Lutetium oxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

Remarks:

Minor procedural deviations. These deviations were considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Correction factor: None (as this would be close to 1).
Final preparation of a solid: According to CiToxLAB France/Study No. 44484 VAS describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study. The dose formulations containing lutetium oxide and prepared at 2 mg/mL and 200 mg/mL in PEG 400 were found to be homogenous and stable after 10 days at room temperature.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in a vehicle

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. This species and strain of rat are recognised as appropriate for general and reproduction toxicity studies. General and reproduction/developmental historical data for this species (same strain and source) are available. This animal model has been proved to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 90 rats (42 males and 48 females), Sprague-Dawley, RjHan: SD (Rats CD®) (SPF quality); Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males approximately 10 weeks old, females approximately 11 weeks old
- Weight at study initiation: (P) males: mean body weight of 449 g (range: 427 g to 493 g), females: mean body weight of 280 g (range: 251 g to 307 g)
- Fasting period before study: no data
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages, (Tecniplast 2154, 940 cm²) with stainless steel lids, containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order not to jeopardise gestation and to avoid aggressive behaviour between males around the time of mating. Towards the end of gestation and during lactation, the females and their litters were provided with autoclaved wood shavings (SICSA, Alfortville, France) as nesting material. Each cage contained a nylabone and a rat hut for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): Free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 68211320 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): Free access to bottles containing tap water (filtered with a 0.22 μm filter)
- Acclimation period: Males were acclimated to the study conditions for a period of 8 days before treatment. Females were acclimated to the study conditions for a period of 6 days before the beginning of estrous cycle monitoring during the pre-treatment period. Two supplementary males in the study and two supplementary females per group were acclimated to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2017-02-23 To: 2017-05-08 (last female)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- In the dose formulation stability study, the dose formulations containing lutetium oxide prepared at 2 mg/mL and 200 mg/mL in PEG 400 were found to be homogenous and stable after 10 days at room temperature. Therefore dose formulation preparation frequency was every 10 days.
- The dose formulations were maintained under delivery conditions (at room temperature) throughout the administration procedure.
- Storage condition of control dose formulation: at room temperature
- The control dose formulation was stirred just before administration and the test item dose formulations for at least 30 minutes before administration. Before stirring, a spatula was used to scrape the test item from the bottom of the test item formulation flasks.
- The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed by CiToxLAB France, Study No. 44484 VAS
- Concentration in vehicle: 0 (0 mg/kg bw/day); 22 mg/mL (110 mg/kg bw/day); 66 mg/mL (330 mg/kg bw/day); 200 mg/mL (1000 mg/kg bw/day)
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no.: MKBG7718V and MKCB9186
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: During the night, each female was placed with the same male until mating occurred (in this study, mating occurred within one week).
- Proof of pregnancy: Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c.
- After successful mating each pregnant female was caged as follows: Individually housed, except during mating and lactation, in polycarbonate cages, (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Towards the end of gestation and during lactation, the females and their litters were provided with autoclaved wood shavings (SICSA, Alfortville, France) as nesting material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical technique: Inductively Coupled Plasma Mass Spectrometry detection (ICP-MS)
Principle and validation of the method: Analytical method provided by the Sponsor and validated at CiToxLAB France (CiToxLAB France/Study No. 44484 VAS) prior to dose formulation analysis.

Determination of test item concentrations in dose formulations:
- Once in week 1 and 5.
- A sample was taken from control and test item dose formulations from all dose groups and analysed using the validated method.

Acceptance criterion:
- Measured concentration = nominal concentration ± 15% (85-115%).

Results:
- The test item concentrations in the administered dose formulations analysed in week 1 and week 5 remained within an acceptable range of variations (+0.8% to +3.8%) when compared to the nominal values (nominal concentrations +/-15%).

Duration of treatment / exposure:

Males: for 2 weeks before pairing, during the mating period (one week), until euthanasia (at least 6 weeks in total)
Females: for 2 weeks before mating, during the mating period (one week), during gestation, during lactation until day 13 p.p. inclusive, until euthanasia for females that did not deliver
Pups: were not given the dose formulations directly, but could potentially be exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces

Frequency of treatment:

Once a day, 7 days per week, at approximately the same time of the day, with a maximum of 7 hours between the earliest and latest administration.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 12 weeks (males) or 13 weeks (females)

Dose / conc.:

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

Remarks:

control group, group 1

Dose / conc.:

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

Remarks:

group 2

Dose / conc.:

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

Remarks:

group 3

Dose / conc.:

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

Remarks:

group 4

No. of animals per sex per dose:

10 animals/sex/dose; 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2-week dose range finding study (CiToxLAB France/Study No. 44485 TSR) performed in the same species. In that study, three groups of male and female Sprague-Dawley rats received the test item daily, by oral administration (gavage) at dose levels of 110, 330 or 1000 mg/kg/day for 2 weeks. Another group received the vehicle only (PEG 400), under the same experimental conditions. As there were no obvious test item effects on clinical signs, food consumption, body weights or necropsy results, the same dose levels were used for the main study: 1000 mg/kg/day was selected as the high dose level for the present study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 110 and 330 mg/kg/day).

- Rationale for animal assignment (if not random): During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a stratified procedure based on body weight (these data are not presented in the report), so that the average body weight of each group was similar. In addition, only females with regular estrous cycles were allocated to the groups.

- Details on route of administration: The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item. The oral route is a mode of administration recommended by the Regulatory Authorities for this type of study. The dose formulations were administered by oral gavage using a plastic syringe fitted with a plastic gavage tube.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day (i.e., during dose formulation administration), until the day of necropsy, at approximately the same time of day, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study (day of necropsy)
- Detailed clinical examinations were performed on all animals.
- Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self mutilation, walking backwards) were also recorded. The day of onset and disappearance of any observed sign was checked.

