2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 Apr 2022 to 22 May 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Test material form:

solid: nanoform

Details on test material:

Yellow powder
Batch 200007P040
Storage conditions: ambient (room temperature)
Expiry date: 01-Feb-2030

- Shape of particles: spherical
- Surface area of particles: 46.6 m2/g
- Crystal structure: crystalline
- Coating: none

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- CAS No.: 76199-85-4
- Batch identification: 200007P040
- Content: 92.5% (based on 7.5 g/100 g Soxhlet extractables) w (pigment) = 91.7 g/100 g
- Homogeneity: Given
- Storage stability: Expiry date: 01 Feb 2030, the stability of the test substance under storage conditions over the test period was guaranteed by the
sponsor, and the sponsor holds this responsibility
- Physical state/ appearance: Solid/ yellow
- Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: body weight of the pregnant animals on day 0 varied between 162.6 – 221.2 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of food and water consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals. Dust-free wooden bedding was used in this study. As nesting material, compacted fibers of softwood was offered toward the end of gestation in all pregnant females. For enrichment, wooden gnawing blocks and play tunnel were offered. The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.
- Diet: ad libitum, mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland
- Water: ad libitum, potable tap water in water bottles
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26-28 Apr 2022 (supply of animals and beginning of acclimatization of the three cohorts) To: 16-18 May 2022 (sacrifice of the three cohorts)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC suspension in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Rate of preparation of test substance: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with vehicle in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
- Concentration in vehicle: 0, 1, 3, 10 g/100 mL, respectively
- Amount of vehicle (if gavage): standard dose volume of 10 mL/kg body weight was used for each test group, the calculation of the administration volume was based on the most recent individual body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study.
Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

Stability: The stability of the test substance in 0.5% CMC suspension in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study with the same batch.

Homogeneity analyses of the test substance preparations: The homogeneous distribution of the test substance in the vehicle (0.5% CMC suspension in deionized water) was demonstrated.

Concentration control analyses of the test substance preparations: The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant: Animals were paired by the breeder ans supplied on GD 0 (= detection of vaginal plug/sperm). The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

Duration of treatment / exposure:

The test substance preparations were administered to the animals from implantation to one day prior to the expected day of parturition (GD 6 to GD 19). The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water) in the same way.

Frequency of treatment:

daily, always at approximately the same time in the morning

Duration of test:

The test substance preparations were administered to the animals from implantation to one day prior to the expected day of parturition (GD 6 to GD 19). On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined and fetuses were removed from the uterus and investigated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low-dose level

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid-dose level

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High-dose level

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0-5 and 20), during treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20
- The body weight change of the animals was calculated based on the obtained results

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 under isoflurane anesthesia by decapitation, in randomized order
- Organs examined: After the dams had been sacrificed, they were necropsied and assessed for gross pathology, special attention being given to the reproductive organs
- The following organ weights were determined in all dams sacrificed on schedule: thyroid glands (with parathyroid glands) (fixed), all paired organs were weighed together (left and right)
- The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution from all dams, sacrificed on schedule: thyroid glands (with parathyroid glands)
- Fixation of thyroid glands from all dams was followed by histotechnical processing (H&E staining) and examination by light microscopy

OTHER:
Corrected (net) body weight gain: the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, on GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order
The uteri and the ovaries were removed and the following data were recorded:
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes, classified as live fetuses and dead implantations
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ sampling and weights) were conducted by technicians unaware of treatment group in order to minimize bias.

Blood sampling:

Blood samples were taken from all females by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood and serum examinations were carried out in a randomized sequence. The parameters depicted in table 1 in "Any other information on materials and methods incl. tables" were determined with EDTA-K3 as anticoagulant using a particle counter. An automatic analyzer was used to examine the clinicochemical parameters (see table 2). The concentrations of TSH were determined by radioimmunassay (RIA), using commercially available RIA test kits and a Gamma-Counter (table 3).

Fetal examinations:

- External examinations: Yes: At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.
- Soft tissue examinations: Yes: The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
- Skeletal examinations: Yes: The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
- Anogenital distance of all live rodent pups: Yes, anogenital index = (anogenital distance [mm]) / (cubic root of fetal weight [g])

Statistics:

Please refer to tables 4-6 in "Any other information on materials and methods incl. tables".

