Germanium

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 sub-acute inhalation toxicity of germanium metal powder in rats

Author:

Arts JHE, Reuzel PGJ, Falke HE and Beems RB

Year:

1990

Bibliographic source:

Food Chem Toxicol. Vol. 28, No. 8, pp. 571-579

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1988

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

EC Number:

231-164-3

EC Name:

Germanium

Cas Number:

7440-56-4

Molecular formula:

Ge

IUPAC Name:

germanium

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): germanium powder
- Physical state: grey powder
- Analytical purity: 99.8%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF-reared, Wistar derived rats (strain code Bor:WISW) delivered by Winkelmann, Versuchstierzucht GmbH &
Co KG, Borchen, FR
- Age at study initiation: 5 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. 1.8 - ca. 2.4 µm

Remarks on MMAD:

MMAD / GSD: dose 9.9 mg/m3; MMAD: 1.8 , GSD: 1.7
dose 65.1 mg/m3; MMAD: 2.4, GSD: 1.8
dose 251.4 mg/m3; MMAD: 1.8, GSD: 1.8

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- System of generating particulates/aerosols: An aerosol was generated by delivering appropriate quantities of the test material by an AccuRate dry material feeder (series 300, AccuRate, Whitewater, WI, USA) to an atomizer (Institute's design). Next, the aerosol was passed through a cyclone (Institute's design) for separating the larger particles from the aerosol. The aerosol was subsequently diluted with clean air before entering the inhalation chamber.
- Particle size distribution: Mean particle size was between 2.0 and 2.4 µm determined with an 11-stage cascade impactor.
-Exposure chambers. The modified H 1000 multi-tiered inhalation chambers for the sub-acute study (Hazleton Systems Inc., Aberdeen, MD, USA) were constructed of stainless-steel with glass doors on two sides, which allowed observation of the animals during exposure. The normal capacity of the chambers was reduced to an effective exposure volume of about 1 m 3. The rats were housed in a cage unit consisting of 24 individual cages. The chambers were operated at a negative pressure of l~J, mm H20 to prevent leakage of the test material. Ports in the walls allowed sampling of the test atmosphere. During exposure the rats were housed at 18.0 + 0.1 C and at a relative humidity of 55 + 2% . The flow-rate through the chambers was between 25 and 35 m3/hr.
-- Method of holding animals in test chamber: rats were housed in an animal room under conventional conditions, five per cage, separated according to sex, in stainless-steel cages with wiremesh
floors and front
- Temperature, humidity, pressure in air chamber: 22 + 3"C and at a relative humidity of 30-70%, A 12-hr light/dark cycle was
maintained
- Air change rate: changed about ten times per hour.


TEST ATMOSPHERE
- Analytical method used: The actual mass concentration of germanium 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.:

9.9 mg/m³ air

Doses / concentrations 2

Dose / conc.:

65.1 mg/m³ air

Doses / concentrations 3

Dose / conc.:

251.4 mg/m³ air

No. of animals per sex per dose:

The control group and top-concentration group consisted of 10 male and 10 female rats each, the other two groups of 5 males and 5 females each.
The control and the high-concentration group were divided into a main group and a satellite group of 5 males and 5 females each.

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 liver, kidneys, nose, trachea and larynx of all control rats and rats exposed to the high concentration and on the lungs of all animals of the main groups and of the recovery groups
BODY WEIGHT: Yes
HAEMATOLOGY: haematological and biochemical variables were measured in rats in the main groups after 28-30 days, and in rats in the recovery groups after a further 26-31 days of observation. The haematological variables determined at the end of the
exposure period were haemoglobin concentration, packed cell volume, erythrocyte count, and total and differential leucocyte counts. Total and differential leucocyte counts were also determined in rats of the recovery groups.
CLINICAL CHEMISTRY: haematological and biochemical variables were measured in rats in the main groups after 28-30 days, and in rats in the recovery groups after a further 26-31 days of observation.

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 using a Cobas-Bio centrifugal analyser in plasma obtained from heparinized blood samples at the end of the exposure period: albumin, alkaline phosphatase, total bilirubin, calcium, chloride, creatinine*,
;,-glutamyltransferase, glucose*, aspartate aminotransferase (ASAT)*, alanine aminotransferase (ALAT)*, inorganic phosphate, potassium, sodium, total protein and urea*. The parameters marked with an asterisk were also measured in rats in the recovery
groups.
URINALYSIS: Urinalysis was carried out in rats of the main groups at day 28. Volume and density were determined, and protein, glucose, occult blood and ketones were measured using test strips (Boehringer, Mannheim, FRG). The sediment in pooled samples of each group was examined microscopically. Volume and density were also determined at day 26 of the recovery period.