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of treatment (day 1), then once a week until euthanasia; females: on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver) and on days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period.
- The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
- During the mating period, food consumption was not measured for males or females.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from at least five males and five females euthanised on Day 14 p.p., from each group, on the day of euthanasia. Some blood samples coagulated, therefore, blood samples from additional animals were taken in order to obtain results from five animals/sex/group as far as possible.
- Blood samples were collected from the orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 9:30 a.m.
- Anaesthetic used for blood collection: yes, light isoflurane anesthesia
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: at least five males and five females from each group
- Parameters examined: erythrocytes (RBC), mean cell volume (MCV), packed cell volume (PCV), hemoglobin (HB), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLT), leucocytes (WBC), differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), large unstained cells (LUC), and monocytes (M)), reticulocytes (RTC), blood coagulation parameters: prothrombin time (PT), fibrinogen (FIB), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from at least five males and five females euthanised on Day 14 p.p., from each group, on the day of euthanasia. Some blood samples coagulated, therefore, blood samples from additional animals were taken in order to obtain results from five animals/sex/group as far as possible.
- Blood samples were collected from the orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 9:30 a.m.
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: at least five males and five females from each group
- Parameters examined: sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (PHOS), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), total proteins (PROT), albumin (ALB), albumin/globulin ratio (A/G), bile acids (BIL.AC).

THYROID HORMONES: Yes
- Blood samples were taken in the first half of the morning (between 7:30 and 10 a.m.) into tubes containing K3-EDTA as anticoagulant.
- Blood samples were taken at termination on Day 14 p.p. from all F0 females and at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anaesthesia).
- Blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into two separate tubes (when possible at least 125 μL in the first tube and the remaining plasma in the second tube) and frozen at -80°C.
- The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined by Luminex MAP® technology for F0 males sampled at termination.
- Plasma samples obtained on Day 14 p.p. from F0 females are kept at -80°C. In agreement with the Sponsor, no analyses were performed for these animals.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once over a 60-minute period
- Battery of functions tested: motor activity

IMMUNOLOGY: No

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- The first five males and the first five surviving females to be euthanised on Day 14 p.p. from each group, were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.
- All animals were observed in the cage, in the hand and in the standard arena.
- Detailed clinical examinations: the following parameters were assessed and graded: in the cage: touch escape or ease of removal from the cage; in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis); in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia
- Reactivity to manipulation and different stimuli: the following measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature

MORTALITY AND MORBIDITY: Yes
- Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day (early in the morning and close to the end of the working day) during the treatment period, including weekends and public holidays. Attention was paid to humane end-points.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning, during the 2 weeks of the pre-treatment period, from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated, and on Day 14 p.p. before euthanasia, to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

During microscopic examination, special attention was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Litter size: Total litter size and sex of each pup were recorded as soon as possible after birth (Day 1 p.p.).
- Any gross external malformations were noted.
- Clinical signs: All pups were observed at least once daily for clinical signs, abnormal behaviour and external abnormalities.
- Body weight: The body weight of each live pup was recorded on Day 1, 4, 8 and 13 p.p.
- The litters were observed daily in order to note the number of live, dead and cannibalised pups.
- Anogenital distance (AGD) was measured in pups on Day 1 p.p. The AGD was normalised to the cube root of body weight recorded on Day 1 p.p.
- Number of nipples and of areolae in male pups was determined on Day 12 p.p.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: On completion of the treatment period (i.e. after at least 6 weeks of treatment in total), after at least 14 hours fasting (maximum 24 hours with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination.
- Maternal animals: On completion of the treatment period, on day 14 p.p., after at least 14 hours (maximum 24 hours) fasting (with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination. F0 females which did not deliver were euthanised by the same way without overnight fasting on Day 24 or 25 p.c. (after a body weight recording to check for a possible un-noticed delivery).

GROSS PATHOLOGY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- A complete macroscopic post mortem examination was performed on the prematurely euthanised female K20543 (group 4). In addition, the number of corpora lutea and implantation sites were recorded.
- A complete macroscopic post-mortem examination was performed on all F0 animals including the female euthanised prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanised as scheduled on Day 14 p.p. and for females euthanised on Day 24 or 25 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY
- The body weight of each animal euthanised as scheduled (after the end of the mating period for males or on Day 14 p.p. for lactating females) was recorded before euthanasia. The following organs of the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 14 p.p. of each group were weighed wet as soon as possible after dissection: adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), epididymides (all animals), heart, kidneys, liver, pituitary gland, prostate (dorso-lateral and ventral) (all animals), seminal vesicles (including coagulating glands) (all animals), spleen, testes (all animals), thymus.
- The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
- The following tissues from the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 14 p.p. of each group and female K20543 prematurely euthanised were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in modified Davidson's fixative): macroscopic lesions (all animals), adrenals, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, Cowper's (i.e. bulbo-urethral) glands (all animals), duodenum, epididymides (all animals), esophagus, eyes with Harderian glands, femoral bone with articulation, glans penis (all animals), gut-associated lymphoid tissue (GALT), heart, ileum, jejunum, kidneys, levator ani plus bulbo cavernous muscle complex (all animals), liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area (all animals), ovaries (with oviducts) (all animals), pituitary gland, prostate (dorso-lateral and ventral) (all animals), rectum, sciatic nerve, seminal vesicles (all animals), skeletal muscle, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes (all animals), thymus, thyroids with parathyroids (all animals), trachea, urinary bladder, uterus (horns and cervix) (all animals), vagina (all animals).
- All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). This tissue processing was performed at CiToxLAB France.
- A microscopic examination was performed on: all tissues listed above from the first five euthanised-as-scheduled males and the first five females euthanised on Day 14 p.p. of the control and high dose groups (groups 1 and 4); all macroscopic lesions of all groups; all tissues listed above from the female euthanised prematurely; reproductive organs from animals that did not conceive (males K20012, K20018 and K20025, females K20515, K20522 and K20531) to investigate possible causes.
- Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE
- All surviving pups on day 13 p.p. were euthanised by an intraperitoneal injection of sodium pentobarbital, followed by exsanguination when the thyroids were sampled.
- Pups selected for blood sampling on Day 4 p.p. (and any other pups not selected for further observation) were euthanised by decapitation under isoflurane anaesthesia on Day 4 p.p.

GROSS NECROPSY
- Found dead and prematurely euthanised pups were submitted for a detailed external examination (including orifices and buccal cavity) after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. No tissues were preserved.
- Pups euthanised on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pup/sex/litter. No other tissues were preserved.

HISTOPATHOLOGY
- Thyroids with parathyroids of the selected pup/sex/litter euthanised on Day 13 p.p. were preserved in 10% buffered formalin. In agreement with the Sponsor and in absence of significant effects on this organ in adults or on thyroid hormones, their thyroids with parathyroids were not examined microscopically.