Indices:

Conception rate (in %) = (number of pregnant animals) / (number of fertilized animals) x 100

Preimplantation loss (in %) = (number of corpora lutea – number of implantations) / (number of corpora lutea) x 100

Postimplantation loss (in %) = (number of implantations – number of live fetuses) / (number of implantations) x 100

Historical control data:

Please see the attachments in "Overall remarks, attachments".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All females of the test substance treated groups (test group 1-3; 100, 300 or 1000 mg/kg bw/d) showed yellowish discolored feces during the treatment period until terminal sacrifice (GD 20). It was initially observed on GD 7 in the mid- and high-dose groups and on GD 8 in the low-dose group. This feces discoloration mirrors the presence of the yellowish test substance in the gastrointestinal tract but was assessed as of no toxicological relevance.
Piloerection was recorded for one mid-dose female (300 mg/kg bw/d) on GD 19 and 20, and for all high-dose females (1000 mg/kg bw/d) from GD 10 onwards until terminal sacrifice.
Additionally, for one high-dose female (1000 mg/kg bw/d) hypothermia (after treatment) was recorded on GD 10-12.
One low-dose female (100 mg/kg bw/d) had a palpable mass in the mammary line on GD 18-20. Gross pathological examination revealed an enlarged lymph node in the abdominal cavity (see gross pathology findings). Since this was an isolated event in one single dam, it was assessed as incidental and not treatment-related and adverse.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only pregnant dams were used for the calculations of body weight and body weight change.
As a consequence of the lower food consumption, the mean body weights (BW; table 7 in "Any other information on results incl. tables") of the high-dose dams (1000 mg/kg bw/d) were statistically significantly lower from GD 8 onwards until scheduled sacrifice on GD 20 (up to 10% below the concurrent control group). The body weight change (BWC; table 8) of the high-dose dams was also distinctly lower at the beginning of the treatment period, attaining statistical significance on GD 6-13 (on GD 6-8, the dams even lost weight (- 2.3 g** [p≤0.01] vs. 6.6 g in control). If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the high-dose dams gained about 35% or 24% less weight than the controls (attaining statistical significance). The mean BW of the mid-dose dams (300 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period. However, the BWC of the dams in test group 2 was statistically significantly reduced during GD 6-8 (about 80% below controls) but recovered afterwards. If calculated for the entire treatment period (GD 6-19), the mid-dose dams gained a comparable weight to the controls. The mean BW and the average BWC of the low-dose dams (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of corrected (net) body weight gain.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically significantly lower in test group 3 (1000 mg/kg bw/d - about 69% below control). Furthermore, the carcass weight of the high-dose dams was statistically significantly reduced in comparison to the control group (about 10% below controls).
The corrected body weight gain of test groups 1 and 2 (100 and 300 mg/kg bw/d) revealed no differences of any biological relevance to the corresponding control group. Moreover, mean carcass weights of test groups 1 and 2 remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Only pregnant dams were used for the calculations of mean maternal food consumption.
In test group 3 (1000 mg/kg bw/d), the mean food consumption was statistically significantly lower during the entire treatment period (up to -42% (GD 8-10) below control). If calculated for GD 6-19, the high-dose dams consumed about 29% less food than the controls. The mean food consumption in test group 2 (300 mg/kg bw/d) was statistically significantly lower on GD 6-13 (up to 16% below control) but recovered afterwards and even exceeded the control value at the end of the treatment period. If calculated for the entire treatment period (GD 6-19), the mid-dose dams consumed about 8% less food than the controls. The mean food consumption of the dams in test group 1 (100 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At gestation day 20 in dams of test groups 2 and 3 (300 and 1000 mg/kg bw/d) absolute reticulocyte and platelet counts were significantly increased. Absolute reticulocyte counts were already significantly increased in dams of test group 1 (100 mg/kg bw/d). However, the values of both parameters were within historical control ranges (dams, absolute reticulocytes 115.3- 181.0 Giga/L, platelets 