Sacrifice and pathology:

pathology: At the end of the exposure period (day 30) or the recovery period (day 61) in the sub-acute study, the rats were killed by exsanguination from the abdominal aorta under ether anaesthesia. They were autopsied and examined for gross pathological changes. From rats in the subacute study the adrenals, heart, kidneys, liver, spleen, testes, thyroid and lungs with trachea and larynx were weighed. Tissue samples of these organs and also 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 #m and stained with haematoxylin and eosin. Full microscopic examination was carried out on the liver, kidneys, nose, trachea and larynx of all control rats and rats exposed to the high concentration and on the lungs of all animals of the main groups and 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, body weight or mortality

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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):

At the end of the exposure period, haematological variables were similar in all groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In females, blood urea was significantly increased in the high-concentration group and creatinine levels were significantly increased in the mid- and high-concentration groups in comparison with the controls. Males of the high-concentration group showed a significantly decreased fasting blood glucose level and relatively high ASAT and ALAT levels . At the end of the recovery period, there were no treatment-related differences between controls and rats of the high-concentration group, since the decreased ASAT level found in females of the high-concentration group was considered to be the consequence of a very high level in female controls and, therefore, was not considered to be of toxicological relevance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In females, urine volume and density showed slight, but significant, changes at the end of the exposure period. Urine volume reached a significantly increased level in the high-concentration group, and urine density was significantly decreased in the midconcentration group. The finding of a significantly decreased urine density in males of the low-concentration group was considered to be fortuitous, since other parameters of kidney function were unaffected in these rats and this change was limited to this group only. At the end of the recovery period, urine volume was significantly increased in males of the high-concentration group.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were distinctly concentration-related increases in absolute and relative lung weights in rats of the mid- and high-concentration groups at the end of the exposure period. At the end of the recovery period, lung weights were still significantly increased in rats of the highconcentration group, although the difference was less pronounced than at the end of the exposure period

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross examination at autopsy at the end of the exposure period revealed a greyish fur and/or tail in rats of the high-concentration group, and to a lesser extent in rats of the mid-concentration group. The mediastinai lymph nodes were enlarged and/or greyish in several females of the highconcentration group and in a single female of the mid-concentration group. Greyish lungs were observed in all treatment groups. The greyish discoloration was still seen in females of the high--
concentration recovery group, albeit to a lesser degree.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed: accumulation of particulate material in the lungs of all treated groups, accumulation of alveolar macrophages in the mid- and high-concentration groups, and alveolitis mainly in the high-concentration group.
After the 4-wk recovery period, in exposed rats of both sexes, lung weights were still increased and histopathological changes were present, but to a lesser extent than at the end of the exposure period

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

9.9 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

2

Dose descriptor:

LOAEL

Effect level:

65.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

3

Dose descriptor:

NOAEL

Effect level:

254.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

urinalysis

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

65.1 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

65.1 mg/m³ air

System:

urinary

Organ:

kidney

Treatment related:

no

Dose response relationship:

no

Relevant for humans:

no

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

1) Kidney effects:
slight changes in the creatinine content of the blood plasma and a reduced specific gravity of the urine were observed at 65.1 mg / m³. The creatinine content is considered as a sensitive indicator of renal function and the kidney is a major target organ of the toxic effect of germanium, however these slight changes at 65.1 mg/m3 occurred only in one sex and not dose-dependently and were only marginal. Histopathological changes in the kidney did not occur in this study even at higher concentrations. For systemic effects, the concentration of 251.4 represents a NOAEC

2) Lung effects: concerning effects on the lung: the LOAEC for histopathological lung effects is 65.1 mg / m³ and the NOAEC is 9.9 mg / m³.

In the similar study with germanium dioxide (Arts et al., 1994) no histopathological changes were found in the lungs at 309 mg / m³, apart from the relative weight gain). The two studies on germanium powder and germanium dioxide show even greater differences in effect concentrations (germanium powder histopathological effects at 65.1 mg / m³, germanium dioxide no histopathological findings, only weight gain at 309 mg / m³). There is also no supportive information that the endpoint (lung) has human relevance in the low-concentration range (Swennen et al., 2000 -Epidemiological survey of workers exposed to inorganic germanium compounds).

Executive summary:

A study was conducted to determine the effects of sub-acute exposure of the test material on the respiratory system in Wistar rats.

Four groups of five male and five female rats were exposed to 0, 9.9, o5.1 or 251.4 mg/m 3 for 6 hr/day, 5 days/wk for 30 days. Two additional (recovery) groups of five male and five female rats exposed

to 0 or 251.4 mg/m 3 were kept untreated for 31 days after exposure. At the end of the treatment period, fasling blood glucose was decreased in males exposed to the high concentration. In females of this group,

blood creatinine and urea levels, and urine volumes were increased, but urine density was decreased. Increased blood creatinine levels and urine volume and decreased urine density were also observed in

females exposed to 65.1 mg/m 3. The absolute and relative lung weights were increased in rats in the mid and high-concentration groups. Histopathological examination revealed: accumulation of particulate

material in the lungs of all treated groups, accumulation of alveolar macrophages in the mid- and high-concentration groups, and alveolitis mainly in the high-concentration group. After the 4-wk recovery

period, urine volume was increased in males that had been exposed to germanium. In exposed rats of both sexes, lung weights were still increased and histopathological changes were present, but to a lesser extent than at the end of the exposure period. It was concluded by Arts et al 190, that the no-adverse-effect level in the 4-wk study was 9.9 mg/m 3 air.