Germanium dioxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Acute and subacute inhalation toxicity of germanium dioxide in rats.

Author:

Arts JH, Til HP, Kuper CF, de Neve R and Swennen B

Year:

1994

Bibliographic source:

Food Chem Toxicol. 32(11):1037-1046

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Germanium dioxide

EC Number:

215-180-8

EC Name:

Germanium dioxide

Cas Number:

1310-53-8

Molecular formula:

GeO2

IUPAC Name:

Germanium dioxide

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): germanium dioxide (GeO2)
- Physical state: hexagonal and amorphous quality, white powder
- Analytical purity: 99% or more
- Impurities (identity and concentrations): hexagonal: total metal impurity concentration less than 10ppm; Cl<500ppm; amorphous: NH3, Cl<0.2%, Fe<5ppm, Cu, Ni, Cr, Mn, Co<1ppm

Specific details on test material used for the study:

only hexagonal form was tested in the RDT study. In the acute tox test both hexagonal and amorphous form was tested.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan CPB (Austerlitz, the Netherlands)
- Age at study initiation: 10 weeks
- Weight at study initiation: mean body weight: M: 197g, F: 138g
- Housing: individually in wire-mesh stainless steel cages
- Diet : cereal based Institute's stock diet ad libitum
- Water : ad libitum
- Acclimation period: acclimatized to the laboratory conditions in the inhalation facilities until the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Mass median aerodynamic diameter (MMAD):

ca. 1.2 - ca. 2 µm

Remarks on MMAD:

MMAD / GSD: dose 16 mg/m3 : MMAD: 1.2 µm, GSD: 1.9µm
dose 72mg/m3: MMAD: 1.6 µm, GSD: 1.8µm
dose 309mg/m3: MMAD: 2.0 µm, GSD: 1.7µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: equipment according to the ASHRAE standard
- System of generating particulates/aerosols: aerosol was generated by dispersing the test material into the air using equipment according to the ASHRAE standard. Two ASHRAE trays were used, one for generation of the high concentration, the other one for the mid concentration test atmosphere. Both trays were placed in a hood. The aerosols were subsequently diluted with clean air before entering the inhalation chamber. The low concentration test atmosphere was trapped from the inlet tube of the high concentration chamber, using an air mover and dilution before entering the low concentration chamber.
- Particle size distribution: determined with an I l-stage cascade impactor (Institute's design)
- Temperature, humidity, pressure in air chamber: 21-22 C, mean relative humidity of 42-56%, flow rate through chambers was between 17 and 34m3/hr

TEST ATMOSPHERE
- Analytical method used: The actual mass concentration of germanium dioxide in the test atmosphere was determined by gravimetry.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

none

Duration of treatment / exposure:

4 wk

Frequency of treatment:

6h/day, 5day/wk for 4 wk

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

16 mg/m³ air

Doses / concentrations 2

Dose / conc.:

72 mg/m³ air

Doses / concentrations 3

Dose / conc.:

309 mg/m³ air

No. of animals per sex per dose:

5 rats of each sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

none

Positive control:

None

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
-pathology: adrenals, heart, kidneys, liver, spleen, testes, thyroid and lungs with trachea and larynx were weighed. Tissue samples of these organs and of the nose were preserved in a 4% aqueous, neutral phosphate buffered formaldehyde solution. After fixation, the noses were decalcified in nitric acid. Organs and tissues were embedded in paraffin wax. sectioned at 5 ltm and stained with haematoxylin and eosin. Kidneys were also stained with periodic acid Schiff reagent. Full microscopic examination was carried out on the adrenals, heart, spleen, thyroid, liver, kidneys, nose, lungs and trachea and larynx of all control and high concentration rats, on the kidneys of all animals of the intermediate groups, and on the kidneys and lungs of all animals of the recovery groups.
BODY WEIGHT: Yes
HAEMATOLOGY: haematological variables were measured in all fasted rats towards the end of treatment (day 23) and after a further 28 days of observation in fasted rats of the recovery groups. The variables included haemoglobin concentration, packed cell volume (haematocrit), erythrocyte and thrombocyte count, prothrombin time, and total and differential leucocyte counts.
CLINICAL CHEMISTRY: Biochemical variables were measured in rats of the main groups at the end of treatment (day 28) and in recovery rats after another 33 days of observation.
The following biochemical variables were measured in plasma obtained from heparinized blood samples at the end of the exposure period (Cobas-Bio centrifugal analyser): albumin, alkaline phosphatase, total bilirubin, calcium, chloride, creatininc, 7-glutamyltransferase, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), inorganic phosphate, potassium, sodium, total protein and urea. Fasting blood glucose was determined towards the end of treatment (day 23) and after a further 28 days of observation in rats fasted for 16 hr and deprived of water for 24 hr. Albumin, total bilirubin, creatinine, ASAT, ALAT, total protein and urea were also measured in rats of the recovery groups.
URINALYSIS: Urinalysis was carried out in all rats on day 23 and in rats of the recovery groups after another 28 days of observation. Rats were deprived
of water for 24 hr and of food during the last 16 hr of this period. Urine was collected during the last 16 hr of the deprivation period. Analyses included protein, glucose, occult blood, ketones, urobilinogen, bilirubin and pH (using test strips: Boehringer), and urinary volume and density. The sediment was also examined microscopically in pooled samples of recovery rats

Sacrifice and pathology:

pathology: adrenals, heart, kidneys, liver, spleen, testes, thyroid and lungs with trachea and larynx were weighed. Tissue samples of these organs and of the nose were preserved in a 4% aqueous, neutral phosphate buffered formaldehyde solution. After fixation, the noses were decalcified in nitric acid. Organs and tissues were embedded in paraffin wax. sectioned at 5 ltm and stained with haematoxylin and eosin. Kidneys were also stained with periodic acid Schiff reagent. Full microscopic examination was carried out on the adrenals, heart, spleen, thyroid, liver, kidneys, nose, lungs and trachea and larynx of all control and high concentration rats, on the kidneys of all animals of the intermediate groups, and on the kidneys and lungs of all animals of the recovery groups.

