Niobium hydride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2020 to 16 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Development Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 87.3650

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Niobium hydride

EC Number:

237-769-9

EC Name:

Niobium hydride

Cas Number:

13981-86-7

Molecular formula:

HNb

IUPAC Name:

niobium(1+) hydride

Test material form:

solid

Remarks:

Silver grey solid

Details on test material:

- Storage conditions: At room temperature.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: At initiation of dosing, males were 10 - 11 weeks old and females were 13 - 14 weeks old.
- Weight at study initiation: Males weighed between 270 and 308 g and females weighed between 200 and 240 g.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labelled with a colour-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet: Ad libitum throughout the study, except during designated procedures.
- Water: Freely available to each animal via water bottles.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pretest period (females) or 7 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
- Food: Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analysed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: Target temperatures of 18 to 24 °C was maintained. The actual daily mean temperature during the study period was 19 to 21 °C.
- Humidity: Target relative target humidity of 40 to 70 % was maintained. The actual daily mean relative humidity of 51 to 79 %. The humidity values that were outside the targeted range occurred for 33 days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod: A 12-hour light/ 12-hour dark cycle was maintained.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Specific gravity 0.92.

Details on exposure:

- The dose volume for each animal was based on the most recent body weight measurement.
- The doses were given using a plastic feeding tube.
- The dosing formulations were stirred continuously during dose administration.
- A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
- Dose volume: 5 mL/kg
- Dose concentration:
100 mg/kg/day group: 20 mg/mL
300 mg/kg/day group: 60 mg/mL
1 000 mg/kg/day group: 200 mg/mL

PREPARATION OF DOSING SOLUTIONS:
- Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 2 hours after adding the vehicle to the test material. Formulations were prepared protected from light.
- Test material dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (adjustment factor 0.92). No correction was made for the purity/composition of the test material. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

- Analysis of test material in vehicle for concentration, homogeneity, and stability was not performed, as no feasible analytical method was available. In theory metal ions, such as in the test material, could be analysed by ICP-MS, if the test material could be dissolved in an aqueous solution. However, the test material did not dissolve in aqueous solutions, nor did it dissolve in concentrated nitric acid, nitric acid/hydrogen peroxide or concentrated hydrochloric acid. Moreover, a visibly stable suspension could be prepared only in corn oil for the administration to rats. A corn oil formulation could not be dissolved in aqueous solutions and therefore, no quantitative analysis was performed.
During the current study, the test material dosing formulations were prepared with corn oil. The test material is a transition metal hydride. It is binary metal hydride, which is known not to dissolve in any solvent. As the substance is a metal compound and does not dissolve in concentrated nitric acid, nitric acid/hydrogen peroxide or concentrated hydrochloric acid, the substance was also not expected to react with any oil-like substance like corn oil and thus was expected to be stable in corn oil for the period from formulation till administration.
- In addition, to limit the impact, the test material preparations were performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 2 hours after completion of the preparation of the test material. Additionally, test material formulations were prepared protected from light. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Details on mating procedure:

- M/F ratio per cage: 1:1 basis within the same treatment group, avoiding sibling mating, after 14 days of treatment.
- Length of cohabitation: For one couple (Male No. 26, Female No. 66), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated 12 days after the actual mating date. The actual mating date was designated Day 0 post-coitum.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged: Individually.

Duration of treatment / exposure:

- A minimum of 28 days.
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 - 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 - 48 days.
- The first day of dosing was designated as Day 1.

Frequency of treatment:

Once daily

Duration of test:

A minimum of 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Five per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of administration was selected this is a possible route of human exposure during manufacture, handling or use of the test material. The dose levels were selected based on the results of a 10-Day Dose Range Finder with oral administration of the test material in rats (Test Facility Reference No. 20236631), which identified no dose-limiting adversity up to the limit dose of 1 000 mg/kg bw/day. Therefore, the highest dose was selected based on the OECD 422 guideline limit dose and the intermediate and low dose were selected in an attempt to produce graded responses to the test material.
- Rationale for animal assignment: Animals were randomly assigned to groups at arrival. Males and females were randomised separately. As this study was a combined reproductive and repeated dose toxicity test, a total of 40 females were selected at randomisation before initiation of the pretest phase. Females were evaluated for regular oestrous cyclicity before allocation. Any selected female classified as not having regular oestrous cycles during the pretest phase (6 females in total) was replaced before initiation of dosing by one of the 8 additional females having regular oestrous cycles. A total of 40 females with regular oestrous cycles continued in the study. The eight supernumerary or irregularly cycling females were removed from the study, and their oestrous cycle results were kept in the raw data but not reported.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Cage side observations checked: Throughout the study, animals were observed for general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning during the first administration of the test material and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables. Clinical observations were conducted in a standard arena beginning before the first administration of the test material and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS:
- Animals surviving until scheduled euthanasia were weighed, and deeply anesthetised using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Scheduled necropsies were conducted on the following days: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14 - 16. Females which failed to deliver (Nos. 43, 65, 64, 70, 76 and 77): With evidence of mating: Post-coitum Day 25 - 27. All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. 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 and the number of corpora lutea was recorded in addition.

