implantations

Table 2 Summary of Body Weights: Gestation

Sex: Female Day Relative to Mating (Litter A)
  0 6 9 12 15 18 21 24 27 29
0 mg/kg/day (Group 1) Mean 3636.6 3592.1 3644.8 3699.4 3766.7 3776.8 3815 3855.4 3868.4 3916.3
SD 319.9 279.1 275.8 265 246.3 262.5 272.9 245.2 234.9 244.5
N 22 22 21 21 21 21 21 21 21 21
20 mg/kg/day (Group 2) Mean 3670.8 3612.2 3665.7 3720.3 3788 3808.3 3843.3 3871.3 3920.4 3978.2
SD 349.3 319.4 315.6 319.5 312.3 328.5 300 298.6 279.6 270.3
N 21 21 21 21 21 21 21 21 21 21
%Diff 0.9 0.6 0.6 0.6 0.6 0.8 0.7 0.4 1.3 1.6
60 mg/kg/day (Group 3) Mean 3575 3510.8 3550.5 3585.6 3634.2 3655.7 3647.2 3682.9 3723.1 3798.6
SD 335.7 321.6 305.1 305.7 291.1 271.3 286.1 282.3 276.7 264
N 21 21 20 20 20 20 19 18 18 18
%Diff -1.7 -2.3 -2.6 -3.1 -3.5 -3.2 -4.4 -4.5 -3.8 -3
200 mg/kg/day (Group 4) Mean 3669.8 3588.9 3458.3 3472.5 3541.8 3562.1 3620.6 3690.5 3757.7 3797.2
SD 370.3 338.1 342.8 354 353.4 357.7 337.8 350.7 339.7 350.3
N 21 21 20 20 16 16 15 15 15 15
%Diff 0.9 -0.1 -5.1 -6.1 -6 -5.7 -5.1 -4.3 -2.9 -3

Anova & Dunnett

Table 3 Summary of Body Weight Gains (g): Gestation

Sex: Female Day Relative to Mating (Litter A)
  6 ¿ 9 [G] 9 ¿ 12 [G1] 12 ¿ 15 [G] 15 ¿ 18 [G] 18 ¿ 21 [G] 21 ¿ 24 [G] 24 ¿ 27 [G] 27 ¿ 29 [G] 6 ¿ 29 [G1]
0 mg/kg/day (Group 1) Mean 67.5 54.7 67.3 10 38.2  40.4 13.0 13 47.9 339
SD 41.9 46.2 72.2 65.4 50.1 51.3 79.5 44.2 184.2
N 21 21 21 21 21 21 21 21 21
20 mg/kg/day (Group 2) Mean 53.4 54.7 67.7 20.2 35 28 49.1 57.8 366
SD 53.5 40.3 90.6 52.9 58.8 72.7 66.7 53.6 210.3
N 21 21 21 21 21 21 21 21 21
60 mg/kg/day (Group 3) Mean 23.1*  35.1 48.6 21.5 -11* 54.4 40.2 75.5 306.1
SD 38.9 38.3 74.7 70 41.3 44.5 69.5 32.4 128.1
N 20 20 20 20 19 18 18 18 18
200 mg/kg/day (Group 4) Mean -128.9**  14.2*  39.1 20.3 27.5 69.9 67.3 39.5 206.3
SD 61.9 63.3 84.6 72.7 61.4 65.8 58 56.9 159.4
N 20 20 16 16 15 15 15 15

15

[G] - Anova & Dunnett; * = p = 0.05; ** = p = 0.01

[G1] - Kruskal-Wallis & Dunn; * = p = 0.05

Table 4 Summary of Food Consumption (Food Mean Daily Consumption (g/animal/day))