ORGAN WEIGTHS
- The body weight of one selected pup/sex/litter (or of two pups from the same sex when there was only one sex in the litter) euthanised on Day 13 p.p. was recorded before euthanasia. In agreement with the Sponsor and in absence of significant effects on this organ in adults or on thyroid hormones, their thyroids with parathyroids were not weighed.

THYROID HORMONES: Yes
- Blood samples were taken in the first half of the morning (between 7:30 and 10 a.m.) into tubes containing K3-EDTA as anticoagulant.
- Blood samples were taken on Day 4 p.p. from two pups/litter culled (chosen by manual randomisation). Approximately 0.25 mL blood was taken per pup, to obtain approximately 0.5 mL of blood in total. Blood was collected by decapitation under isoflurane anaesthesia and then pooled per litter.
- Blood samples were taken at termination on Day 13 p.p. from two pups/litter (chosen by manual randomisation). Approximately 0.25 mL blood was taken per pup, to obtain approximately 0.5 mL of blood in total. Blood was collected from the vena cava immediately after sacrifice and then pooled per litter.
- Blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into two separate tubes (when possible at least 125 μL in the first tube and the remaining plasma in the second tube) and frozen at -80°C.
- The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined by LC-MS/MS for pups sampled on Day 13 p.p.
- Plasma samples obtained on Day 4 p.p. from pups are kept at -80°C. In agreement with the Sponsor, no analyses were performed for these animals.

Statistics:

Body weight, food consumption and reproductive data:
- Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hematology and blood biochemistry:
- A sequence was used for the statistical analyses of hematology and blood biochemistry data. According to the sequence, the Dunn test, Dunnett test or Mann-Whitney/Wilcoxon test were used to analyse the data.

Organ weight:
- PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to a sequence. At the end of the sequence, the data were analysed using either Dunn test or Dunnett test.

Reproductive indices:

The following parameters were calculated:
- pre-implantation loss = (Number of corpora lutea - Number of implantation sites)/Number of corpora lutea x 100
- post-implantation loss (calculated with Excel) = (Number of implantation sites - Number of live pups)/Number of implantation sites x 100
- mating index = Number of mated animals/Number of paired animals x 100
- fertility index = Number of pregnant female partners/Number of mated pairs x 100
- gestation index = Number of females with live born pups/Number of pregnant females x 100
Data of non-pregnant females are not included in group mean calculations such as body weight, body weight change, food consumption.

Offspring viability indices:

- live birth index = Number of live born pups/Number of delivered pups x 100
- viability index on Day 4 p.p. = Number of surviving pups on Day 4 p.p./Number of live born pups x 100
- lactation index on Day 13 p.p. = Number of surviving pups on Day 13 p.p./Number of surviving pups on Day 4 p.p. x 100
- AGD/cube root of body weight ratio (calculated with Excel): AGD/³√Body weight

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Ptyalism was noted at 1000 mg/kg bw/day in five males (on 5 to 12 days around the middle of the treatment period) and three females (on 5 to 13 days; in gestation mainly), while two control females only (no males) had ptyalism (on 1 or 2 days). This finding was considered as non-adverse (not severe clinical sign) and of minor toxicological relevance. A relationship between the test item treatment and the presence of ptyalism during lactation in female K20533 for 6 days at 330 mg/kg bw/day cannot be excluded.
- Other clinical signs observed in test item groups (chromodacryorrhea, chromorhinorrhea, area of hair loss, cutaneous lesion, scab, loud breathing, reflux at dosing, soiled body parts and short/broken teeth) were considered to be incidental (noted in isolated animals, with no dose-relationship and/or commonly observed in this species and strain).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- No unscheduled deaths in males in any group.
- No unscheduled deaths in females at 0, 110 and 330 mg/kg bw/day.
- At 1000 mg/kg bw/day, female K20543 was prematurely sacrificed during lactation following the death (cannibalism, death or premature euthanasia) of its entire litter on Day 4 p.p. (except one pup found dead before). On the same date, soiled head and limbs were noted for this dam, which lost 6% of body weight from Day 1 p.p. There were no findings in the dam at necropsy or microscopy explaining the premature death of the litter.
- In absence of similar event in the study, this death was considered to be unlikely related to the test item treatment and was considered incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.

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):