Organ Weights – F0-Generation
- The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
- Brain, Cervix (weighed together with the uterus), Epididymis (paired organ weight), Gland, adrenal (paired organ weight), Gland, coagulation (paired organ weight; weighed together with the seminal vesicles), Gland, parathyroid (weighed together with the thyroid), Gland, prostate, Gland, seminal vesicle (paired organ weight), Gland, thyroid (paired organ weight), Heart, Kidney (paired organ weight), Liver, Ovaries (paired organ weight), Spleen, Testes (paired organ weight), Thymus, Uterus.

- Organs Weighed at Necropsy for all Remaining Animals (incl. Males that Failed to Sire and Females that Failed to Deliver): Epididymis (paired organ weight), Gland, coagulations, (weighed together with seminal vesicles), Gland, parathyroid (weighed together with the thyroid), Gland, prostate, Gland, seminal vesicle (paired organ weight), Gland, thyroid (paired organ weight), Testes (paired organ weight).

HISTOPATHOLOGY: Yes
- Representative samples of the tissues identified below were collected from all animals and preserved in 10 % neutral buffered formalin (neutral phosphate buffered 4 % formaldehyde solution) unless otherwise indicated.
Tissue Collection and Preservation for all Selected Animals:
Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (eight levels)
Cervix
Epididymides (preserved in modified Davidson's fixative and transferred to formalin after fixation for at least 24 h)
Esophagus
Eye (preserved in modified Davidson's fixative and transferred to formalin after fixation for at least 24 h).
Gland, adrenal
Gland, coagulation
Gland, Harderian (preserved in modified Davidson's fixative and transferred to formalin after fixation for at least 24 h).
Gland, lacrimal
Gland, mammary
Gland, parathyroid (Only collected if present in the routine section of the thyroid).
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optic (Only collected if present in the routine section of the eye. Part of the optic nerve attached to the eye was fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin).
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testes (Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours).
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina

- Tissues that were supposed to be microscopically evaluated per protocol but were not available on the slide (and therefore not evaluated) are listed in Individual Animal Data of the pathology report as not present. These missing tissues did not affect the outcome or interpretation of the pathology portion of the study because sufficient data was available.
Tissue Collection and Preservation for all Remaining Animals (incl. Males that Failed to Sire and Females that Failed to Deliver):
Animal identification
Cervix
Epididymis (Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours).
Gland, coagulation
Gland, mammary
Gland, parathyroid (Only collected if present in the routine section of the thyroid).
Gland, pituitary
Gland, prostate
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Ovaries
Testes (Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours).
Uterus
Vagina

- The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin for selected animals:
Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Tissues identified above (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Remaining animals: Gross lesions/ masses.

Histopathology – F0-Generation: All tissues as defined under Histology – F0-Generation were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.

HAEMATOLOGY
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis.
- Anaesthetic used for blood collection: Yes under anesthesia using isoflurane.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- Parameters checked: Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analysed for:
White blood cell (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cell (RBC), Reticulocytes (absolute), Red blood cell distribution width gated (RDWG), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets.
A blood smear was prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results.
- Coagulation: Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as anticoagulant. Samples were processed for plasma, and plasma was analysed for the parameter Prothrombin time (PT)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Glucose Alkaline phosphatase (ALP), Total protein, Albumin, Total bilirubin, Bile acids, Urea, Glucose, Creatinine, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate (Inorg. Phos).

PLASMA/SERUM HORMONES/LIPIDS
- Time of blood sample collection: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Blood samples at a target volume of 1.0 mL (F0- animals), 0.5 mL (pooled PND 4 pups) and 1.0 mL (PND 14 - 16 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analysed for the parameters. Blood samples were processed for serum, and serum was analysed for total Thyroxine (T4). For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14 - 16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14 - 16.
Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75 °C). Under these storage conditions, samples are stable for 6 months. Any remaining sample were discarded at finalisation.

