Reaction mass of Ammonium bisethanedioato bisaquo niobate(V) (anhydrous form) and Ammonium hydrogen ethanedioate ethanedioic acid (anhydrous form)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(adopted 25 June 2018)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

(adopted August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

diammonium oxalic acid pentaoxo-1,4,6,9-tetraoxa-5-niobaspiro[4.4]nonan-5-uide tetrahydrate hydrogen oxalate

EC Number:

939-559-9

Molecular formula:

Constituent 1: C4H8NNbO11 Constituent 2: C4H7NO8

IUPAC Name:

diammonium oxalic acid pentaoxo-1,4,6,9-tetraoxa-5-niobaspiro[4.4]nonan-5-uide tetrahydrate hydrogen oxalate

Specific details on test material used for the study:

- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about concentration (± 10 %) and stability of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: females: approx. 13 - 14 weeks; males: between 19 weeks and not older than 32 weeks
- Weight at study initiation: males: 378 – 560 g; females: 213 - 281 g
- Fasting period before study: No
- Housing: Full barrier in an air-conditioned room. The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages.
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany).
- Water: Tap water in bottles, ad libitum; sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). The purpose of the acidification is to provide additional protection against microbial contamination. This is a standard practice at BSL BIOSERVICE and should not be associated with any risk for long-term studies.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

sterile water (aqua ad injectionem)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was ground into a fine powder and weighed into a tared plastic vial on a precision balance. Dose formulations were prepared by adding the required volume of vehicle (aqua ad injectionem) to obtain the appropriate final concentration of the test item. Prepared formulations were vortexed and / or stirred until visual solubility was achieved. Based on the results of stability testing the test item formulations were prepared at least every 10 days. The prepared formulation was stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 10, 40 and 140 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw; Dose volumes were adjusted individually based on the body weight most recently measured.
- Lot/batch no.: 902272

A specified procedure is required for cleaning of the gavage apparatus (needle, flexible cannula or tube) after drawing up the test item and before administering the item by gavage as follows. Following completion of filling of the syringe attached to the gavage apparatus with the required amount of test item, the outside of the gavage tube was wiped clean of all test item residue. The gavage tube was then lightly tapped onto a clean towel to remove the remaining droplet from the tip of the tubing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigation of homogeneity and substance concentration was included on samples from various levels (top, middle and bottom) of prepared formulations from all dose groups and from the middle of the control group in the 1st and last study week of treatment (total 20 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 1 mL). The A-samples were analysed at CIP Chemisches Institut Pforzheim GmbH (Eurofins Munich Study Phase No. 186794) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below -15°C at CIP Chemisches Institut Pforzheim GmbH and discarded after completion of the final study report.

The mean recoveries observed for the low dose group ranged between 44.2 - 76.1% of the nominal value (50 mg/kg bw/day), between 77.4 - 99.8% for the mid dose group (200 mg/kg bw/day) and between 70.2 - 94.% for the high dose group (700 mg/kg bw/day) at week 1 and last week. The overall mean recoveries observed in the low dose, medium dose and high dose groups were 71.4%, 88.4%, and 83.9% of the nominal concentrations, respectively. Nominal concentrations were not confirmed for all dose groups, as measured concentrations were not within acceptance criterion of 10%.
Analysis of dose formulations revealed that the measured Niobium concentrations were significantly below the nominal concentrations. Subsequent investigations by Sponsor and at test site revealed that the test item had degraded over the period of storage at room temperature. Hence, based on the measured concentrations, the lowest mean values for each dose level at the two testing occasions were used to determine the actually achieved doses in this study.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 / 2 (nulliparous and non-pregnant)
- Length of cohabitation: 24 hours a day until confirmation of mating by sperm positive vaginal smear
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

daily (7days/week)

Duration of test:

From mating until gestation day 20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

analysed lowest concentration: 6.7 mg/mL; correspond to a nominal dose of 50 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

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

Remarks:

analysed lowest concentration: 31.7 mg/mL; correspond to a nominal dose of 200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

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

Remarks:

analysed lowest concentration: 104.1 mg/mL; correspond to a nominal dose of 700 mg/kg bw/day

No. of animals per sex per dose:

23 mated females / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected in order to demonstrate any dose-related response. Dose levels in this study were chosen based on the data of a dose range finding study with Ammonium Niobium Oxalate in Wistar rats (CBMM Europe BV, Supporting, 2020), a 28-day repeated dose oral toxicity study with Ammonium Niobium Oxalate in Wistar rats (CBMM Europe BV, Key, 2014, RDT) and the corresponding 14-day dose range finding oral toxicity study with Ammonium Niobium Oxalate in Wistar rats (CBMM Europe BV, Supporting, 2013). Mortality of 2/3 females at 1000 mg/kg bw/day was observed in the 14-day dose range finding study for the repeated dose toxicity. Thus, 1000 mg/kg bw/day was not considered as a feasible dose for the present study. No test item-related mortality occurred during the treatment or recovery period in any of the above metntioned studies. No test item-related systemic toxicity was observed in the dose range finer for developmental toxicity until the highest dose tested (600 mg/kg bw/day), besides slight but dose-dependently lower group mean body weights and body weight gain at the end of the gestation period (from gestation days 17 - 20) in test item-treated animals compared to the controls. No signs of foetal toxicity were seen in terms of effect on prenatal data, litter data and fetal external findings up to the highest dose level of 600 mg/kg bw/day. Based on these results and in consultation with the sponsor the dosis were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Inadvertently, mortality was only observed once daily on 24 June 2019 for the animals no. 1-5, 24-27, 47-50 and 70-73.
- Cage side observations checked: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size, changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of mating

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within ± 20% variation. The sperm positive females were weighed on GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption (g/animal/day) of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Sperm positive females were subjected to a caesarean section after sacrificing the animals using anaesthesia (ketamine/xylazine). None of the females showed signs of abortion or premature delivery prior to scheduled terminal sacrifice. At the time of termination or death during the study, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde. Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status. Each gravid uterus with the cervix was weighed. However, the gravid uterus obtained from dead animals was not weighed.
Thyroid / parathyroid glands from all dams were preserved in 4 % neutral-buffered formaldehyde. The weight of thyroid / parathyroid glands was assessed after 24 hours fixation.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.

OTHER:
- Evaluation of Thyroid Hormones
Thyroid hormones levels from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination on respective gestation day 20, blood samples were collected in the morning within 2 hours from the abdominal aorta and were stored under appropriate conditions. Blood samples were assessed for serum levels for thyroid hormones (T3, T4 and TSH) by using ELISA method from all the groups except animal no. 70 and 81 which were euthanised for animal welfare reason and animal no. 59 which was found dead.

- Histopathology
A full histopathological examination was carried out on the preserved thyroid / parathyroid glands from all dams of all dose groups which were sacrificed at the end of the treatment period.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Liestal, Switzerland (test site for histopathology).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The position and number of foetuses in each uterine horn was also recorded.

Fetal examinations:

- External examinations:
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies and the anogenital distance (AGD) was measured. Foetal body weight assessed on gestation day 20 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of foetus weight). The foetus was ascertained as live or dead from reflex shown after touch.
The lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. The limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

- Soft tissue / head examinations:
One half of the foetuses of each litter was examined for soft tissue anomalies by a microdissection technique. After the completion of the external examination, foetuses were transferred to plastic bottles containing Bouin’s solution for later soft tissue / visceral and craniofacial examination (internal structures including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique). At least three weeks after completion of female caesarean sections, foetuses from Bouin’s solution were transferred to plastic bottles containing 95% ethanol. The soft tissue / visceral evaluation of the body and craniofacial examination of the heads of the same foetuses of 20 litters per group were performed.
The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.

The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off centre and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence as well as position of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo-ventricular septum.

Craniofacial examination: The head of the foetus was subjected to 5 - 7 transverse sections in order to examine the internal structures of the head including the symmetry of the external nares, nasal conchae, nasal septum, palate, as well as the development of the cerebellum and brain stem.

- Skeletal examinations:
The remaining half of foetuses from each litter was processed by Alcian blue and Alizarin staining and 20 litters per group were examined for skeletal alterations.

The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centres. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centres and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones and cartilages. Any deviation from the normal development was recorded for each foetus.

The soft tissue / visceral, craniofacial and skeletal examinations were performed at AnaPath GmbH, AnaPath Services, Liestal, Switzerland.

