Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
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

Constituent 1
Chemical structure
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 species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The test facility has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test material. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
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
Details on route of administration:
- 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
Vehicle:
corn oil
Remarks:
Specific gravity 0.92.
Details on oral exposure:
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.
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
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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

Observations and examinations performed and frequency:
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.

FOOD EFFICIENCY: No

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- 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: Yes
- 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: Yes
- 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.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
These tests were performed after dosing, after completion of clinical observations. Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6 - 13).
- Dose groups that were examined: 5 males and 5 females.
- Battery of functions tested: The following tests were performed: Hearing ability (HEARING) (Score 0 = normal/ present, score 1 = abnormal/ absent). Pupillary reflex (PUPIL L/R) (Score 0 = normal/ present, score 1 = abnormal/ absent). Static righting reflex (STATIC R) (Score 0 = normal/ present, score 1 = abnormal/ absent). Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal. Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterised by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- 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, coagulationa, (weighed together with seminal vesicles), Gland, parathyroid (weighed together with the thyroid), Gland, prostate, Gland, seminal vesicle (paired organ weight), Gland, thyroid (paired organ weigth), 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. 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).
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.
Other examinations:
Parental Variables
Body Weight Gains: Calculated against the body weight on Day 1 (pre-mating and lactation periods) or Day 0 (post-coitum period).
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.
Statistics:
See below.

Results and discussion

Results of examinations

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.
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:
effects observed, non-treatment-related
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.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material-related effects at any dose.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Dose Range Finder (DRF) Results









































Parameter



500 mg/kg/day Group



1 000 mg/kg/day Group



Mortality



No mortality



No mortality



Clinical Appearance



No findings



Hunched posture at 1 and 3 h after dosing in Animal 6 on Day 2; and directly and 1 h after dosing in all animals on Day 4.



Body Weight



Normal body weight gain for Animals 2 and 3 between Days 1 – 5 and 5 – 10.


Slight body weight loss (up to 3 %) for Animal 1 between Days 1 – 10.



Normal body weight gain for Animals 4 and 5, and no body weight gain for Animal 6 between Days 1 – 5.


Normal body weight gain for all animals between Days 5 – 10.



Food Consumption



Normal



Normal



Macroscopic Examination



No findings noted



No findings noted



Organ Weights



Normal liver and kidney weights.



Lover and kidney weights (absolute and relative to body weight) slightly lower than normal.



Based on the results of the dose range finder, selected dose levels for the Main study were 100, 300 and 1 000 mg/kg/day. Since no clear peak effect of occurrence of clinical signs was observed in the dose range finder, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.


 


 


Main Study Results


Body Weights Summary (Grams)

































































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Day 1


Week 1



Mean



287



296



289



29



St. Dev.



7.8



8.2



7.9



12.9



N



10



10



10



10



Day 8


Week 2



Mean



300



310



300



307



St. Dev.



14.5



11.6



13.4



13.9



N



10



10



10



10



Mating period



Day 1


Week 1



Mean



312



321



313



323



St. Dev.



17.1



13.2



15.4



14.3



N



10



10



10



10



Day 8


Week 2



Mean



322



334



326



334



St. Dev.



17.6



14.0



16.6



15.3



N



10



10



10



10



Day 15


Week 3



Mean



333



348



340



349



St. Dev.



19.9



13.3



17.7



18.5



N



10



10



10



10



 


Body Weights Summary (Grams)

































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Day 1


Week 1



Mean



16



216



219



219



St. Dev.



9.0



5.0



10.1



10.6



N



10



10



10



10



Day 8


Week 2



Mean



221



223



222



226



St. Dev.



9.9



6.5



10.



12.5



N



10



10



10



10



Mating period



Day 1


Week 1



Mean



224



224



226



229



St. Dev.



10.0



8.1



11.5



14.4



N



10



10



10



10



Day 8


Week 2



Mean



 



 



-



 



St. Dev.



 



 



-



 



N



 



 



0 x



 



Day 15


Week 3



Mean



 



 



-



 



St. Dev.



 



 



-



 



N



 



 



0 x



 



X Excluded data


 


Body Weight Summary: F0 Generation (Grams)





































































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Post coitum



Day 0



Mean



228



226



225



229



St. Dev.



10.5



6.4



8.0



12.1



N



9



10



6



10



Day 4



Mean



239



237



237



242



St. Dev.



10.4



6.6



9.0



14.8



N



9



10



6



10



Day 7



Mean



249



243



243



250



St. Dev.



12.6



6.8



8.8



15.7



N



9



10



6



10



Day11



Mean



259



256



256



262



St. Dev.



11.4



7.1



71



17.9



N



9



10



6



10



Day 14



Mean



268



267



268



270



St. Dev.



12.1



8.5



9.3



17.8



N



9



10



6



10



Day 17



Mean



291



288



292



289



St. Dev.



14.0



11.4



8.3



23.7



N



9



10



6



10



Day 20



Mean



328



325



329



316



St. Dev.