Sex: Female Day Relative to Mating (Litter A)
  6 ¿ 9 [G] 9 ¿ 12 [G1] 12 ¿ 15 [G] 15 ¿ 18 [G] 18 ¿ 21 [G] 21 ¿ 24 [G] 24 ¿ 27 [G] 27 ¿ 29 [G] 6 ¿ 29 [G1]
0 mg/kg/day (Group 1) Mean 134.14 131.38 93.03 83.79 113.21 106.05 80.27 91.33 104.71
SD 23.9 23.58 45.74 42.23 35.48 34.94 37.14 33.54 24.07
N 21 21 21 21 21 21 21 21 21
20 mg/kg/day (Group 2) Mean 131.56 126.08 94.49 87.75 120.84 98.95 81.38 98.83 105.25
SD 28.46 27.7 37.46 45.81 27.94 36.34 40.77 29.52 21.98
N 21 21 21 21 21 21 21 21 21
%Diff -1.93 -4.04 1.57 4.72 6.74 -6.69 1.38 8.21 0.52
60 mg/kg/day (Group 3) Mean 117.67  109.70*  65.9 69.75 93.58 95.52 87 103.11 93.76
SD 19.01 24.18 37.09 37.95 39.31 28.06 36.11 28.73 21.31
N 20 20 20 20 19 18 18 18 18
%Diff -12.28 -16.5 -29.16 -16.76 -17.34 -9.93 8.38 12.9 -10.46
200 mg/kg/day (Group 4) Mean 36.27**  53.68**  37.19**  49.10*  91.22 106.47 103.04 96.13 72.88**
SD 28.85 37.73 25.25 25.83 35.95 24.71 16.75 22.83 12.97
N 20 20 16 16 15 15 15 15 15
%Diff -72.96 -59.14 -60.03 -41.4 -19.42 0.4 28.37 5.26  -30.40

[G] - Anova & Dunnett: * = p = 0.05; ** = p = 0.01

[G1] - Kruskal-Wallis & Dunn

Table 5 Summary of Maternal Performance, Mortality, Ovarian and Uterine Examinations and Litter Observations

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
Group Size - Females   22 22 22 22
Number of Females Pregnant [f] N+ve 22 21 21 21
% 100 95.5 95.5 95.5
Female with Live Fetuses [f] N+ve 22 21 19 19
% 100 100 90.5 90.5
Total Resorptionst [f] N+ve 0 0 2 2
% 0 0 9.5 9.5
Female with all Nonviable [f] N+ve 0 0 2 2
% 0 0 9.5 9.5
Terminal Euthanasia [f] N+ve 21 22 19 15*
% 95.5 100 86.4 68.2*
Unscheduled Death/Euthanasia [f] N+ve 1 0 3 7*
% 4.5 0 13.6 31.8*
Unscheduled Euthanasia [f] N+ve 1 * 0 2 7
% 4.5 0 9.1 31.8*
Delivered [f] N+ve 0 0 1 0
% 0 0 4.5 0
Female with Live Fetuses N+ve 21 21 18 15
% 100 100 100 100
Number of Corpora Lutea [k] Mean  11.5 10.3 10.3 10.9
SD 2.8 1.7 1.9 1.6
N 21 21 18 15
%Diff - -10 -10 -5.3
Number of Implantations [k] Mean  9.6 9 9.4 9.9
SD 3.5 2 2.5 2.1
N 21 21 18 15
%Diff - -5.9 -1.8 2.6
Pre-implantation Loss (%)[k] Mean  16.45 11.48 8.76 9.09
SD 21.04 16.13 18.33 13.98
N 21 21 18 15
%Diff - -30.21 -46.75 -44.72
Total Number of Fetuses [k] Mean  9 8.4 9.2 9.5
SD 3.5 2 2.5 2.1
N 21 21 18 15
%Diff - -6.4 2.4 5.7
Number of Live Fetuses [k] Mean  9 8.3 9.2 9.5
SD 3.5 2 2.5 2.1
N 21 21 18 15
%Diff - -6.9 2.4 5.7
Number of Dead Fetuses [k] Mean  0 0 0 0
SD 0 0 0 0
N 21 21 18 15
%Diff - - - -
Number of Early Resorptions [k] Mean  0.5 0.5 0.3 0.2
SD 0.9 0.7 0.6 0.4
N 21 21 18 15
%Diff - 0 -47 -61.8
Number of Late Resorptions [k] Mean  0.1 0.1 0 0.2
SD 0.7 0.4 0 0.4
N 21 21 18 15
%Diff -      
Total Number of Resorptions [k] Mean  0.7 0.7 0.3 0.4
SD 1.1 0.7 0.6 0.6
N 21 21 18 15
%Diff - 0 -58.3  -40
Post-implantation Loss (%) [k] Mean  6.91 8.59 2.83 4.43
SD 11.31 11.68 6.46 6.89
N 21 21 18 15
%Diff - 24.44 -58.95 -35.8
Number of Live Male Fetuses [k] Mean  4.7 4.1 4.3 5.1
SD 2.3 1.8 1.7 2.1
N 21 21 18 15
%Diff - -11.2 -8.3 10
Number of Live Female Fetuses [k] Mean  4.3 4.2 4.9 4.3
SD 1.8 1.7 1.8 1.6
N 21 21 18 15
%Diff - -2.2 14.1 1.1
Live Male Fetus/Litter (%) [k] Mean  51.39 49.06 46.62 52.93
SD 10.88 16.72 12.84 16.38
N 21 21 18 15
%Diff - -4.54 -9.29 2.99
Live Female Fetuses/Litter (%) [k] Mean  48.61 50.35 53.38 47.07
SD 10.88 17.43 12.84 16.38
N 21 21 18 15
%Diff - 3.58 9.82 -3.16
Mean Fetal Weight males (g) [G] Mean  39.55 42.3 38.57 35.35
SD 7.29 3.94 6.49 4.29
N 21 21 18 15
%Diff - 6.97 -2.47 -10.6

