Digadolinium trioxide

Endpoint summary

Description of key information

Repeated dose toxicity - oral

The key 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. Based on these results, the test substance is not classified as STOT RE.

Repeated dose toxicity - inhalation/dermal

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

A key 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

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:

experimental study

Adequacy of study:

supporting study

Study period:

1963

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The test item was administered to guinea pigs at a dose level of 2000 mg/kg every other day for a month via oral gavage. A control group was included.

GLP compliance:

not specified

Limit test:

yes

Species:

guinea pig

Strain:

not specified

Details on species / strain selection:

No further data

Sex:

not specified

Details on test animals or test system and environmental conditions:

no further data

Route of administration:

oral: gavage

Details on route of administration:

no further data

Vehicle:

other: edulcorated starch paste

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dust was mixed with paste and delivered via syringe to the animal's mouth.

VEHICLE
Justification for use and choice of vehicle (if other than water): starch paste for palatabilty of test substance.
:

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

During the experiment, each animal received 16-18 g of test item.

Duration of treatment / exposure:

30 days

Frequency of treatment:

every other day

Dose / conc.:

2 000 other: mg/kg bw, every other day

No. of animals per sex per dose:

no data

Control animals:

yes, concurrent no treatment

Details on study design:

no data

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Not specified

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined: Hemoglobin, erythrocytes and leukocytes, differential blood count; blood coagulability (prothrombin index).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined: Study of state of the liver and processes of protein metabolism: amino acids in blood serum, protein levels and enzyme levels in blood serum.

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Study of state of the liver and processes of protein metabolism: amino acids in urine

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Not specified

HISTOPATHOLOGY: Yes
- Microscopic investigation of the structure of the internal organs of animals killed after completion of dosing.

Other examinations:

no data

Statistics:

yes, no further data

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Statistical analysis of the blood data revealed no significant effects from the exposure to the test item.
No further data.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Statistical analysis of the blood data revealed no significant effects from the exposure to the test item.
No further data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No further data.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of internal organs revealed no significant findings. No further data.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

no further data

Dose descriptor:

NOAEL

Effect level:

>= 2 000 other: mg/kg bw, every other day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Interpretation of results: study not used for classification due to deficiencies of the study.

The degree of toxicity of the the test item to guinea pigs over a 30 day exposure period was found to be negligible, when administering a dose of 2000 mg/kg bw every other day via oral gavage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 008 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1966

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were exposed to 30 mg/m3 gadolinium oxide for periods from 20 to 120 days.

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

other: CFW strain

Sex:

not specified

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

< 1 µm

Remarks on MMAD:

The mean particle size was determined by sampling the chamber atmosphere with a point to plane electrostatic precipitator. The samples were obtained directly on an electron microscope grid, examined under an electron microscope, and fields photographed at random. The greatest diameter of each particle which was horizontal with the top of the electron photomicrograph was determined by means of the Zeiss particle size analyser. The percentage of the total aerosol mass made up of particles of the desired size was determined by finding the mass distribution using a cascade impactor. This impactor consists of four progressively finer air jets impinging on a series of glass disks coated with stopcock grease, and finally on a filter paper between the fourth jet and the vacuum line. The gadolinium oxide collected at each stage was estimated by the arsenazo method. In addition, particle sizes at each impactor stage were determined by exposing electron microscope grids in the line of deposition at each disk for 3 sec at a flow rate of 17.5 liters per minute. The values obtained from these procedures gave an estimate of approximately 70% of the total aerosol mass in the form of particles of gadolinium oxide less than 1.0 µm in mean diameter. Mean particle size was determined to be 0.312 µm (+/-0.287 µm).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chambers were designed in accordance with a method described by Cook and Fisher (1961). The design requirements for the inhalation system were set by Davison (1963). The generation equiment which introduced dust into the chambers has been shown by Cook, Fisher, and Davison, to give an even distribution of dust.
- Air flow rate: The air flow was held onstant at 8 cu ft/min and the generator gear set to give the desired concentration of gadolinium oxide.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily samples were taken by withdrawing 16.8 cu ft of chamber air per hour through a cellulose filter for 15 minutes. The amount of gadolinium oxide on each filter was determined by the arsenazo color reaction of Fritz et al. (1958).

Duration of treatment / exposure:

20-120 days

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

256 mice total

Control animals:

yes, concurrent no treatment

Details on study design:

At the end of each 20-day exposure period, 20 to 30 mice, selected equally from both sexes, were removed and held for the duration of their lives.
Six to ten mice of both sexes per group served as non-exposed controls.

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Upon removal from exposure chamber. The procedure was repeated on survivors at one year and at 18 months.
- How many animals: 20% ot the total number of animals, random, both sexes equally represented.
- Parameters examined: total leukocyte counts, hemoglobin level, packed cell volume, differential leukocyte counts, clotting times

Sacrifice and pathology:

HISTOPATHOLOGY: Yes
- Representative tissues were fixed in 10% neutral formalin.
- Sections were stained with hematoxylin and eosin, the Van Gieson connective-tissue stain and the Von Kossa stain for calcium.
- The finding of pulmonary calcification was not expected, and led to a method of grading based on the microscopic evaluation of the amount of calcification present in the alveolar septa and walls of the small vessels. The most marked degree of calcification was graded 4+, while slight traces were given values of +/- 0.5, with intermediate values of +1, +2, and +3.