GENERAL REPRODUCTION DATA - F0 GENERATION
- From the mating period onwards, the following parameters were recorded for each female: Male number paired with, mating date, confirmation of pregnancy and delivery day.
- Females were allowed to litter. Postnatal Day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
- Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Blood sampling:

Blood of F0-animals was collected on the day of scheduled necropsy.

Fetal examinations:

Pups that died or were euthanised before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated

Statistics:

See below.

Indices:

For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): (Number of females mated / Number of females paired) x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating
(Fertility index (%): Number of pregnant females / Number of females mated) x 100

Gestation index (%):
(Number of females with living pups on Day 1 / Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100

Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100

Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Historical control data:

Historical control data for Wistar Han rats; F0-males (period 2017 - 2020): Reticulocytes (10E9/L) mean = 221.5; P5 – P95 = 164.25-288.35 (n=400).

Historical control data for Wistar Han rats; F0-females (period 2017 - 2020): Prothrombin time (sec): mean = 17.6; P5 – P95 = 15.9-19.5 (n=382).

Historical control data for Wistar Han rats; F0-females (period 2017 - 2020): Albumin: mean = 30.5; P5 – P95 = 27.50-37.20 (n=401).

Historical control data for Wistar Han rats; F0-females (period 2016 - 2020): Implantation sites (N): mean = 12.3; P5 – P95 = 6.0-16.0 (n=1511).

Historical control data for Wistar Han rats; F0-females (period 2016 - 2020): Gestation index (%): mean = 98; P5 – P95 = 89-100 (n=145).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations.
Hunched posture was noted on multiple days (Days 24 - 27) during the last week of treatment in one male treated at 1 000 mg/kg/day, in one female at 300 mg/kg over Days 29 - 41 of treatment and in several females at 1 000 mg/kg/day over Days 20 - 41 of treatment. As clinical signs were transient and occurred in a few animals only, these were considered incidental and not toxicologically relevant.
Salivation seen incidentally after dosing among some animals among all dose groups was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs that were noted during the treatment period (i.e. scabs, wounds, alopecia and piloerection) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test material-related changes in body weights and body weight gain were observed in males and females up to 1 000 mg/kg/day.
The slightly lower body weight (gain) on post-coitum Day 20 in females at 1 000 mg/kg/day was attributed to two females with implantation sites only.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A lower haemoglobin concentration was noted in females treated at 300 and 1 000 mg/kg/day (0.94x and 0.93x of control, and 4/5 and 4/5 values were below the range of the concurrent control values, respectively).
The decreased number of reticulocytes in males treated at 100 mg/kg/day was considered to be unrelated to treatment with the test material as this change was considered the result of slightly high control values which had no dose-related trend.
It should be noted that the apparent decrease in white blood cell and lymphocyte counts in females at 1 000 mg/kg/day was mainly attributed to low individual values of Animal No. 75. As these findings were noted for one animal only it was considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.
The higher prothrombin time (PT) of females treated at 100, 300 and 1 000 mg/kg/day were considered to have arisen as a result of slightly low control values
and therefore considered to be of no toxicological significance with mean PT for treatment groups remaining within the historical control range.
Historical control data for Wistar Han rats; F0-males (period 2017 - 2020): Reticulocytes (10E9/L) mean = 221.5; P5 – P95 = 164.25-288.35 (n=400).
Historical control data for Wistar Han rats; F0-females (period 2017 - 2020): Prothrombin time (sec): mean = 17.6; P5 – P95 = 15.9-19.5 (n=382).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment with the test material up to 1 000 mg/kg/day.
A lower mean albumin concentration noted in females at 1 000 mg/kg/day (0.94x of control) was considered to have arisen as a result of slightly high control values and therefore considered to be of no toxicological significance.
The decreased mean potassium concentration in males at 100 and 300 mg/kg/day was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Historical control data for Wistar Han rats; F0-females (period 2017 - 2020): Albumin: mean = 30.5; P5 – P95 = 27.50-37.20 (n=401).

