Germanium

Endpoint summary

Description of key information

Repeated dose oral toxicity:

Animal repeated dose oral toxicity studies after exposure to GeO2 are demonstrating nephrotoxicity and myopathy. The study reporting the most sensitive (most relevant) data point (Sanai et al., 1991) was used: LOAEL= 0.0375g/kg BW/d inducing systemic toxicity by weight loss, anemia, and hypo-proteinemia and renal dysfunction (indicated by the increase of blood urea nitrogen and the decrease of creatinine clearance). In this 24-week feeding study, a dose-dependent effect of GeO2 is demonstrated in rats.

The lowest LOAEL, which was reported by Yim et al., 1999 (0.01g/kg BW/d), was not retained for further assessment since this effect concentration was solely based on the subcellular effects mitochondrial myopathia.

Repeated dose inhalation toxicity:

28d repeated dose inhalation (Arts et al., 1990): In a guideline study, conducted to GLP, administration of rats to Germanium metal powder by inhalation 6h/d and 5d/wk for 4wk at doses of 0, 9.9, 65.1, or 251.4 mg/m3. From this study local effects in the lungs resulted in a NOAEC of 9.9 mg Ge/m3 and a LOAEC of 65.1 mg Ge/m3. For systemic effects, the concentration of 251.4 represents a NOAEC because the slight changes in creatinin levels (sensitive indicator of renal function) found at this concentration occurred only in one sex and was not dose-dependent. Histopathological changes in the kidney did not occur in this study even at higher concentrations.

Repeated dose dermal toxicity: No repeated dose toxicity study by the dermal route were identified. Testing by the dermal route has been waived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

see section 13 in IUCLID for read-across justification report

Principles of method if other than guideline:

In a 40 weeks pair-feeding study the dose dependency of GeO2 induced nephrotoxicity was investigated experimentally in rat groups orally treated for 24 weeks with high, moderate or low doses of GeO2 and in an untreated group.
It is not according to OECD guideline 408 for which the normal standard exposure period is of 13weeks

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Source of test material: Sumitomo Metal Mining Co. Japan

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kyushu University Animal Center, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 140-170g
- Fasting period before study:no information
- Housing: no information
- Diet (e.g. ad libitum): the amount of food administered was adjusted to the level of the group with minimal ingestion
- Water (e.g. ad libitum): no information
- Acclimation period:no information

ENVIRONMENTAL CONDITIONS
- Temperature (°C):no information
- Humidity (%):no information
- Air changes (per hr):no information
- Photoperiod (hrs dark / hrs light):no information

Route of administration:

oral: feed

Details on route of administration:

feeding study: GeO2 was mixed with powdered regular chow

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

daily

Frequency of treatment:

24 weeks

Dose / conc.:

37.5 other: mg/kg/day

Dose / conc.:

75 other: mg/kg/day

Dose / conc.:

150 other: mg/kg/day

No. of animals per sex per dose:

37.5 mg/kg/day: n=4
75 mg/kg/day : n=6
150 mg/kg/day : n=5
control group: n=8

Control animals:

yes, concurrent no treatment

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no information

BODY WEIGHT: Yes
- Time schedule for examinations: every 4 weeks in all the dose groups

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
In order to maintain an equal calorie and protein intake, the amount of food administered was adjusted to the level of the group with minimal ingestion

HAEMATOLOGY: Yes
hematocrit and blood urea nitrogen (BUN) were examined every 4 weeks in all the groups and also at week 10 in the high dose and control groups


URINALYSIS: Yes
24h urinary protein excretion, creatinin clearance (Ccr), serum total protein, albumin and urinalysis were examined at an early stage (weeks 5-16), an intermediate stage (weeks 17-28) and a late stage (weeks 29-40) of the experiment

Sacrifice and pathology:

HISTOPATHOLOGY: Yes

Statistics:

Data are expressed as mean +/- SD. Statistical differences were calculated using the one-way analysis of variance among groups and the unpaired t test with Bonferroni's method

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

GeO2-H group: appeared inactive and listless after week 8, 1 rats died from azotemia at week 10

Mortality:

mortality observed, treatment-related

Description (incidence):

GeO2-H group: appeared inactive and listless after week 8, 1 rats died from azotemia at week 10

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

GeO2-H: BW (93±8g): significantly lower than control group ( 185±5g) at week 10 (p<0.001); GeO2-M: BW( 103±12g): sign lower than control group (177±5g) at week 24(p<0.001); GeO2-L: BW(171.4g) sign lower than control group at week 40 (205±6) (p<0.001)

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):

Hematocrit: GeO2-H: 38.8±2.6%): significantly lower than control group ( 46.3±2%) at week 10 (p<0.001); GeO2-M: slightly but sign reduced at wks 12 and 16and at week 36 in GeO2-L when compared to that in the control group (p<0.05 for both groups)