[f] - Fisher's Exact: * = p = 0.05

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett

Table 6 Summary of Fetal Malformations - Individual Descriptions

Dose Level (mg/kg/day) Female No. Fetus No. Malformation(s)#
0 4 L6 Forepaw, Both, Hyperflexion (E)
Forepaw, Left, Ectrodactyly (E)
Forepaw, Right, Misshapen (E)
Trunk, Omphalocele (E)
Aortic arch, Narrow (V)
Heart, Musc Ventricular Septal Defect (V)
Lung Lobe, Right medial, Fused (V)
Forepaw phalanges, 1 or more, Absent (S)
Metacarpal, 1 or more, Absent (S)
Radius, Left, Absent (S)
Sternebra, 1 or more, Absent (S)
L7 Rib, 1 or more, Fused (S)
R11 Lumbar vertebra, 1 or more, Supernumerary (S)
8 L6 Heart, Large (V)
11 L2 Sternebra, 1 or more, Fused, Severe (S)
12 L4 Diaphragm, Hernia (V)
20 24 L2@ Abdomen, Distended (E)
Heart, Large (V)
25 R6 Lumbar vertebra, 1 or more, Supernumerary (S)
36 R7 Lumbar vertebra, 1 or more, Supernumerary (S)
41 L1 Diaphragm, Hernia (V)
43 R9 Lumbar vertebra, 1 or more, Supernumerary (S)
60 56$ R14 Trunk, Omphalocele (E)
60 L5 Aortic arch, Dilatation (V)
Ductus arteriosus, Narrow (V)
Ventricular wall, Left, Thick (V)
Pulmonary trunk, Narrow (V)
R12 Lumbar vertebra, 1 or more, Supernumerary (S)
61 L1 Heart, Musc Ventricular Septal Defect (V)
L4 Lumbar vertebra, 1 or more, Supernumerary (S)
200 74 R7 Intestine, Distended (V)
80 L1 Forepaw, Left, Hyperflexion (E)
Trunk, Gastroschisis (E)
Lateral ventricle, Left, Dilatation, Severe (V)*
Lumbar centrum, 1 or more, Fused (S)
Lumbar vertebra, 1 or more, Hemivertebra (S)

#: Including external (E), visceral (V) and skeletal (S) examinations.

*: Cephalic examimation.

@: Dead fetus.$: Unscheduled necropsy.