Statistics:

Body weight (during exposure period): one-way analysis of covariance using pre-exposure (day 0) weights as the covariate; if group means were
significantly different (P < 0.05), individual pairwise comparisons were made using Dunnett's multiple comparison tests.
Body weights (during the recovery period): two-sample t-test.
Organ weights, and haematological, urinalytical and clinicochemical data (obtained during the exposure period): analysed for each sex by one-way analysis of
variance (ANOVA). If significant differences among the means were indicated (P < 0.05), Dunnett's test was performed to determine which exposed groups
differed from the control.
Two-sample t-tests were applied to data obtained during the recovery period instead. In case of group mean differences (P < 0.05), pairwise comparisons between control and exposed groups were determined by Mann-Whitney U-tests. Mann-Whitney U-tests were applied during the recovery period instead. Incidences of histopathological changes were analysed by Fisher's exact probability test. All pairwise comparisons were two tailed. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no exposure-related changes in condition, health, behaviour or mortality

Mortality:

no mortality observed

Description (incidence):

no exposure-related changes in condition, health, behaviour or mortality

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were significantly lower in rats of the high concentration group during almost the entire study, recovery period included

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:

effects observed, treatment-related

Description (incidence and severity):

decreases in haematocrit (females) and thrombocyte count (both sexes), and increases in neutrophil count (both sexes) and white blood cell count (females)

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary volume was elevated, and urine density and pH were lowered in both sexes.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative weights of kidneys, spleen, heart and lungs were higher than in controls.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

At the end of the treatment period , changes were observed only in rats of the high concentration group
CLINICAL SIGNS AND MORTALITY: no exposure-related changes in condition, health, behaviour or mortality

BODY WEIGHT AND WEIGHT GAIN: decreased body weight

HAEMATOLOGY: decreases in haematocrit (females) and thrombocyte count (both sexes), and increases in neutrophil count (both sexes) and white blood cell count (females)

CLINICAL CHEMISTRY: decreased fasting blood glucose (females), decreased total protein concentration (both sexes),
increased plasma alanine aminotransferase and aspartate aminotransferase activities (females). increased plasma urea nitrogen (males) and increased plasma bilirubin level (females) were observed

URINALYSIS: urinary volume was elevated, and urine density and pH were lowered in both sexes.

ORGAN WEIGHTS: Relative weights of kidneys, spleen, heart and lungs were higher than in controls.

HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopical examination revealed treatment-related changes in the kidneys, consisting of an increased occurrence of proximal as well as distal basophilic tubules in high concentration rats of both sexes, in addition, tubules with hypertrophic
epithelial cells were observed. Increased occurrence of proximal basophilic and tubules with hypertrophic cells concomitant with distal tubular and collecting tubular vacuolation of epithelial cells was still present in the tubular cells of exposed rats at the end of the recovery period. PAS-positive granules were present both at the end of treatment at 309 mg/m3 and after recovery; the granules were larger at the end of the recovery period than just after treatment. No treatment-related histopathological changes were observed in the adrenals, heart, nasal cavity, larynx, trachea, lungs, liver, spleen and thyroid.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

72 mg/m³ air

Sex:

male/female

Basis for effect level:

other: effects on growth rate, kidney

2

Dose descriptor:

LOAEL

Effect level:

309 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

309 mg/m³ air

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Considering the observed effects on growth, kidneys in rats exposed to 309 mg/m3, it is concluded that the no-toxic-effect level in the present study is 72 mg/m3

Executive summary:

A study was conducted to determine the effects of short term exposure of the test material on the respiratory system in Wistar rats.

The test material was administered by inhalation 6 h /d and 5 d/wk for 4wk at 0, 16, 72, or 309 mg/m3 to groups of 5 rats/sex/dose. Two additional groups of 5 rats per sex, exposed either to 0 or to 309 mg/m3. were kept for a 33,.day post-exposure period. At the end of the treatment period, changes were observed only in rats of the high concentration group: these changes were decreased body weight gain (both sexes), decreases in haematocrit (females) and thrombocyte count (both sexes), and increases in neutrophil count (both sexes) and white blood cell count (females). On clinical chemistry evaluation, decreased fasting blood glucose (females), decreased total protein concentration (both sexes), increased plasma alanine aminotransferase and aspartate aminotransferase activities (females). increased plasma urea nitrogen (males) and increased plasma bilirubin level (females) were observed. In addition, urinary volume was elevated, and urine density and pH were lowered in both sexes. Relative weights of kidneys, spleen, heart and lungs were higher than in controls. Microscopic examination revealed effects on renal tubular epithelium. Effects on growth, kidneys, and liver were still present at the end of the 33-day recovery period.

It was concluded that the no-adverse-effect-level in the 4-wk study using hexagonal germanium dioxide was 72 mg/m3.