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

daily urinary protein excretion did not reveal any abnormalities in any of the groups. Urinary excretion and renal tissue content of Ge were significantly elevated in the GeO2-H

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

vacuolar degeneration and depositions of granules positive for periodic acid-Schiff in distal tubules were predominant in the higher dose group of GeO2

Histopathological findings: neoplastic:

not examined

Dose descriptor:

LOAEL

Effect level:

37.5 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: systemic toxicity and dose dependant renal dysfunction

Critical effects observed:

not specified

none

Conclusions:

GeO2 induced nephrotoxicity develops dose dependently

Executive summary:

In a 40-week pair-feeding study, a a dose-dependent effect of GeO2 is demonstrated in rats. This study

also demonstrated that the higher the dose the shorter the exposure duration required to develop the adverse effects. A lowest observed adverse effect dose of 37.5 mg/kg body wt/day of GeO2 or 26 mg/kg body wt/day of Ge was established for decreased growth, anemia, renal dysfunction, and renal tubular degeneration accompanied with elevated urinary and renal germanium.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

37.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Several repeated dose oral toxicity studies on GeO2 are available, but the study of Sanai et al (1991) was used since reporting the most sensitive and most relevant data point.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

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

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

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

Dose / conc.:

9.9 mg/m³ air

Dose / conc.:

65.1 mg/m³ air

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

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.

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

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

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

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

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

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

none

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

251.4 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and of high quality (klimisch score=1).

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

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

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

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

Dose / conc.:

9.9 mg/m³ air

Dose / conc.:

65.1 mg/m³ air

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

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.

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

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

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

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

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

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

none

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

65.1 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and of high quality (klimisch score=1).

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

Mode of Action Analysis / Human Relevance Framework

No data identified

Additional information

The repeated dose inhalation toxicity of germanium metal powder was evaluated in rats in a study performed to OECD guideline 412 (Arts et al 1990).

Wistar rats (5 male and 1 female/group each) had a whole body exposure to 0; 9.9; 65.1 and 251.4 mg Ge/m3 at 6h/d, 5d/week for 4wks. Two additional groups of 5 rats per sex were exposed to 0 and 251.4 mg/m3 respectively, followed by 31d without exposure.

At the end of the treatment period, fasting 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.

Justification for classification or non-classification

Repeated dose toxicity: inhalation:

Based on the repeated dose inhalation study (Arts et al., 1990) with for systemic effects (kidney effects) a NOAEL = 251.4 mg/m3 and no LOAEL could be defined, a classification for STOT RE is not required according to the EU CLP criteria (EU 1272/2008).

Repeated dose toxicity: oral:

A substance is considered to be a STOT RE (oral) cat 2 if 10 < effect conc < 100mg/kg BW/d. However these references values refer to effects seen in a standard 90-day toxicity study. Similar to Haber’s rule for inhalation (effective dose is directly proportional to exposure concentration and the duration of exposure), the LOAEL (Sanai et al., 1991) based on 24w experiment (LOAEL = 37.5 mg/kg BW/d) was converted to the 13w standard (LOAEL = 69.4 mg/kg BW/d), resulting in a STOT RE cat 2 classification according to the EU CLP criteria (EU 1272/2008).

In a WoE approach the STOT RE2 (oral) classification is checked and justified starting from the repeated dose inhalation rat study of Arts et al, 1994 and applying route to route extrapolation to the oral route.

Arts et al, 1994 demonstrated for GeO2 a NOAEL = 72 mg/m3 and a LOAEL = 309 mg/m3.

Taking into account the respiratory volume of a rat being 0.2 l/min/rat, the daily respiratory volume = 0.2 l/min/rat x 60min/h x 6h/d = 72.0 l

NOAEL = 72 mg/m3 x 72.0 l = 5.184 mg/d

LOAEL = 309 mg/m3 x72.0 l= 22.248 mg/d

Taking into account the body weights of the mid (72 mg/m3) and high dose groups (309 mg/m3) for males being respectively 321.9g and 251.1 g and for females being respectively 186.5 and 150.1 g leads to the following calculated doses:

males:

NOAEL = 5.184 mg/d / 0.3219kg = 16.1 mg/kg/d

LOAEL= 22.248 mg/d /0.2511kg = 88.6 mg/kg/d

females:

NOAEL = 5.184 mg/d / 0.1865kg = 27.8 mg/kg/d

LOAEL= 22.248 mg/d /0.1501kg = 148.2 mg/kg/d

The calculated LOAEL (males and females) also triggers STOT RE cat 2 classification as the effect concentrations are < 300 mg/kg BW/d (for a 28 -days toxicity study).