Table 7 Summary of 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
  Number of Fetuses Examined 188 176 165 142
  Number of Fetuses Evaluated 191 179 165 145
  Number of Litters Examines 21 21 18 15
  Number of Litters Evaluated 21 21 18 15
Paw          
Forepaw, Both, Hyperflexion - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Forepaw, Left, Hyperflexion - Malformation Fetuses N (%) 0(0.00) 0(0.00)  0(0.0) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Paw/digit          
Forepaw, Left, Ectrodactyly - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Forepaw, Right, Misshapen - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Trunk          
Abdomen, Distended - Malformation Fetuses N (%) 0(0.00) 1(0.60) 0(0.00) 0(0.00)
   Litters N (%) 0(0.00) 1(4.8) 0(0.00) 0(0.00)
Trunk, Gastroschisis - Malformation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Trunk, Omphalocele - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
    0 mg/kg/day Group 1 20 mg/kg/day Group 2 60 mg/kg/day Group 3 200 mg/kg/day Group 4
  Number of Fetuses Examined 96 87 82 72
  Number of Fetuses Evaluated 191 179 165 145
  Number of Litters Examines 21 21 18 15
  Number of Litters Evaluated 21 21 18 15
Brain          
Lateral ventricle, Left, Dilatation, Severe - Malformation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(1.11)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
    0 mg/kg/day Group 1 20 mg/kg/day Group 2 60 mg/kg/day Group 3 200 mg/kg/day Group 4
  Number of Fetuses Examined 188 176 165 142
  Number of Fetuses Evaluated 191 179 165 145
  Number of Litters Examines 21 21 18 15
  Number of Litters Evaluated 21 21 18 15
Aortic arch          
Aortic arch, Dilatation - Malformation Fetuses N (%) 0(0.00) 0(0.00) 1(0.43) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)
Aortic arch, Narrow - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Artery          
Artery, Supernumerary - Variation Fetuses N (%) 0(0.00) 0(0.00) 1(0.62) 2(2.41)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 2(13.3)
Diaphragm          
Diaphragm, Hernia - Malformation Fetuses N (%) 1(0.68) 1(0.60) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 1(4.8) 0(0.00) 0(0.00)
Ductus arteriosus          
Ductus arteriosus, Narrow - Malformation Fetuses N (%) 0(0.00) 0(0.00) 1(0.43) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)
Gallbladder/bile duct          
Gallbladder, Bilobed - Variation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Gallbladder, Cyst - Variation Fetuses N (%) 1(0.43) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Gallbladder, Small - Variation Fetuses N (%) 4(1.64) 2(1.28) 1(0.43) 1(0.83)
   Litters N (%) 3(14.3) 2(9.5) 1(5.6) 1(6.7)
Gallbladder content, Abnormal consistency - Incidental Fetuses N (%) 0(0.00) 1(0.48) 0(0.00) 0(0.00)
   Litters N (%) 0(0.00) 1(4.8) 0(0.00) 0(0.00)
General          
Abdomen, Fluid filled - Variation Fetuses N (%) 1(0.34) 1(0.60) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 1(4.8) 0(0.00) 0(0.00)
Gonad          
Ovary, Both, Discolored - Incidental Fetuses N (%) 1(0.68) 0(0.00) 0(0.00) 2(1.11)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 1(6.7)
Ovary, Left, Discolored - Incidental Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 0(0.00)
Oviduct, Left, Cyst - Variation Fetuses N (%) 1(0.43) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Oviduct, Right, Cyst - Variation Fetuses N (%) 1(0.48) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Heart          
Atrium, Right, Large - Variation Fetuses N (%) 1(0.34) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Heart, Large - Malformation Fetuses N (%) 1(0.34) 1(0.60) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 1(4.8) 0(0.00) 0(0.00)
Heart, Musc Ventricular Septal Defect - Malformation Fetuses N (%) 1(0.37) 0(0.00) 1(0.51) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 1(5.6) 0(0.00)
Ventricle, Left ventricle, Large - Variation Fetuses N (%) 0(0.00) 0(0.00) 1(0.43) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)
Ventricular wall, Left, Thick - Malformation Fetuses N (%) 0(0.00) 0(0.00) 1(0.43) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)
Innominate artery          
Innominate artery, Absent - Variation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Intestine          
Intestine, Distended - Malformation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.74)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Kidney          
Renal papilla, Right, Absent - Variation Fetuses N (%) 1(0.34) 2(1.12) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 2(9.5) 0(0.00) 0(0.00)
Liver          
Lobe, Caudate process, Cyst - Variation Fetuses N (%) 0(0.00) 1(0.48) 1(0.51) 0(0.00)
   Litters N (%) 0(0.00) 1(4.8) 1(5.6) 0(0.00)
Lobe, Left medial, Supernumerary - Variation Fetuses N (%) 1(1.19) 0(0.00) 0(0.00) 1(0.83)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 1(6.7)
Lobe, Papillary process, Cyst - Variation Fetuses N (%) 1(1.19) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Lung          
Lobe, Accessory, Absent - Variation Fetuses N (%) 1(0.37) 2(1.19) 5(2.90) 1(0.74)
   Litters N (%) 1 (4.8)  2(9.5)  2(11.1) 1(6.7)
