Tris[oxalato(2-)]digadolinium

Endpoint summary

Description of key information

Repeated dose toxicity - oral

No studies on oral repeated dose toxicity are available for gadolinium oxalate. The endpoint is therefore covered using a study performed with the related substance gadolinium oxide. The key read across study (Papineau, 2017) was performed according to OECD guideline 422 and conform GLP requirements. In this study, gadolinium oxide was administered by oral gavage to male and female Sprague-Dawley rats, starting 2 weeks before mating, during mating and (for females) throughout gestation and until day 5 post-partum, at the dose levels of 110, 330 or 1200 (until day 17 of the study)/1008 (from day 18 of the study until end of exposure) mg/kg bw/day. In this study, the NOAEL for parental systemic toxicity was considered to be higher than or equal to 1008 mg/kg bw/day based on the absence of adverse findings related to the test item at the highest dose level. This is equivalent to a NOAEL higher than or equal to 1609 mg/kg bw/day when recalculated to gadolinium oxalate taking into account the gadolinium content of both substances. Based on these results, neither gadolinium oxide nor gadolinium oxalate need to be classified as STOT RE. The justification for read across is attached to IUCLID Section 13.

Repeated dose toxicity - inhalation/dermal

No key studies were identified for repeated dose toxicity after inhalation or dermal exposure to gadolinium oxalate.

A key read across study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (Column 2, Annex VIII, Section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation or dermal route of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-08-02 to 2017-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 March 1996

Deviations:

yes

Remarks:

From the beginning of the study until Day 17, group 4 animals were administered the dose level of 1200 mg/kg bw/day instead of 1000 mg/kg bw/day. From Day 18 onwards until the end of dosing the normal dose was administered.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Correction factor: None (as this would be close to 1).
Final preparation of a solid: According to CiToxLAB France/Study No. 43935 VAS describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in the study.

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 82 rats (41 males and 41 females), Sprague-Dawley, RjHan: SD (Rats CD®) (SPF quality); Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males approximately 10 weeks old, females approximately 9 weeks old
- Weight at study initiation: (P) males: 470 g (range: 438 g to 495 g); females: 277 g (range: 259 g to 310 g)
- Fasting period before study: no data
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages, (Tecniplast 2154, 940 cm²) with stainless steel lids, containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order not to jeopardise gestation and to avoid aggressive behaviour between males around the time of mating. Towards the end of gestation and during lactation, the females and their litters were provided with autoclaved wood shavings (SICSA, Alfortville, France) as nesting material. Each cage contained a nylabone and a cocoon for the environmental enrichment of the animals. The cages were placed in numerical order on the racks
- Diet (e.g. ad libitum): Free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 9705482 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): Free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: The animals were acclimated to the study conditions for a period of 8 days before the beginning of the treatment period. One supplementary animal of each sex was acclimated to permit the selection and/or replacement of individuals. Upon arrival at CiToxLAB France, the animals were given a clinical examination to ensure that they were in good condition.

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

IN-LIFE DATES: From: 2016-08-10 To: 2016-09-25

Route of administration:

oral: gavage

Details on route of administration:

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

Vehicle:

methylcellulose

Remarks:

0.5% aqueous solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- In the dose formulation stability study, the dose formulations containing gadolinium oxide prepared at 2 mg/mL and 240 mg/mL in 0.5% (w/w) methylcellulose aqueous solution (4000 cps) were found to be homogeneous and stable after 19 days at room temperature. Therefore dose formulation preparation frequency was every 19 days.
- The dose formulations were maintained under delivery conditions (at room temperature) throughout the administration procedure.
- Storage condition of control dose formulation: in the refrigerator set at +5°C
- The control dose formulation was stirred just before administration and the test item dose formulations were stirred for at least 15 minutes before administration.
- The formulations were maintained under continuous magnetic stirring throughout the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed by CiToxLAB France for CiToxLAB France/Study No. 43936 TSR
- Concentration in vehicle: 0 (0 mg/kg bw/day); 22 mg/mL (110 mg/kg bw/day); 66 mg/mL (330 mg/kg bw/day), 240 mg/mL (1200 mg/kg bw/day until day 17 of the study, 1008 mg/kg bw/day from day 18 of the study) (see below Details on study design / Dose selection rationale for further explanation)
- Amount of vehicle (if gavage): 5 mL/kg/day with the exception of group 4 animals for which the dosage-volume was 4.2 mL/kg/day from Day 18 of the study in order to achieve a dose level of 1008 mg/kg/day.
- Lot/batch no. (if required): SLBM3894V
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical technique: Inductively Coupled Plasma Mass Spectrometry detection (ICP-MS)
Principle and validation of the method: Analytical method provided by the Sponsor and validated at CiToxLAB France (CiToxLAB France/Study No. 43935 VAS) prior to dose formulation analysis.

Determination of test item concentrations in dose formulations:
- Once in the first and last week of treatment
- A sample (1 sample and 2 reserve samples) was taken from control and test item dose formulations from all dose groups and analysed using the validated method. Reserve samples were destroyed as results were in the range of the nominal concentration (85-115%).

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

Results:
- The test item concentrations in the administered dose formulations analysed in week 1 and the last week of treatment remained within an acceptable range of variations (-7.0% to +3.9%) when compared to the nominal values (nominal concentrations +/-15%).
- A small peak was detected on the vehicle chromatograms at the test item retention time. As its area (measured at 427.47 and 513.12 cps, respectively, in the first and the last week of treatment) was below the LOQ area (measured at 1378.32 and 2139.62 cps), this peak was considered as negligible. Its concentration could be estimated to be below 0.01 mg/mL (corresponding to the LOQ concentration x dilution factor of the vehicle (50000)).

Duration of treatment / exposure:

Males: for 2 weeks before mating, during the mating period (up to 3 weeks), until euthanasia (5 weeks in total)
Females: for 2 weeks before mating, during the mating period (up to 3 weeks), during gestation, during lactation until day 5 p.p. inclusive, until euthanasia for females that did not deliver

Frequency of treatment:

Once a day, 7 days per week, at approximately the same time of the day, with a maximum of 6 hours between the earliest and latest administration.

Dose / conc.:

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

Remarks:

control group, group 1

Dose / conc.:

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

Remarks:

group 2

Dose / conc.:

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

Remarks:

group 3

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Remarks:

group 4, 1200 mg/kg bw/day until day 17 of the study, 1008 mg/kg bw/day from day 18 of the study until the end of dosing (see below Details on study design / Dose selection rationale for further explanation)

No. of animals per sex per dose:

10 animals/sex/dose; 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous dose range finding study (CiToxLAB France/Study No. 43936 TSR) performed in the same species. In that study, three groups of Sprague-Dawley rats received the test item daily, by oral administration (gavage) at dose levels of 110, 330 or 1000 mg/kg/day for 2 weeks. Another group received the vehicle control, 0.5% (w/w) methylcellulose aqueous solution, under the same experimental conditions. The dosage volume was 5 mL/kg. As no relevant findings were observed during the study, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level. Therefore, 1000 mg/kg/day was selected as the high dose level for the present study. The low dose and mid dose were selected using a ratio representing approximately a 3- to 3.6-fold interval (i.e. 110 and 330 mg/kg/day).
However, dose formulations of the high dose level were prepared at 240 mg/mL for a volume of administration of 5 mL/kg/day by error (instead of 200 mg/mL as required in the study plan). Therefore high dose animals received 1200 mg/kg bw/day instead of 1000 mg/kg bw/day during the dose range finding toxicology study (CiToxLAB France/Study No. 43936 TSR) and during the first 17 days of the current study (CiToxLAB France/Study No. 43937 RSR).
- Rationale for animal assignment (if not random): During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a stratified procedure based on body weight (these data are not presented in the report), so that the average body weight of each group was similar.