17.3



18.5



12.1



35.2



N



9



10



7



10



Lactation



Day 1



Mean



357



356



253



261



St. Dev.



16.0



11.7



7.5



20.8



N



9



10



7



8



Day 4



Mean



261



263



266



271



St. Dev.



12.9



11.1



13.2



21.5



N



9



10



7



8



Day 7



Mean



269



269



269



275



St. Dev.



11.5



8.6



16.8



18.9



N



9



10



7



8



Day 13



Mean



282



283



287



289



St. Dev.



13.0



16.7



18.9



15.9



N



9



10



7



8



 


Body Wight Gain (%) Summary

































































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Day 1


Week 1



Mean



0



0



0



0



St. Dev.



0.0



0.0



0.0



0.0



N



10



10



10



10



Day 8


Week 2



Mean



4



5



4



6



St. Dev.



2.8



2.0



2.9



1.8



N



10



10



10



10



Mating period



Day 1


Week 1



Mean



9



8



8



12



St. Dev.



4.0



3.2



4.2



3.0



N



10



10



10



10



Day 8


Week 2



Mean



12



13



13



15



St. Dev.



4.6



3.5



4.4



3.7



N



10



10



10



10



Day 15


Week 3



Mean



16



17



18



20



St. Dev.



5.1



3.4



4.7



4.5



N



10



10



10



10



 

































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Day 1


Week 1



Mean



0



0



0



0



St. Dev.



0.0



0.0



0.0



0.0



N



10



10



10



10



Day 8


Week 2



Mean



2



3



2



3



St. Dev.



3.2



2.9



2.3



3.4



N



10



10



10



10



Mating period



Day 1


Week 1



Mean



4



4



3



4



St. Dev.



4.5



4.0



3.5



3.3



N



10



10



10



10



Day 8


Week 2



Mean



 



 



-



 



St. Dev.



 



 



-



 



N



 



 



0 x



 



Day 15


Week 3



Mean



 



 



-



 



St. Dev.



 



 



-



 



N



 



 



0x



 



x Explanations for excluded data are listed in the tables of the individual values


 


Body Weight Gain (%) Summary: F0 Generation





































































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Post coitum



Day 0



Mean



0



0



0



0



St. Dev.



0.0



0.0



0.0



0.0



N



9



10



6



10



Day 4



Mean



5



5



5



6



St. Dev.



1.2



1.4



0.9



2.7



N



9



10



6



10



Day 7



Mean



8



7



8



9



St. Dev.



1.8



1.4



1.8



2.7



N



9



10



6



10



Day11



Mean



14



13



13



15



St. Dev.



1.9



1.3



2.0



3.5



N



9



10



6



10



Day 14



Mean



18



18



19



18



St. Dev.



0.9



1.8



16



3.1



N



9



10



6



10



Day 17



Mean



28



27



29



26



St. Dev.



2.7



3.0



1.9



5.2



N



9



10



6



10



Day 20



Mean



44



44



47



38



St. Dev.



5.3



6.4



3.4



10.2



N



9



10



6



10



Lactation



Day 1



Mean



0



0



0



0



St. Dev.



0.0



0.0



0.0



0.0



N



9



10



7



8



Day 4



Mean



2



3



5



4



St. Dev.



2.8



3.1



3.3



2.0



N



9



10



7



8



Day 7



Mean



5



5



6



5



St. Dev.



4.5



2.8



4.9



2.1



N



9



10



7



8



Day 13



Mean



10



11



13



11



St. Dev.



5.2



6.1



5.1



6.5



N



9



10



7



8



 


Food Consumption Summary (g/animal/day)



























































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-Mating



Days 1 – 8


Weeks 1 - 2



Mean



18



20



18



19



St. Dev.



0.8



0.3



1.5



0.4



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



17



18



18



19



St. Dev.



0.2



0.3



0.2



0.3



N (cage)



2



2



2



2



Mean of means over pre-mating period



Mean



18



19



18



19



Mating Period



Days 1 – 8


Weeks 1 - 2



Mean



16



18



17



17



St. Dev.



0.2



0.8



0.7



0.9



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



17



18



18



18



St. Dev.



0.4



1.1



0.4



0.1



N (cage)



2



2



2



2



Mean of means over mating period



Mean



17



18



17



18



 





























































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-Mating



Days 1 – 8


Weeks 1 - 2



Mean



15



15



15



16



St. Dev.



1.1



1.2



1.0



0.7



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



14



14



14



15



St. Dev.



0.4



1.0



1.8



1.6



N (cage)



2



2



2



2



Mean of means over pre-mating period



Mean



14



14



14



15



Mating Period



Days 1 – 8


Weeks 1 - 2



Mean



 



-



 



 



St. Dev.



 



-



 



 



N (cage)



 



0



 



 



 


Food Consumption Summary (g/animal/day): F0 Generation









































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Post Coitum



Days 0 – 4



Mean



13



13



13



13



St. Dev.



1.6



1.5



1.7



2.3



N



9



10



6



10



Days 4 – 7



Mean



15



14



14



14



St. Dev.



