Digadolinium trioxide

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1965

Report date:

1965

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1967

Report date:

1967

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Digadolinium trioxide

EC Number:

235-060-9

EC Name:

Digadolinium trioxide

Cas Number:

12064-62-9

Molecular formula:

Gd2O3

IUPAC Name:

Digadolinium trioxide

Test material form:

solid: particulate/powder

Details on test material:

Source: Michigan Chemical Company, St. Louis, Michigan
Purity: 99.9%
Spectrographic analysis revealed the presence of no other rare earth. The only contaminant present was 100 ppm calcium.
Appearance: solid, finely pulverised powder with unknown particle size

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.

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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).

Applicant's summary and conclusion

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.