Statistics:

A 2-way classification test for independence was applied to the differences in pulmonary calcification between exposure groups, with a 5% significance level.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

In general, the control groups had life spans which averaged longer than those of the exposed groups by two to eight months, except in the 80-day exposure group, where there was a heavy early mortality in the control mice due to infectious hepatitis and to murine leukemia. There was no significant decrease in life spans with increasing exposure times.
During exposure, there were no deaths due to pneumonia in controls, however, there were some deaths from pneumonia among exposed mice, the most of this fatal pneumonia occuring between 2 to 5 weeks of exposure. The authors concluded that these results suggest that exposure to gadolinium oxide dust may have increased the susceptibility of the mice to pneumonia.
Nevertheless, during the post-exposure holding period, the percentage of deaths due to pneumonia was only slightly higher in the exposed mice (22%) than in control mice (14%).
See Table 1 and Table 2 attached to this entry in section "Attached background material".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A statistical analysis of weight gains failed to show any significant differences in the exposed mice as compared to the controls.

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:

no effects observed

Description (incidence and severity):

A statistical analysis of hematologic data failed to show any significant differences in the exposed mice as compared to the controls.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Localised accumulations of metal-containing macrophages were observed in lungs of mice exposed for 20 days or more. There was no appreciable reaction observed around such localisations, or at most, minimal interstitial thickening was observed. These localisations were most commonly located in the periphery of the lungs, often immediately under the pleura, and also around bronchioles. Macrophages were usually located free within the alveoli. Less commonly, metal-containing macrophages were found in the interstitium of the alveolar septa. Metal was always present in the tracheobronchial lymph nodes. It was concentrated in the reticuloendothelial cells of the medulla.
Pulmonary calcification was observed in a variable percentage of mice exposed for the different time intervals, and was not found in the lungs of any control mice. Neither the presence nor the severity of pulmonary calcification was associated with significant shortening of life spans. Pulmonary calcification required several months to manifest itself, but did not become more severe with increasing exposure to gadolinium oxide.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Primary lung tumors were commonly found at necropsy in both control (25.5%) and exposed mice (27.5%). Most of these were bronchial adenomas, but a few possessed criteria of malignancy. This suggests that in mice, gadolinium oxide is not a lung tumor accelerator.

Other effects:

not examined

Details on results:

Although some gadolinium oxide dust was moved from the alveoli to the regional lymph nodes, the large amounts left in the lungs several months post-exposure suggest that the mouse has an inadequate clearing mechanism for this compound.

Conclusions:

Interpretation of results: study not used for classification due to deficiencies of the study.

In this study, mice were exposed to gadolinium oxide (30 mg/m3) via inhalation for 20 to 120 days. No effects were observed on body weight or the haemotological parameters studied. A trend towards reduced life span was observed among exposed mice, partly due to increased mortality as a result of pneumonia, which was not observed in the respective control mice. However, the results seem to be confused by the occurrence of hepatitis and murine leukemia in control mice for the 80-day exposure group, and almost equal occurrence of pneumonia during the post-exposure period in both exposed and control mice. Consequently, it cannot be safely concluded that the observed effects on life span were (only) due to exposure to gadolinium oxide, as there might have been some issues with bacterial/viral contamination during the study, affecting the observed results.
Further, from 20 days of exposure onwards, macrophages with ingested gadolinium oxide were observed in the lungs. Gadolinium oxide was always observed in the tracheobronchial lymph nodes. Also, pulmonary calcification was observed in a variable percentage of mice exposed for different periods, however, pulmonary calcification was not associated with significant shortening of life spans. Since large amounts of dust were left in the lungs several months post-exposure, the authors concluded that the mouse has an inadequate clearing mechanism for this compound. When interpreting these results, it must be kept in mind that gadolinium oxide with a median particle size of 0.312 µm and MMAD < 1 µm was used in the study, which is rather low compared to what is currently prescribed by OECD, and moreover not reflecting the typical particle size of the products on the EU market. Also, the exposure concentration of 30 mg/m3 is not considered representative for potential exposure at the working place these days. The exposure concentration and low particle size may indeed have resulted in lung overload conditions for the exposed mice, explaining the observed accumulation of dust in the lungs due to inadequate clearing.

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Only a disregarded study (Ball and Van Gelder, 1966, Klimisch 3) has been identified for this endpoint.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1966

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were exposed to 30 mg/m3 gadolinium oxide for periods from 20 to 120 days.

