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EC number: 237-769-9 | CAS number: 13981-86-7
- Life Cycle description
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
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Endpoint summary
Administrative data
Description of key information
Repeated Dose Toxicity - Oral Route (Doomen-van den Hoven, 2021)
Under the conditions of the study, the NOAEL for the test material was > 1 000 mg/kg/day.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- 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
- 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
- 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. - 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
- 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. - 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.
- 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.
- 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.
- Critical effects observed:
- no
- 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.
Reference
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
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Single study available on the registered substance conducted according to OECD test guidelines and in compliance with GLP.
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Repeated Dose Toxicity - Oral Route (Doomen-van den Hoven, 2021)
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.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does not require classification with respect to repeated dose oral toxicity.
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