1.9



1.3



2.2



2.8



N



9



10



6



10



Days 7 – 11



Mean



17



16



16



17



St. Dev.



1.7



1.6



2.2



2.1



N



9



10



6



10



Days 11 – 14



Mean



19



18



18



18



St. Dev.



1.6



1.3



1.1



2.2



N



9



10



6



10



Days 14 – 17



Mean



21



20



21



20



St. Dev.



1.8



1.8



2.6



2.5



N



9



10



6



10



Days 17 – 20



Mean



22



22



22



21



St. Dev.



2.1



1.7



2.1



2.5



N



9



10



6



10



Mean of means



Mean



18



17



17



17



Lactation



Days 1 – 4



Mean



25



32



31



28



St. Dev.



6.7



16.5



5.3



5.4



N



8



9



6



8



Days 4 – 7



Mean



33



34



36



35



St. Dev.



7.6



7.1



5.3



5.0



N



9



10



7



8



Days 7 – 13



Mean



44



47



49



48



St. Dev.



11.9



13.9



6.0



4.8



N



9



10



7



8



Mean of means



Mean



34



37



39



37



 


Relative Food Consumption Summary (g/kg bw/day)



























































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Days 1 – 8


Weeks 1 - 2



Mean



61



63



61



63



St. Dev.



2.1



0.7



4.1



1.3



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



58



58



60



60



St. Dev.



0.2



1.4



1.6



0.8



N (cage)



2



2



2



2



Mean of means over pre-mating period



Mean



60



61



60



62



Mating Period



Days 1 – 8


Weeks 1 - 2



Mean



51



53



51



52



St. Dev.



0.0



2.2



2.2



2.9



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



50



52



52



51



St. Dev.



0.5



2.8



0.8



0.9



N (cage)



2



2



2



2



Mean of means over mating period



Mean



51



52



52



51



 





























































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Pre-mating



Days 1 – 8


Weeks 1 - 2



Mean



66



66



67



71



St. Dev.



5.1



3.9



3.4



1.0



N (cage)



2



2



2



2



Days 8 – 15


Weeks 2 - 3



Mean



65



63



63



65



St. Dev.



1.9



3.0



7.1



4.9



N (cage)



2



2



2



2



Mean of means over pre-mating period



Mean



66



64



65



68



Mating Period



Days 1 – 8


Weeks 1 - 2



Mean



 



-



 



 



St. Dev.



 



-



 



 



N (cage)



 



0



 



 



 


Relative Food Consumption Summary (g/kg bw/day): F0 Generation









































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Post Coitum



Days 0 – 4



Mean



56



53



54



55



St. Dev.



5.9



5.6



5.7



7.9



N



9



10



6



10



Days 4 – 7



Mean



62



58



57



57



St. Dev.



5.7



4.8



8.7



9.2



N



9



10



6



10



Days 7 – 11



Mean



66



63



62



64



St. Dev.



6.0



5.7



7.3



5.9



N



9



10



6



10



Days 11 – 14



Mean



69



67



68



68



St. Dev.



3.7



4.8



3.7



5.0



N



9



10



6



10



Days 14 – 17



Mean



72



69



71



69



St. Dev.



4.9



5.7



7.5



4.1



N



9



10



6



10



Days 17 – 20



Mean



67



68



66



67



St. Dev.



3.3



4.5



4.3



3.3



N



9



10



6



10



Mean of means



Mean



65



63



63



63



Lactation



Days 1 – 4



Mean



97



120



113



103



St. Dev.



26.3



55.2



17.2



15.7



N



8



9



6



8



Days 4 – 7



Mean



121



125



133



129



St. Dev.



28.1



24.5



13.1



13.0



N



9



10



7



8



Days 7 – 13



Mean



158



163



171



167



St. Dev.



41.8



42.4



12.3



13.5



N



9



10



7



8



Mean of means



Mean



125



136



139



133



 


Functional Observations





















































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Hearing


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Pupil L


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Pupil R


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Static R score 0/1



Mean



0



0



0



0



N



5



5



5



5



Grip Force


Gram



Mean



1223



1258



1223



1131



St. Dev.



69



95



124



47



N



5



5



5



5



Grip Hind Gram



Mean



817



761



742



732



St. Dev.



50



78



62



91



N



5



5



5



5



 





















































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Hearing


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Pupil L


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Pupil R


Score 0/1



Mean



0



0



0



0



N



5



5



5



5



Static R score 0/1



Mean



0



0



0



0



N



5



5



5



5



Grip Force


Gram



Mean



962



918



936



960



St. Dev.



111



139



89



137



N



5



5



5



5



Grip Hind Gram



Mean



383



321



387



340



St. Dev.



48



72



60



63



N



5



5



5



5



 


Motor Activity Test Summary: Males


































Total Movements



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Mean



3144



3023



3067



3416



N



5



5



5



5



St. Dev.



919



664



416



767



 


Motor Activity Test Summary: Males


































Ambulations



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Mean



693



759



633



714



N



5



5



5



5



St. Dev.