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

other: CFW strain

Sex:

not specified

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

< 1 µm

Remarks on MMAD:

The mean particle size was determined by sampling the chamber atmosphere with a point to plane electrostatic precipitator. The samples were obtained directly on an electron microscope grid, examined under an electron microscope, and fields photographed at random. The greatest diameter of each particle which was horizontal with the top of the electron photomicrograph was determined by means of the Zeiss particle size analyser. The percentage of the total aerosol mass made up of particles of the desired size was determined by finding the mass distribution using a cascade impactor. This impactor consists of four progressively finer air jets impinging on a series of glass disks coated with stopcock grease, and finally on a filter paper between the fourth jet and the vacuum line. The gadolinium oxide collected at each stage was estimated by the arsenazo method. In addition, particle sizes at each impactor stage were determined by exposing electron microscope grids in the line of deposition at each disk for 3 sec at a flow rate of 17.5 liters per minute. The values obtained from these procedures gave an estimate of approximately 70% of the total aerosol mass in the form of particles of gadolinium oxide less than 1.0 µm in mean diameter. Mean particle size was determined to be 0.312 µm (+/-0.287 µm).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chambers were designed in accordance with a method described by Cook and Fisher (1961). The design requirements for the inhalation system were set by Davison (1963). The generation equiment which introduced dust into the chambers has been shown by Cook, Fisher, and Davison, to give an even distribution of dust.
- Air flow rate: The air flow was held onstant at 8 cu ft/min and the generator gear set to give the desired concentration of gadolinium oxide.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily samples were taken by withdrawing 16.8 cu ft of chamber air per hour through a cellulose filter for 15 minutes. The amount of gadolinium oxide on each filter was determined by the arsenazo color reaction of Fritz et al. (1958).

Duration of treatment / exposure:

20-120 days

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

256 mice total

Control animals:

yes, concurrent no treatment

Details on study design:

At the end of each 20-day exposure period, 20 to 30 mice, selected equally from both sexes, were removed and held for the duration of their lives.
Six to ten mice of both sexes per group served as non-exposed controls.

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: not specified

DETAILED CLINICAL OBSERVATIONS: not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Upon removal from exposure chamber. The procedure was repeated on survivors at one year and at 18 months.
- How many animals: 20% ot the total number of animals, random, both sexes equally represented.
- Parameters examined: total leukocyte counts, hemoglobin level, packed cell volume, differential leukocyte counts, clotting times

Sacrifice and pathology:

HISTOPATHOLOGY: Yes
- Representative tissues were fixed in 10% neutral formalin.
- Sections were stained with hematoxylin and eosin, the Van Gieson connective-tissue stain and the Von Kossa stain for calcium.
- The finding of pulmonary calcification was not expected, and led to a method of grading based on the microscopic evaluation of the amount of calcification present in the alveolar septa and walls of the small vessels. The most marked degree of calcification was graded 4+, while slight traces were given values of +/- 0.5, with intermediate values of +1, +2, and +3.

Statistics:

A 2-way classification test for independence was applied to the differences in pulmonary calcification between exposure groups, with a 5% significance level.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

In general, the control groups had life spans which averaged longer than those of the exposed groups by two to eight months, except in the 80-day exposure group, where there was a heavy early mortality in the control mice due to infectious hepatitis and to murine leukemia. There was no significant decrease in life spans with increasing exposure times.
During exposure, there were no deaths due to pneumonia in controls, however, there were some deaths from pneumonia among exposed mice, the most of this fatal pneumonia occuring between 2 to 5 weeks of exposure. The authors concluded that these results suggest that exposure to gadolinium oxide dust may have increased the susceptibility of the mice to pneumonia.
Nevertheless, during the post-exposure holding period, the percentage of deaths due to pneumonia was only slightly higher in the exposed mice (22%) than in control mice (14%).
See Table 1 and Table 2 attached to this entry in section "Attached background material".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A statistical analysis of weight gains failed to show any significant differences in the exposed mice as compared to the controls.

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:

no effects observed

Description (incidence and severity):

A statistical analysis of hematologic data failed to show any significant differences in the exposed mice as compared to the controls.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Localised accumulations of metal-containing macrophages were observed in lungs of mice exposed for 20 days or more. There was no appreciable reaction observed around such localisations, or at most, minimal interstitial thickening was observed. These localisations were most commonly located in the periphery of the lungs, often immediately under the pleura, and also around bronchioles. Macrophages were usually located free within the alveoli. Less commonly, metal-containing macrophages were found in the interstitium of the alveolar septa. Metal was always present in the tracheobronchial lymph nodes. It was concentrated in the reticuloendothelial cells of the medulla.
Pulmonary calcification was observed in a variable percentage of mice exposed for the different time intervals, and was not found in the lungs of any control mice. Neither the presence nor the severity of pulmonary calcification was associated with significant shortening of life spans. Pulmonary calcification required several months to manifest itself, but did not become more severe with increasing exposure to gadolinium oxide.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Primary lung tumors were commonly found at necropsy in both control (25.5%) and exposed mice (27.5%). Most of these were bronchial adenomas, but a few possessed criteria of malignancy. This suggests that in mice, gadolinium oxide is not a lung tumor accelerator.

Other effects:

not examined

Details on results:

Although some gadolinium oxide dust was moved from the alveoli to the regional lymph nodes, the large amounts left in the lungs several months post-exposure suggest that the mouse has an inadequate clearing mechanism for this compound.

Conclusions:

Interpretation of results: study not used for classification due to deficiencies of the study.