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment with the test material up to 1 000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test material up to 1 000 mg/kg/day.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals up to 1 000 mg/kg/day.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related alterations in organ weights.
There was a statistically significant lower brain weight in females at 300 and 1 000 mg/kg/day (absolute only, -5 % for both treatment groups). There was no macroscopic or microscopic correlate for this slightly lower brain weight in the females examined and was therefore regarded of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Other effects:

no effects observed

Description (incidence and severity):

- Food consumption: Food consumption before or after correction for body weight was similar to the control level up to 1 000 mg/kg/day.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The number of implantation sites was decreased in females at 1 000 mg/kg/day (mean 9.0
compared to 13.1 in the control group), which was attributed to the low numbers of
implantation sites in 4/10 pregnant females (Nos. 76 and 77 with each one implantation site
only, and Nos. 74 and 79 with seven and six implantation sites, respectively). Whilst the
mean number of implantation sites was also below the historical control mean
(12.3), the value remained within the overall range (individual data) in the historical control data (6 to 16 implantation sites).


The total number of offspring born compared to the total number of uterine implantations was
considered not to be affected by treatment with the test material.
Post-implantation survival index (total number of offspring born as percentage of total
number of uterine implantation sites) was 78, 92, 97 and 93 % for the control, 100, 300 and
1 000 mg/kg/day groups, respectively. The relatively low post implantation survival index in
the control group could be mainly attributed to the very low individual post implantation
survival indices of two females (Nos. 45 and 48; i.e. 33 % and 7 %, respectively).
Historical control data for Wistar Han rats; F0-females (period 2016-2020): Implantation sites (N): mean = 12.3; P5 – P95 = 6.0-16.0 (n=1511).

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was considered not to be affected by treatment with the test material. The live
birth indices were 92, 95, and 100 % for the control, 100, 300 and 1 000 mg/kg/day groups, respectively.
Seven pups of the control group (Litter Nos. 45 and 48) and 5 pups at 100 mg/kg/day (Litter
No. 55) were found dead at first litter check. No toxicological relevance was attributed to
these dead/missing pups since the mortality incidence did not show a dose-related trend and
remained within the range considered normal for pups of this age.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was not affected by treatment with the test material. The mean duration of
gestation was 21.6, 21.6, 21.1 and 21.3 days for the control, 100, 300 and 1 000 mg/kg/day
groups, respectively.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Gestation index was decreased in females at 1 000 mg/kg/day (80 % compared to 100 % in the
control group). This was attributed to Female Nos. 76 and 77, which both had one
implantation site only neither had a successful pregnancy and below the
range of historical control data.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No toxicologically relevant parental toxicity was noted up to 1 000 mg/kg/day.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test material.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was considered not to be affected by treatment with the test material. The live
birth indices were 92, 95, 100 and 100 % for the control, 100, 300 and 1 000 mg/kg/day
groups, respectively.
Seven pups of the control group (Litter Nos. 45 and 48) and 5 pups at 100 mg/kg/day (Litter
No. 55) were found dead at first litter check. No toxicological relevance was attributed to
these dead/missing pups since the mortality incidence did not show a dose-related trend and
remained within the range considered normal for pups of this age.

The number of live offspring on Day 4 before culling compared to the number of offspring on
Day 1 was not affected by treatment with the test material.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test material.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Litter size was considered not affected by treatment with the test material.
Live litter sizes at first litter check were 9.4, 10.2, 12.6 and 10.5 living pups/litter for the
control, 100, 300 and 1 000 mg/kg/day groups, respectively.
A relatively low mean litter size was noted in the control group and in females treated at 100
and 1 000 mg/kg/day, which was mainly attributed to two litters with a relatively low number
of pups (1 - 6 pups) in each of these groups.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalised for body weight) in male and female pups was
considered not to be affected by treatment with the test material.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Viability indices (number of live offspring on PND 4 before culling as percentage of number
of live offspring on PND 1) were 98, 100, 100 and 100 % for the control, 100, 300 and
1 000 mg/kg/day groups, respectively.
Two pups of the control group (Litter No. 49) were missing on PND 2. Pups missing were
most likely cannibalised. No toxicological relevance was attributed to these dead/missing
pups since the mortality incidence did not show a dose-related trend and remained within the
range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to
treatment with the test material up to 1 000 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to
treatment with the test material up to 1 000 mg/kg/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to
treatment with the test material up to 1 000 mg/kg/day.

Other effects:

no effects observed

Description (incidence and severity):

Areola/Nipple Retention: Treatment up to 1 000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups were nipples observed at PND 13.

