Yttrium trinitrate

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:

2015-10-12 to 2017-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2015-07-07 to 2015-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley (SD) rat was the species and strain of choice because it is accepted by many regulatory authorities and international guidelines as a recommended test system. There is ample experience and historical background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 40 Hsd: Sprague Dawley (SD) rats (20 males and 20 females)
- Age at delivery: 27 - 29 days
- Weight at arrival: 88 - 95 grams for males and 88 - 94 grams for females
- Fasting period before study: no data
- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulphone solid bottomed cages as indicated in the relevant SOP (SOP/ANI/001). Nesting material was provided inside suitable bedding bags and was changed at least twice a week. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): A commercially available powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water (e.g. ad libitum): ad libitum, via water bottles
- Acclimation period: Approximately 2 weeks, under test conditions, during which time the health status of the animals was assessed by thorough observations. A health check was performed by a veterinarian.
- There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2015-07-07 To: 2015-07-28

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man. Administration via diet was selected due to a too low pH in solution with water-based vehicles.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was formulated using powdered diet, by a premix followed by dilution with further quantity of diet and mixing. The formulation was prepared separate for each group/sex. Inclusion levels (ppm) were reviewed during the study, in order to obtain nominal dose levels of 250, 500 and 1000 mg/kg/day.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diet was prepared up to 3-4 days according to available stability data supplied by the Laboratory LAUS which performed the validation study (stability in diet up to 10 days at room temperature in the range of 250-15000 ppm).
- Mixing appropriate amounts with (Type of food): Powdered rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy).
- Storage temperature of food: no data
- Concentrations were calculated and expressed in terms of active ingredient (71.3%).
- The concentrations of test item in the diet were adjusted as required in order to maintain the required dose level for each treated group.
- Initial dietary concentrations were predicted from growth and food intake data collected during the acclimatisation period. The dietary inclusion levels were reviewed twice weekly (on the day of body weight measurements) and new diets supplied the day after body weight measurements.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in diet

No. of animals per sex per dose:

4 males and 4 females/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): On the day of allocation (7 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs were recorded.
- Cage side observations were included.
- All clinical signs were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day before treatment commenced (day -1), twice weekly thereafter (i.e. days 4, 7, 11, 14) and just prior to necropsy (day 15).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food consumed by each cage of rats was recorded twice weekly following allocation. The group mean daily intake per rat was calculated.
- Twice weekly the group mean achieved intake of test item was calculated from the group mean body weight and food consumption data and the dietary inclusion levels of the test item.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 2 of treatment (day 15) prior to necropsy
- Samples of blood were withdrawn from the abdominal vena cava.
- Anaesthetic used for blood collection: yes (light isofluorane anaesthesia)
- Animals fasted: yes, overnight fasting
- How many animals: all surviving animals
- Parameters examined: haematocrit (%), haemoglobin (g/dL), red blood cell count (x 10^6/µL), reticulocyte count (% or x 10^9/L), mean red blood cell volume (fL), mean corpuscular haemoglobin (pg), mean corpuscular haemoglobin concentration (g/dL), white blood cell count (x 10^3/µL), differential leucocyte count, neutrophils (% or x 10^3/L), lymphocytes (% or x 10^3/L), eosinophils (% or x 10^3/L), basophils (% or x 10^3/L), monocytes (% or x 10^3/L), large unstained cells (% or x 10^3/L), platelets (x 10^3/µL), coagulation: prothrombin time (sec)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 2 of treatment (day 15) prior to necropsy
- Samples of blood were withdrawn from the abdominal vena cava.
- Animals fasted: yes, overnight fasting
- How many animals: all surviving animals
- Parameters examined: alkaline phosphatase (U/L), alanine aminotransferase (U/L), aspartate aminotransferase (U/L), urea (mg/dL), creatinine (mg/dL), glucose (mg/dL), total bilirubin (mg/dL), total cholesterol (mg/dL), total protein (g/dL), albumin (g/dL), sodium (mmol/dL), potassium (mmol/dL), calcium (mmol/dL), chloride (mmol/dL).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
MORTALITY:
- Time schedule: All animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals that completed the scheduled test period, were killed by exsanguination under isofluorane anaesthesia (by inhalation) and were subjected to necropsy after weighing, supervised by a pathologist.
- The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative.
- From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed, tissues were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol): abnormalities/gross lesions, adrenal glands, bone marrrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Payer's patches), kidneys, liver, lungs, lymph nodes - mesenteric, lymph nodes - cervical, ovaries, oviducts, parathyroid glands, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal column, spinal cord, spleen, stomach, testes, thymus (where present), thyroid, trachea, urinary bladder, uterus - cervix
- The ratios of organ weight to body weight and organ weight to brain weight were calculated for each animal.

