Germanium dioxide

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 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., 1994): In a guideline study, conducted to GLP, administration of rats to Germanium dioxide by inhalation 6h/d and 5d/wk for 4wk at doses of 0,16, 72, or 309 mg/m3, had adverse effects at the highest dose especially on growth, kidneys and liver. A NOAEL of 72mg/m3 was established.

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

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

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.

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

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

Dose / conc.:

16 mg/m³ air

Dose / conc.:

72 mg/m³ air

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

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.

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.

Dose descriptor:

NOAEL

Effect level:

72 mg/m³ air

Sex:

male/female

Basis for effect level:

other: effects on growth rate, kidney

Dose descriptor:

LOAEL

Effect level:

309 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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

none

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

72 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

Endpoint conclusion

Endpoint conclusion:

no study available

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 dioxide was evaluated in rats in a study performed to OECD guideline 412 (Arts et al 1994). Germanium dioxide was administered by inhalation 6h/d and 5d/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 to evaluate recovery, persistence or delayed occurrence of the toxic effects. At the end of the treatment period, changes were observed only in rats of the high concentration group especially on growth, kidneys and liver.

Growth rate was markedly inhibited during the exposure period in both sexes. No progression in growth during the recovery period was noticed in this group either in males or in females.

The effects on the kidneys were characterized by increases in relative kidney weight, in plasma urea levels, increased urinary volumes, decreased urinary densities and decreased urinary pH, and treatment-related

histopathological changes in the kidneys, consisting of basophilic tubules which are considered indicative of tubular degeneration and regeneration. Renal dysfunction was still present at the end of the recovery period since none of the affected parameters had returned to normal.

Although the study did not show liver enlargement or treatment related microscopical hepatic changes, clinical chemistry did reveal a number of changes that may be indicative of liver damage. These changes

included decreases in fasting blood glucose and total plasma protein, and increases in plasma ASAT and ALAT activities and plasma bilirubin levels in the high-concentration group in females at the end of the exposure period. Plasma protein was also decreased in males of this group. All changes indicative of liver damage, except for the decreased blood glucose level had disappeared at the end of the recovery period.

Justification for classification or non-classification

Repeated dose toxicity: inhalation:

Based on the repeated dose inhalation study (Arts et al., 1994) with LOAEL = 309 mg/m3, GeO₂can be categorized as a STOT RE cat 2 according to the EU CLP criteria (EU 1272/2008). A substance (dust/mist/fume) is classified as a STOT RE (inhalation) cat 2 if 60 < effect concentration < 600 mg/m3 (for a 28-day toxicity study)

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).