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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The key study for this endpoint is a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, performed according to OECD guideline 422 and conform GLP requirements (Rossiello, 2017; Klimisch 1). The test substance was administered daily to male and female Sprague-Dawley rats (10 animals/sex/group) at dose levels of 0, 250, 500 and 1000 mg yttrium trinitrate/kg bw/day via diet. Males were dosed during 32-33 days, females during 41-44 days. No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated. Therefore the NOAEL for systemic toxicity in parent animals was considered to be higher than or equal to 1000 mg/kg bw/day. No adverse effects on sexual function and fertility were observed at any of the dose levels investigated.

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.

Link to relevant study records
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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0, 250, 500 and 1000 mg yttrium trinitrate/kg bw/day via their diet (OECD 422; Rossiello, 2017). The vehicle used was powdered rodent diet and the test solutions were prepared weekly and administered once a day.

No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (nominal dose levels of 250, 500 and 1000 mg yttrium trinitrate/kg bw/day, corresponding to mean achieved dose levels of 271, 547 and 1096 mg/kg bw/day for males and 323, 584 and 1160 mg/kg bw/day for females). Therefore the NOAEL for systemic toxicity in parent animals was considered to be higher than or equal to 1000 mg/kg bw/day.

Oestrous 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 the control group on Day 4 post partum.

Lower mean percentage of males was seen on Day 4 post partum in the high-dose group in comparison with 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 item-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 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.

Although the effects observed in the mid- and high-dose group on live litter size, litter weight, mean pup weight and cumulative pup loss on Day 4 post partum 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.

This study was considered reliable without restriction (Klimisch 1) and was assigned key status for endpoint coverage.

Effects on developmental toxicity

Description of key information

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (performed according to OECD guideline 422; Rossiello, 2017; Klimisch 1), a NOAEL for effects on the progeny of 250 mg/kg bw/day was established based on observed effects on live litter size, litter weight, mean pup weight and cumulative pup loss on Day 4 post partum. These effects were not or not clearly progressive with increasing dose, neither were there accompanying toxic effects observed (for instance in histopathology). There were no indications of teratogenicity either. No further studies are considered required on developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Apart from the OECD 422 screening study, a supporting developmental neurotoxicity study is available (Li et al., 2015; Klimisch 2; see IUCLID Section 7.9.1). In this study, female rats were exposed from gestation day 6 onwards to postnatal day 21 to 5, 15 or 45 mg/kg bw/day of an yttrium trinitrate aqueous solution. The pups were followed up to postnatal day 70. No toxicologically relevant effects related to the treatment have been observed in maternal animals or their offspring over the course of the study. The NOAEL was therefore considered to be higher than or equal to 45 mg/kg bw/day, i.e. the highest dose tested. Based on the results of this study, there are no indications for yttrium (administered as yttrium trinitrate) to be a developmental neurotoxicant.

Justification for classification or non-classification

Based on the results of the OECD 422 study, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL >= 1000 mg/kg bw/day

Reproductive performance NOAEL = 250 mg/kg bw/day

NOAEL for effects on progeny = 250 mg/kg bw/day

Based on the results of this study, yttrium trinitrate is not to be classified as reproductive toxicant according to the CLP Regulation.

Additional information