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EC number: 215-180-8 | CAS number: 1310-53-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
Skin irritation/corrosion:
The in vitro skin irritation test in the EPISKIN model (OECD 439) with germanium dioxide (Orovecz, CiToxLAB Hungary 2017) demonstrate a lack of irritation potential
Eye irritation:
Based on the in vitro eye irritation assay in the isolated chicken eyes test (OECD 438) (Orovecz, CiToxLAB Hungary 2017) , germanium dioxide is not classified as a severe irritant and not classified as non-irritant. Further histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed slight erosion of the corneal epithelium in 5/6 sections and very slight severity in one case. At the top epithelial layer, necrosis with very slight degree in 3/6 sections, moderate in 1/6 section and severe in 2/6 sections was seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes.
Based on the in vitro eye irritation assay, Reconstructed Human Cornea-like Epithelium (RhCE) Test method (OECD 492) (Richez, CiToxLAB France 2017), germanium dioxide is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- version 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- skin obtained from plastic surgery from multiple donors
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (Manufacturer: SkinEthic, France
- Tissue batch number(s):16-EKIN-023
- Expiry Date: 13 June 2016
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25.1-26.8°C
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1wahsing step: rinsing thoroughly with PBS 1x solution (0.9%)
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570nm
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (see any other info on mat and meth) - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 10mg
VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required
NEGATIVE CONTROL: PBS
- Concentration (if solution): 50µl
POSITIVE CONTROL: SDS 5%
- Concentration (if solution): 50µl - Duration of treatment / exposure:
- 15 minutes (± 0.5 min)
- Duration of post-treatment incubation (if applicable):
- 42h
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 83.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this in vitro skin irritation test in the EPISKIN model with germanium dioxide the results indicated that the test item is Non Irritant
- Executive summary:
Disks of EPISKINTM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution atin an incubator with 5% CO2protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.
PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units /control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.
Following exposure with GERMANIUM DIOXIDE, the mean cell viability was 83.3% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN model test with GERMANIUM DIOXIDE(Batch number: 968), the results indicate that the test item is non-irritant to skin.
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Data waiving:
- other justification
- Justification for data waiving:
- other:
Referenceopen allclose all
The results of the optical density (OD) measured at 570 nm of each sample and the
calculated relative viability % values are presented below:
Table: Optical Density mean (OD) and the calculated relative viability % of the samples
Substance |
Optical Density (OD) |
Viability (% RV) |
|||
|
Measured |
Blank corrected |
|||
Negative Control: |
1 |
0.697 |
0.649 |
90.1 |
|
Phosphate buffered saline |
2 |
0.792 |
0.744 |
103.4 |
|
|
3 |
0.815 |
0.767 |
106.5 |
|
|
mean |
-- |
0.720 |
100.0 |
|
Positive Control: |
1 |
0.082 |
0.034 |
4.8 |
|
5% (w/v) SDS solution |
|
2 |
0.096 |
0.048 |
6.7 |
|
3 |
0.082 |
0.034 |
4.8 |
|
|
mean |
-- |
0.039 |
5.4 |
|
Test Item: |
1 |
0.691 |
0.643 |
89.4 |
|
GERMANIUM DIOXIDE |
2 |
0.612 |
0.564 |
78.3 |
|
|
3 |
0.639 |
0.591 |
82.1 |
|
|
mean |
-- |
0.600 |
83.3 |
Notes:
1. Mean blank value was 0.048.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three
decimal places).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- version 26th July 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied: 30mg - Duration of treatment / exposure:
- 10 seconds
- Duration of post- treatment incubation (in vitro):
- up to 240 minutes (at 30, 75, 120, 180 and 240 min) after post-treatment rinse for the cornea thickness and cornea opacity
Fluorescein retention was measured at base line (t=0) and at 30 minutes after rinse. - Number of animals or in vitro replicates:
- 3 test item treated eyes, 3 positive control treated eyes and 1 negative control eye
- Details on study design:
- SELECTION AND PREPARATION OF ISOLATED EYES
Eyes selection:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes:
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period (45-60min), a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No corneal thickness changes (0.0 %) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.