706.917 Giga/L). Therefore, these alterations were regarded as incidental and not treatment-related. In dams of test group 3 (1000 mg/kg bw/d) absolute and relative lymphocyte and absolute large unstained cell (LUC) counts were significantly increased. Consistently to the higher relative lymphocyte counts, relative neutrophil counts were significantly decreased. Absolute lymphocyte and neutrophil counts in dams of test group 3 were within historical control ranges whereas absolute LUC counts were slightly above this range (dams, absolute lymphocytes 2.58-4.35 Giga/L, absolute neutrophils 1.50-2.23 Giga/L, absolute LUC 0.01-0.02 Giga/L). However, relative LUC counts were so small (0.5%) that the chance to further evaluate these cells in a manual differential blood cell counts (1 of 200 cells) was very low. Therefore, the alterations of lymphocyte and neutrophil counts were regarded as incidental and not treatment-related, whereas the increase of absolute LUC counts was assessed as non-adverse, if at all treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 20 in dams of test group 3 (1000 mg/kg bw/d) total bilirubin values were significantly increased whereas total protein, albumin and globulin values were significantly decreased. These alterations were regarded as treatment-related and adverse.
Total protein, albumin and globulin values were already significantly lower in dams of test group 2 (300 mg/kg bw/d) compared to study controls. Albumin values were within the historical control range whereas total protein and globulin values were slightly below their ranges (dams, total protein 56.50-63.74 g/L, albumin 31.14-35.55 g/L, globulins 24.39-29.68 g/L). In fact, the total protein level decrease was based on lower globulin values, because they were the sum of globulins and albumin. Therefore, only one liver parameter, i.e. globulins, was changed in dams of test group 2. Therefore, this change was regarded as treatment-related but not adverse.
The following significant changes were regarded as incidental and not treatment-related because the values were within historical control ranges: increased alanine aminotransferase (ALT) activities and cholesterol values in dams of test group 3 (1000 mg/kg bw/d); increased inorganic phosphate levels in dams of test groups 2 and 3 (300 and 1000 mg/kg bw/d; dams ALT 0.72-1.38 μkat/L, cholesterol 1.82-2.43 mmol/L, inorganic phosphate 1.21-1.99 mmol/L). In dams of test groups 1 and 3 (100 and 1000 mg/kg bw/d) triglyceride levels were significantly decreased, but the change was not dose dependent. Therefore, the mentioned alterations in this section were regarded as incidental and not treatment-related.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In dams of test group 3 (1000 mg/kg bw/d) T3 and T4 levels were significantly decreased. However, the values of both parameters were within historical control ranges (dams, T3 0.56- 1.31 nmol/L, T4 23.17-38.52 nmol/L) whereas at least T4 values in study controls were in the upper part of this range. Neither any thyroid weight change nor any relevant histologic finding in the thyroid gland were observed. Therefore, T3 and T4 decreases in dams of test group 3 were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights and mean organ weights.
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Absolute weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights: All mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A yellowish discolored content of the stomach as well as of the entire intestine was recorded in 7 (out of 25) low-dose females and in all mid- and high-dose females, respectively. These discolorations mirrored the presence of the test substance in the gastrointestinal tract. They were considered to be non-adverse, causing no toxic effects by themselves.
Additionally, three spontaneous findings were noted in individual females of test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d). An empty stomach was seen in control female, while one low-dose female had an enlarged lymph node at the abdominal cavity. A diaphragmatic hernia was recorded for one mid-dose female. Since these findings occurred in single females without a dose-response relationship, they were assessed as incidental and not treatment-related.
No gross lesions were observed in any of the test groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 1, a minimal increase in the incidence of altered colloid was noted in the thyroid glands. As no organ weight changes were present and no dose-response relationship was observed, this finding was considered incidental and not treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Maternal developmental toxicity