In this study, mice were exposed to gadolinium oxide (30 mg/m3) via inhalation for 20 to 120 days. No effects were observed on body weight or the haemotological parameters studied. A trend towards reduced life span was observed among exposed mice, partly due to increased mortality as a result of pneumonia, which was not observed in the respective control mice. However, the results seem to be confused by the occurrence of hepatitis and murine leukemia in control mice for the 80-day exposure group, and almost equal occurrence of pneumonia during the post-exposure period in both exposed and control mice. Consequently, it cannot be safely concluded that the observed effects on life span were (only) due to exposure to gadolinium oxide, as there might have been some issues with bacterial/viral contamination during the study, affecting the observed results.
Further, from 20 days of exposure onwards, macrophages with ingested gadolinium oxide were observed in the lungs. Gadolinium oxide was always observed in the tracheobronchial lymph nodes. Also, pulmonary calcification was observed in a variable percentage of mice exposed for different periods, however, pulmonary calcification was not associated with significant shortening of life spans. Since large amounts of dust were left in the lungs several months post-exposure, the authors concluded that the mouse has an inadequate clearing mechanism for this compound. When interpreting these results, it must be kept in mind that gadolinium oxide with a median particle size of 0.312 µm and MMAD < 1 µm was used in the study, which is rather low compared to what is currently prescribed by OECD, and moreover not reflecting the typical particle size of the products on the EU market. Also, the exposure concentration of 30 mg/m3 is not considered representative for potential exposure at the working place these days. The exposure concentration and low particle size may indeed have resulted in lung overload conditions for the exposed mice, explaining the observed accumulation of dust in the lungs due to inadequate clearing.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1965

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

Guinea pigs were exposed to gadolinium oxide via inhalation for 40, 80 or 120 days. After exposure, lung tissues were evaluated histologically and pulmonary function was assessed.

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

The test material was reduced to a moderately uniform particle diameter by two stages of grinding.
In the first stage the oxide was pulverised in a Pica blender mill using stainless steel vials and balls for 8 min.
The second stage consisted of manually regrinding the powder suspended in absolute ethanol in a mortar with a ceramic pestle.
The alcohol was removed from the mixture by burning, until a dry powder remained.
The powder was then stored in a desiccator until use.

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: not specified
- Weight at study initiation: 817 +/- 52 g (males); 744 +/- 51 g (females)
- Fasting period before study: not specified
- Housing: After removal from the exposure chambers, animals were housed in a closed animal room in a standard cage unit containing pine shavings. Same through-put air flow as during the experiment.
- Diet (e.g. ad libitum): Ad libitum when not in exposure chamber, diet not specified.
- Water (e.g. ad libitum): Ad libitum when not in exposure chamber, source not specified.
- No food or water available during exposure.
- Acclimation period: Yes, length not specified. At least until no weight loss was observed in the animals.

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS: not specified

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

< 0.563 µm

Geometric standard deviation (GSD):

0.531

Remarks on MMAD:

92% of the mass of gadolinium oxide collected from the exposure chamber was of particles smaller than 0.563 +/- 0.531 µm in diameter. The mean particle diameter was determined to be 0.22 +/- 0.21 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure chamber: During exposure, the animals were segregated by sex and housed in cages constructed of expanded stainless steel mesh.
- Exposure apparatus: The chambers were designed in accordance with the specifications set by Davison (1963) and described by Cook (1961).
- Air flow through the chambers: 8 ft3/min
- System of generating particulates/aerosols: The suspension of the dust in the air was accomplished by the use of a Wright dust feed mechanism. It operated by scraping dust from a cylinder tightly packed with gadolinium oxide, then removing the dust with a stream of air. The dust-laden air was injected into the main stream of chamber air in the throat of the chamber just below an upstream air filter. This assured that the aerosol was the only foreign material in the chamber atmosphere. The dust was introduced into the top of the chamber in such a manner that the incoming air gave an even distribution in all parts of the chamber as demonstrated by Cook (1961).
- Temperature, humidity, pressure in air chamber: Temperature not specified. Dehumidified air was necessary to prevent clogging of the scraper plate of the dust generator. This was accomplished via a water trap at the air compressor unit, an air filter, and a dehumidifying unit in the line before the air passed the scraper blade.
- Method of particle size determination: The percentage of the total aerosol mass made up of particles of the desired size was determined by studying the mass distribution using a Casella cascade impactor. This impactor consists of four progressively finer air jets impinging in series on glass discs coated with stopcock grease, and finally a filter unit between the fourth stage and the line to the vacuum pump. A time of 30 seconds was used for collection of samples. The method described for determining chamber aerosol concentration was used for the quantitative determination of gadolinium oxide impacted at each stage of the filter. Hot, dilute nitric acid (1:1) melted the stopcock grease and released the particles so that they could go into solution. The particle sizes at each impactor stage were determined by exposing electron microscope grids in the line of deposition at each stage. Sampling for 3 seconds was found to be optimal. In order to determine the mean particle diameter of the generated aerosol, the chamber atmosphere was sampled with a point-to-plane electrostatic precipitator. Precipitation was accomplished by drawing chamber air through the instrument at a flow rate of 2.5 ft3/hr. The particles were deposited on an electron microscope grid overlaid with a carbon film where they could be photographed with the electron microscope. The maximum diameter horizontal with the top or bottom edge of the electron photomicrograph was determined with a particle size analyser. The obtained figures were based on 1000 particles photographed at random from the grid and represented that portion of the population that could be seen and measured at a magnification of 4125x.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken daily from the chambers. Sampling was accomplished by drawing 16.8 ft3/hr of chamber air across a cellulose acetate filter for 15 min. The arsenazo method was used, as described by Fritz et al. (1958), for determining the amount of gadolinium oxide on each filter. The test item was eluted from the filter with 50% nitric acid. This was diluted to a total volume of 50 mL with deinonised water, and a 1-mL aliquot was used for analysis. The sample was buffered with a triethanolamine buffer, purified arsenazo reagent added, and the pH adjusted to 8.2 with NH4OH. The resulting color was measured against a reagent blank, prepared with an unexposed filter, at 570 µm in a spectrophotometer. The concentration of the test item during each exposure period was approximately 20 mg/m3 (20.64 +/- 3.37, 20.82 +/- 6.98 and 20.77 +/- 6.18 mg/m3 for the 40-, 80- and 120-day exposure periods, respectively).