Positive control:

no

Observations and examinations performed and frequency:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of treatment (day 1), then once a week until sacrifice; females: on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 p.c. (post-coitum) and on days 1 and 5 p.p.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each male was measured once a week, from the first day of treatment until the start of the mating period.
- The quantity of food consumed by each female was measured once a week, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p.
- During the mating period, food consumption was not measured for males or females.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: the first five males on the day of euthanasia and the first five females euthanised on Day 6 p.p. (all groups)
- Blood samples were collected from the retro-orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 11 a.m.
- Anaesthetic used for blood collection: yes, light isoflurane anesthesia
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females (all groups)
- Parameters examined: erythrocytes (RBC), mean cell volume (MCV), packed cell volume (PCV), hemoglobin (HB), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLT), leucocytes (WBC), differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), large unstained cells (LUC), monocytes (M), reticulocytes (RTC), blood coagulation parameters: prothrombin time (PT), fibrinogen (FIB), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the first five males on the day of euthanasia and the first five females euthanised on Day 6 p.p. (all groups)
- Blood samples were collected from the retro-orbital sinus of each animal into tubes containing the appropriate anticoagulant between 8 a.m. and 11 a.m.
- Animals fasted: yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females (all groups)
- Parameters examined: sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (PHOS), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), alanine, aminotransferase (ALAT), aspartate, aminotransferase (ASAT), total proteins (PROT), albumin (ALB), albumin/globulin ratio (A/G), bile acids (BIL.AC).

URINALYSIS: No

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

IMMUNOLOGY: No

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- The first five males and the first five females euthanised on Day 6 p.p. from each group were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 5 p.p. after euthanasia of the pups.
- All animals were observed in the cage, in the hand and in the standard arena.
- Detailed clinical examinations: the following parameters were assessed and graded: in the cage: touch escape or ease of removal from the cage; in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis); in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia
- Reactivity to manipulation and different stimuli: the following measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature

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

Sacrifice and pathology:

SACRIFICE
- Male animals: On completion of the treatment period, after the end of the mating period (at least 5 weeks of treatment in total), after at least 14 hours (maximum 24 hours) fasting (with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination.
- Maternal animals: On completion of the treatment period, on day 6 p.p., after at least 14 hours (maximum 24 hours) fasting (with water available), all surviving F0 animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and euthanised by exsanguination. The following F0 females were euthanised by inhalation of carbon dioxide gas followed by cervical dislocation without overnight fasting: females H21786 (group 3) and H21799 (group 4) which did not deliver: on day 25 or 26 p.c., respectively (after a body weight recording to check for a possible un-noticed delivery). During the gestation period, prematurely sacrificed female H21773 (group 2) was euthanised by inhalation of carbon dioxide gas followed by cervical dislocation.

GROSS PATHOLOGY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- A complete macroscopic post mortem examination was performed on the prematurely sacrificed female H21773 (group 2). The pregnancy status was determined and the numbers of corpora lutea and implantation sites were recorded and classified as live or dead concepti, early or late resorptions or scars.
- A complete macroscopic post-mortem examination was performed on all F0 animals including the female euthanised prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanised as scheduled on Day 6 p.p. and for females euthanised on Day 25 or 26 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY
- The body weight of each animal euthanised as scheduled (after the end of the mating period for males or on Day 6 p.p. for females) was recorded before euthanasia. The following organs of the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 6 p.p. of each group were weighed wet as soon as possible after dissection: adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), epididymides, heart, kidneys, liver, spleen, testes, thymus.
- The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.
- The following tissues from the first 5 euthanised-as-scheduled males and the first 5 females euthanised on Day 6 p.p. of each group and female H21773 prematurely euthanised were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in modified Davidson's fixative): macroscopic lesions (all animals), adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides (all animals), esophagus, gut-associated lymphoid tissue (GALT), heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, ovaries (with oviducts) (all animals), prostate (all animals), rectum, sciiatic nerve, seminal vesicles (all animals), spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes (all animals), thymus, thyroids with parathyroids, trachea, urinary bladder, uterus (horns and cervix) (all animals), vagina (all animals).
- All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). This tissue processing was performed at CiToxLAB France.
- A microscopic examination was performed on: all tissues listed above from the first five euthanised-as-scheduled males and the first five females euthanised on Day 6 p.p. of the control and high dose groups (groups 1 and 4); all macroscopic lesions of all groups; all tissues listed above from the female euthanised prematurely, reproductive organs from group 3 female H21786 and group 4 female H21799 that did not conceive to investigate possible causes.
- Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Other examinations:

No further data

Statistics:

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

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

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

Clinical signs:

no effects observed

Description (incidence and severity):

- Piloerection associated with pallor of extremities was noted over the first three days of the lactation period in 1/9 females given 1200/1008 mg/kg/day. Ptyalism was sporadically recorded in 2/10 control males, 2/10 males given 110 or 330 mg/kg/day and 1/10 males given 1200/1008 mg/kg/day. As the clinical signs were of isolated occurrence or commonly observed when a test item is administered by gavage (ptyalism), they were considered not to be adverse.

- Other findings (reddish/brownish vaginal discharge, chromodacryorrhea, chromorhinorrhea, areas of hair loss, scab on head or neck, cutaneous lesion(s) on neck, nodosities on hindlimb, opacity of left eye), were considered to be not treatment related as they were noted in control animals, are commonly observed in this species and strain, were of isolated occurrence and/or resulted from the blood sampling procedure.

Mortality:

no mortality observed

Description (incidence):

- There were no unscheduled deaths in males during the study.
- Females that did not deliver: Female H21786, given 330 mg/kg/day, was sacrificed on Day 25 p.c. and female H21799, given 1200/1008 mg/kg/day, was sacrificed on Day 26 p.c. These females were found to be non-pregnant at necropsy. No microscopic alterations were observed in these females, which were both cycling.
- Female H21773 given 110 mg/kg/day was prematurely sacrificed on Day 19 p.c. for poor clinical conditions related to a mammary adenocarcinoma. This isolated finding in a low dose animal was considered to be spontaneous (i.e., unrelated to digadolinium trioxide). There were no test item-related findings in the mammary gland from high dose animals. This female was pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All the differences from controls (namely higher mean white blood cell count (including higher neutrophil, eosinophil, basophil, lymphocyte and large unstained cell counts) in high dose males and females at all dose levels, lower mean hemoglobin concentration and packed cell volume in intermediate dose males, prolonged prothrombin and activated partial thromboplastin times in males at all dose levels, higher mean monocyte count in intermediate dose females, lower mean red blood cell count in high dose females, and higher mean cell hemoglobin concentration in low and intermediate dose females), were slight, not dose related and/or due to lower mean control values. Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Higher mean inorganic phosphorus level together with higher mean urea level in high dose females were considered to be of only minor importance as the differences were of low magnitude, did not correlate with any other biochemistry findings and were not associated with any histological changes.
- All the other differences (namely higher mean glucose level in high dose males, lower mean cholesterol and biliary acid levels in males at all dose levels, higher mean cholesterol and biliary acid levels in high dose females and higher mean total bilirubin level in intermediate and high dose females) were minimal, of opposite trends and/or not dose related. Therefore, they were considered to be of no toxicological significance.

The main differences in blood biochemistry parameters when compared with control values are presented in the section "Any other information on results incl. tables".