- No test item-related effects in males.
- In females at 1000 mg/kg bw/day and when compared with controls, the mean numbers of lymphocytes (6.93 G/L vs. 8.16 G/L) and white blood cells (11.72 G/L vs. 13.43 G/L) tended to be slightly lower. As this observation was not noted in males, was observed in 2/5 females only, as the observed values of these two females remained comparable to controls of similar studies, and in absence of correlating effects in pathology, an effect of the test item was considered to be unlikely.
- There were no effects at 110 and 330 mg/kg bw/day.
- The statistical significance of mean LUC counts in test item-treated groups was considered to be not biologically significant in view of the way of the change (0.15, 0.10, 0.11 and 0.11 G/L at 0, 110, 330 and 1000 mg/kg bw/day, respectively, p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- In males, when compared to controls, mean triglyceride levels tended to be higher in a dose-related manner in test item-treated groups (0.75, 0.99, 1.00 and 1.02 mmol/L at 0, 110, 330 and 1000 mg/kg bw/day, respectively). However, the differences were slight, not statistically significant and individual data were generally comparable to controls of similar studies. Therefore an effect of the test item was considered to be unlikely.
- In females, when compared with controls, mean cholesterol level was higher at 1000 mg/kg bw/day (3.50 mmol/L vs. 2.59 mmol/L, p<0.05), as well as aspartate aminotransferase (ASAT: 144 U/L vs. 108 U/L, p<0.05). These effects were attributed to the test item treatment and considered as not adverse in view of the slight magnitude of differences from controls.
- Female K20539 having the highest cholesterol and ASAT levels also had the highest alanine aminotransferase and triglyceride levels. The relationship with the test item treatment was unclear.
- Thyroid hormone levels: At 1000 mg/kg bw/day, the trend towards higher mean TSH level and lower mean T4 level in males was considered to be of no toxicological significance in absence of statistical significance and correlating effects at pathology, and in view of the slight differences from control means.
- There were no effects at 110 and 330 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Compared with control animals, treated males had a trend towards dose-related lower mean landing foot splay. However, there were no other signs indicating problems of gait or equilibrium during FOB or signs of neurologic dysfunction. Moreover, there was a moderate inter- and intra-variability. A relationship to the test item treatment was considered to be excluded at 110 and 330 mg/kg bw/day, and doubtful at 1000 mg/kg bw/day.
- In females, no differences from controls were considered to be test item-related as there was no dose relationship, they were noted in limited incidence and/or they did not correlate with clinical signs or other FOB results.
- Besides, almost all females were surprisingly noted in mydriasis when the animals were evaluated in the hand, while males were all in myosis, while they are in myosis in similar studies. This event was not considered to be due to a problem at the eyes because the females had no problems during the two other eye tests (visual stimulus response, and especially papillary reflex where myosis was possible with a light stimulus).
- An overview of the results mentioned above is given in Table 1 in the Section "Any further information on results incl. tables" in the entry under Section 7.5.1 (repeated dose toxicity: oral) for this study.
- In absence of correlating clinical signs and FOB findings and in view of the slight to marked inter-variability, no test item related effects on motor activity were evidenced.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- All microscopic observations were spontaneous in nature and not related to test item treatment. In particular, there were no treatment related changes in the thyroids and in any of the reproductive organs.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related effects on estrous cycles during the first 2 weeks of treatment.
- At 1000 mg/kg bw/day, there were two females with 3 consecutive days of estrus once during the first 2 weeks of treatment, which was not noted in controls or in pre-treatment. Taken into account the limited number of affected females and the slight duration of the increased estrus, a relationship with the test item treatment was considered unlikely.
- At the end of the treatment period, females were in diestrus (sometimes listed as metestrus) as expected at mid lactation with the exception of three control females and one female treated at 110 mg/kg bw/day which were noted in proestrus or estrus on Day 14 p.p. At necropsy, this correlated as expected with mucification of the vaginal epithelium for all females observed microscopically at 1000 mg/kg bw/day. Mucification was also observed in four out of the five control females examined microscopically although proestrus or estrus was noted for three out of the four females at vaginal lavage. The fifth control female was noted with diestrus at vaginal lavage but was in proestrus at sacrifice according to microscopic examination.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

- Moderate testicular hypospermatogenesis and epididymal azoospermia were observed in male No. K20025 (330 mg/kg bw/day). These findings show an absence of production of sperm, which is the reason why this animal did not conceive. Such a finding is an occasion that could also be observed in control rats. In the absence of testicular findings at the high dose, it is considered fortuitous and not test item treatment-related.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating and fertility data:
- There were no test item-related effects on mating and fertility data.
- All pairs mated in less than 5 days as expected.
- There were two pairs whose female was not pregnant at 110 mg/kg bw/day and 1 at 330 mg/kg bw/day. At 330 mg/kg bw/day, the absence of gestation was due to the absence of sperm production in male K20025. At 110 mg/kg bw/day, female K20515 had congenital abnormalities unrelated to treatment (agenesis of the right ovary, uterine horn, kidney, adrenal and ureter) which may be the reason of no gestation. Female No. K20522 (group 2) was in diestrus. There were no changes to explain why this female did not conceive.
- These events were not considered to be test item-related (no similar pattern and no dose-relationship).
- An overview of the results is shown in Table 1 in the Section "Any other information on results incl. tables".

Delivery data:
- There were no effects on mean duration of gestation, mean pre-implantation loss rate and the number of pups delivered.
- At 1000 mg/kg bw/day, mean post-implantation loss rate tended to be higher when compared to control animals. This was particularly due to one female which had 86.7% of post-implantation loss. Two other females had a rather high post-implantation loss (29.4% and 36.8%) but one control female also (37.5%). At this dose, females also had higher mean number of corpora lutea, with no statistical significance. An effect of the test item treatment on both parameters was unclear but considered as non-adverse in absence of statistical significance and in view of the slight magnitude of difference.
- An overview of the results is shown in Table 2 in the Section "Any other information on results incl. tables".

No further data.

Key result

Dose descriptor:

NOAEL

Remarks:

for parental systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive performance (mating, fertility and delivery)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- None of the findings were considered to be due to the test item treatment.
- At 1000 mg/kg bw/day, almost all findings were observed in one litter only: K20543, which was entirely dead by Day 4 p.p. (cannibalism, death or premature euthanasia). Dehydration was observed in all its pups from Day 2 p.p. On Day 4 p.p. the surviving pups were all cold to the touch, had no milk in the stomach and lost weight, and some of them were also thin, blue and/or had increased-in-size abdomen. The found dead pups on Day 4 p.p. had also no milk in the stomach.
In absence of similar event in the study, these deaths were considered to be unlikely related to the test item-treatment and incidental.
- An overview of pup clinical signs is given in Table 3 in the Section "Any other information on results incl. tables".

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related effects on pup mortality and viability at 110 and 330 mg/kg/day.
- At 1000 mg/kg bw/day, the low viability index was due to one litter (K20543) dead by Day 4 p.p. following severe clinical signs (see above). Except for this litter, there was only one other pup found dead in the group. This litter death being at an isolated incidence and as there were no comparable signs in the other females of the same group, it was considered to be unlikely that these observations were treatment related.
- An overview of viability index and prematurely dead pups is given in Table 4 and Table 5 in the Section "Any other information on results incl. tables", respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day on Day 1 p.p., males tended to have a lower mean body weight than controls, which was less evident in females. On Day 4 p.p., differences from controls became more pronounced in both sexes and were at least partially due to litter K20543 which lost weight. Afterwards the differences tended to disappear. This effect on mean pup body weight towards the beginning of the treatment period was considered to be test item-related but non-adverse in view of the absence of statistical significance.
- At 110 and 330 mg/kg/day when compared with controls, differences from controls were rarely dose-related and/or too low. Moreover, there was no statistical significance. A test item effect was considered unlikely.
- An overview of pup mean body weight and pup mean body weight gains is given in Table 6 in the Section "Any other information on results incl. tables", respectively.