245



237



207



172



 


Motor Activity Test Summary: Females


































Total Movements



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Mean



3600



4208



4020



3797



N



5



5



5



5



St. Dev.



857



885



1458



1107



 


Motor Activity Test Summary: Females


































Ambulations



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Mean



889



1098



1001



904



N



5



5



5



5



St. Dev.



260



370



519



306



 


Summary of Haematology Values: F0 Generation


Day 30 Relative to Start Date


























































































































































































































































Male



WBC


(10^9/L)


[G]



NEUT


(10^9/L)


[G]



LYMPH


(10^9/L)


[G]



MONO


(10^9/L)


[GI]



EOS


(10^9/L)


[G]



BASO


(10^9/L)


[G]



LUC


(10^9/L)


[G]



RBC


(10^9/L)


[G]



RETIC


(10^9/L)


[G]



RDWG


(%)


[G]



HGB


(g/L)


[G]



HCT


(L/L)


[G]



0 mg/kg/day


Group 1



Mean



7.242



1.206



5.798



0.108



0.068



0.014



0.052



8.930



300.32



12.22



158.0



0.4524



SD



1.850



0.711



1.404



0.036



0.016



0.009



0.022



0.810



36.96



0.60



6.3



0.0160



N



5



5



5



5



5



5



5



5



5



5



5



5



100 mg/kg/day


Group 2



Mean



6.460



0.954



5.312



0.090



0.058



0.016



0.030



8.444



238.2*



12.38



155.2



0.4322



SD



1.890



0.120



1.958



0.029



0.011



0.011



0.016



0.447



19.26



0.54



9.1



0.0245



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.89



0.79



0.92



0.83



0.85



1.14



0.58



0.95



0.79



1.01



0.98



0.96



300 mg/kg/day


Group 3



Mean



7.804



1.198



6.366



0.104



0.066



0.014



0.052



8.752



257.76



12.34



156.2



0.4506



SD



2.036



0.524



1.827



0.013



0.043



0.009



0.025



0.661



25.62



0.59



8.6



0.024



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.08



0.99



1.10



0.96



0.97



1.00



1.00



0.98



0.86



1.01



0.99



1.00



1 000 mg/kg/day


Group 4



Mean



6.804



1.128



5.456



0.102



0.054



0.016



0.050



8.330



252.30



13.40



154.2



0.4378



SD



1.996



0.535



1.563



0.029



0.017



0.005



0.026



0.467



43.24



0.98



9.4



0.0315



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.94



0.94



0.94



0.94



0.79



1.14



0.96



0.93



0.84



1.10



0.98



0.97



[G] - Anova & Dunnett: * = p ≤ 0.05


 


Summary of Haematology Values: F0 Generation


Day 30 Relative to Start Date


























































































































Male



MCV


(fL)


[G]



MCH


(pg)


[GI]



MCHC


(g/L)


[GI]



PLT


(10^9/L)


[G]



0 mg/kg/day


Group 1



Mean



50.86



17.78



349.4



690.8



SD



3.11



1.05



2.6



57.4



N



5



5



5



5



100 mg/kg/day


Group 2



Mean



51.18



18.36



358.6



646.6



SD



0.97



0.22



7.8



111.3



N



5



5



5



5



tCtrl



1.01



1.03



1.03



0.94



300 mg/kg/day


Group 3



Mean



51.62



17.90



346.8



745.0



SD



2.36



0.84



2.6



79.4



N



5



5



5



5



tCtrl



1.01



1.01



0.99



1.08



1 000 mg/kg/day


Group 4



Mean



52.64



18.54



352.4



713.2



SD



4.29



1.29



5.0



122.5



N



5



5



5



5



tCtrl



1.03



1.04



1.01



1.03



[G] - Anova & Dunnett


[G1] - Kruskal-Wallis & Dunn


 


Summary of Haematology Values: F0 Generation


Day 51 Relative to Start Date


























































































































































































































































Female



WBC


(10^9/L)


[G]



NEUT


(10^9/L)


[G]



LYMPH


(10^9/L)


[G]



MONO


(10^9/L)


[GI]



EOS


(10^9/L)


[G]



BASO


(10^9/L)


[G]



LUC


(10^9/L)


[G]



RBC


(10^9/L)


[G]



RETIC


(10^9/L)


[G]



RDWG


(%)


[G]



HGB


(g/L)


[G]



HCT


(L/L)


[G]