Duration of treatment / exposure:

Groups were exposed for 40, 80 or 120 days (i.e. three groups).

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

20 mg/m³ air

No. of animals per sex per dose:

6 of each sex per exposure period, same amount of animals in the respective control groups

Control animals:

yes, concurrent no treatment

Details on study design:

Experiment designed as a three way factorial, with treatment, duration of exposure, and sex as the three factors.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout treatment period

Sacrifice and pathology:

GROSS PATHOLOGY: Not specified
HISTOPATHOLOGY: Yes, lung tissue was examined.
- After the inflation tests, the lungs were placed in 10% formalin and were later sectioned and stained with hematoxylin and eosin for histologic evaluation.

Other examinations:

Pulmonary function:
- A measure of elasticity was chosen as the comparison parameter for pulmonary function.
- Elastance is the reciprocal of compliance.
- Compliance is the volume change per unit of pressure change.
- Compliance was measured in lungs removed in inflated state from the thoracic cavity of experimental animals.
- The lungs were inflated at a constant rate and the relationship of intrapulmonary pressure to volume of air injected was recorded.

Statistics:

The results of the inflation tests were analysed by means of a three-way factorial design with unequal numbers of observations per subclass. The mean of each subclass was calculated and analysed. Homogeneity of variance by Bartlett's test was non-significant at the 0.20 level.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Changes in the exposed lungs included alveolar cell hypertrophy, septal wall thickening, lymphoid hyperplasia and macrophage proliferation.
- No changes were observed in the lungs from control animals.
- Severity of the lesions increased as exposure time increased.
- The predominant changes after 40 days of exposure were mild swelling and proliferation of septal cells and nodular lymphocytic hyperplasia. Microscopic observations of the stained slides revealed a definite difference between control and exposed lung sections.
- At 80 days of exposure, a more extensive parenchymal cell reaction was evident. The predominant finding was the presence of numerous septal and alveolar macrophages. These alveolar macrophages or dust cells are desquamated alveolar cells. Alveolar epithelial cells become alveolar macrophages by a process of hypertrophy and hyperplasia of intact cells. This process in itself would contribute to a thickened septal wall, and could possibly interfere with normal pulmonary function. When a comparison was made between 40 and 80 days of exposure, there was an apparent general increase in nodular lymphocytic hyperplasia.
- After 120 days of exposure, histological changes included further thickening of alveolar walls, presence of numerous macrophages and nodular lymphocytic hyperplasia. The heavy infiltration of lymphocytic nodules is speculated to be great enough to physically hinder normal expansion of the lung.
- The sequential change in histologic appearance as exposure time was increased correlated with the conclusions from the statistical analysis of the slopes of the elastance curves. Thickness of the alveolar walls and numbers of lymphocytic elements increased with greater exposure time as did the slopes of the elastance curves.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pulmonary function:
- Lungs exposed to gadolinium oxide had increased elastance (and decreased compliance) compared to lungs from unexposed control guinea pigs.
- The effect was related linearly to length of exposure.
- Analysis of variance demonstrated a significant (p<0.01) treatment versus duration interaction, indicating that exposed lungs reacted differently than the control lungs over the three periods of exposure.
- There was no effect attributable to sex.

Details on results:

- One factor other than thickened alveolar walls and lymphoid hyperplasia should be mentioned as a possible cause of changes in elastance of the lungs. Guinea pigs have far more airway smooth muscle than other species (Miller, 1947). Radford and Lefcoe (1955) state that with bronchoconstriction some of the small bronchioles may close off entirely and when closed, larger pressures are required to open them. An acute reflex of bronchoconstriction, such as observed for charcoal dust in cats (Widdicombe et al., 1962), may be caused due to stimulation of receptors in the airways. This phenomenon was not investigated in this study.
- There was an absence of pulmonary fibrosis throughout the sections of exposed lung tissue. This was also observed in other similar studies with rare earths (Schepers et al., 1955; Davison, 1963; Reece, 1965; Talbot et al., 1965).

Conclusions:

Interpretation of results: study not used for classification due to deficiencies of the study.