Clinical Biochemistry (T4 Levels): Serum T4 levels in male and female PND 14 - 16 pups were considered not to be affected by treatment with the test material.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The effect on gestation index reflects a fertility effect rather than a developmental change.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Main Study Results

Developmental Data

F0 Generation - Lactation

		Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1 000 mg/kg/day
Litters	Total	9	10	7	8
Duration of gestation	Mean (+)	21.6	21.6	21.1	21.3
	St. Dev.	0.9	0.7	0.4	0.5
	N	9	10	7	8
Dead pups at first litter check	Litters affected (#)	2	1	0	0
	Total	7	5	0	0
	Mean (+)	0.8	0.5	0.0	0.0
	St. Dev.	2.0	1.6	0.0	0.0
	N	9	10	7	8
Living pups at first litter check	% of males/females (#)	44/56	44/56	50/50	48/52
	Total	85	102	88	84
	Mean (+)	9.4	10.2	12.6	10.5
	St. Dev.	4.4	4.3	1.6	3.1
	N	9	10	7	8
Postnatal loss	% of living pups	2.4	0.0	0.0	0.0
	Litters affected (#)	1	0	0	0
	Total (#)	2	0	0	0
	Mean (+)	0.2	0.0	0.0	0.0
	St. Dev.	0.7	0.0	0.0	0.0
	N	9	10	7	8
Culled pups	Total	22	33	32	24
Living pups day 4 p.p.	Total	61	69	56	60
	Mean (+)	6.8	6.9	8.0	7.5
	St. Dev.	2.5	2.3	0.0	0.9
	N	9	10	7	8
Breeding loss days 5 – 13 p.p	% of living pups at day 4	0.0	0.0	1.8	0.0
	Litters affected (#)	0	0	1	0
	Total (#)	0	0	1	0
	Mean (+)	0.0	0.0	0.1	0.0
	St. Dev.	0.0	0.0	0.4	0.0
	N	9	10	7	8
Living pups days 13 p.p	% of males/females (#)	43/57	48/52	49/51	48/52
	Total	61	69	55	60
	Mean (+)	6.8	6.9	7.9	7.5
	St. Dev.	2.5	2.3	0.4	0.9
	N	9	10	7	8

+/++ Steel-test significant at 5 % (+) or 1 % (++) level # / ## Fisher's Exact test significant at 5 % (#) or 1 % (##) level

 

Developmental Data

	Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1 000 mg/kg/day
Number of offspring born	92	107	88	84
Total number of uterine implantation sites	118	116	91	90
Number of live offspring on Day 1 after littering	85	102	88	84
Number of offspring on Day 4 (before culling)	83	102	88	84
Number of live offspring on Day 4 (after culling)	61	69	56	60
Number of live offspring on Day 13 after littering	61	69	56	60
Post implantation survival index	78	92	97	93
Live birth index	92	95	100	100
Viability index	98	100	100	100
Lactation index	100	100	98	100

 

Body Weight of Pups (gram): F0 Generation Lactation

Day	Sex		Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1 000 mg/kg/day
1	M	Mean	6.2	6.7	6.2	6.6
		St. Dev.	0.5	0.8	0.3	0.8
		N	9	9	7	8
	F	Mean	5.9	6.4	5.8	6.3
		St. Dev.	0.5	0.7	0.3	0.6
		N	8	10	7	8
	M + F	Mean	6.0	6.5	6.0	6.4
		St. Dev.	0.5	0.7	0.3	0.7
		N	9	10	7	8
4	M	Mean	9.7	10.0	9.1	9.9
		St. Dev.	1.0	1.5	0.4	1.4
		N	9	9	7	8
	F	Mean	9.3	9.7	8.6	9.6
		St. Dev.	1.0	1.3	0.5	1.1
		N	8	10	7	8
	M + F	Mean	9.4	9.8	8.8	9.8
		St. Dev.	0.9	1.3	0.4	1.3
		N	9	10	7	8
7	M	Mean	15.7	16.4	15.5	16.2
		St. Dev.	1.6	2.0	0.6	1.9
		N	9	9	7	8
	F	Mean	156	16.0	14.8	16.0
		St. Dev.	0.7	1.8	0.9	1.4
		N	8	10	7	8
	M + F	Mean	15.3	16.2	15.1	16.1
		St. Dev.	1.5	1.8	0.6	1.6
		N	9	10	7	8
13	M	Mean	29.7	31.6	30.1	31.3
		St. Dev.	2.8	2.9	1.5	2.9
		N	9	9	7	8
	F	Mean	30.1	31.0	29.1	30.9
		St. Dev.	1.8	2.6	1.4	2.4
		N	8	10	7	8
	M + F	Mean	29.5	31.3	29.5	31.1
		St. Dev.	2.8	2.6	1.1	2.6
		N	9	10	7	8