0 mg/kg/day


Group 1



Mean



5.810



1.664



3.946



0.098



0.076



0.005



0.022



7.284



272.96



12.90



141.8



0.4220



SD



0.939



0.210



0.812



0.031



0.030



0.004



0.008



0.353



50.30



0.39



5.7



0.0193



N



5



5



5



5



5



5



5



5



5



5



5



5



100 mg/kg/day


Group 2



Mean



5.024



1.274



3.582



0.084



0.066



0.006



0.016



7.116



035.00



12.98



135.4



0.4016



SD



0.733



0.327



0.635



0.017



0.057



0.005



0.005



0.400



47.31



1.18



3.0



0.0068



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.86



0.77



0.91



0.86



0.87



0.75



0.73



0.98



0.86



1.01



0.95



0.95



300 mg/kg/day


Group 3



Mean



5.204



1.624



3.366



0.106



0.090



0.006



0.014



6.94



258.84



13.70



133.4*



0.4004



SD



1.229



0.517



0.752



0.057



0.053



0.005



0.005



0.623



29.06



1.72



6.0



0.0186



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.90



0.98



0.85



1.08



1.18



0.75



0.64



0.95



0.95



1.06



0.94



0.95



1 000 mg/kg/day


Group 4



Mean



4.604



1.310



3.124



0.086



0.064



0.004



0.018



6.890



276.78



12.84



132.4*



0.3958



SD



0.434



0.128



0.481



0.017



0.046



0.005



0.011



0.216



29.58



0.73



5.1



0.00137



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.79



0.79



0.79



0.88



0.84



0.50



0.82



0.95



1.01



1.00



0.93



0.94



[G] - Anova & Dunnett


* = p ≤ 0.05


[G1] - Kruskal-Wallis & Dunn


 


Summary of Haematology Values: F0 Generation


Day 51 Relative to Start Date


























































































































Female



MCV


(fL)


[G]



MCH


(pg)


[GI]



MCHC


(g/L)


[GI]



PLT


(10^9/L)


[G]



0 mg/kg/day


Group 1



Mean



57.94



19.48



336.6



687.6



SD



1.30



0.61



6.9



134.7



N



5



5



5



5



100 mg/kg/day


Group 2



Mean



56.58



19.04



337.0



759.4



SD



2.72



0.76



6.0



128.7



N



5



5



5



5



tCtrl



0.98



0.98



1.00



1.10



300 mg/kg/day


Group 3



Mean



58.28



19.42



333.4



520.2



SD



4.28



1.28



8.6



98.7



N



5



5



5



5



tCtrl



1.01



1.00



0.99



1.19



1 000 mg/kg/day


Group 4



Mean



57.44



19.20



334.4



735.8



SD



1.72



0.46



6.8



67.3



N



5



5



5



5



tCtrl



0.99



0.99



0.99



1.07



 


Summary of Coagulation Values: F0 Generation


Day 30 Relative to Start Date


























































































Male



PT


(sec)


[G]



APTT


(sec)


[G]



0 mg/kg/day


Group 1



Mean



16.48



14.00



SD



1.23



3.82



N



5



5



100 mg/kg/day


Group 2



Mean



17.36



15.10



SD



1.25



5.21



N



5



5



tCtrl



1.05



1.08



300 mg/kg/day


Group 3



Mean



16.68



13.60



SD



0.71



2.33



N



4



4



tCtrl



1.01



0.97



1 000 mg/kg/day


Group 4



Mean



17.10



16.44



SD



1.56



3.46



N



5



5



tCtrl



1.04



1.17



[G] - Anova & Dunnett


 


Summary of Coagulation Values: F0 Generation


Day 51 Relative to Start Date


























































































Female



PT


(sec)


[G]



APTT


(sec)


[G]



0 mg/kg/day


Group 1



Mean



15.70



15.84



SD



0.44



1.69



N



5



5



100 mg/kg/day


Group 2



Mean



16.46*



16.16



SD



0.54



1.62



N



5



5



tCtrl



1.05



1.02



300 mg/kg/day


Group 3



Mean



16.42*



16.44



SD



0.43



1.92



N



5



5



tCtrl



1.05



1.04



1 000 mg/kg/day


Group 4



Mean



16.76**



16.14



SD



0.27



2.86



N



5



5



tCtrl



1.07



1.02



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


 


Summary of Clinical Chemistry Values: F0 Generation


Day 30 Relative to Start Date


























































































































































































































































Male



ALT


(U/L)


[G]



AST


(U/L)


[G]



ALP


(U/L)


[GI]



TPROT


(g/L)


[GI]



ALB


(g/L)


[GI]



BILEAC


(umol/L)


[G]



TBIL


(umol/L)


[GI]



UREA


(mmol/L)


[GI]



CREAT


(umol/L)


[G]



GLUC


(mmol/L)


[GI]



CHOL


(mmol/L)


[GI]



NA


(mmol/L)


[GI]