In this study, guinea pigs were exposed via inhalation to gadolinium oxide (20 mg/m3) for 40, 80 or 120 days. After each exposure period, the lungs of exposed and control animals were removed from the thoracic cavity to assess pulmonary function (elastance) followed by histological examination. Changes in the lungs (e.g., alveolar cell hypertrophy, septal wall thickening, lymphoid hyperplasia and macrophage proliferation) were observed in exposed animals, the severity of the lesions increasing with exposure time, whereas no changes were observed in the lungs from control animals. Also, lungs exposed to gadolinium oxide appeared to have increased elastance (decreased compliance) compared to lungs from unexposed control guinea pigs. This effect was also related linearly to length of exposure. Moreover, the sequential change in histologic appearance as exposure time was increased correlated with the conclusions from the statistical analysis of the slopes of the elastance curves. Thickness of the alveolar walls and numbers of lymphocytic elements increased with greater exposure time as did the slopes of the elastance curves. Lung fibrosis was not observed.
When interpreting the results of this study, it should be kept in mind that a very low particle size was used (92% of the particles (by mass) was smaller than 0.563 µm and mean particle size was 0.22 µm), which is rather low compared to current prescriptions by OECD and which cannot be considered representative for the products currently on the EU market. Further, the exposure concentration of 20 mg/m3 is also not representative for potential occupational exposure these days. Altogether, the results of the study suggest that the animals were most likely exposed under lung overload conditions, explaining the histological observations made in the lungs of exposed animals and the consequent effect on pulmonary function. Further, the study did not report on any mortalities or other evidence of systemic toxicity, limiting its use for endpoint coverage.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1965

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Principles of method if other than guideline:

White mice (CFW) were exposed to gadolinium oxide via inhalation (ca. 30 mg/m3) for 20, 40, 60, 80, 100 or 120 days. After exposure, blood samples were taken for analysis, mice were euthanatised, gross pathological observations were made and tissues harvested for histopathological examination.

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

- Test material as received was a finely pulverised powder of unknown particle size.
- The test material was reduced to a moderately uniform particle size by two grindings. The first was with a blender mill using stainless steel vials and balls for 8 min. The second stage consisted of suspending the powder in absolute ethanol and manually pulverising it in a mortar with a ceramic pestle. The alcohol was removed from the mixture by burning and a satisfactory dry dust remained. It was stored in a desiccator until used.

Species:

mouse

Strain:

other: CFW

Details on species / strain selection:

no data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: When the mice were not being exposed in the inhalation chambers, they were housed 10 per cage in plastic boxes in a separate animal isolation room.
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

DETAILS OF FOOD AND WATER QUALITY: No data

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

< 1 µm

Remarks on MMAD:

Approximately 70 percent of the total mass of dust generated was contained in particles less than 1 micron in diameter.
Mean particle size was determined to be 0.312 +/- 0.287 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: As designed and described by Davison (1963) and Cook and Fisher (1961).
- Method of holding animals in test chamber: Stainless steel 12 x 8 x 23 inch cages of 3/8 inch expanded steel fabric to allow free passage of air. Even distribution of dusts throughout the chambers and cages has been demonstrated by Cook and Fisher (1961) and by Davison (1963).
- System of generating particulates/aerosols: The oxide was suspended in the chamber air by using a Wright dust feed generator which introduced the dust into the throat of the inhalation chamber.
- Air flow rate: held constant at 8 cu ft. per min
- Method of particle size determination: The chamber atmosphere was sampled with a point to plane electrostatic precipitator. The samples were obtained directly on a parlodion coated, 200 mesh, copper, electron microscope grid which had been overlaid with a carbon film. Chamber air was drawn through the precipitator at a flow rate of 2.5 cubic feet per hour. A voltage of 7 KV producing 4 to 5 micro-amps of current across the air gap for 7 seconds was used. The grids were examined in an electron microscope and random fields photographed. The maximum diameter of each particle horizontal with the top of the electron photomicrograph was determined with a particle size analyser. Mean particle size was based on 1000 gadolinium oxide particles photographed at random. They represent that portion of the population that could be seen and measured at a magnification of 10000x. In order to determine what percent of the total mass of aerosol generated consisted of particles less than 1 micron in diameter, the mass distribution was determined with a Casella cascade impactor. The flow rate of chamber air through the impactor was regulated to 17.5 liters per minute and samples collected on glass cover-slips coated with stop-cock grease for 30 seconds. The arsenazo method of Fritz et al. (1958) was used to determine the concentration of gadolinium collected at each stage. To determine the size of the particles collected at each stage of the impactor, electron microscope grids were mounted and the chamber air sampled for 3 seconds at a flow rate of 17.5 liters per minute.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily samples were taken from the chamber air by withdrawing 16.8 cu ft of chamber air per hour through a cellulose filter for 15 minutes. The amount of test item on each filter was determined by the method described by Fritz et al. (1958). This method uses arsenazo as the color-forming agent and is not highly pH dependent.

Duration of treatment / exposure:

20, 40, 60, 80, 100 and 120 days

Frequency of treatment:

6 hrs/day, 5 days/week

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

10 males, 10 females per group and per control

Control animals:

yes, concurrent no treatment

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of each 20-day exposure period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: All, 20 exposed animals (10 males, 10 females) and 20 control animals (idem) at the end of each 20-day exposure period.
- Parameters examined: total leukocyte counts, hemoglobin, packed cell volume, differential leukocyte counts

CLINICAL CHEMISTRY: Not specified

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: The exposed mice and respective control mice were euthanised after each 20-day exposure period. Gross pathological observations were made.
HISTOPATHOLOGY: Tissues of exposed mice and respective control mice were harvested for histopathological examinations after each 20-day exposure period. Lungs and other tissues were examined, however, the other tissues were not specified.