Details on maternal toxic effects:

Reproduction data:
Only pregnant dams with scheduled sacrifice (GD 20) were used for the summary of reproduction data.
The conception rate was 92% in test groups 0 and 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age.
A 100% postimplantation loss was observed in high-dose female (1000 mg/kg bw/d). Twelve early resorptions were recorded. Complete litter loss in single animals is a common finding and since only one dam was affected, this isolated finding was regarded as incidental and without relation to the treatment.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weight of test group 3 was statistically significantly lower in comparison to the concurrent control group (3.1 g** vs. 3.6 g in control, all viable fetuses). However, the highdose value was well within the historical control range (HCD both sexes: mean 3.6 g [2.4 – 4.3]). The observed reduced fetal body weights of the high-dose group (1000 mg/kg bw/d) corresponded to a delay in ossification as indicated by a broad range of skeletal variations (see below). The findings in the high-dose fetuses occurred in presence of maternal toxicity and were in the same order of magnitude as reduced food consumption and body weight gain of the dams. Therefore, they were assessed as treatment-related but not as adverse.
The mean fetal weights of test groups 1 and 2 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

The anogenital distance of the male fetuses of test group 3 (1000 mg/kg bw/d) was reduced in comparison to the concurrent control group (-19%; attaining statistical significance) and outside the historical control range (HCD ag distance: mean 2.8 mm [2.3 – 3.4]).
Furthermore, the anogenital index of the male fetuses in test group 3 was statistically significantly reduced (mean 1.50, 14% below control). However, this value was within the historical control range (HCD ag index: mean 1.85 [1.46 – 2.17]).
The anogenital distance and anogenital index in the female fetuses in test group 3 as well as in all male and female fetuses in test groups 1 and 2 were comparable to the concurrent control values.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external malformations:
An external malformation was recorded in one female high-dose fetus. This fetus had an associated soft tissue malformation. Since this was an isolated event and ‘anasarca’ can be found in the historical control data at comparable incidences, it was assessed as neither treatment-related nor adverse. The overall incidences (tables 9 and 10) of external malformations were comparable to those found in the historical control data.

For fetal external variations and external unclassified observations, see result chapter "Other effects".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal malformations:
Skeletal malformations were detected in one fetus of the low-dose group, three fetuses of the mid-dose group and in two fetuses of the high-dose group, as shown in tables 15 and 16.
‘Misshapen basisphenoid’ occurred in one single fetus of test group 1 and can be found in the historical control data, thus, it was assessed as incidental.
The findings ‘shortened scapula’ (mean% 0.0/0.0/1.3/1.4 ) and ‘shortened humerus’ (mean% 0.0/0.0/0.7/0.8) occurred in single fetuses of test groups 2 and 3, twice as combination in one fetus. Both findings can be found in the historical control data at comparable incidences, thus, it was regarded to be incidental and not treatment-related.
The incidence of ‘bent rib’ (mean% 0.0/0.0/2.0*/0.0 [p  0.05]) was statistically significantly increased in test group 2. Since this mid-dose value was within the historical control range (HCD: mean% 0.1 [0.0 – 2.0]) and the increase was not dose related, it was assessed as incidental.
The addition of skeletal malformations resulted in a slightly increased affected fetuses per litter incidence in test group 2 which attained statistical significance. However, due to the lack of dose-response relationship and the fact, that the overall incidences of all test groups were well within the historical control range (HCD total skeletal malformations: mean% 1.1 [0.0 – 3.9]), this increase was considered to be spontaneous in origin and not treatment-related.

For fetal skeletal variations and skeletal unclassified observations, see result chapter "Other effects".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Weight of placentae:
The mean placental weight of test group 3 (1000 mg/kg bw/d) was statistically significantly lower in comparison to the concurrent control group. However, the high-dose mean value (0.37 g**, both sexes combined [p≤0.01]) was within the historical control range (HCD both sexes: mean 0.47 g [0.36 - 0.97]). Notably, for the dams, reduced food consumption and body weight gain was observed in the high dose group.
The mean placental weights of the low- and mid-dose groups (100 and 300 mg/kg bw/d) were comparable to the corresponding control group.

Fetal external variations:
No external variations were recorded.

Fetal external unclassified observations:
One external unclassified observation, placentae fused, was recorded in one mid-dose fetus without relation to dose (table 11). In addition, this finding can be found in the historical control data, thus, it was considered as not treatment-related.

Fetal soft tissue malformations:
Soft tissue malformations were detected in single fetuses of test groups 0, 2 and 3, as shown in tables 12 and 13. One high-dose fetus had an associated external malformation. These findings were single events in single fetuses and both can be found in the historical control data at comparable incidences. Thus, they were assessed as not treatment-related.
The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Fetal soft tissue variations:
Three soft tissue variations were detected: short innominate and dilated ureter in test groups 0 and 1, respectively, and dilated renal pelvis in test groups 0-2 (table 14). The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were assessed as not treatment-related.

Fetal soft tissue unclassified observations:
No soft tissue unclassified observations were recorded.

Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose (table 17). The overall incidences of skeletal variations were comparable to the historical control data. All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table 18. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in italicized bold types. As can be seen in table 18, the finding ‘incomplete ossification of sacral arch (cartilage present)’ was not related to dose and/or the mean values were clearly inside the historical control ranges. Therefore, this finding was assessed as not treatment-related.
The findings ‘unossified sternebra (unchanged cartilage)’ and ‘wavy rib’ were statistically significantly increased in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The mid-dose values were clearly within the historical control range, respectively. All other skeletal variations recorded in the table 18 were statistically significantly increased in test group 3 (1000 mg/kg bw/d) and outside the respective historical control ranges.

Fetal skeletal unclassified cartilage observations:
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (table 19). The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to the dose.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations:
External, soft tissue and skeletal malformations were noted in one control, one low-dose, four mid-dose and three high-dose fetuses (0, 100, 300 and 1000 mg/kg bw/d). Four fetuses carried more than one malformation: for one male mid-dose fetus shortened scapula and bent rib was recorded, while one male mid-dose fetus showed a shortened scapula, bent rib and shortened humerus. One female high-dose fetus had anasarca combined with a situs inversus and for one male high-dose fetus a shortened scapula and a shortened humerus was recorded. The finding ‘bent rib’ which was seen in a multiple malformed fetus in test group 2 occurred also in two other fetuses of the same test group. Further malformations, i.e. hydronephrosis and misshapen basisphenoid, were observed in individual fetuses. These findings were single cases which occurred without relation to treatment and can be found in the historical control data. Since no ontogenetic pattern was recognizable in the other offspring of the test groups, these findings were regarded as not treatment-related. The total incidences of malformations are summarized in table 20.
External variations did not occur in any of the fetuses in this study. Three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dosing (table 21). The majority of individual variations were equally distributed about the different test groups, if normal biological variation was taken into account, and can be found in the historical control data at a comparable frequency. The findings ‘unossified sternebra (unchanged cartilage)’ and ‘wavy rib’ were statistically significantly increased in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The mid-dose values were clearly within the historical control range, respectively, and thus, evaluated as of no toxicological relevance. However, ‘incomplete ossification of supraoccipital (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘incomplete ossification of nasal (unchanged cartilage)’, ‘basioccipital hole(s)’, ‘incomplete ossification of thoracic centrum (unchanged cartilage)’, ‘supernumerary thoracic vertebra’, ‘incomplete ossification of sacral arch (cartilage present)’, ‘unilateral ossification of sternebra (unchanged cartilage)’ and ‘incomplete ossification of pubis’ were statistically significantly increased in test group 3 (1000 mg/kg bw/d) and outside the respective historical control ranges. These findings indicated a slight delay in ossification, which is known to be reversible. Particularly, no changes in the underlying cartilages and no abnormalities in the respective bone structures were observed which qualifies them to be of relatively low toxicological concern. Ossification of skull bones and vertebral arches occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). In this study, distinct maternal toxicity was observed at the highest-dose level. In addition, the observed skeletal variations did not influence the overall rate of fetal variations (table 21). Overall, the above-mentioned findings in the high-dose group (1000 mg/kg bw/d) were assessed as minor effects indicating a developmental delay and were, therefore, assessed as treatment-related but not as adverse. Soft tissue unclassified observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the external unclassified observation and skeletal unclassified cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings did not suggest any relation to treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See "Remarks"

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 7: Mean maternal body weights during gestation [in g]