 

Anogenital Distance and Nipple Retention: F0 Generation Lactation

		Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1 000 mg/kg/day
Anogenital dist. M mm	Mean	2.67	2.72	2.94	2.78
	St. Dev.	0.24	0.08	0.31	0.33
	N	9	9	7	8
Anogenital dist. F mm	Mean	0.94	0.90	0.98	0.97
	St. Dev.	0.13	0.10	0.14	0.09
	N	8	10	7	8
Number of nipples	Mean	0.00	0.00	0.00	0.00
	St. Dev.	0.00	0.00	0.00	0.008
	N	9	9	7

# / ## Fisher's Exact test significant at 5 % (#) or 1 % (##) level

+/++ Steel-test significant at 5 % (+) or 1 % (++) level

 

Corrected Anogenital Distance Summary

		Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1 000 mg/kg/day
PND 1 Norm. analog. Dist. M mm	Mean	1.46	1.45	1.60	1.49
	St. Dev.	0.12	0.05	0.17	0.21
	N	9	9	7	8
PND 1 Norm. analog. Dist. F mm	Mean	0.52	0.48	0.55	0.53
	St. Dev.	0.07	0.06	0.09	0.06
	N	8	10	7	8

+/++ Steel-test significant at 5 % (+) or 1 % (++)

 

Summary of Thyroid Hormone Values: F1 Generation

Day 14 Relative to Birth Date

Male		T4 (ng/mL) [G]
0 mg/kg/day Group 1	Mean	66.70
	SD	23.95
	N	9
100 mg/kg/day Group 2	Mean	77.41
	SD	10.35
	N	9
	tCtrl	1.16
300 mg/kg/day Group 3	Mean	70.37
	SD	11.67
	N	7
	tCtrl	1.06
1 000 mg/kg/day Group 4	Mean	97.16
	SD	13.49
	N	8
	tCtrl	1.01

[G] - Anova & Dunnett

 

Summary of Thyroid Hormone Values: F1 Generation

Day 14 Relative to Birth Date

Female		T4 (ng/mL) [G]
0 mg/kg/day Group 1	Mean	62.60
	SD	12.55
	N	8
100 mg/kg/day Group 2	Mean	72.87
	SD	7.11
	N	11
	tCtrl	1.16
300 mg/kg/day Group 3	Mean	67.37
	SD	9.58
	N	7
	tCtrl	1.08
1 000 mg/kg/day Group 4	Mean	70.20
	SD	14.18
	N	8
	tCtrl	1.12

[G] - Anova & Dunnett

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the developmental NOAEL was 1 000 mg/kg/day, as the effect on gestation index reflects a fertility effect rather than a developmental change.

Executive summary:

The developmental toxicity of the test material was assessed according to OECD test Guideline 422 and in compliance with GLP.

The objectives of this study were to determine the potential toxic effects of the test material when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300, 1 000 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Study No. 20236631). The study design was as follows. Test groups consisting of 10 males and 10 females were administered with 100, 300 or 1 000 mg/kg/day of test material. A control group receive the vehicle only.

The following parameters and end points were evaluated in this study: Mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: Mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14 - 16 pups)).

Analysis of test material in vehicle for concentration, homogeneity, and stability was not performed in this study. No feasible analytical method was available, since the test material did not dissolve in any vehicle/solution at all. Due to these test material characteristics and procedures of test formulation preparation/administration in this study (formulations were prepared daily protected from light, considered to be homogeneous after visual inspection and stirred continuously during dose administration within 2 hours after preparation), this GLP exception was considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Parental Results:

No toxicologically relevant parental toxicity was noted up to 1 000 mg/kg/day.

The decreased haemoglobin concentration in females at 300 and 1 000 mg/kg/day was considered not to be toxicologically relevant, since these changes were not associated with any corroborating findings or any pathological alterations.

No mortality and no toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, clinical laboratory investigations, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels), macroscopic examination, organ weights, and microscopic examination).

Developmental Results:
A lower gestation index was noted in females at 1 000 mg/kg/day, which was attributed to two pregnant females with no litters (both had one implantation site only but no successful pregnancy).
No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).
Under the conditions of the study, the developmental NOAEL was 1 000 mg/kg/day as the effect on gestation index reflects a fertility effect rather than a developmental change.  The NOAEL for maternal effects was > 1 000 mg/kg/day.