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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2019 - 18 Jul 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Testing was initiated as requested in ECHA decision TPE-D-2114359620-51-01/F. 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 in such a way that it does not require an unnecessary number of animals to accomplish its objectives. At this time, 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: yellow liquid
- Storage conditions: At room temperature
Specific details on test material used for the study:
Purity/Composition correction factor: 2.08 (based on solid content);
Stability in water for at least 6 hours at room temperature under normal laboratory light conditions was confirmed over the concentration range 1.0 to 200 mg/mL, and stability in water for at least 7 days in the refrigerator was confirmed over the concentration range 1.0 to 200 mg/mL, Charles River Project 518371.

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 188 - 278 g
- The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating) at the test facility
- Fasting period before study: No
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm)
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 36-53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 January 2019 To: 07 February 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. For the high dose group (1000 mL/kg bw/day) pure test item was used. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Charles River Study 518371). For verification of concentration and homogeneity duplicate sets of samples (approximately 500 mg) were analysed. Concentration results were regarded to be acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Homogeneity results were regarded to be acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Charles River Study 518371) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Not relevant, females were mated at the breeder.
Duration of treatment / exposure:
15 days (from Day 6 to Day 20 post-coitum, inclusive)
Frequency of treatment:
Once daily
Duration of test:
Animals were sacrificed Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a Combined 28-day Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test (OECD 422) by oral gavage of Dehyton® DC in rats (Charles River Study 518366, summarizd in Section 7.5.1), the preliminary results of a 90-day study (Charles River Study 20164357, summarizd in Section 7.5.1). It was attempted to produce graded responses to the test item. To minimize the risk of regurgitation of the test item risk, it was decided to lower the dosing volume by administering undiluted test item to the high-dose group in the current study. The lower dose groups was treated with the same volume of formulations of the test item in water to exclude any dose volume related effects. Final dose volume was 1.796 mL/kg bw, the dose volume for each animal was based on the most recent body weight measurement. To minimize foam formation, the dosing formulations were not stirred continuously during dose administration, but were swirled shortly before dosing instead.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for general health/mortality and moribundity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, cage debris was examined to detect premature birth

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

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

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
- The uterus and thyroid glands were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for the animal euthanized in extremis. Organ to body weight ratio (using the body weight on Day 21 post-coitum) was calculated.
- Histopathologic examination was performed on thyroid glands from all control and high dose animals.

OTHER:
- Blood was collected from all surviving females on day of sacrifice (rats not fasted o/n) to analyse Triiodothyronine (T3), Thyroid-Stimulating Hormone (TSH) and Thyroxine (T4) levels
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea;
- The weight of the (gravid) uterus (not for animals found dead or sacrificed before planned necropsy);
- The number of implantation sites;
- The number and distribution of live and dead fetuses;
- The number and distribution of embryo-fetal deaths;
- The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Fetal examinations:
Gross external examination was performed of the dam that was sacrificed before planned necropsy to detect late resorptions, recognizable fetuses, or normal implantations in development..

Litters of females surviving to scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are regarded 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).

Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and its weight (not for fetuses of animals sacrificed before planned necropsy) was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.

The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1972). The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique (1965). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (1926). Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing, this was concluded not to be of influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

The numbers of fetuses (litters) available for morphological examination were 229 (22), 229 (22), 214 (20) and 227 (22) in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. External examination was performed for all fetuses, visceral examination was performed for approximately half of the fetuses of all groups, and skeletal examination was performed for the other half of fetuses. Moreover, as during eviscerating of the fetuses prior to skeletal staining prominent visceral malformations were noticed in one fetus at 100 mg/kg bw/day and one fetus at 1000 mg/kg bw/day, that were both selected for skeletal examination, these fetuses were also subjected to a visceral examination. Besides that,
skeletal examination was added for one fetus at 100 mg/kg/day (A043-04) to further examine an externally noted malformation. One dam was sacrificed pre-term,the morphological examination of her fetuses was reported separately
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum;
Corrected Body Weight Gains: Body weight recorded on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus;
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum;
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/number of corpora lutea)*100;
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/number of implantation sites)*100.