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:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormones: In absence of statistical significance and in view of the limited differences from control means and/or the poor dose-relationship, there was no toxicologically significant effect in mean T4 and TSH concentrations. An overview of thyroid hormone levels is given in Table 7 in the Section "Any other information on results incl. tables".

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related findings at post-mortem examination of the pups.
There were no macroscopic findings to explain why the litter of female No. K20543 (1000 mg/kg bw/day) was either found dead or sacrificed prematurely.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no microscopic findings to explain why the litter of female No. K20543 (1000 mg/kg bw/day) was either found dead or sacrificed prematurely.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio:
- There were no test item-related effects on the percentage of male pups at birth which was comparable among groups and close to 50%.

Anogenital distance:
- There were no effects on AGD in males.
- When compared with controls, AGD in females was statistically significantly higher at 110, 330 and 1000 mg/kg bw/day as indicated by the ratios AGD to cube root of body weight. However, taking into account the absence of dose relationship and the standard deviations and knowing that data were comparable to contemporaneous studies, a test item treatment-related effect was considered unlikely. For an overview see Table 8 under the Section "Any other information on results incl. tables".

Nipples and areolae numbers:
- There were no test item-related effects on number of nipples and of areolae in male pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No further data

Key result

Dose descriptor:

NOAEL

Remarks:

effects on progeny

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Table 1. Mating and fertility data

Dose level (mg/kg bw/day)	0	110	330	1000
Number of animals paired (M+F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mating index	100%	100%	100%	100%
Mean number of days taken to mate	1.8	2.3	2.5	2.8
Number of pregnant females	10	8	9	10
Fertility index	100%	80%	90%	100%
Number of females with live born pup(s)	10	8	9	10
Gestation index	100%	100%	100%	100%

Table 2. Delivery data

Dose level (mg/kg bw/day)	0	110	330	1000
Number of females which delivered	10	8	9	10
Mean duration of gestation (days)	22.0	21.9	22.1	21.9
Mean number of corpora lutea	14.2	14.4	13.8	16.7
Mean number of implantations	14.1	13.9	13.6	16.7
Mean pre-implantation loss (%)	0.7	3.8	3.7	0.0
Mean number of pups delivered	12.1	12.4	12.2	12.9
Mean post-implantation loss (%)	13.7	11.0	9.0	23.1

Table 3. Pup clinical signs

Dose level (mg/kg bw/day)	0	110	330	1000
Absence of milk in stomach	1 (1)	1 (1)	2 (1)	9 (2) c
Desquamation (back)			1 (1)
Dehydration				14 (1) b
Hematoma (abdomen/back/head)	1 (1)	1 (1) a	1 (1)	3 (1) b
Wound and/or scab (back/forelimb)		1 (1) a		1 (1) b
Blood in anal region		1 (1) a
Cold to the touch				6 (1) b
Thin appearance				3 (1) b
Blue appearance				2 (1) b
Increase in size abdomen				2 (1) b
Generalised pallor		1 (1)
Total of pups affected (litters affected/total of litters)	2 (2/10)	3 (3/8)	4 (3/9)	15 (2/10)

Number of pups (litters) affected.

a: pup K20514-7

b: litter K20543

c: all pups from litter K20543 except one

Table 4. Viability and lactation indexes

Dose level (mg/kg bw/day)	0	110	330	1000
Viability index on Day 4 p.p. (%)	97.5	96.0	97.3	87.6 a
Lactation index on Day 13 p.p. (%)	100	100	100	100

a: 99.1% when excluding litter K20543

Table 5. Prematurely dead pups

Dose level (mg/kg bw/day)	0	110	330	1000
Number of cannibalised pups (litters affected)	2 (2)	2 (2)		6 (1)
Number of found dead pups (litters affected)	1 (1)	2 (2)	3 (2)	4 (2)
Number of prematurely euthanised pups (litters affected)				6 (1)
Total of prematurely dead pups (litters affected/total litters)	3 (3/10)	4 (3/8)	3 (2/9)	16 (2/10)

Table 6. Mean pup body weight and pup mean body weight gains

Sex	Male				Female
Dose level (mg/kg bw/day)	0	110	330	1000	0	110	330	1000
Day 1 p.p.	8.4	8.3	8.2	7.9	7.9	7.8	7.7	7.6
(difference from control in %)		-1	-2	-6		-1	-3	-4
Day 4 p.p. (preculling)	12.5	11.9	11.9	10.9	11.9	11.5	11.1	10.7
(difference from control in %)		-5	-5	-13		-3	-7	-10
Day 4 p.p. (postculling)	12.6	11.9	12.1	11.0	11.8	11.6	11.1	10.8
(difference from control in %)		-6	-4	-13		-2	-6	-8
Day 8 p.p.	22.9	21.5	22.0	21.3	21.9	21.0	20.5	20.9
(difference from control in %)		-6	-4	-7		-4	-6	-5
Day 13 p.p.	37.6	35.3	36.2	36.1	36.0	34.1	34.2	36.0
(difference from control in %)		-6	-4	-4		-5	-5	0
Days 1-4 p.p.	4.1	3.6	3.7	3.0	4.0	3.7	3.4	3.1
Days 4-13 p.p.	24.9	23.4	24.1	24.6	24.2	22.5	23.1	24.6

Table 7. Mean thyroid hormone levels (+/- SD) in litter plasma on Day 13 p.p.

Dose level (mg/kg bw/day)	0	110	330	1000
T4 (ng/mL)	31.9 +/- 4.06	30.8 +/- 3.38 (-3%)	30.6 +/- 2.58 (-4%)	29.9 +/- 3.55 (-6%)
TSH (pg/mL)	1428 +/- 511	1355 +/- 349 (-5%)	1217 +/- 275 (-15%)	1588 +/- 510 (+11%)

Table 8. Anogenital distance data for pups (mean +/- SD)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	110	330	1000	0	110	330	1000
AGD (mm) on Day 1 p.p.	5.19 +/- 0.60	5.02 +/- 0.57 (-3%)	5.18 +/- 0.46 (-0%)	5.07 +/- 0.51 (-2%)	2.73 +/- 0.49	3.05** +/- 0.59 (+12%)	2.91 +/- 0.36 (+7%)	2.94 +/- 0.40 (+8%)
Body weight on Day 1 p.p.	8.4	8.3 (-1%)	8.2 (-2%-	7.9 (-6%)	7.9	7.8 (-1%)	7.7 (-3%)	7.6 (-4%)
Ratio AGD/^3√body weight (mm/g^1/3)	2.55 +/- 0.29	2.51 +/- 0.26 (-2%)	2.57 +/- 0.21 (+1%)	2.55 +/- 0.25 (+0%)	1.37 +/- 0.24	1.55** +/- 0.31 (+13%)	1.48* +/- 0.18 (+8%)	1.51** +/- 0.20 (+10%)