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- There were no differences between test item-treated and control groups in motor activity (horizontal movements and rearing).
- There were no test item-related behavioural or neurologic abnormalities.
- Higher mean landing foot splay values (102 mm vs. 85 mm in controls) and higher mean rectal temperature (38.0°C vs. 37.0°C in controls) were noted in males given 1200/1008 mg/kg/day. These findings were considered to be of no toxicological importance as they did not correlate with any other findings (landing foot splay) or were within physiological values (rectal temperature).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no changes in organ weights considered to be related to the administered test item.
- When compared with controls, the mean absolute and relative to body testis weights were statistically significantly decreased in animals (-13%) at the highest dose. Although not statistically significant, a decrease in absolute and relative epididymis weight (-9%) was also observed. These decreases were mainly due to the low epididymides and testes weights in male H21715. In this animal, these weight changes correlated with marked tubular atrophy/degeneration in the testes, as well as reduced sperm content in the epididymides.
- A minor decrease in absolute and relative thymus weights were also observed in animals of both sexes treated at the highest dose. However, given the low magnitude, lack of statistical significance and of macroscopic or microscopic correlates, they were not considered to be directly related to the test item.
- Other organ weight changes were not considered to be related to the test item as they were too small in amplitude, not statistically significant, had no gross or microscopic correlates, and/or were not dose related in magnitude. Furthermore, they reflected the usual range of individual variations.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- There were no test item-related macroscopic findings.
- Herein, most macroscopic findings had no significant histologic correlates or correlated with common histologic findings in control rats, and were considered incidental.
- In male H21715 (treated at 1200/1008 mg/kg/day), a bilateral decrease in the size of the testes and epididymides was observed. Being an isolated occurrence (i.e., in the absence of similar changes in other high dose males), this finding was considered to be spontaneous.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- No microscopic changes that could be related to treatment were observed.
- In a single high dose male (H21715), there was marked bilateral tubular atrophy/degeneration, along with markedly reduced sperm content in the epididymides. In the absence of similar changes in other high dose males, this isolated change was considered incidental and unrelated to the test item administration.
- In the other males, qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle. Furthermore, the numbers of corpora lutea, of growing follicles and atretic follicles were similar in treated and control females.
- There were no microscopic alterations in these females that were prematurely sacrificed for failure to deliver, and they both presented histologic signs of normal estrous cycling. Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No further data

Key result

Dose descriptor:

NOAEL

Remarks:

for parental systemic toxicity

Effect level:

>= 1 008 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Blood biochemistry

The table below presents the main differences in blood biochemistry parameters when compared with control values:

	Blood biochemistry
Sex	Male				Female
Dose level	0	110	330	1200/1008	0	110	330	1200/1008
(mg/kg/day)
Inorganic phosphorus (mmol/L)	2.32	2.23	2.34	2.34	2.52	2.39	2.76	2.99**
Urea (mmol/L)	7.3	6.8	6.5	6.7	7.0	6.9	7.6	8.1
Statistically significant: **	: p < 0.01

Conclusions:

Interpretation of results: GHS criteria not met

The test item, digadolinium trioxide, was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating and (for females) throughout gestation and until Day 5 post-partum, at the dose-levels of 110, 330 or 1200/1008 mg/kg/day.
Based on the experimental conditions of the study the No Observed Adverse Effect Level (NOAEL) was considered to be higher than or equal to 1008 mg/kg/day based on the absence of adverse findings related to treatment with the test item at this high dose level.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data from the related substance gadolinium oxide is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

for parental systemic toxicity

Effect level:

>= 1 609 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

anhydrous gadolinium oxalate

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Based on the results of Papineau (2017) obtained with the related substance gadolinium oxide. Result (>= 1008 mg/kg bw/day) recalculated to gadolinium oxalate based on gadolinium content of both substances.