Lobe, Right medial, Fused - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Pulmonary trunk          
Pulmonary trunk, Narrow - Malformation Fetuses N (%) 0(0.00) 0(0.00) 1(0.43) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)
Spleen          
Spleen, Discolored - Incidental Fetuses N (%) 0(0.00) 1(0.68) 0(0.00) 0(0.00)
   Litters N (%) 0(0.00) 1(4.8) 0(0.00) 0(0.00)
Spleen, Small - Variation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Spleen, Supernumerary - Variation Fetuses N (%) 2(0.95) 3(1.21) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 2(9.5) 0(0.00) 0(0.00)
Ureter          
Ureter, Right, Retrocaval - Variation Fetuses N (%) 5(2.24)  5(2.86)  5(2.90)  1(0.61)
   Litters N (%) 4(19.0) 4(19.0)  4(22.2)  1(6.7)
Forelimb          
Forepaw phalanges, 1 or more, Absent - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Forepaw phalanges, 1 or more, Unossified - Variation Fetuses N (%) 2(1.12)  3(1.64)  2(1.02)  11(7.74)
   Litters N (%) 2(9.5)  3(14.3)  2(11.1)  4(26.7)
Metacarpal, 1 or more, Absent - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Metacarpal, 1 or more, Unossified - Variation Fetuses N (%) 0(0.00) 1(0.68) 0(0.00) 0(0.00)
   Litters N (%) 0(0.00) 1(4.8) 0(0.00) 0(0.00)
Radius, Left, Absent - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Ulna, Left, Misshapen - Variation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Hindlimb          
Hindpaw phalanges, 1 or more, Unossified - Variation Fetuses N (%) 23(10.08)  19(11.21)  23(12.01)  34(25.46)
   Litters N (%) 9(42.9)  6(28.6)  10(55.6)  12(80.0)
Talus, Both, Unossified - Variation Fetuses N (%) 9(3.53)  2(1.01)  1(0.51) 6(3.95)
   Litters N (%) 5(23.8)  2(9.5)  1(5.6)  4(26.7)
Talus, Left, Unossified - Variation Fetuses N (%) 1(0.37)  0(0.00)  1(0.46)  3(2.07)
   Litters N (%) 1(4.8)  0(0.0)  1(5.6)  3(20.0)
Talus, Right, Unossified - Variation Fetuses N (%) 1(0.34) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Pelvic girdle          
Ilium, Both, Misaligned - Variation Fetuses N (%) 4(2.01) 3(1.48)  6(3.32)  5(3.54)
   Litters N (%) 3(14.3)  3(14.3)  4(22.2)  5(33.3)
Pubis, Both, Unossified - Variation Fetuses N (%) 1(0.34) 0(0.00) 0(0.00) 1(0.74)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 1(6.7)
Pubis, Left, Unossified - Variation Fetuses N (%) 2(0.68) 0(0.00) 0(0.00) 1(0.61)
   Litters N (%) 2(9.5) 0(0.00) 0(0.00) 1(6.7)
Rib          
Costal cartilage, 1 or more, Fused - Variation Fetuses N (%)   0(0.00) 0(0.00) 0(0.00)
   Litters N (%)   0(0.00) 0(0.00) 0(0.00)
Rib, 1 or more, Fused - Malformation Fetuses N (%)   0(0.00) 0(0.00) 0(0.00)
   Litters N (%)   0(0.00) 0(0.00) 0(0.00)
Sternebra          
Sternebra, 1 or more, Absent - Malformation Fetuses N (%) 1(0.37) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Sternebra, 1 or more, Branched - Variation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Sternebra, 1 or more, Fused, Severe - Malformation Fetuses N (%) 1(0.48) 0(0.00) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8) 0(0.00) 0(0.00) 0(0.00)
Sternebra, 1 or more, Fused - Variation Fetuses N (%) 2(1.02) 0(0.00) 2(1.16) 1(1.67)
   Litters N (%) 2(9.5) 0(0.00) 2(11.1) 1(6.7)
Sternebra, 1 or more, Misaligned - Variation Fetuses N (%) 3(1.73) 0(0.00) 3(1.62) 1(0.61)
   Litters N (%) 2(9.5) 0(0.00) 2(11.1)  1(6.7)
Sternebra, 1 or more, Unossified - Variation Fetuses N (%) 37(16.94) 43(23.10)  39(21.99)  38(24.93)
   Litters N (%) 12(57.1) 16(76.2)  14(77.8)  12(80.0)
Sternebra, 1 or more, Incomplete ossification - Variation Fetuses N (%) 26(13.00) 17(10.26) 28(15.76)  9(6.35)
   Litters N (%) 11(52.4) 11(52.4)  10(55.6)  5(33.3)
Xiphoid cartilage, Hole - Variation Fetuses N (%) 2(1.27) 1(0.60) 0(0.00) 0(0.00)
   Litters N (%) 2(9.5) 1(4.8) 0(0.00) 0(0.00)
Supernumerary rib          
Cervical, 1 or more, Short - Variation Fetuses N (%) 3(1.93) 2(1.07)  2(1.16) 0(0.00)
   Litters N (%) 2(9.5) 2(9.5)  2(11.1) 0(0.00)
Thoracolumbar, 1 or more, Full - Variation Fetuses N (%) 97(49.95)  88(52.53)  70(38.87)  103(72.32)
   Litters N (%) 20(95.2)  19(90.5)  16(88.9)  15(100.0)
Thoracolumbar, 1 or more, Short - Variation Fetuses N (%) 50(28.97)  39(23.58)  29(20.96)  21(14.99)
   Litters N (%) 18(85.7)  17(81.0)  14(77.8)  11(73.3)
Vertebra          
Caudal vertebra, 1 or more, Incomplete ossification - Variation Fetuses N (%) 1(0.53)  2(1.16) 0(0.00) 0(0.00)
   Litters N (%) 1(4.8)  2(9.5) 0(0.00) 0(0.00)
Lumbar centrum, 1 or more, Fused - Malformation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Lumbar vertebra, 1 or more, Hemivertebra - Malformation Fetuses N (%) 0(0.00) 0(0.00) 0(0.00) 1(0.56)
   Litters N (%) 0(0.00) 0(0.00) 0(0.00) 1(6.7)
Lumbar vertebra, 1 or more, Misaligned - Variation   0(0.00) 0(0.00) 0(0.00) 1(0.56)
    0(0.00) 0(0.00) 0(0.00) 1(6.7)
Lumbar vertebra, 1 or more, Supernumerary - Malformation Fetuses N (%) 1(0.37)  3(1.67)  2(0.93) 0(0.00)
   Litters N (%) 1(4.8)  3(14.3)  2(11.1) 0(0.00)
Thoracic centrum, 1 or more, Incomplete ossification - Variation Fetuses N (%) 0(0.00) 0(0.00) 1(0.62) 0(0.00)
   Litters N (%) 0(0.00) 0(0.00) 1(5.6) 0(0.00)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