Particular attention was paid to the reproductive tract which was examined for signs of altered development. External foetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent / cryptorchidism was noted in male foetuses.
Inadvertently, the foetal evaluation (sex, body weight, external observation and AGD) was not performed for the pubs no. 1-10 of female no. 66.

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p < 0.05 is considered as statistically significant).

Indices:

No indices calculated.

Historical control data:

The following historical control data for prenatal developmental toxicity studies (OECD 414) using the Wistar Rat were provided from the years 2011 - 2019 in the Annex of the study report: uterine data, litter weight data, fetal external examination, fetal visceral examination, fetal craniofacial examination, fetal skeletal examination.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two dams from the high dose group were sacrificed in moribund condition on gestation day 18. The cause of mortality could not be determined but it cannot be excluded that these two mortalities were related to treatment with the test item.
One dam from the mid dose group was found dead on gestation day 20. At necropsy fluid filled lung was observed indicative of gavaging error which was not considered to be related to the treatment with the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

- Mortality
Two dams from the high dose group were sacrificed in moribund condition on respective gestation day 18 due to animal welfare reasons. On the day of moribund sacrifice, major clinical signs observed in females 70 and 81 were reduced spontaneous activity, half eyelid closure, piloerection and abnormal breathing. At necropsy, enlarged and gas filled stomach was observed in female 81. No macroscopic findings were observed at necropsy in female no. 70. Individual body weight and food consumptions were found to be comparable and increased during the gestation days in both dams. The cause of mortality could not be determined and but it cannot be excluded that these two mortalities were related to treatment with the test item.
One dam from mid dose group found dead on gestation day 20. No clinical signs were observed in this animal throughout the study as well as before death. However, at necropsy fluid filled lung was observed indicative of gavaging error which was not considered to be related to the treatment with the test item.
All other animals survived till the end of the scheduled terminal sacrifice of gestation day 20.

- Clinical Observations (Please refer also to the attached document / Table 1):
In terminally sacrificed females, predominant transient clinical signs observed on few days during the treatment period of the study included slight to moderate increased salivation (1/23 in mid dose and 3/23 in high dose), moving the bedding (1/23 in mid dose and 15/23 in high dose), reduced spontaneous activity (1/23 in high dose), piloerection (3/23 in high dose), abnormal breathing (1/23 in mid dose and high dose) and chromodacryorrhea (1/23 in low dose).
There were also low incidences of hairless areas on various body parts (2/23 in control, and 3/23 in high dose) observed in isolated females of control and high dose group and cough / sneezing in 2/23 high dose females.
Specific clinical signs observed in high dose group moribund female (female no. 70) between gestation days 17 - 18 before moribund sacrifice on gestation day 18 were slight reduction in spontaneous activity, moderate piloerection, half eyelid closure, abnormal breathing, moving the bedding, severe piloerection and nasal discharge. In another high dose group moribund female (female no. 81) sacrificed on gestation day 18, predominant clinical signs observed were moving the bedding (gestation days 11 - 17), half eyelid closure, severe reduction in spontaneous activity and abnormal breathing on gestation day 18.
As moving the bedding was noted mainly immediately after test item administration and just for a short period, this transient sign was considered to be due to a local reaction to the test item rather than an adverse systemic effect. Piloerection was observed on very few days without any consistency in few females of high dose group. In the light of no effect observed on body weight development and food consumption in treatment groups, all clinical signs observed in terminally sacrificed treatment group females were incidental and considered to be of no toxicological relevance or non-adverse in nature.

- Body Weight Development:
The mean body weight remained unaffected by treatment with the test item and increased as expected with the progress of the study in all treated groups throughout the study period, as compared to control. No statistical significance was achieved for differences in body weight or body weight gain in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the controls.

- Food Consumption:
Statistical analysis of food consumption data revealed statistical significantly lower group mean food consumption during gestation day 5 - 8 in high dose animals (22 % lower) when compared with the control.
However, this finding could be attributed to the very low individual food consumption value of a single female of the high dose group. Since no effect on body weight development was observed, this statistically significant effect on food consumption during gestation days 5 - 8 in the high dose group was not considered to be toxicologically relevant.

