3a,4,7,7a-tetrahydro-4,7-methanoindene

Developmental toxicity / teratogenicity

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

Report date:

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day

Doses / concentrations 4

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.