Applicant's summary and conclusion

Conclusions:
The maternal and developmental No Observed Adverse Effect Levels (NOAEL) for DCPD were established to be 20 mg/kg/day.
Executive summary:

In this OECD 414 test guideline study, rabbits were dosed orally with 20, 60 and 200 mg/kg/day DCPD. Treatment with DCPD resulted in a significant level of maternal toxicity at 200 mg/kg/day and five females were euthanised in extremis between post-coitum Days 13 and 18 due to severely reduced or no food consumption for at least 7 consecutive days and a significant body weight loss. These females also showed erected fur, hunched posture, thin appearance and/or reduced feces production. Additionally, one female at 200 mg/kg/day was sacrificed in extremis on Day 13 post-coitum with paralysis of both the hindlegs. This finding was correlated with a dislocation of lumbar vertebra no. 7 and accumulation of dark red fluid in the same region at necropsy. No explanation was found for the dislocated vertebra. However, given the convulsions observed for another female at 200 mg/kg/day, this animal may have had convulsions as well that could have caused the dislocation of the vertebra. As such, a relationship with the test item could not be excluded. At 60 mg/kg/day, one female was sacrificed in extremis on Day 23 post-coitum, based on similar findings as observed for the females at 200 mg/kg/day (severely reduced food consumption, body weight loss and reduced feces production). Lower fetal body weights were noted at 200 mg/kg/day, mainly in male fetuses. These changes were considered related to signs of delayed ossification (unossified fore and hindpaw phalanges) noted at the same dose level. There were 2 females of the 60 and 200 mg/kg/day groups each with early resorptions only. At the incidence observed, a relationship with the test item could not be excluded. It should be noted that finding might be related to severe maternal toxicity, as these 4 females were all preterm sacrifices. The maternal and developmental No Observed Adverse Effect Levels (NOAEL) for DCPD were established to be 20 mg/kg/day based on test item-related mortality and reduced food intake and body weight loss starting at 60 mg/kg/day and the incidence of females with resorptions only starting at 60 mg/kg/day as well as lower fetal body weights combined with delayed skeletal development at 200 mg/kg/day, respectively.