			TEST GROUP 0 0 mg/kg bw/d		TEST GROUP 1 100 mg/kg bw/d	TEST GROUP 2 300 mg/kg bw/d	TEST GROUP 3 1000 mg/kg bw/d
DAY	0	MEAN	194.2	D	197.0	193.8	192.4
		S.D.	8.95		13.92	10.99	8.81
		N	23		25	25	23
DAY	1	MEAN	206.9	D	210.9	207.6	205.2
		S.D.	9.61		14.03	12.19	9.17
		N	23		25	25	23
DAY	3	MEAN	216.3	D	220.3	215.8	213.1
		S.D.	10.70		14.82	12.36	9.62
		N	23		25	25	23
DAY	6	MEAN	225.7	D	229.8	226.3	222.9
		S.D.	11.85		15.76	12.85	10.93
		N	23		25	25	23
DAY	8	MEAN	232.3	D	235.8	227.6	220.6*
		S.D.	12.09		16.68	13.66	9.37
		N	23		25	25	23
DAY	10	MEAN	240.3	D	244.1	235.8	221.0**
		S.D.	13.34		16.70	13.55	6.88
		N	23		25	25	23
DAY	13	MEAN	254.3	D	259.9	249.1	228.0**
		S.D.	14.38		18.32	14.71	9.84
		N	23		25	25	23
DAY	15	MEAN	265.1	D	271.4	260.5	237.5**
		S.D.	14.61		19.22	15.64	9.56
		N	23		25	25	23
DAY	17	MEAN	282.4	D	290.6	278.7	253.2**
		S.D.	16.68		19.87	16.96	10.82
		N	23		25	25	23
DAY	19	MEAN	305.4	D	314.7	302.5	274.4**
		S.D.	18.16		21.15	19.13	15.32
		N	23		25	25	23
DAY	20	MEAN	318.5	D	326.4	315.6	287.3**
		S.D.	18.37		22.54	21.01	17.52
		N	23		25	25	23

Statistics: D = Dunnett-test (two-sided); * : p <= 0.05, ** : p <= 0.01

 

 

Table 8: Mean maternal body weight change during gestation [in g]

					TEST GROUP 0 0 mg/kg bw/d		TEST GROUP 1 100 mg/kg bw/d	TEST GROUP 2 300 mg/kg bw/d	TEST GROUP 3 1000 mg/kg bw/d
DAYS	0	TO	1	MEAN	12.7	D	13.9	13.7	12.8
				S.D.	3.00		3.48	4.40	3.42
				N	23		25	25	23
DAYS	1	TO	3	MEAN	9.5	D	9.4	8.2	7.9
				S.D.	3.36		3.01	2.63	3.19
				N	23		25	25	23
DAYS	3	TO	6	MEAN	9.4	D	9.5	10.5	9.9
				S.D.	3.13		2.94	3.09	3.47
				N	23		25	25	23
DAYS	6	TO	8	MEAN	6.6	D	6.0	1.3**	-2.3**
				S.D.	2.28		3.23	3.74	5.78
				N	23		25	25	23
DAYS	8	TO	10	MEAN	8.0	D	8.3	8.2	0.3**
				S.D.	3.11		3.96	1.93	4.71
				N	23		25	25	23
DAYS	10	TO	13	MEAN	14.0	D	15.8	13.3	7.0**
				S.D.	3.18		3.28	3.76	8.25
				N	23		25	25	23
DAYS	13	TO	15	MEAN	10.8	D	11.6	11.4	9.5
				S.D.	2.11		2.60	3.67	5.67
				N	23		25	25	23
DAYS	15	TO	17	MEAN	17.3	D	19.2	18.2	15.7
				S.D.	3.77		4.45	3.53	5.94
				N	23		25	25	23
DAYS	17	TO	19	MEAN	22.9	D	24.1	23.8	21.2
				S.D.	3.98		4.90	4.54	8.32
				N	23		25	25	23
DAYS	19	TO	20	MEAN	13.2	D	11.7	13.1	12.9
				S.D.	2.87		4.07	4.80	4.81
				N	23		25	25	23

Statistics: D = Dunnett-test (two-sided); * : p <= 0.05, ** : p <= 0.01

 

 

Table 9: Individual fetal external malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	79-06 Fa)	Anasarca

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional soft tissue malformation

 

 

Table 10: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 259	25 278	25 290	22 259
Fetal incidence	N (%)	0.0	0.0	0.0	1 (0.4)
Litter incidence	N (%)	0.0	0.0	0.0	1 (4.5)
Affected fetuses/litter	Mean%	0.0	0.0	0.0	0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 11: Total external unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 259	25 278	25 290	22 259
Fetal incidence	N (%)	0.0	0.0	1 (0.3)	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.0)	0.0
Affected fetuses/litter	Mean%	0.0	0.0	0.3	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 12: Individual fetal soft tissue malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	6-04 M	Hydronephrosis
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	53-14 M	Situs inversus
3 (1000 mg/kg bw/d)	79-06 Fa)	Situs inversus

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional external malformation

 

 