Statistical significance:*: p<0.05, **: p<0.01

Conclusions:

In an OECD 422 study, the test item lutetium oxide was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 13 p.p., at dose levels of 110, 330 and 1000 mg/kg bw/day. Based on the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be at least 1000 mg/kg bw/day based on the absence of adverse effects at this dose level and the non-adverse findings in clinical signs and blood biochemistry,
- the NOAEL for reproductive performance (mating, fertility and delivery) was considered to be at least 1000 mg/kg bw/day based on the absence of adverse effects on mating, fertility and delivery data,
- the NOAEL for toxic effects on progeny was considered to be at least 1000 mg/kg bw/day based on the absence of adverse effects in pups and on the reduction in mean pup weight towards the beginning of the lactation period.
Based on the results of this study, lutetium oxide does not need to be classified for reproductive toxicity under the CLP Regulation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

Remarks:

Minor procedural deviations. These deviations were considered not to have compromised the validity or integrity of the study. See "Any further information on materials and methods incl. tables" for further details.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lutetium oxide

EC Number:

234-764-3

EC Name:

Lutetium oxide

Cas Number:

12032-20-1

Molecular formula:

Lu2O3

IUPAC Name:

Lutetium oxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): dilutetium trioxide
- Physical state: solid
- Appearance: white (yellowish) powder

Specific details on test material used for the study:

Correction factor: None (as this would be close to 1).
Final preparation of a solid: According to CiToxLAB France/Study No. 44484 VAS describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study. The dose formulations containing lutetium oxide and prepared at 2 mg/mL and 200 mg/mL in PEG 400 were found to be homogenous and stable after 10 days at room temperature.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in a vehicle

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. This species and strain of rat are recognised as appropriate for general and reproduction toxicity studies. General and reproduction/developmental historical data for this species (same strain and source) are available. This animal model has been proved to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 90 rats (42 males and 48 females), Sprague-Dawley, RjHan: SD (Rats CD®) (SPF quality); Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males approximately 10 weeks old, females approximately 11 weeks old
- Weight at study initiation: (P) males: mean body weight of 449 g (range: 427 g to 493 g), females: mean body weight of 280 g (range: 251 g to 307 g)
- Fasting period before study: no data
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages, (Tecniplast 2154, 940 cm²) with stainless steel lids, containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order not to jeopardise gestation and to avoid aggressive behaviour between males around the time of mating. Towards the end of gestation and during lactation, the females and their litters were provided with autoclaved wood shavings (SICSA, Alfortville, France) as nesting material. Each cage contained a nylabone and a rat hut for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): Free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 68211320 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): Free access to bottles containing tap water (filtered with a 0.22 μm filter)
- Acclimation period: Males were acclimated to the study conditions for a period of 8 days before treatment. Females were acclimated to the study conditions for a period of 6 days before the beginning of estrous cycle monitoring during the pre-treatment period. Two supplementary males in the study and two supplementary females per group were acclimated to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2017-02-23 To: 2017-05-08 (last female)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item. The oral route is a mode of administration recommended by the Regulatory Authorities for this type of study. The dose formulations were administered by oral gavage using a plastic syringe fitted with a plastic gavage tube.

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- In the dose formulation stability study, the dose formulations containing lutetium oxide prepared at 2 mg/mL and 200 mg/mL in PEG 400 were found to be homogenous and stable after 10 days at room temperature. Therefore dose formulation preparation frequency was every 10 days.
- The dose formulations were maintained under delivery conditions (at room temperature) throughout the administration procedure.
- Storage condition of control dose formulation: at room temperature
- The control dose formulation was stirred just before administration and the test item dose formulations for at least 30 minutes before administration. Before stirring, a spatula was used to scrape the test item from the bottom of the test item formulation flasks.
- The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed by CiToxLAB France, Study No. 44484 VAS
- Concentration in vehicle: 0 (0 mg/kg bw/day); 22 mg/mL (110 mg/kg bw/day); 66 mg/mL (330 mg/kg bw/day); 200 mg/mL (1000 mg/kg bw/day)
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no.: MKBG7718V and MKCB9186
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical technique: Inductively Coupled Plasma Mass Spectrometry detection (ICP-MS)
Principle and validation of the method: Analytical method provided by the Sponsor and validated at CiToxLAB France (CiToxLAB France/Study No. 44484 VAS) prior to dose formulation analysis.

Determination of test item concentrations in dose formulations:
- Once in week 1 and 5.
- A sample was taken from control and test item dose formulations from all dose groups and analysed using the validated method.

Acceptance criterion:
- Measured concentration = nominal concentration ± 15% (85-115%).

Results:
- The test item concentrations in the administered dose formulations analysed in week 1 and week 5 remained within an acceptable range of variations (+0.8% to +3.8%) when compared to the nominal values (nominal concentrations +/-15%).

Duration of treatment / exposure:

Males: for 2 weeks before pairing, during the mating period (one week), until euthanasia (at least 6 weeks in total)
Females: for 2 weeks before mating, during the mating period (one week), during gestation, during lactation until day 13 p.p. inclusive, until euthanasia for females that did not deliver

Frequency of treatment:

Once a day, 7 days per week, at approximately the same time of the day, with a maximum of 7 hours between the earliest and latest administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control group, group 1

Doses / concentrations 2

Dose / conc.:

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

Remarks:

group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

group 4

No. of animals per sex per dose:

10 animals/sex/dose; 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2-week dose range finding study (CiToxLAB France/Study No. 44485 TSR) performed in the same species. In that study, three groups of male and female Sprague-Dawley rats received the test item daily, by oral administration (gavage) at dose levels of 110, 330 or 1000 mg/kg/day for 2 weeks. Another group received the vehicle only (PEG 400), under the same experimental conditions. As there were no obvious test item effects on clinical signs, food consumption, body weights or necropsy results, the same dose levels were used for the main study: 1000 mg/kg/day was selected as the high dose level for the present study. The low dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 110 and 330 mg/kg/day).