HISTOPATHOLOGY: No

Other examinations:

No

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant or treatment-related clinical signs were recorded during the study.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment related effects were seen on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- No toxicologically relevant differences in food consumption were noted between control and treated groups.
- The achieved dosages throughout the study were calculated to be 260, 634 and 1221 mg/kg/day for males and 322, 582 and 1187 mg/kg/day for females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- No changes of toxicological relevance were recorded. Statistically significant decreases between control and treated females (haemoglobin and monocytes) were recorded in animals dosed with 250 and/or 500 mg/kg/day, therefore considered unrelated to treatment.
- No changes were recorded in coagulation parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- A statistically significant decrease of alanine aminotransferase was recorded in males dosed with 250 and 1000 mg/kg/day (26% and 29%, respectively). In addition, urea was decreased in females dosed with 1000 mg/kg/day (28%). Due to the absence of dose-relation, the direction and/or the severity of changes, the above findings were considered of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- No relevant differences were noted in the organ weights (absolute and relative organ weights to terminal body weight or to brain weight) and terminal body weights, with the exception of a slight statistically significant decrease in relative liver weight (-13%) seen in low dose males, and hence considered not treatment-related or toxicologically relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Animals killed at termination did not show relevant macroscopic changes that could be treatment related. However, enlarged spleen was observed in two high dose males sacrificed at final sacrifice. The remaining observed changes are suggested to be incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated SD rats of the same age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No further data

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

anhydrous yttrium trinitrate

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The purpose of this study was to investigate the toxicity of yttrium trinitrate in a preliminary 2-week dietary study in rats which received medicated diet at nominal dose levels of 250, 500 and 1000 mg/kg/day.
The achieved dosages troughout the study were calculated to be 260, 634 and 1221 mg/kg/day for males and 322, 582 and 1187 mg/kg/day for females.
No mortality occurred during the study.
No significant effects were seen in the in vivo parameters (clinical signs, body weight and food consumption).
Statistically significant decreases were observed in haemoglobin and monocytes when comparing control and females dosed at 250 and/or 500 mg/kg/day. Statistically significant decreases in alanine aminotransferase in males dosed at 250 and 1000 mg/kg/day and in urea in females dosed at 1000 mg/kg/day, were observed. All abovementioned changes were considered not toxicologically relevant.
No relevant differences were noted in the organ weights and terminal body weights.
No relevant macroscopic changes were seen in treated animals compared to controls. No clear significance could be attributed to the enlarged spleen, seen in 2 high dose males, in the absence of other correlated changes.
Based on the results obtained in this study, the administration of the test item was considered well tolerated up to the high nominal dose level of 1000 mg/kg/day. Doses of 250, 500 and 1000 mg/kg/day could be used in the subsequent OECD 422 study.

Cross-reference 2

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:

2015-10-12 to 2017-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other 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:

adopted on 22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities, recognised as appropriate for general and reproduction toxicity studies and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 194-220 g males; 170-184 g females
- Fasting period before study: no
- Housing:
Pretest: Animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
Mating: Animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
Post-mating: The males were housed individually and remained in the same cages as during the mating; the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet (e.g. ad libitum): ad libitum, commercially available laboratory rodent diet
- Water (e.g. ad libitum): ad libitum, drinking water to each cage via water bottles.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours each day

Route of administration:

oral: feed

Vehicle:

other: powdered rodent diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was formulated using powdered diet, by a premix (using mortar and pestle) followed by dilution with further quantity of diet and mixing. When necessary, the formulation was prepared separated for each group/sex. Inclusion levels (ppm) were reviewed during the study, in order to obtain nominal dose levels of 250, 500 and 1000 mg/kg/day. Fresh diets were prepared at weekly intervals according to available stability data supplied by the Laboratory LAUS which performed the validation study. Concentrations were calculated and expressed in terms of active ingredient (71.3%).
- The concentrations of test item in the diet were adjusted as required in order to maintain the required dose level of each treated group. Initial dietary concentrations were predicted from growth and food intake data collection during the acclimatisation period. The dietary inclusion levels were reviewed at weekly intervals, the day after body weight measurements.


VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance was administered orally via diet
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): not specified
- Purity: 99.4%

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: After mating, the males were housed individually and remained in the same cages as during the mating; the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. The female was paired with the same male until positive identification occurred or 14 days had elapsed. For female no. 49, the mating was inadvertently not detected. However the pregnancy status became evident and the female was individually caged and gave birth.
- Proof of pregnancy: A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method and the proposed method of formulation had been validated in a separate study (LAUS Study No. 14111201G926) to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation at room temperature for 10 days was satisfactory in the concentration range of 250 - 15000 ppm. Samples of the formulations were prepared at the Test Facility during pre-treatment period and on four occasions during the study and were analysed to check the homogeneity and concentration.
Two replicates of 5 g were drawn from group 1 and group 3 preparations while 6 replicates of 5 g were drawn from group 2 and group 4 preparations (2 from the top, 3 from the middle and 1 from the bottom of the formulated diet sample) during the pre-treatment, weeks 3, and 7. During weeks 1 and 5, formulations were prepared separately by sex, therefore, for each sampling date a total of 16 or 24 specimens were prepared for analysis. For each specimen, an additional back up sample of 10 g was retained at the test facility. Samples were labelled as follows: study number, sampling date, nominal concentration and replicate number.