- Thyroid Hormones and Thyroid Weight:
No statistically significant and / or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels for all treated groups and values were comparable to those of the control.
Statistical analysis of post fixed thyroid / parathyroid weights from all dams revealed no statistically significant differences in the absolute and relative (to body weight) thyroid / parathyroid weights of the dose groups when compared to the control group.

- Pathology:
No gross pathological changes of toxicological significance were observed during the macroscopic examination of the females of the low, mid and high dose groups as compared to controls.
In fact, some changes were reported in single animals, which rather were incidental than due to the treatment.

- Histopathology:
There were no histopathological findings observed in thyroid glands that could be related to treatment with test item up to 521 mg/kg bw/day.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, number of corpora lutea, implantation sites, early and late resorptions, number of live and dead foetuses, number of male and female foetuses, sex ratio (% males), and percentual pre- and post-implantation loss in treatment groups when compared to the controls. However, group mean net weight change (adjusted maternal weight - gestation day 0 body weight) was observed to be statistically significantly lower in high dose group when compared to the control group. This effect on lower net weight change in high dose group was attributed to negative individual values from 2 high dose females and values from the remaining high dose animals were comparable to controls; therefore, this effect was not considered to be of toxicological relevance.

Successful mating resulted in 20/23 pregnancies in the low dose, 23/23 in mid dose and 20/23 in the high dose group compared to 18/23 pregnancies in the control group. Taking into account the 3 cases of mortality previously mentioned, these pregnancy rates led to 20, 22, 19 and 18 pregnant females at caesarean section in the low dose, mid dose, high doseand control group, respectively (at terminal scheduled sacrifice). The variation in pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 78.27% in the control group compared to treatment groups (86.96% in low dose, 95.65% in mid dose and 82.61% in high dose) was considered to be a biological variation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no test item-related effects of toxicological relevance observed for the mean fetal weight, male and female fetal weight on litter basis (group mean of individual litter mean) or individual basis (sum of weight of all foetuses in group divided by total number of foetuses in respective group) in any of the treatment groups when compared to the control. All these values were found to be within the historical control range in the strain used in this study. 

In males and females, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected and was comparable to control.
All male foetuses were checked for indication of incomplete testicular descent / cryptorchidism and evaluation revealed completion of testicular descent in all male foetuses from all groups.

- External Examination:
There were no external abnormalities observed in foetuses from any of the dose groups as compared to control.

- Visceral Examination:
Visceral findings observed in the dose groups were at frequencies generally comparable to controls. As the observed findings were either minor variations and / or without any dose dependency and consistency, no toxicological relevance was attributed to these findings and they were considered to be spontaneous in nature.
Statistical analysis of visceral data revealed statistically significantly lower group litter incidences for malpositioned umbilical artery in low dose and dilated renal pelvis in mid dose and high dose group when compared to control. As these litter incidences almost were lower in these treatment groups compared to the control group, they had no toxicological relevance.

- Craniofacial Examination:
Craniofacial examination revealed a few predominant findings in foetuses of the low and high dose group (hole in brain, small brain and increased perimeningeal space) at low frequencies generally comparable to the control. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for litter incidence of any of these findings.

- Skeletal Examination:
Statistical analysis of skeletal data revealed no statistical significance for litter incidences in dose groups when compared with the control.
Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification of skull – parietal - bilateral (47.4% compared to 22.2% in controls), skull - hyoid body, (21.1% compared to 5.6% in controls), skull – right zygomatic arch (10% compared to 0% in controls), 4th sternebra (21.1% compared to 5.6% in controls), 5th sternebra (52.6% compared to 38.9% in controls) and unossified caudal vertebral centrum (36.8 compared to 11.1% in controls) were observed in the high dose group when compared to the control group.
The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the high dose group were either marginally higher or within historical control data range. Generally, delayed ossification is not regarded to persist postnatally and/or to be not associated with long-term consequences on survival, general growth and development; therefore the finding is not considered to be adverse.
There was no statistical significance and no indication of a test item-related trend in the type and / or litter incidences of other skeletal findings and they were therefore considered to be spontaneous in nature. Frequencies for litter incidences of few skeletal findings were even less in the treated groups compared to the controls. Therefore, these findings are not considered to be treatment-related but rather solely spontaneous in nature.

 

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 521 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up to and included the highest dose tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The test substance had no effect on intrauterine development.