0 mg/kg/day


Group 1



Mean



81.0



83.7



156.3



62.20



38.62



52.13



2.38



6.15



29.3



10.742



1.686



144.2



SD



12.9



10.8



36.4



2.93



1.60



14.62



0.16



1.31



1.5



1.029



0.065



0.4



N



5



5



5



5



5



5



5



5



5



5



5



5



100 mg/kg/day


Group 2



Mean



74.3



87.9



138.3



59.38



36.74



39.83



2.30



6.18



30.9



9.492



1.426



144.0



SD



12.5



15.0



24.0



1.17



0.90



25.04



0.28



0.56



2.3



1.127



0.210



1.2



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



0.92



1.05



0.88



0.95



0.95



0.76



0.97



1.00



1.06



0.88



0.85



1.00



300 mg/kg/day


Group 3



Mean



149.5



181.4



173.8



62.48



38.92



67.52



2.56



6.61



28.3



9.132



1.666



144.2



SD



141.1



220.7



38.3



2.58



1.64



9.09



0.26



0.78



5.3



1.350



0.151



0.8



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.85



2.17



1.11



1.00



1.01



1.30



1.08



1.07



0.97



0.52



0.99



1.00



1 000 mg/kg/day


Group 4



Mean



92.6



84.3



154.2



63.34



38.52



40.36



2.50



5.87



30.9



9.640



1.728



143.8



SD



24.0



12.2



18.9



2.36



1.83



9.10



0.19



0.75



2.4



2.134



0.372



1.8



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.14



1.01



0.99



1.02



1.00



0.77



1.05



0.95



1.06



0.90



1.02



1.00



[G] - Kruskal-Wallis & Dunn [G1] - Anova & Dunnett


 


Summary of Clinical Chemistry Values: F0 Generation


Day 30 Relative to Start Date










































































































































Male



K


(mmol/L)


[GI]



CL


(mmol/L)


[GI]



CA


(mmol/L)


[GI]



PHOS


(mmol/L)


[GI]



T4


(mmol/L)


[GI]



0 mg/kg/day


Group 1



Mean



4.49



105.6



2.652



1.988



40.77



SD



0.21



1.3



0.073



0.258



7.96



N



5



5



5



5



10



100 mg/kg/day


Group 2



Mean



3.94**



105.6



2.532



1.846



4.02



SD



0.12



1.1



0.073



0.206



11.12



N



5



5



5



5



10



tCtrl



0.88



1.00



0.95



0.93



1.08



300 mg/kg/day


Group 3



Mean



3.93**



104.8



2.724



1.920



44.96



SD



0.21



1.1



0.159



0.197



8.14



N



5



5



5



5



10



tCtrl



0.87



0.99



1.03



0.97



1.10



1 000 mg/kg/day


Group 4



Mean



4.23



1.06



2.690



2.096



44.30



SD



0.22



0.7



0.099



0.219



7.87



N



5



5



5



5



10



tCtrl



0.94



1.00



1.01



1.05



1.09



[G] - Anova & Dunnett: ** = p ≤ 0.01


 


Summary of Clinical Chemistry Values: F0 Generation


Day 51 Relative to Start Date


























































































































































































































































Female



ALT


(U/L)


[G]



AST


(U/L)


[G]



ALP


(U/L)


[GI]



TPROT


(g/L)


[GI]



ALB


(g/L)


[GI]



BILEAC


(umol/L)


[G]



TBIL


(umol/L)


[GI]



UREA


(mmol/L)


[GI]



CREAT


(umol/L)


[G]



GLUC


(mmol/L)


[GI]



CHOL


(mmol/L)


[GI]



NA


(mmol/L)


[GI]



0 mg/kg/day


Group 1



Mean



118.5



90.0



249.6



56.48



34.64



24.87



2.28



9.06



23.1



8.304



1.950



140.6



SD



12.2



8.3



121.9



2.27



1.29



20.07



0.47



0.77



2.8



1.167



0.329



2.7



N



5



5



5



5



5



5



5



5



5



5



5



5



100 mg/kg/day


Group 2



Mean



128.0



93.3



157.9



53.88



33.06



25.45



2.13



8.82



24.7



8.316



1.878



140.8



SD



42.3



10.0



35.0



2.08



1.27



18.83



0.32



1.15



2.4



2.126



0.259



1.6



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.08



1.04



0.75



0.95



0.95



1.02



0.95



0.96



1.07



1.00



0.96



1.00



300 mg/kg/day


Group 3



Mean



119.2



93.5



211.0



54.82



33.76



21.42



2.12



9.68



22.0



7.530



1.734



141.4



SD



8.5



6.4



103.7



0.88



0.52



9.48



0.49



0.91



2.6



1.341



0.311



0.9



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.01



1.04



0.85



0.97



0.97



0.86



0.93



1.07



0.95



0.91



0.89



1.01



1 000 mg/kg/day


Group 4



Mean



122.5



94.8



184.0



53.30



32.62*



18.87



2.12



9.42



24.3



7.844



1.930



139.8



SD



29.6



11.0



76.4



1.98



1.01



15.66



0.38



0.73



3.3



1.205



0.430



0.4



N



5



5



5



5



5



5



5



5



5



5



5



5



tCtrl



1.03



1.05



0.74



0.94



0.94



0.76



0.93



1.04



1.05



0.94



0.99



0.99



[G] - Anova & Dunnett: * = p ≤ 0.05 [G1] - Kruskal-Wallis & Dunn


 


Summary of Clinical Chemistry Values: F0 Generation


Day 51 Relative to Start Date


























































































































Female



K


(mmol/L)


[GI]



CL


(mmol/L)


[GI]



CA


(mmol/L)


[GI]