Duration of treatment / exposure:

Males: 32-33 days; 2 weeks prior to pairing, during pairing with females until the day before necropsy
Females: 41-44 days; 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post partum
Pups were not exposed 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:

Animals were dosed once a day, 7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1; control

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2; mean achieved dose 271 mg/kg/day (males), 323 mg/kg/day (females)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 3; mean achieved dose 574 mg/kg/day (males), 584 mg/kg/day (females)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4; mean achieved dose 1096 mg/kg/day (males), 1160 mg/kg/day (females)

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Nominal dose levels of 250, 500 and 1000 mg/kg/day were selected in agreement with the Sponsor, based on a previous preliminary non-GLP compliant study (refer to cross-reference Rossiello, 2015).
- Rationale for animal assignment (if not random): On the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremis of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs were recorded.
- A parturition check was performed from day 20 to day 25 post coitum. Female no. 67 of group 4, which did not give birth after 25 days of post coitum period, was sacrificed on day 26 post coitum and was found not pregnant at necropsy. Gestation length was calculated as the time between the day of succesful mating (day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered day 0 post coitum.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter. Each animal was removed from the home cage and observed in an open arena.
- Parameters: Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Males were weighed on the day of allocation, on the day before treatment commenced, weekly thereafter and just prior to necropsy.
- Females were weighed on the day of allocation, on the day before treatment commenced, weekly thereafter up to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: Weekly during the premating period starting from allocation. During the mating period the food consumption was recorded daily. After mating for male animals, food consumption was recorded daily until sacrifice. Individual food consumption for the females was measured daily during gestation starting from Day 0 post coitum and daily during post partum period starting from Day 1 post partum.
- Achieved dosage: The group mean achieved intake of test item was calculated for males (before pairing and from pairing up to the end of the study) and for females (before pairing, during post coitum including high dose female not pregnant and excluding mid dose female with mating not detected and during post partum) from the group mean body weight and food consumption data and the dietary inclusion levels of the test item.
The following formula was used: Achieved dosage (mg/kg/day) = (ppm (mg/kg) x mean daily food consumption (g/day)) / mean period body weight (g).
For females during post coitum and during post partum periods, the ppm were changed within the interval of body weight measurement, therefore the calculation was done for single animals and for different day intervals.

HAEMATOLOGY: Yes
- As part of the sacrificial procedure, after recording the terminal body weight, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
- Parameters assessed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time (as coagulation parameter).

CLINICAL CHEMISTRY: Yes
- As part of the sacrificial procedure, after recording the terminal body weight, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyl transferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

FUNCTIONAL OBSERVATIONS:
- Grip strength and sensory reactivity to stimuli: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength.
- Motor activity assessment: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device.

MORTALITY
- Throughout the study, all parental animals were checked early in each working day in the morning and in the afternoon. At weekends and public holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post-mortem examinations to be carried out during the working period of that day. A complete necropsy was performed.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the anomalies of the oestrous cycle and the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Litter observations:

STANDARDISATION OF LITTERS
- As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. If possible, defects or cause of death were evaluated.

PARAMETERS EXAMINED
- Live pups were individually weighed on Days 1 and 4 post partum.
- Litter observation (counting and mortality check) was performed once daily for all litters.
- All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

Postmortem examinations (parental animals):

SACRIFICE
Animals selected for blood collection were killed by exsanguination under isofluorane anaesthesia.
Animals not selected for blood collection were killed under carbon dioxide asphyxiation.
- Parental males: Males were killed after the mating of all females and after 32-33 days of treatment.
- Parental females: The females with live pups were killed on Day 4 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. Female no. 67 of group 4 which did not give birth after 25 days of post coitum period was sacrificed on day 26 post coitum and was found not pregnant at necropsy.

GROSS PATHOLOGY: Yes
- The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
- Changes were noted, organs weighed and tissue samples preserved.
- All females were examined also for number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
- For all animals completing the scheduled test period.
- Organs were dissected free of fat and weighed.
- Ratios of organ weight to body weight were calculated for each animal.
- Organs: Adrenal glands, Brain, Epididymides, Glans penis, Heart, Kidneys, Liver, Ovaries with oviducts, Parathyroid glands, Prostate gland, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)

HISTOPATHOLOGY
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 µm thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 µm thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
- Samples of the following tissues and organs were collected and fixed: abnormalities, Adrenal glands, bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Clitoris, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum including Peyer's patches, Kidneys, Liver, Lung with mainstem bronchi, Lymph nodes - cervical, Lymph nodes - mesenteric, Nasal cavity, Oesophagus, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles with coagulation glands, Spinal column, Spinal cord - cervical, mid-thoracic, lumbar, Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus-cervix, Vagina

Postmortem examinations (offspring):

SACRIFICE
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.

GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined.

Statistics:

Group mean values were calculated for all parameters.
Data from females with total resorption or non-pregnant were excluded from group mean calculations as considered appropriate by the study director.
The following statistical methods were used to analyse the data:
- Standard deviations were calculated as appropriate.
- For continuous variables, the significance of the differences amongst group means was assessed by Dunnett's-test or modified t-test, depending on the homogeneity of the data.
- The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
- Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
- The criterion for statistical significance was p<0.05.

Reproductive indices:

For each group, the following calculations were performed:
For males:
- Copulatory index (%) = (Number of animals mated/Number of animals paired) x 100
- Fertility index (%) = (Number of males which induced pregnancy/Number of males paired) x 100
For females:
- Copulatory index (%) = (Number of animals mated/Number of animals paired) x 100
- Fertility index (%) = (Number of pregnant females/Number of females paired) x 100
For males and females:
- Precoital interval = mean number of days between pairing and mating

Offspring viability indices:

For females:
- Pre-implantation loss was calculated as a percentage from the formula = ((number of corpora lutea - number of visible implantation)/number of corpora lutea) x 100
- Pre-birth loss was calculated as a percentage from the formula = ((number of visible implantations - total litter size at birth)/number of visible implantations) x 100
- Pup loss at birth was calculated as a percentage from the formula = ((total litter size at birth - live litter size)/total litter size) x 100
- Cumulative pup loss on day 4 post partum was calculated as a percentage from the formula = ((total litter size at birth - live litter size at day 4)/total litter size at birth) x 100
- Sex ratios were calculated at birth and on day 4 post partum and are presented as the percentage of males per litter

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related effects were observed in treated males and females throughout the study. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on body weight were seen in males. The slight increases in body weight seen in treated female groups, when compared to controls, during the pre-pairing period, statistically significant in the low dose group, were considered of no toxicological significance.
However, transient statistically significant variations in body weight gain were recorded in the mid-dose males and in the low- and mid-dose females before pairing, and in the low- and high-dose females during gestation, with no dose relation, therefore considered not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Food consumption: No toxicologically relevant effects on food consumption were observed in either males or females of all test item-treated groups when compared to controls. Slight increases in food consumption were seen on occasions troughout the study in mid- and high-dose animals.
- Achieved dosage: The calculated mean achieved dosages for 2 weeks of treatment of the pre-pairing period were 270, 539 and 1108 mg/kg/day for males and 289, 579 and 1135 mg/kg/day for females. The achieved dosages for males from mating up to the end of the study were 271, 555 and 1084 mg/kg/day. During the post coitum and post partum periods, females had achieved dosages of 279, 562 and 1127 mg/kg/day and 401, 611 and 1217 mg/kg/day, respectively. During the entire treatment period, the mean achieved dosages were 271, 547 and 1096 mg/kg/day for males and 323, 584 and 1160 mg/kg/day for females, with respect to the nominal dose levels of 250, 500 and 1000 mg/kg/day.

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):

- One high dose female (no. X0070071) showed marked lymphopenia (83% below mean control data). Due to the low incidence, this change cannot be conclusively attributed to treatment.
- The other statistically significant differences between control and treated animals, such as decrease of reticulocytes in mid-dose males, and increase of reticulocytes in high-dose females were not consistent between sexes and/or dose-related, therefore considered unrelated to treatment.
- No change was recorded in the coagulation test.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Statistically significant fluctuations of some biochemical parameters were observed, such as: decrease of alkaline phosphatase in low-dose males (26%), decrease of creatinine and potassium in mid-dose males (25% and 4%, respectively) and increase of glucose in high-dose females (32%). Due to the absence of dose-relation or other related findings, the above changes were considered of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity and sensory reaction to stimuli: No relevant differences were noted in all parameters investigated between control and treated groups, with the exception of a statistically significant increase in the grip strength observed in mid-dose group. In the absence of dose-relationship or other affected parameters, this change was considered incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted.
The histopathological changes, reported in control and treated animals, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the prevailing experimental conditions.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) did not show differences related to treatment.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- All males and all females mated. One high dose male (no. 68) did not induce pregnancy.
- Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) did not show differences related to treatment.
- Gestation periods were similar between treated animals and controls being of 22 days in all groups (mean value).
- No relevant differences were observed in the number of corpora lutea and implantations, pre-implantation and pre-birth loss between control and treated females.
- Lower live litter size (-26% and -30%), litter weight (-30% and -20%) and mean pup weight (-21% and -15%) and a higher cumulative pup loss (60% and 67%) were seen in mid- and high-dose groups, respectively, when compared to controls, on Day 4 post partum.
- A number of females with total litter loss on Day 4 post partum was seen in all groups comprising control, with similar incidence.
- Sex ratios at birth did not show differences between treated and control groups.
- Lower mean percentage of males was seen on Day 4 post partum, in the high-dose group (-32%) compared to the control group.