Other examinations:

No data

Statistics:

No data

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

no effects observed

Description (incidence and severity):

There were no significant differences found between the blood evaluations of the control and exposed mice.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lungs of exposed mice progressed through a sequence of changes as the exposure time increased from 20 through 120 days.
- 20 days exposure: Slight thickening of of septal walls, mild swelling and proliferation of septal cells, peribronchiolar and periarteriolar lymphocytic hyperplasia, and occasional macrophages containing dust particles were seen. No lesions in other organs were found (not specified which organs were studied).
- 40 days exposure: Septal thickening to 3 or more cells in some areas. Some pneumonia was diagnosed grossly and later verified microscopically. The lesions were focal and did not resemble typical lesions of broncho-pneumonia in mice in that the primary sites were not in the most dependent portions of the lobes. The lesions were patchy and did not show a general progression from a site of origin.
- 60 days exposure: The alveolar walls presented an increased thickness due to both swelling of the mural epithelium and an accumulation of dust-laden macrophages.
- 80 days exposure: The general pattern of impaction of dust with subsequent phagocytosis continued, until as much as 25% of the lung tissue appeared to be filled with dust-laden cells. Peribronchiolar and periarteriolar lymphocytic hyperplasia were present in most lungs examined.
- 100 days exposure: Dust-laden macrophages were extensive in lung tissue. At this time the first histological evidence of dust translocation could be visualised. Macrophages containing dust particles were observed in the tracheobroncial lymph nodes. Accumulations of macrophages were present in multiple foci throughout the lymph node sections examined. High power microscopic examination revealed typical foamy dust-laden macrophages similar in appearance to those seen in the lung parenchyma. Multinuclear giant cells containing dust particles were also seen, probably evidencing coalescence of two or more macrophages.
- 120 days exposure: The lungs were severely impacted. Many alveoli appeared to be completely occluded with the macrophage proliferation. Multinuclear giant cells were present in the lung fields as well as in the tracheobronchial lymph nodes. Staining of the histological sections with Gomori's one step tri-chrome stain verified the absence of any extensive fibrosis.
- Control animals: The lungs and other tissues harvested from the control mice at each 20-day interval were free from pathological lesions. An occasional lung field from control mice sections taken at 20 and 40 days did show slight periarteriolar lymphocytic hyperplasia but was not seen following 40 days of exposure. The complete freedom of lesions in these lung sections is interpreted as an influence of the strict isolation provided to the mice in their animal isolation room as well as the influence of breathing dust particle free air for 6 hours of each day while they were in the control inhalation chamber.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Conclusions:

Interpretation of results: study not used for classification due to deficiencies of the study.

Gadolinium oxide is relatively inert and non-toxic because it elicits no acute inflammatory response or any chronic fibrosis in mice after 120 days of exposure by inhalation to gadolinium oxide dust (30 mg/m3). The only response seems to be the physiological mechanism present in the lungs for engulfment of dust particles. The lung parenchyma responds only by swelling of the epithelial cells and engulfment of the dust by macrophages. The macrophages were observed to translocate the dust to the tracheobronchial lymph nodes, after 100 days of exposure or more. No lesions were seen in organs other than the lungs and tracheobronchial lymph nodes. It is however not specified which other organs were studied. It is assumed that gadolinium was not soluble enough to be absorbed into the blood stream at amounts high enough to affect other organs histologically. The blood parameters studied showed no difference between exposed and control animals.
When interpreting the results of this study, it should be kept in mind that a very low particle size was used (70% of the particles (by mass) was smaller than 1 µm and mean particle size was 0.22 µm), which is rather low compared to current prescriptions by OECD and which cannot be considered representative for the products currently on the EU market. Further, the exposure concentration of 30 mg/m3 is also not representative for potential occupational exposure these days. Altogether, the results of the study suggest that the animals were most likely exposed under lung overload conditions, explaining the histological observations made in the lungs of exposed animals. Further, the study did not report on any mortalities or other evidence of systemic toxicity, limiting its use for endpoint coverage.

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Only a disregarded study (Ball and Van Gelder, 1966) and two supporting studies (Talbot et al., 1965; Abel and Talbot, 1965, also described in Abel and Talbot, 1967) have been identified for this endpoint. These three studies were considered not reliable (Klimisch 3) due to significant methodological deficiencies and/or inability to cover the endpoint.

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 test item 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 study was considered as the key study for endpoint coverage.

Another study was identified in scientific literature, in which guinea pigs were exposed for 30 days to a dose of 2000 mg/kg bw every other day via oral gavage (Mogilevskaya and Roshchina, 1964). This study reported that the degree of toxicity of the test item to guinea pigs over the 30-day exposure period was negligible. Statistical analysis of blood data revealed no significant effects from exposure to the test item. Microscopic examination of internal organs did not reveal any significant findings either. Because this study only contains limited information on test set-up and observations, the study was assigned a Klimisch reliability score of 4 (not assignable) and was considered to yield supporting information only.