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/(number of viable fetuses/litter))*100.
Historical control data:
Historical Control Data on Crl:WI(Han) (outbred, SPF-Quality), Gestation Day 21 from studies performed 2014 - 2018 are included in the report and attached in the summary.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted in animals treated up to 1000 mg/kg bw/day. Salivation was seen after dosing among animals treated at 300 and 1000 mg/kg bw/day with increasing incidence and severity (mild to moderate) with increasing dose levels. This finding was regarded as not toxicologically relevant, taking into account the nature of the effect and its time of occurrence (i.e. after dosing). It was regarded as a physiological response rather than a sign of systemic toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was related to treatment with the test item. At 300 mg/kg bw/day, one female was euthanized in extremis on Day 16 post-coitum for animal welfare reasons as it presented with a severe protruding spine. During veterinary examination, the animal also showed signs of severe pain, including restlessness, fear, abnormal posture, hunched posture, abnormal gait, vocalization, thickened area of the back
and hypersensitivity to touch of the back area. At necropsy, the spine was noted to be misshapen in the abdominal region, corresponding to the findings at clinical observation and were therefore not related to treatment with the test item. No other abnormalities were noted. The animal was pregnant, and carried normal developing fetuses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the study period.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly lower serum levels of thyroid stimulating hormone (TSH) were noted at 300 and 1000 mg/kg bw/day (0.76x and 0.74x of controls, respectively), however without reaching statistical significance and within the historical control range. This slight decrease was mainly caused by 4 animals in the control group, for which TSH serum levels were above the historical control range (Historical control data for pregnant Wistar Han rats TSH (μLU/mL); mean (P5-P95): 0.407 (0.1270-0.7600), n=52; Total T3 (ng/dL); mean (P5-P95): 64.7 (51.00-84.50) n=47; Total T4 (μg/dL); mean (P5-P95): 2.12 (1.430-3.090), n=50), data for this study are included as attachment. This slight difference in TSH levels was therefore regarded to be unrelated to the test-item. Serum levels of total T3 and T4 were regarded to be unaffected by treatment up to 1000 mg/kg/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights and thyroid:body weight ratios of treated animals were similar to those of control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were regarded to have arisen as a result of treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in the thyroid glands of rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The number of early resorptions slightly increased upon increasing dose levels. However, no statistical significance was reached and all values remained within historical control data. Therefore, this slight increase was regarded to be unrelated to the test item.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At 300 mg/kg bw/day, one female was not pregnant. The pregnancy rate was within the normal range that could be expected for rats of this age and strain, therefore, this single occurrence was regarded to be unrelated to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed up to and including the highest dose tested (1000 /kg bw/day)

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal body weight (both sexes) was unaffected by treatment up to 1000 mg/kg bw/day.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg bw/day.
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.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on external morphology following treatment up to 1000 mg/kg bw/day. At 100 mg/kg/day, two fetuses were externally malformed. One fetus had an omphalocele and another fetus had no lower jaw and had a cleft palate. Skeletal examination substantiated the findings of the latter fetus, whereby the lower jaw was anomalous and appeared to be only visible after staining. Due to the single occurrence and occurrence at the low dose level, these malformations were regarded to be of spontaneous origin. No external variations were observed in animals treated up to 1000 mg/kg bw/day.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on skeletal morphology following treatment up to 300 mg/kg bw/day. Skeletal examination revealed four different malformations, in addition to the underlying malformations in the fetus discussed before at 100 mg/kg bw/day. Bent limb bones occurred in two control fetuses, and in two fetuses at 100 and 1000 mg/kg bw/day, respectively. In addition, one fetus had fused skull bones and a vertebral anomaly without associated rib anomaly. A vertebral anomaly occurred in a single fetus of the control and 100 mg/kg bw/day groups as well. At 100 mg/kg bw/day, one fetus was observed with sternoschisis. All malformations were also previously noted among historical control fetuses and due to the low incidences and group distribution, they were regarded not to be test item-related. Among skeletal variations, 7th cervical ossification site showed a remarkable increase at 1000 mg/kg bw/day. Mean litter incidences were 1.5%, 5.2%, 4.6% and 11.3% per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at the high dose was not statistically significant, but was outside the historical control data range (Historical Control Data Prenatal Developmental Studies Rat GD21 includes 49 studies, performed between 2014 and 2018. In total 6219 (1070) fetuses (litters) were skeletally examined. 7th Cervical ossification site(s); mean (p5-p95): 3.8% (0.0-8.7%)). Therefore, a test item-related effect cannot be excluded. All other variations were either limited to the control group only or occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, these variations were regarded not to be test item-related.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral malformations occurred in two fetuses at 100 mg/kg bw/day and one fetus each at 300 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day, one fetus had a right-sided aortic arch, ventricular septum defect and no eyes. At 300 mg/kg bw/day, one fetus was also affected with a ventricular septum defect and was in addition observed with the absence of the ductus arteriosus, situs inversus and abnormal lung lobation. The latter two findings were also observed at 100 mg/kg bw/day in one fetus together with three cardiovascular malformations (ventricular septum defect, interrupted aortic arch and retro-esophageal ductus arteriosus). The other affected fetus at 100 mg/kg bw/day had abnormal lung lobation and transposition of the great vessels.
Although no dose-response was observed in any of the cardiovascular malformations, a test item-related effect could not be excluded, as in case of right-sided aortic arch the incidence was above historical control data range (Historical Control Data Prenatal Developmental Studies Rat GD21 includes 49 studies, performed between 2014 and 2018. In total 6234 (1071) fetuses (litters) were viscerally examined. Aortic arch right sided: mean (p5-p95): 0.0% (0.0-0.3%); summary incidence: 2 (2) fetuses (litters); Absence of the eye: mean (p5-p95): 0.2% (0.0-1.5%); summary incidence: 7 (7) fetuses (litters); Abnormal lobation of the lung: mean (p5-p95): 0.1% (0.0-0.8%); summary incidence: 4 (4) fetuses (litters); Situs inversus: mean (p5-p95): 0.2% (0.0-1.1%); summary incidence: 14 (14) fetuses (litters); Ventricular septum defect: mean (p5-p95): 0.0% (0.0-0.0); summary incidence: 1 (1) fetuses (litters)); (aortic arch right-sided: 0.9% at 1000 mg/kg/day; ventricular septum defect: 0.9% at 100, 300 and 1000 mg/kg/day) or were previously not observed in any of the control fetuses in studies performed at this Test Facility between 2014 and 2018 (interrupted aortic arch, retro-esophageal ductus arteriosus, absent ductus arteriosus and transposition of the great vessels).
The remaining malformations were regarded to be of spontaneous origin: the incidences of absence of the eyes (0.6% at 1000 mg/kg/day), situs inversus (0.6% and 1.3% at 100 and 300 mg/kg/day, respectively) and abnormal lobation of the lung (1.5% and 1.3% at 100 and 300 mg/kg/day, respectively) remained within historical control ranges or were only slightly above. In addition, no dose-related trend could be established for the latter two findings. Only one visceral variation (small supernumerary liver lobes) was observed in this study. At the low incidence it occurred, this was regarded not to be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal ano-genital distance (both sexes) noted after treatment up to 1000 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
visceral malformations
Remarks on result:
other: The incidence and severity of the effects do not increase dose-dependently, therefore it cannot be excluded that the effects are not related to test item exposure.