Analytical verification: Results:
Samples of formulations were sent to the test site LAUS during pre-treatment, week 1 and the last week (week 5, both sexes present). Since measurements of week 1 and of the last week were not acceptable, repetitions in week 3 and week 7 (last week with only females present) were performed and were found acceptable. Generally, high heterogeneity of the analytical replicates was found, probably due to clumps and aggregates easily formed in the diet by the hygroscopic test item. However, the heterogeneity found in the diet is not a concern regarding the administered dose since the analytical sampling carried out in LAUS laboratory was 1 g which is a fairly small amount compared to the daily food consumption of the animals (approximately 30 g).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

anhydrous yttrium trinitrate

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

anhydrous yttrium trinitrate

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test compound-related effects: small pups, pups cold to touch and/or with apparently no food intake were generally observed in all groups including controls without substantial differences.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Lower live litter size (-26% and -30%) and a higher cumulative pup loss (60% and 67%) were seen in mid- and high-dose groups, respectively, when compared to the control group, on Day 4 post partum. A number of females with total litter loss on Day 4 post partum was seen in all groups comprising control, with similar incidence.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter weight (-30% and -20%) and mean pup weight (-21% and -15%) were seen in mid- and high-dose groups, respectively, when compared to controls, on day 4 post partum.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings in decedent pups and in pups sacrificed on day 4 post partum:
- Autolysis of all organs was observed for the majority of the decedent pups, in all groups.
- For few pups no milk in stomach or dark staining on the abdominal region or stomach distended with abnormal content (gas), were recorded.
- No milk in stomach was the only sign recorded at necropsy on day 4, in some pups in all groups.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

anhydrous yttrium trinitrate

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

no

Relevant for humans:

not specified

Hematology

The table below presents the main differences in hematology parameters when compared with control values

HEMATOLOGY
	Male				Female
dose level (mg/kg/day)	0	250	500	1000	0	250	500	1000
lymphocytes 10^3/µL	9.116	5.432	6.908	8.464	5.394	4.304	4.843	3.456
reticulocytes %	2.430	2.114	1.888*	2.462	3.772	4.366	5.195	5.828*

*p<0.05

**p<0.01

Clinical biochemistry

The table below presents the main differences in clinical biochemistry when compared with control values

CLINICAL BIOCHEMISTRY
	Male				Female
dose level (mg/kg/day)	0	250	500	1000	0	250	500	1000
alkaline phosphatase U/L	355.80	264.72*	338.50	297.30	138.18	126.26	112.00	126.00
creatinine mg/dL	0.428	0.342	0.322*	0.358	0.312	0.308	0.348	0.278
potassium mmol/L	3.920	3.764	3.752*	3.768	3.698	3.378	3.242	3.454
glucose mg/dL	154.36	139.34	157.28	168.08	115.36	108.20	122.50	152.56**

*p<0.05

**p<0.01

Organ weight

The table below presents the main differences in absolute and relative organ weight when compared with control values

ORGAN WEIGHT
	Male				Female
dose level (mg/kg/day)	0	250	500	1000	0	250	500	1000
testes - absolute (g)	3.2854	3.4387	3.2125	3.5784*
testes - relative	0.7342	0.7697	0.7609	0.8263*
epididymides - absolute (g)	1.2482	1.2914	1.2620	1.3815
epididymides - relative	0.2785	0.2895	0.2974	0.3186
seminal vesicles - absolute (g)	1.6663	1.4891	1.4471	1.4425
seminal vesicles - relative	0.3699	0.3326	0.3423	0.3334
adrenal - absolute (g)	0.0639	0.0636	0.0644	0.0708	0.0781	0.0670	0.0759	0.0731
adrenal - relative	0.0143	0.0142	0.0152	0.0163	0.0276	0.0221	0.0258	0.0243
heart - absolute (g)	1.559	1.519	1.428	1.536	1.124	1.101	1.126	1.177
heart - relative	0.347	0.340	0.334	0.353	0.395	0.363	0.381	0.392

*statistically significant (Dunnett's test)

Reproductive performance

REPRODUCTIVE PERFORMANCE
	at birth			on day 1 post partum		on day 4 post partum
	total litter size	live litter size	pup loss (%)	litter weight (g)	mean pup weight (g)	live litter size	cumulative loss (%)	litter weight (g)	mean pup weight (g)
0 mg/kg/day	14.20	14.00	1.33	84.98	6.48	9.00	35.52	86.10	7.75
250 mg/kg/day	14.90	14.80	0.67	92.30	6.41	9.60	37.71	85.30	7.64
500 mg/kg/day	14.90	14.80	0.67	84.47	6.26	6.70	57.00	60.30	6.14
1000 mg/kg/day	15.78	15.78	0.00	96.27	6.29	6.33	59.38	69.02	6.57

statistical analysis: Kruskal Wallis test

Conclusions:

In this study performed according to OECD guideline 422, treatment with yttrium trinitrate via the diet in male and female Sprague Dawley rats at dose levels of 250, 500 and 1000 mg/kg bw/day revealed no adverse systemic effects in parent animals, resulting in a NOAEL >= 1000 mg/kg bw/day. At the mid- and high-dose however, lower live litter size, litter weight, mean pup weight and a higher cumulative pup loss were seen when compared to the control group on Day 4 post partum. Although these effects were not or not clearly progressive with increasing dose and because there were no accompanying toxic effects observed (for instance in histopathology), a NOAEL for reproductive performance of the parent animals and for effects on the progeny was set at 250 mg/kg bw/day.

Executive summary:

In this GLP-compliant OECD 422 study, yttrium trinitrate was administered once daily orally to Sprague-Dawley rats of both sexes (10 animals/sex/group) via diet at 0, 250, 500 or 1000 mg/kg/day (nominal concentration). Males were treated for 2 weeks prior to pairing, during pairing with females until the day before necropsy, for a total of 32-33 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post partum, for 41-44 days. No significant changes and no treatment-related effects were detected in males and females during the in vivo phase.

Oestrus cycle, pre-coital intervals, copulatory and fertility indices did not show intergroup differences. No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss and pre-birth loss between control and treated groups. Gestation length was similar between groups.

Reproductive outcomes of dams (number and weight of pups and the percentage of live pups) and sex ratio of pups on day 1 post partum were also comparable between groups. Lower live litter size, litter weight and mean pup weight and a higher cumulative pup loss were seen in mid- and high-dose groups, when compared to controls on day 4 post partum. Lower mean percentage of males was seen on day 4 post partum, in the high-dose group compared to the control group. A number of females with total litter loss on day 4 was seen in all groups comprising the control group, with similar incidence.

There were no test compound-related effects at the clinical signs of pups. Necropsy findings in decedent pups and in pups sacrificed on day 4 post partum did not reveal any treatment-related effect. No relevant changes were detected at post mortem examination in treated animals, when compared to the control animals. No treatment-related changes were seen in organs/tissues evaluated nor in the abnormalities detected in all groups at post mortem examination. No alterations were detected in the spermatogenic cycle within the different stages.

In conclusion, no treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated. No adverse effects on sexual function and fertility were observed at any of the dose levels investigated. Effects on developmental parameters and lactation were noted at the mid- and high-dose level. On the basis of the results obtained in this study, the NOAEL for systemic toxicity was considered to be >= 1000 mg/kg/day for males and females (corresponding to approximately 1096 mg/kg/day for males and 1160 mg/kg/day for females). For reproductive performance and effects on progeny, the NOAEL was considered to be 250 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

adopted on 22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Yttrium trinitrate

EC Number:

233-802-6

EC Name:

Yttrium trinitrate

Cas Number:

10361-93-0

Molecular formula:

N3O9Y

IUPAC Name:

yttrium trinitrate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: yttrium trinitrate hexahydrate
- Molecular formula: Y(NO3)3.6H2O
- Molecular weight: 383.01 g/mol
- Appearance: powder
- Storage condition of test material: room temperature (20 ± 5°C) in the dark under dry conditions
- Further information on test material confidential.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities, recognised as appropriate for general and reproduction toxicity studies and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 194-220 g males; 170-184 g females
- Fasting period before study: no
- Housing:
Pretest: Animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changes at least twice a week.
Mating: Animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
Post-mating: The males were housed individually and remained in the same cages as during the mating; the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet (e.g. ad libitum): ad libitum, commercially available laboratory rodent diet
- Water (e.g. ad libitum): ad libitum, drinking water to each cage via water bottles.
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours each day

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test item was administered orally, via the diet. The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

other: powdered rodent diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was formulated using powdered diet, by a premix (using mortar and pestle) followed by dilution with further quantity of diet and mixing. When necessary, the formulation was prepared separated for each group/sex. Inclusion levels (ppm) were reviewed during the study, in order to obtain nominal dose levels of 250, 500 and 1000 mg/kg/day. Fresh diets were prepared at weekly intervals according to available stability data supplied by the Laboratory LAUS which performed the validation study. Concentrations were calculated and expressed in terms of active ingredient (71.3%).
- The concentrations of test item in the diet were adjusted as required in order to maintain the required dose level for each treated group. Initial dietary concentrations were predicted from growth and food intake data collection during the acclimatisation period. The dietary inclusion levels were reviewed at weekly intervals, the day after body weight measurements.


VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance was administered oral via diet
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): not specified
- Purity: 99.4%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method and the proposed method of formulation had been validated in a separate study (LAUS Study No. 14111201G926) to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation at room temperature for 10 days was satisfactory in the concentration range of 250 - 15000 ppm. Samples of the formulations were prepared at the Test Facility during pre-treatment period and on four occasions during the study and were analysed to check the homogeneity and concentration.
Two replicates of 5 g were drawn from group 1 and group 3 preparations while 6 replicates of 5 g were drawn from group 2 and group 4 preparations (2 from the top, 3 from the middle and 1 from the bottom of the formulated diet sample) during the pre-treatment, weeks 3, and 7. During weeks 1 and 5, formulations were prepared seperately by sex, therefore, for each sampling date a total of 16 or 24 specimens were prepared for analysis. For each specimen, an additional back up sample of 10 g was retained at the test facility. Samples were labelled as follows: study number, sampling date, nominal concentration and replicate number.

Duration of treatment / exposure:

Males: 32-33 days; 2 weeks prior to pairing, during pairing with females until the day before necropsy
Females: 41-44 days; 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post partum

Frequency of treatment:

Animals were dosed once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1; control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2; mean achieved dose 271 mg/kg/day (males), 323 mg/kg/day (females)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 3; mean achieved dose 574 mg/kg/day (males), 584 mg/kg/day (females)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4; mean achieved dose 1096 mg/kg/day (males), 1160 mg/kg/day (females)

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Nominal dose levels of 250, 500 and 1000 mg/kg/day were selected in agreement with the Sponsor, based on a previous preliminary non GLP compliant study (refer to cross-reference Rossiello, 2015).
- Rationale for animal assignment (if not random): on the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremis of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal intial group mean body weights.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter. Each animal was removed from the home cage and observed in an open arena.
- Parameters: observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (eg lachrymation, piloerection, pupil size, unusual respiratory pattern)

BODY WEIGHT: Yes
- Males were weighed on the day of allocation, on the day before treatment commenced, weekly thereafter and just prior to necropsy.
- Females were weighed on the day of allocation, on the day before treatment commenced, weekly thereafter up to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: Weekly during the pre-mating period starting from allocation. During the mating period the food consumption was recorded daily. After mating for male animals, food consumption was recorded daily until sacrifice. Individual food consumption for the females was measured daily during gestation starting from Day 0 post coitum and daily during post partum period starting from Day 1 post partum.
- Achieved dosage: The group mean achieved intake of test item was calculated for males (before pairing and from pairing up to the end of the study) and for females (before pairing, during post coitum including high dose female not pregnant and excluding mid dose female with mating not detected and during post partum) from the group mean body weight and food consumption data and the dietary inclusion levels of the test item.
The following formula was used: Achieved dosage (mg/kg/day) = (ppm (mg/kg) x mean daily food consumption (g/day)) / mean period body weight (g).
For females during post coitum and during post partum periods, the ppm were changed within the interval of body weight measurement, therefore the calculation was done for single animals and for different day intervals.

HAEMATOLOGY: Yes
- As a part of the sacrificial procedure, after recording the terminal body weight, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
- Parameters assessed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time (as coagulation parameter).

CLINICAL CHEMISTRY: Yes
- As a part of the sacrificial procedure, after recording the terminal body weight, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyl transferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

FUNCTIONAL OBSERVATIONS:
- Grip strength and sensory reactivity to stimuli: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip stregth.
- Motor activity assessment: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device.

OTHER:
- MORTALITY: Throughout the study, all parental animals were checked early in each working day in the morning and in the afternoon. At weekends and public holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post-mortem examinations to be carried out during the working period of that day. A complete necropsy was performed.

Sacrifice and pathology:

SACRIFICE
Animals selected for blood collection were killed by exsanguination under isofluorane anaesthesia. Animals not selected for blood collection were killed under carbon dioxide asphyxiation.
- Parental males: Males were killed after the mating of all females and after 32-33 days of treatment.
- Parental females: The females with live pups were killed on day 4 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. Female no. 67 of group 4 which did not give birth after 25 days of post coitum period was sacrificed on day 26 post coitum and was found not pregnant at necropsy.

GROSS PATHOLOGY: Yes
- Clinical history of males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
- Changes were noted, organs weighed and tissue samples preserved.

HISTOPATHOLOGY: Yes
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 µm thickness and stained with haematoxylin and eosine. In addition, testes and epididymides were cut at 2-3 µm thickness and stained with periodic acid schiff.
- The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
- Samples of the following tissues and organs were collected and fixed: abnormalities, Adrenal glands, bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Clitoris, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum including Peyer's patches, Kidneys, Liver, Lung including mainstem bronchi, Lymph nodes - cervical, Lymph nodes - mesenteric, Nasal cavity, Oesophagus, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles with coagulation glands, Spinal column, Spinal cord - cervical, midthoracic, lumbar, Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus-cervix, Vagina

ORGAN WEIGHTS
- For all animals completing the scheduled test period.
- Organs were dissected free of fat and weighed.
- Ratios of organ weight to body weight were recorded.
- Organs: Adrenal glands, Brain, Epididymides, Glans penis, Heart, Kidneys, Liver, Ovaries with oviducts, Parathyroid glands, Prostate gland, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus-cervix