PHOS


(mmol/L)


[GI]



0 mg/kg/day


Group 1



Mean



4.58



106.6



2.148



0.772



SD



0.48



3.0



0.043



0.287



N



5



5



5



5



100 mg/kg/day


Group 2



Mean



4.48



104.8



2.472



1.514



SD



0.75



3.7



0.082



0.529



N



5



5



5



5



tCtrl



0.98



0.98



1.02



1.96



300 mg/kg/day


Group 3



Mean



4.66



105.8



2.506



1.408



SD



0.44



1.5



0.080



0.873



N



5



5



5



5



tCtrl



1.02



0.99



1.04



1.82



1 000 mg/kg/day


Group 4



Mean



4.70



105.6



2.518



1.588



SD



0.14



1.9



0.055



0.624



N



5



5



5



5



tCtrl



1.03



0.99



1.04



2.06



[G] - Anova & Dunnett


 


Macropscopic Findings Summary


End of Treatment








































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Animals examined



10



10



10



10



Animals without findings



9



10



7



9



Animals affected



1



0



3



1



Liver:



 



 



 



 



- Focus/foci



0



0



1



0



- Hardened



0



0



1



0



- Discolouration



0



0



1



0



Pancreas- gelatinous



0



0



1



0



Testes - agenesis



0



0



1



0



Epididymides – Agenesis



0



0



1



0



Pituitary gland – Cyst(s)



1



0



0



0



Mesenteric lymph node discolouration



0



0



1



0



Skin – Scab formation



0



0



0



1



 


 
































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Animals examined



10



10



10



10



Animals without findings



8



6



8



7



Animals affected



2



4



2



3



Uterus



 



 



 



 



-Thickened



0



0



1



0



-Contents



0



0



0



1



Clitoral glands – focus/foci



1



0



0



0



Placenta – contains fluid



0



0



0



1



Thyroid gland



 



 



 



 



-Reduced in size



0



1



0



0



-Discolouration



0



0



1



0



Thymus - foci



0



2



0



0



Mesenteric lymph node



 



 



 



 



-Enlarged



0



0



1



0



-Discolouration



0



0



0



1



Mandibular lymph node - discolouration



1



0



1



0



Skin



 



 



 



 



-Alopecia



1



0



0



0



-Wound(s)



0



0



0



1



-Scab formation



0



1



0



0



Body cavities – contains fluid



0



0



0



1



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


 


Organ Weights Summary (grams)











































































































































































































































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Bodyweight (g)



Mean



317



330



323



331



St. Dev.



20



12



16



16



N



10



10



10



10



Brain



Mean



1.97



1.96



1.96



1.97



St. Dev.



0.05



0.07



0.04



0.04



N



5



5



5



5



Heart



Mean



0.935



0.927



0.927



0.970



St. Dev.



0.070



0.021



0.062



0.104



N



5



5



5



5



Liver



Mean



8.06



7.74



8.09



8.44



St. Dev.



1.09



0.46



0.48



1.10



N



5



5



5



5



Thyroids



Mean



0.0142



0.0168



0.0142



0.0167



St. Dev.



0.0022



0.0027



0.0029



0.0033



N



10



10



10



10



Thymus



Mean



0.285



0.310



0.330



0.326



St. Dev.



0.054



0.027



0.113



0.083



N



5



5



5



5



Kidneys



Mean



2.07



2.09



2.08



2.25



St. Dev.



0.19



0.20



0.12



0.34



N



5



5



5



5



Adrenals



Mean



0.056



0.065



0.058



0.061



St. Dev.



0.012



0.014



0.005



0.014



N



5



5



5



5



Spleen



Mean



0.617



0.563



0.557



0.608



St. Dev.



0.122



0.096



0.076



0.147



N



5



5



5



5



Testes



Mean



3.35



3.32



3.12



3.29



St. Dev.



0.36



0.28



0.25



0.36



N



10



10



9



10



Prostate gland



Mean



0.763



0.847



0.817



0.749



St. Dev.



0.107



0.103



0.130



0.159



N



10



10



10



10



Epididymides



Mean



1.042



1.045



1.034



1.083



St. Dev.



0.098



0.078



0.073



0.097



N



10



10



9



10



Seminal vesicles



Mean



1.318



1.259



1.187



1.281



St. Dev.



0.146



0.113



0.181



0.108



N



10



10



10



10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Organ/Body Weight Ratios (%) Summary











































































































































































































































































































Males



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Bodyweight (g)



Mean



317



330



323



331



St. Dev.



20



12



16



16



N



10



10



10



10



Brain



Mean



0.62



0.60



0.61



0.60



St. Dev.



0.03



0.01



0.05



0.04



N



5



5



5



5



Heart



Mean



0.293



0.282



0.290



0.294



St. Dev.



0.018



0.010



0.029



0.018



N



5



5



5



5



Liver



Mean



2.53



2.36



2.52



2.55



St. Dev.



0.36



0.15



0.07



0.22



N



5



5



5



5



Thyroids



Mean



0.0045



0.0051



0.0044



0.0050



St. Dev.