Table 13: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 124	25 132	25 139	22 122
Fetal incidence	N (%)	1 (0.8)	0.0	1 (0.7)	1 (0.8)
Litter incidence	N (%)	1 (4.3)	0.0	1 (4.0)	1 (4.5)
Affected fetuses/litter	Mean%	0.6	0.0	0.5	0.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 14: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 124	25 132	25 139	22 122
Fetal incidence	N (%)	5 (4.0)	6 (4.5)	1 (0.7)	0.0
Litter incidence	N (%)	3 (13)	5 (20)	1 (4.0)	0.0
Affected fetuses/litter	Mean%	3.8	4.0	0.8	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 15: Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	34-11 M	Misshapen basisphenoid
2 (300 mg/kg bw/d)	58-11 M	Shortened scapula, bent rib
	59-01 M	Shortened scapula, bent rib, shortened humerus
	62-01 F	Bent rib
3 (1000 mg/kg bw/d)	83-01 M	Shortened scapula, shortened humerus
	88-13 M	Shortened scapula

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

 

Table 16: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 135	25 146	25 151	22 137
Fetal incidence	N (%)	0.0	1 (0.7)	3 (2.0)	2 (1.5)
Litter incidence	N (%)	0.0	1 (4.0)	3 (12)	2 (9.1)
Affected fetuses/litter	Mean%	0.0	0.7	2.0*	1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

 

 

Table 17: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 135	25 146	25 151	22 137
Fetal incidence	N (%)	134 (99)	142 (97)	146 (97)	137 (100)
Litter incidence	N (%)	23 (100)	25 (100)	25 (100)	22 (100)
Affected fetuses/litter	Mean%	99.4	97.3	96.8	100.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 18: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of supraoccipital; unchanged cartilage	22.7	19.5	27.7	56.4**	21.6 (9.1 - 32.4)
Incomplete ossification of skull; unchanged cartilage	12.2	13.3	16.2	26.3*	10.1 (2.1 - 21.5)
Incomplete ossification of nasal; unchanged cartilage	0.7	0.6	2.7	11.7**	0.9 (0.0 - 4.1)
Basioccipital hole(s)	0.0	0.0	0.0	2.4*	0.4 (0.0 - 1.5)
Incomplete ossification of thoracic centrum; unchanged cartilage	0.7	0.7	0.0	7.0*	0.4 (0.0 - 2.0)
Supernumerary thoracic vertebra	1.5	9.9*	3.4	12.7**	5.6 (3.0 - 10.3)
Incomplete ossification of sacral arch; cartilage present	0.0	0.0	2.6*	1.6	0.8 (0.0 - 2.7)
Unossified sternebra; unchanged cartilage	2.7	5.8	9.0*	26.0**	5.2 (0.0 - 13.8)
Unilateral ossification of sternebra; unchanged cartilage	2.1	0.0	2.7	8.1*	0.6 (0.0 - 2.2)
Wavy rib	0.9	9.9	10.9**	18.2**	4.7 (0.7 - 14.7)
Incomplete ossification of pubis; cartilage present	0.0	0.6	0.0	3.7*	0.3 (0.0 - 1.0)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])     ** = p <= 0.01 (Wilcoxon-test [one-sided])

 

 

 

Table 19: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 135	25 146	25 151	22 137
Fetal incidence	N (%)	105 (78)	116 (79)	98 (65)	100 (73)
Litter incidence	N (%)	23 (100)	25 (100)	25 (100)	22 (100)
Affected fetuses/litter	Mean%	77.6	81.0	65.1	73.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

 

Table 20: Total fetal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 259	25 278	25 290	22 259
Fetal incidence	N (%)	1 (0.4)	1 (0.4)	4 (1.4)	3 (1.2)
Litter incidence	N (%)	1 (4.3)	1 (4.0)	4 (16)	3 (14)
Affected fetuses/litter	Mean%	0.3	0.4	1.3	1.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Table 21: Total fetal variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	23 259	25 278	25 290	22 259
Fetal incidence	N (%)	139 (54)	148 (53)	147 (51)	137 (53)
Litter incidence	N (%)	23 (100)	25 (100)	25 (100)	22 (100)
Affected fetuses/litter	Mean%	53.6	53.3	50.8	52.9

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose level of 300 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is 1000 mg/kg bw/d. The test substance is considered not teratogenic in Wistar rats under the test conditions employed in this study.