Statistics:

Group mean values were calculated for all parameters.
Data from females with total resorption or non-pregnant were excluded from group mean calculations as considered appropriate by the study director.
The following statistical methods were used to analyse the data:
- Standard deviations were calculated as appropriate.
- For continuous variables, the significance of the differences amongst group means was assessed by Dunnett's-test or modified t-test, depending on the homogeneity of the data.
- The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
- Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
- The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related effects were observed in treated males and females throughout the study. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on body weight were seen in males. The slight increases in body weight seen in treated female groups, when compared to controls, during the pre-pairing period, statistically significant in the low dose group, were considered of no toxicological significance.
However, transient statistically significant variations in body weight gain were recorded in the mid-dose males and in the low and mid-dose females before pairing, and in the low and high dose females during gestation, with no dose relation, therefore considered not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Food consumption: No toxicologically relevant effects on food consumption were observed in either males or females of all test item-treated groups when compared to controls. Slight increases in food consumption were seen on occasion troughout the study in mid- and high dose animals.
- Achieved dosage: The calculated mean achieved dosages for 2 weeks of treatment of the pre-pairing period were 270, 539 and 1108 mg/kg/day for males and 289, 579 and 1135 mg/kg/day for females. The achieved dosages for males from mating up to the end of the study were 271, 555 and 1084 mg/kg/day. During the post coitum and post partum periods, females had achieved dosages of 279, 562 and 1127 mg/kg/day and 401, 611 and 1217 mg/kg/day, respectively. During the entire treatment period, the mean achieved dosages were 271, 547 and 1096 mg/kg/day for males and 323, 584 and 1160 mg/kg/day for females, with respect to the nominal dose levels of 250, 500 and 1000 mg/kg/day.

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):

- One high dose female (no. X0070071) showed marked lymphopenia (83% below mean control data). Due to the low incidence, this change cannot be conclusively attributed to treatment.
- The other statistically significant differences between control and treated animals, such as decrease of reticulocytes in mid-dose males, and increase of reticulocytes in high dose females were not consistent between sexes and/or dose-related, therefore considered unrelated to treatment.
- No change was recorded in the coagulation test.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Statistically significant fluctuations of some biochemical parameters were observed, such as: decrease of alkaline phosphatase in low dose males (26%), decrease of creatinine and potassium in mid-dose males (25% and 4%, respectively) and increase of glucose in high dose females (32%). Due to the absence of dose-relation or other related findings, the above changes were considered of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity and sensory reaction to stimuli: No relevant differences were noted in all parameters investigated between control and treated groups, with the exception of a statistically significant increase in the grip strength observed in mid-dose group. In the absence of dose-relationship or other affected parameters, this change was considered incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Terminal body weight was unaffected by treatment in both sexes.
- Statistically significant increases in absolute and relative testes weight (9% and 13%, respectively), slight increases in absolute and relative epididymides weight (11% and 14%, respectively) and slight decreases in absolute and relative seminal vescicles weight (-13% and -10%, respectively), were seen in high dose males; decreases in absolute and relative (statistically significant ) adrenals weight (-14% and -20%) and statistically significant decrease in relative heart weight (-8%) were seen in low dose females. Due to the absence of related histopathological findings and/or the lack of dose-relationship, these changes were not considered of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The most remarkable changes observed at post mortem examination in treated animals, when compared with controls, were:
- Red/dark red mucoid contents of the stomach in two high dose females without any evident correlation at histopathology.
- Increased size of the adrenals of two high dose males and one mid-dose male. However, this finding did not correspond to any histopathological changes of both cortex and medulla. As morphology is comparable to control animals, a direct treatment-related impact is not considered to be present.
The remaining macroscopic observations were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted.
The histopathological changes, reported in control and treated animals, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions of the test.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

anhydrous yttrium trinitrate

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

Analytical verification: results

Samples of formulations were sent to the test site LAUS during pre-treatment, week 1 and the last week (week 5, both sexes present). Since measurements of week 1 and of the last week were not acceptable, repetitions in week 3 and week 7 (last week with only females present) were performed and were found acceptable. Generally, high heterogeneity of the analytical replicates was found, probably due to clumps and aggregates easily formed in the diet by the hygroscopic test item. However, the heterogeneity found in the diet is not a concern regarding the administered dose since the analytical sampling carried out in LAUS laboratory was 1 g which is a fairly small amount compared to the daily food consumption of the animals (approximately 30 g).

Applicant's summary and conclusion

Conclusions:

The toxicological potential of yttrium trinitrate after repeated oral exposure was investigated in an OECD 422 study. In this study, treatment with yttrium trinitrate via the diet in male and female Sprague Dawley rats at dose levels of 250, 500 and 1000 mg/kg bw/day revealed no adverse effects. Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be higher than or equal to 1000 mg/kg bw/day, i.e. the highest dose tested.
Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.