- Rationale for animal assignment (if not random): During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a stratified procedure based on body weight (these data are not presented in the report), so that the average body weight of each group was similar. In addition, only females with regular estrous cycles were allocated to the groups.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day (i.e., during dose formulation administration), until the day of necropsy, at approximately the same time of day, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study (day of necropsy)
- Detailed clinical examinations were performed on all animals.
- Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self mutilation, walking backwards) were also recorded. The day of onset and disappearance of any observed sign was checked.

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of treatment (day 1), then once a week until euthanasia; females: on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver) and on days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period.
- The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
- During the mating period, food consumption was not measured for males or females.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from at least five males and five females euthanised on Day 14 p.p., from each group, on the day of euthanasia. Some blood samples coagulated, therefore, blood samples from additional animals were taken in order to obtain results from five animals/sex/group as far as possible.
- Blood samples were collected from the orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 9:30 a.m.
- Anaesthetic used for blood collection: yes, light isoflurane anesthesia
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: at least five males and five females from each group
- Parameters examined: erythrocytes (RBC), mean cell volume (MCV), packed cell volume (PCV), hemoglobin (HB), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLT), leucocytes (WBC), differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), large unstained cells (LUC), and monocytes (M)), reticulocytes (RTC), blood coagulation parameters: prothrombin time (PT), fibrinogen (FIB), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from at least five males and five females euthanised on Day 14 p.p., from each group, on the day of euthanasia. Some blood samples coagulated, therefore, blood samples from additional animals were taken in order to obtain results from five animals/sex/group as far as possible.
- Blood samples were collected from the orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 9:30 a.m.
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: at least five males and five females from each group
- Parameters examined: sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (PHOS), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), total proteins (PROT), albumin (ALB), albumin/globulin ratio (A/G), bile acids (BIL.AC).

THYROID HORMONES: Yes
- Blood samples were taken in the first half of the morning (between 7:30 and 10 a.m.) into tubes containing K3-EDTA as anticoagulant.
- Blood samples were taken at termination on Day 14 p.p. from all F0 females and at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anaesthesia).
- Blood was centrifuged within 2 hours after sampling (approximately 3000g for 10 minutes at +4°C). The plasma was transferred into two separate tubes (when possible at least 125 μL in the first tube and the remaining plasma in the second tube) and frozen at -80°C.
- The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined by Luminex MAP® technology for F0 males sampled at termination.
- Plasma samples obtained on Day 14 p.p. from F0 females are kept at -80°C. In agreement with the Sponsor, no analyses were performed for these animals.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once over a 60-minute period
- Battery of functions tested: motor activity

IMMUNOLOGY: No

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- The first five males and the first five surviving females to be euthanised on Day 14 p.p. from each group, were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.
- All animals were observed in the cage, in the hand and in the standard arena.
- Detailed clinical examinations: the following parameters were assessed and graded: in the cage: touch escape or ease of removal from the cage; in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis); in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia
- Reactivity to manipulation and different stimuli: the following measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature

MORTALITY AND MORBIDITY: Yes
- Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day (early in the morning and close to the end of the working day) during the treatment period, including weekends and public holidays. Attention was paid to humane end-points.

Sacrifice and pathology:

SACRIFICE
- Male animals: On completion of the treatment period (i.e. after at least 6 weeks of treatment in total), after at least 14 hours fasting (maximum 24 hours with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination.
- Maternal animals: On completion of the treatment period, on day 14 p.p., after at least 14 hours (maximum 24 hours) fasting (with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination. F0 females which did not deliver were euthanised by the same way without overnight fasting on Day 24 or 25 p.c. (after a body weight recording to check for a possible un-noticed delivery).

GROSS PATHOLOGY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- A complete macroscopic post mortem examination was performed on the prematurely euthanised female K20543 (group 4). In addition, the number of corpora lutea and implantation sites were recorded.
- A complete macroscopic post-mortem examination was performed on all F0 animals including the female euthanised prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanised as scheduled on Day 14 p.p. and for females euthanised on Day 24 or 25 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY
- The body weight of each animal euthanised as scheduled (after the end of the mating period for males or on Day 14 p.p. for lactating females) was recorded before euthanasia. The following organs of the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 14 p.p. of each group were weighed wet as soon as possible after dissection: adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), epididymides (all animals), heart, kidneys, liver, pituitary gland, prostate (dorso-lateral and ventral) (all animals), seminal vesicles (including coagulating glands) (all animals), spleen, testes (all animals), thymus.
- The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
- The following tissues from the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 14 p.p. of each group and female K20543 prematurely euthanised were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in modified Davidson's fixative): macroscopic lesions (all animals), adrenals, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, Cowper's (i.e. bulbo-urethral) glands (all animals), duodenum, epididymides (all animals), esophagus, eyes with Harderian glands, femoral bone with articulation, glans penis (all animals), gut-associated lymphoid tissue (GALT), heart, ileum, jejunum, kidneys, levator ani plus bulbo cavernous muscle complex (all animals), liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area (all animals), ovaries (with oviducts) (all animals), pituitary gland, prostate (dorso-lateral and ventral) (all animals), rectum, sciatic nerve, seminal vesicles (all animals), skeletal muscle, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes (all animals), thymus, thyroids with parathyroids (all animals), trachea, urinary bladder, uterus (horns and cervix) (all animals), vagina (all animals).
- All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). This tissue processing was performed at CiToxLAB France.
- A microscopic examination was performed on: all tissues listed above from the first five euthanised-as-scheduled males and the first five females euthanised on Day 14 p.p. of the control and high dose groups (groups 1 and 4); all macroscopic lesions of all groups; all tissues listed above from the female euthanised prematurely; reproductive organs from animals that did not conceive (males K20012, K20018 and K20025, females K20515, K20522 and K20531) to investigate possible causes.
- Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Other examinations:

No further data

Statistics:

Body weight, food consumption and reproductive data:
- Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hematology and blood biochemistry:
- A sequence was used for the statistical analyses of hematology and blood biochemistry data. According to the sequence, the Dunn test, Dunnett test or Mann-Whitney/Wilcoxon test were used to analyse the data.