0.0006



0.0009



0.0009



0.0009



N



10



10



10



10



Thymus



Mean



0.089



0.094



0.102



0.098



St. Dev.



0.017



0.008



0.032



0.021



N



5



5



5



5



Kidneys



Mean



0.65



0.64



0.65



0.68



St. Dev.



0.05



0.05



0.02



0.07



N



5



5



5



5



Adrenals



Mean



0.018



0.020



0.018



0.018



St. Dev.



0.004



0.004



0.001



0.004



N



5



5



5



5



Spleen



Mean



0.193



0.171



0.174



0.183



St. Dev.



0.040



0.030



0.023



0.037



N



5



5



5



5



Testes



Mean



1.06



1.01



0.96



0.99



St. Dev.



0.12



0.07



0.09



0.09



N



10



10



9



10



Prostate gland



Mean



0.241



0.257



0.255



0.226



St. Dev.



0.033



0.035



0.049



0.045



N



10



10



10



10



Epididymides



Mean



0.329



0.317



0.319



0.327



St. Dev.



0.035



0.021



0.026



0.023



N



10



10



9



10



Seminal vesicles



Mean



0.416



0.383



0.368



0.387



St. Dev.



0.048



0.042



0.054



0.033



N



10



10



10



10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Organ Weights Summary: End of Treatment (grams)































































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Bodyweight (g)



Mean



272



284



271



281



St. Dev.



20



16



30



20



N



10



10



10



10



Brain



Mean



197



1.94



1.87*



1.87*



St. Dev.



0.04



0.05



0.05



0.08



N



5



5



5



5



Heart



Mean



0.810



0.831



0.782



0.885



St. Dev.



0.072



0.063



0.052



0.050



N



5



5



5



5



Liver



Mean



12.00



11.64



11.44



12.85



St. Dev.



1.28



2.03



1.19



0.79



N



5



5



5



5



Thyroids



Mean



0.0153



0.0162



0.0171



0.0165



St. Dev.



0.0035



0.0034



0.0024



0.0033



N



10



10



10



10



Thymus



Mean



0.190



0.183



0.215



0.210



St. Dev.



0.045



0.056



0.027



0.041



N



5



5



5



5



Kidneys



Mean



1.96



1.87



1.79



2.02



St. Dev.



0.22



0.19



0.08



0.10



N



5



5



5



5



Adrenals



Mean



0.072



0.071



0.069



0.072



St. Dev.



0.006



0.008



0.009



0.012



N



5



5



5



5



Spleen



Mean



0.504



0.491



0.179



0.521



St. Dev.



0.053



0.068



0.014



0.027



N



5



5



5



5



Ovaries



Mean



0.118



0.117



0.109



0.104



St. Dev.



0.014



0.012



0.014



0.015



N



5



5



5



5



Uterus



Mean



0.366



0.373



0.340



0.366



St. Dev.



0.040



0.065



0.054



0.020



N



5



5



5



5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Organ/Body Weight Ratios (%) Summary: End of Treatment































































































































































































































































Females



Group 1


Control



Group 2


100 mg/kg/day



Group 3


300 mg/kg/day



Group 4


1 000 mg/kg/day



Bodyweight (g)



Mean



272



284



271



281



St. Dev.



20



16



30



20



N



10



10



10



10



Brain



Mean



0.7



0.68



0.37



0.64



St. Dev.



0.05



0.06



0.07



0.03



N



5



5



5



5



Heart



Mean



0.290



0.289



0.279



0.303



St. Dev.



0.026



0.008



0.006



0.018



N



5



5



5



5



Liver



Mean



4.31



4.03



4.08



4.40



St. Dev.



0.47



0.48



0.17



0.35



N



5



5



5



5



Thyroids



Mean



0.0056



0.0057



0.0064



0.0059



St. Dev.



0.0014



0.0012



0.0013



0.0011



N



10



10



10



10



Thymus



Mean



0.068



0.065



0.077



0.072



St. Dev.



0.013



0.024



0.010



0.014



N



5



5



5



5



Kidneys



Mean



0.70



0.65



0.64



0.69



St. Dev.



0.05



0.03



0.04



0.05



N



5



5



5



5



Adrenals



Mean



0.026



0.025



0.025



0.025



St. Dev.



0.003



0.001



0.001



0.004



N



5



5



5



5



Spleen



Mean



0.81



0.171



0.172



0.179



St. Dev.



0.022



0.021



0.013



0.013



N



5



5



5



5



Ovaries



Mean



0.042



0.041



0.039



0.036



St. Dev.



0.003



0.005



0.006



0.006



N



5



5



5



5



Uterus



Mean



0.131



0.131



0.123



0.125



St. Dev.



0.015



0.027



0.027



0.007



N



5



5



5



5



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the NOAEL for the test material was > 1 000 mg/kg/day.
Executive summary:

The repeated dose 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.


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.


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.


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).


Under the conditions of the study, the NOAEL for the test material was > 1 000 mg/kg/day.