Executive summary:

In this GLP-compliant prenatal developmental toxicity study, the test was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

 

Regarding clinical examinations, all females of the test substance treated groups (test group 1-3; 100, 300 or 1000 mg/kg bw/d) showed yellowish discolored feces during the treatment period until terminal sacrifice (GD 20). It was initially observed on GD 7 in the mid- and high-dose groups and on GD 8 in the low-dose group. This feces discoloration mirrored the presence of the yellowish test substance in the gastrointestinal tract. An adverse, toxic effect by itself was not assumed.

At the high-dose level (1000 mg/kg bw/d), distinct signs of maternal toxicity were observed such as piloerection from GD 10 onwards in all dams and hypothermia on GD 10-12 in one dam. In addition, reduced mean food consumption (29% below control) and decreased body weight change (35% below control) were seen during the treatment period (GD 6-19). The corrected body weight gain of these high-dose dams was also statistically significantly decreased (69% below control). Due to the variety of affected parameters, the abovementioned findings were assessed as treatment-related and adverse.

Although the dams of the test group 2 (300 mg/kg bw/d) showed a decreased mean food consumption on GD 6-13 (up to 16% below control), they recovered afterwards and even exceeded the control value at the end of the treatment period. Piloerection was seen on GD 19-20 in one dam only. These findings were considered as signs of a beginning maternal toxicity, which were related to the treatment but not yet adverse.

All in all, at 300 and 100 mg/kg bw/d, no treatment-related adverse effects were observed. Regarding clinical pathology, in dams of test group 3 (1000 mg/kg bw/d) significantly increased total bilirubin levels and significantly decreased total protein, albumin and globulin levels, in absence of any signs of anemia, were observed most probably due to an altered liver cell metabolism. These findings were regarded to be treatment-related and adverse.

Regarding pathology, all findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution was noted. However, anogenital distance and anogenital index were decreased in male fetuses (-19% and -14% below control, respectively) of the high-dose group. In addition, lower mean placental weights (0.37 g vs. 0.45 g on control, both sexes combined) and mean fetal body weights (3.1 g vs. 3.6 g in control, all viable fetuses) were observed in this group (1000 mg/kg bw/d). It was assumed that these parameters were possibly affected by treatment, but since almost all of them were within the historical control data regarded to be not adverse. 

The observed reduced fetal body weights of the high-dose group (1000 mg/kg bw/d) corresponded to a delay in ossification as indicated by a broad range of skeletal variations, i.e. ‘unossified sternebra (unchanged cartilage)’, ‘wavy rib’, ‘incomplete ossification of supraoccipital (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘incomplete ossification of nasal (unchanged cartilage)’, ‘basioccipital hole(s)’, ‘incomplete ossification of thoracic centrum (unchanged cartilage)’, ‘supernumerary thoracic vertebra’, ‘incomplete ossification of sacral arch (cartilage present)’, ‘unilateral ossification of sternebra (unchanged cartilage)’ and ‘incomplete ossification of pubis’. Almost all of these variations appeared at high background incidences in the population of the used rat breed, are unlikely to be detrimental for survival or health and are at least to some extent being repaired postnatally, which qualifies them to be of relatively low toxicological concern. Specifically, the ossification of skull bones and vertebral arches occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). In this study, the findings in the high-dose fetuses occurred in presence of maternal toxicity and were in the same order of magnitude as reduced food consumption and body weight gain of the dams. Overall, the above-mentioned findings were considered to be minor, reversible effects indicating a developmental delay and were, therefore, assessed as treatment-related but not as adverse. 

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) provided evidence of maternal toxicity, such as a reduction in food consumption, a decrease in (corrected) body weight gain and changes in clinical pathology parameters (e.g. increased total bilirubin values) at the highest dose level of 1000 mg/kg bw/d. In conclusion, the NOAEL for maternal toxicity is the mid-dose level of 300 mg/kg bw/d.

Overall, there was no evidence for specific adverse effects of the test substance on fetal morphology at any dose. Minor, reversible effects on fetal weight and ossification in the presence of maternal toxicity were indicating a temporary developmental delay and were, therefore, assessed as treatment-related but not as adverse. Thus, the NOAEL for prenatal developmental toxicity is 1000 mg/kg bw/d. The test substance is considered not teratogenic in Wistar rats under the test conditions employed in this study.