Organ weight:
- PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to a sequence. At the end of the sequence, the data were analysed using either Dunn test or Dunnett test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Ptyalism was noted at 1000 mg/kg bw/day in five males (on 5 to 12 days around the middle of the treatment period) and three females (on 5 to 13 days; in gestation mainly), while two control females only (no males) had ptyalism (on 1 or 2 days). This finding was considered as non-adverse (not severe clinical sign) and of minor toxicological relevance. A relationship between the test item treatment and the presence of ptyalism during lactation in female K20533 for 6 days at 330 mg/kg bw/day cannot be excluded.
- Other clinical signs observed in test item groups (chromodacryorrhea, chromorhinorrhea, area of hair loss, cutaneous lesion, scab, loud breathing, reflux at dosing, soiled body parts and short/broken teeth) were considered to be incidental (noted in isolated animals, with no dose-relationship and/or commonly observed in this species and strain).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- No unscheduled deaths in males in any group.
- No unscheduled deaths in females at 0, 110 and 330 mg/kg bw/day.
- At 1000 mg/kg bw/day, female K20543 was prematurely sacrificed during lactation following the death (cannibalism, death or premature euthanasia) of its entire litter on Day 4 p.p. (except one pup found dead before). On the same date, soiled head and limbs were noted for this dam, which lost 6% of body weight from Day 1 p.p. There were no findings in the dam at necropsy or microscopy explaining the premature death of the litter.
- In absence of similar event in the study, this death was considered to be unlikely related to the test item treatment and was considered incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.

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):

- No test item-related effects in males.
- In females at 1000 mg/kg bw/day and when compared with controls, the mean numbers of lymphocytes (6.93 G/L vs. 8.16 G/L) and white blood cells (11.72 G/L vs. 13.43 G/L) tended to be slightly lower. As this observation was not noted in males, was observed in 2/5 females only, as the observed values of these two females remained comparable to controls of similar studies, and in absence of correlating effects in pathology, an effect of the test item was considered to be unlikely.
- There were no effects at 110 and 330 mg/kg bw/day.
- The statistical significance of mean LUC counts in test item-treated groups was considered to be not biologically significant in view of the way of the change (0.15, 0.10, 0.11 and 0.11 G/L at 0, 110, 330 and 1000 mg/kg bw/day, respectively, p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- In males, when compared to controls, mean triglyceride levels tended to be higher in a dose-related manner in test item-treated groups (0.75, 0.99, 1.00 and 1.02 mmol/L at 0, 110, 330 and 1000 mg/kg bw/day, respectively). However, the differences were slight, not statistically significant and individual data were generally comparable to controls of similar studies. Therefore an effect of the test item was considered to be unlikely.
- In females, when compared with controls, mean cholesterol level was higher at 1000 mg/kg bw/day (3.50 mmol/L vs. 2.59 mmol/L, p<0.05), as well as aspartate aminotransferase (ASAT: 144 U/L vs. 108 U/L, p<0.05). These effects were attributed to the test item treatment and considered as not adverse in view of the slight magnitude of differences from controls.
- Female K20539 having the highest cholesterol and ASAT levels also had the highest alanine aminotransferase and triglyceride levels. The relationship with the test item treatment was unclear.
- Thyroid hormone levels: At 1000 mg/kg bw/day, the trend towards higher mean TSH level and lower mean T4 level in males was considered to be of no toxicological significance in absence of statistical significance and correlating effects at pathology, and in view of the slight differences from control means.
- There were no effects at 110 and 330 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Compared with control animals, treated males had a trend towards dose-related lower mean landing foot splay. However, there were no other signs indicating problems of gait or equilibrium during FOB or signs of neurologic dysfunction. Moreover, there was a moderate inter- and intra-variability. A relationship to the test item treatment was considered to be excluded at 110 and 330 mg/kg bw/day, and doubtful at 1000 mg/kg bw/day.
- In females, no differences from controls were considered to be test item-related as there was no dose relationship, they were noted in limited incidence and/or they did not correlate with clinical signs or other FOB results.
- Besides, almost all females were surprisingly noted in mydriasis when the animals were evaluated in the hand, while males were all in myosis, while they are in myosis in similar studies. This event was not considered to be due to a problem at the eyes because the females had no problems during the two other eye tests (visual stimulus response, and especially papillary reflex where myosis was possible with a light stimulus).
- An overview of the results mentioned above is given in Table 1 in the Section "Any further information on results incl. tables".
- In absence of correlating clinical signs and FOB findings and in view of the slight to marked inter-variability, no test item related effects on motor activity were evidenced.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no differences between control and test item-treated groups which could be attributable to test item treatment, even though on rare occasions differences reached statistical significance.
- Marginally decreased spleen and thymus mean weights in all test item-treated male groups and marginally increased spleen and thymus mean weights in all test item-treated female groups did not show a dose relationship and had no histological correlate. These differences were therefore considered spurious.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- All macroscopic observations were spontaneous in nature and not related to test item treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- All microscopic observations were spontaneous in nature and not related to test item treatment. In particular, there were no treatment related changes in the thyroids and in any of the reproductive organs.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No further data

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

for parental systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Detailed clinical examination and reactivity to manipulation and different stimuli - most relevant differences compared with controls.

Sex  Dose level (mg/kg bw/day)	Male 0	Male 110	Male 330	Male 1000	Female 0	Female 110	Female 330	Female 1000
Landing foot splay (mean ± SD; in mm)	103 ± 44	95 ± 27	89 ± 31	69 ± 25	109 ± 28	102 ± 21	101 ± 16	106 ± 28
Mydriasis					5/5	4/5	5/5	3/5
Slight piloerection								1/5
Defecation: excessive quantity or abnormal appearance						1/5

Applicant's summary and conclusion

Conclusions:

Interpretation of results: GHS criteria not met

The test item, lutetium oxide, was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating and (for females) throughout gestation and until Day 13 post-partum, at the dose-levels of 110, 330 or 1000 mg/kg bw/day.
Based on the experimental conditions of the study the No Observed Adverse Effect Level (NOAEL) was considered to be higher than or equal to 1000 mg/kg bw/day based on the absence of adverse findings (i.e. findings of toxicological relevance) related to treatment with the test item at this high dose level.