Repeated dose toxicity - inhalation

A key 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 route of exposure.

Several studies reporting on this endpoint were identified in scientific literature. However, these studies were all assigned a Klimisch reliability score of 3 due to significant methodological deficiencies and/or inability to cover the endpoint. The main findings of these studies and the watch-outs for interpretation are described below.

In a first study, Ball and Van Gelder (1966) exposed mice to gadolinium oxide (30 mg/m3) via inhalation for 20 to 120 days. No effects were observed on body weight or the haemotological parameters studied. A trend towards reduced life span was observed among exposed mice, partly due to increased mortality as a result of pneumonia, which was not observed in the respective control mice. However, the results seem to be confused by the occurrence of hepatitis and murine leukemia in control mice for the 80-day exposure group, and almost equal occurrence of pneumonia during the post-exposure period in both exposed and control mice. Consequently, it cannot be safely concluded that the observed effects on life span were (only) due to exposure to gadolinium oxide, as there might have been some issues with bacterial/viral contamination during the study, affecting the observed results. For this reason, the study is considered as disregarded study. Further, from 20 days of exposure onwards, macrophages with ingested gadolinium oxide were observed in the lungs. Gadolinium oxide was always observed in the tracheobronchial lymph nodes. Also, pulmonary calcification was observed in a variable percentage of mice exposed for different periods, however, pulmonary calcification was not associated with significant shortening of life spans. Since large amounts of dust were left in the lungs several months post-exposure, the authors concluded that the mouse has an inadequate clearing mechanism for this compound. When interpreting these results, it must be kept in mind that gadolinium oxide with a median particle size of 0.312 µm was used in the study, which is rather low compared to what is currently prescribed by OECD, and moreover not reflecting the typical particle size of the products on the EU market. Also, the exposure concentration of 30 mg/m3 is not considered representative because it is much higher than the current potential exposure conditions at the working places. The exposure concentration and low particle size may indeed have resulted in lung overload conditions for the exposed mice, explaining the observed accumulation of dust in the lungs due to inadequate clearing.

In addition, two studies were identified which can be considered as supporting studies. The first study (Talbot et al., 1965) concluded that gadolinium oxide is relatively inert and non-toxic because it elicits no acute inflammatory response or any chronic fibrosis in mice after 20 up to 120 days of exposure by inhalation to gadolinium oxide dust (30 mg/m3). The only response seems to be the physiological mechanism present in the lungs for engulfment of dust particles. The lung parenchyma responds only by swelling of the epithelial cells and engulfment of the dust by macrophages. The macrophages were observed to translocate the dust to the tracheobronchial lymph nodes, after 100 days of exposure or more. No lesions were seen in organs other than the lungs and tracheobronchial lymph nodes. It is however not specified which other organs were studied. It is assumed that gadolinium was not soluble enough to be absorbed into the blood stream at amounts high enough to affect other organs histologically. The blood parameters studied showed no difference between exposed and control animals. In the second study (Abel and Talbot, 1965, also described in Abel and Talbot, 1967), guinea pigs were exposed via inhalation to gadolinium oxide (20 mg/m3) for 40, 80 or 120 days. After each exposure period, the lungs of exposed and control animals were removed from the thoracic cavity to assess pulmonary function (elastance) followed by histological examination. Changes in the lungs (e.g., alveolar cell hypertrophy, septal wall thickening, lymphoid hyperplasia and macrophage proliferation) were observed in exposed animals, the severity of the lesions increasing with exposure time, whereas no changes were observed in the lungs from control animals. Also, lungs exposed to gadolinium oxide appeared to have increased elastance (decreased compliance) compared to lungs from unexposed control guinea pigs. This effect was also related linearly to length of exposure. Moreover, the sequential change in histologic appearance as exposure time was increased correlated with the conclusions from the statistical analysis of the slopes of the elastance curves. Thickness of the alveolar walls and numbers of lymphocytic elements increased with greater exposure time as did the slopes of the elastance curves. Lung fibrosis was not observed. Here too, when interpreting the results of these two studies, it should be kept in mind that a very low particle size was used (Talbot et al. (1965): 70% of the particles (by mass) was smaller than 1 µm and mean particle size was 0.22 µm; Abel and Talbot (1965, 1967): 92% of the particles (by mass) was smaller than 0.563 µm and mean particle size was 0.22 µm), which is rather low compared to current prescriptions by OECD and cannot be considered representative for the products currently on the EU market. Further, the exposure concentrations of 20-30 mg/m3 are also not representative because they are much higher than the current potential exposure conditions at the working places. Altogether, the results of these studies suggest that the animals were most likely exposed under lung overload conditions, explaining the histological observations made in the lungs of exposed animals (and, in Abel and Talbot (1965, 1967), consequent effects on pulmonary function). Further, both studies did not report on any mortalities or other evidence of systemic toxicity, limiting their use for endpoint coverage. Therefore, these studies only yield supporting information. The facts that only loco-regional effects were observed, which seem to be related to the lung overload conditions of exposure, and no fibrosis was observed in both studies, are line with the expectation that gadolinium oxide does not induce systemic effects after repeated exposure via inhalation.

 

Repeated dose toxicity - dermal

A key 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 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 1008 mg/kg bw/day, the test substance 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.