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Cardiovascular malformations (including ventricular septum defects) at 100, 300 and 1000 mg/kg bw/day, in 4 (4) fetuses (litters) in total. As the incidence and severity of the effects do not increase dose-dependently, it cannot be excluded that the effects are not related to test item exposure.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

The data per dose for all dose groups (and for individual rats) in tabular form are included as attached background material. Results of statistical analyses are indicated (where applicable). In addition, historical control data of fetal examinations at this test facility and with this rat strain are attached.

Applicant's summary and conclusion

Conclusions:
A prenatal developmental toxicity study was performed in rats according to OECD/EC guideline 414 and GLP principles. The maternal No Observed Adverse Effect Level (NOAEL) for Dehyton® DC was determined to be at least 1000 mg/kg bw/day. No developmental NOAEL could be determined, as severe cardiovascular malformations were observed at all dose levels. As the incidence and severity of the effects do not increase dose-dependently, it cannot be excluded that the effects are not related to test item exposure.
Executive summary:

A prenatal developmental toxicity study was performed according to OECD/EC guideline 414 and GLP principles. Time-mated female Wistar Han rats were treated with Dehyton® DC from Day 6 to 20 postcoitum, inclusive, by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle (water) alone. Correct dosing was confirmed by chemical analyses showing that the formulations were homogeneous and that the concentrations were accurate. No treatment-related mortality occurred during the study period and no test item-related changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, thyroid hormone levels, thyroid weights, microscopic appearance of the thyroids, and fertility parameters (pregnancy rate, numbers of corpora lutea and implantation sites, and pre-implantation loss).

Visceral examination of fetuses revealed severe cardiovascular malformations at 100, 300 and 1000 mg/kg bw/day, in 4 (4) fetuses (litters) in total. At 1000 mg/kg bw/day, one fetus had a right-sided aortic arch, ventricular septum defect and no eyes. At 300 mg/kg bw/day, one fetus had a ventricular septum defect, absence of the ductus arteriosus, situs inversus and abnormal lung lobation. At 100 mg/kg bw/day, two fetuses were viscerally malformed: one fetus had abnormal lung lobation and transposition of the great vessels, and the other fetus presented itself with situs inversus, abnormal lung lobation, interrupted aortic arch (between right subclavian and right carotid), retro-esophageal ductus arteriosus and a ventricular septum defect. Although no dose-response was observed in any of the cardiovascular malformations, it cannot be excluded that there is a possible test item-related relationship taken the above in consideration. Mean litter incidences of a 7th cervical ossification site were 1.5%, 5.2%, 4.6% and 11.3% per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at the high dose was not statistically significant, but was outside the historical control data range1. Therefore, a test item-related effect cannot be excluded for this type of skeletal variation. No test item-related changes were noted in litter size, post-implantation loss, sex ratio, fetal body weights, fetal ano-genital distance, external and skeletal malformations and variations. In conclusion, based on the results of this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for Dehyton® DC was determined to be at least 1000 mg/kg bw/day. No developmental NOAEL for Dehyton® DC could be determined, as severe cardiovascular malformations were observed at all dose levels. However, as the incidence and severity of the effects do not increase dose-dependently, at this point it cannot be excluded that the effects are not related to test item exposure.