Key result

Critical effects observed:

no

Conclusions:

Interpretation of results: GHS criteria not met

No study is available on oral repeated dose toxicity of gadolinium oxalate. Therefore, read across is performed using a study from the related substance gadolinium oxide. In this study, gadolinium oxide was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating and (for females) throughout gestation and until Day 5 post-partum, at the dose-levels of 110, 330 or 1200/1008 mg/kg/day. Based on the experimental conditions of the study the No Observed Adverse Effect Level (NOAEL) was considered to be higher than or equal to 1008 mg/kg/day based on the absence of adverse findings related to treatment with the test item at this high dose level. This is equivalent to a NOAEL higher than or equal to 1609 mg/kg/day when recalculated to gadolinium oxalate, taking into account the gadolinium content of both substances. The read across justification is attached to IUCLID Section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 609 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity - oral

In a reliable, GLP-compliant study performed according to OECD guideline 422 (Papineau, 2017), the read across substance gadolinium oxide was administered once daily to 3 groups of 10 male and 10 female Sprague-Dawley rats by oral administration (gavage) at dose levels of 110, 330 or 1200 (until day 17 of the study)/1008 (from day 18 of the study until end of exposure) mg/kg bw/day. The dosing period started 2 weeks before mating, continued during mating, and for females, throughout gestation until day 5 post-partum. The test item was administered in the vehicle (0.5% methyl cellulose aqueous solution) under a constant dosage volume of 5 mL/kg bw/day, with the exception of the group receiving the highest dose level, for which the dosage-volume was 4.2 mL/kg bw/day from day 18 of the study onwards in order to achieve a dose level of 1008 mg/kg/day. One other group of 10 males and 10 females received the vehicle alone and acted as a control group.

Actual concentrations of the test item in the dose formulations analysed during the study (weeks 1 and 7) remained within an acceptable range of variations (-7.0% to +3.9%) when compared to the nominal values (nominal concentration +/-15%). There were no unscheduled test item-related deaths. Clinical signs were of isolated occurrence (e.g., piloerection) or commonly observed when test item is administered by gavage (e.g., ptyalism) and were therefore not considered to be adverse. There were no effects on body weight, body weight gain, food consumption, functional observation battery tests or motor activity data in any group or sex. Haematological differences, clinical biochemistry findings, and histopathological findings were not related to treatment with the test item. No test item-related changes were observed during gross pathological examination. Based on the experimental conditions of this study, the NOAEL for parental systemic toxicity was considered to be higher than or equal to 1008 mg/kg bw/day based on the absence of adverse findings related to the test item at this high dose level. This is equivalent to a NOAEL of 1609 mg/kg bw/day when recalculated to gadolinium oxalate taking into account the gadolinium content of both substances. This study is considered as the key study for endpoint coverage. The read across justification is attached to IUCLID Section 13.

Repeated dose toxicity - inhalation/dermal

No information has been identified on the repeated dose toxicity of gadolinium oxalate after inhalation or dermal exposure. A key read across study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (Column 2, Annex VIII, Section 8.6.1).

Anyhow, low exposure to gadolinium oxalate is expected based on the inherent properties of this compound. No vapour pressure value has been determined as gadolinium oxalate does not melt below 300°C. Therefore, inhalation of gadolinium oxalate as a vapour is not likely to occur.

Further, gadolinium oxalate is typically manufactured as wet powder. Thus, the formation of respirable suspended particulate matter is unlikely. Occasionally manufactured dry powders typically have a D50 > 50 μm, which implies that no or only a very limited fraction of the powders may be respirable and capable of reaching the alveolar region of the lungs. Consequently, human exposure by inhalation is considered not significant for this compound.

Finally, the dermal route of exposure is not considered relevant either due the poor water solubility of gadolinium oxalate.

Consequently, it is not necessary to perform a repeated dose toxicity study via the inhalation or dermal route of exposure.

Justification for classification or non-classification

Repeated dose toxicity - oral

Based on the NOAEL value of higher than or equal to 1609 mg/kg bw/day, recalculated from the NOAEL of higher than or equal to 1008 mg/kg bw/day obtained in an OECD 422 study performed with the read across substance gadolinium oxide, gadolinium oxalate is not to be classified for Specific Target Organ Toxicity after Repeated Exposure (STOT RE) according to the CLP Regulation.

Repeated dose toxicity - inhalation

No key repeated dose toxicity study via inhalation is available.

Repeated dose toxicity - dermal

No key repeated dose toxicity study via dermal application is available.