NUMBER OF REPLICATES
3 test item treated eyes, 3 positive control treated eyes and 1 negative control eye
NEGATIVE CONTROL USED
treated with 30µL isotonic saline
SOLVENT CONTROL USED (if applicable): not applicable
POSITIVE CONTROL USED
treated with 30 mg imidazole
APPLICATION DOSE AND EXPOSURE TIME
test substance treated chicken eye: treated with 30 mg germanium dioxide during 10 seconds
OBSERVATION PERIOD
30, 75, 120, 180 and 240 min after post-treatment. The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.
REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: cornea surface was rinsed thoroughly with 20ml isotonic saline
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
- Swelling: Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
- Macroscopic morphological damage to the surface: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Histopathology: Semi-quantitative microscopic evaluation was performed on the cornea in the ICET. The classification of histopathology findings was performed based on two publications, M.K. Prinsen et al./Toxicology in Vitro 25 (2011), 1475-1479), Elodie Cazelle et al./ Toxicology in Vitro 28 (2014), 657-666 and Atlas of histopathological lesions of Isolated Chicken Eyes, M.V.W. Wijnands and M.K. Prinsen, June 2015.
SCORING SYSTEM: see below other info on mat and meth
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment
- Histopathology
DECISION CRITERIA: as indicated in the TG - Irritation parameter:
- percent corneal swelling
- Run / experiment:
- up to 75 minutes
- Value:
- 1.7
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- percent corneal swelling
- Run / experiment:
- up to 240 minutes
- Value:
- 2.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- cornea opacity score
- Value:
- 1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- fluorescein retention score
- Value:
- 1.33
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- histopathological observations
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Remarks:
- Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.
- Conclusions:
- Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.
- Executive summary:
An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).
After the zero reference measurements, the eye was held in horizontal position and 30 mg of test itemwas applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline.
The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline(0.9% (w/v) NaCl solution).
Based on this in vitro eye irritation assay in the isolated chicken eyes test with GERMANIUM DIOXIDE, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.
To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed slight erosion of the corneal epithelium in 5/6 sections and very slight severity in one case. At the top epithelial layer, necrosis with very slight degree in 3/6 sections, moderate in 1/6 section and severe in 2/6 sections was seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, GERMANIUM DIOXIDE was classified as Category 1 (classification of UN GHS) according to criteria published by Prinsen et al in 2011 and Cazelle et al in 2014.
Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- GLP compliance:
- yes (incl. QA statement)
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)
POSITIVE and NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µl - Duration of treatment / exposure:
- 6h
- Duration of post- treatment incubation (in vitro):
- 18h
- Number of animals or in vitro replicates:
- 4 replicates
- Details on study design:
- - Details of the test procedure used: The design of this study was based on the protocol published by MatTek Corporation: "EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals" (adopted version, June 2015) following OECD test guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage (adopted 28 July 2015).
- RhCE tissue construct used, including batch number: The EpiOcular tissue construct is a non-keratinized epithelium composed of stratified normal human keratinocytes in a three-dimensional structure. It models the cornea epithelium with progressively stratified, but not cornified cells. supplier MatTek, Bratislava, Slovak Republic
the EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
Expiry date: the EpiOcular tissues were used within 72 hours of their production.
- Doses of test chemical and control substances used:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)
POSITIVE and NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µl
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a humidified incubator.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable as test item was found in the preliminary test not to have any colouring potential and any direct MTT reducing properties, no additional controls were run during the main test
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The OD was measured at 570 nm using a plate reader.
- Description of the method used to quantify MTT formazan: The cell viability was then assessed by means of the colourimetric MTT reduction assay: Cell viability determination is based on cellular mitochondrial succinate dehydrogenase activity measured (within the mitochondria of viable cells) by the reduction and the conversion of a yellow dye, MTT [3 (4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], into a blue formazan salt. The formazan precipitate is then extracted using isopropanol and quantified by spectrophotometry. For each test item, the mean Optical Density of two treated tissues is determined and expressed as a relative percentage of viability of the negative control.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The results of the study were considered acceptable if the following criteria are fully met:
the mean cOD of the negative controls is between 0.8 and 2.5,
relative mean viability of the positive control is < 50% of the relative mean viability of the negative control,
the difference of viability between the two tissue replicate is < 20%.
- Acceptable variability between tissue replicates for positive and negative controls: All acceptance criteria for the negative and positive controls were fulfilled.
- Acceptable variability between tissue replicates for the test chemical: yes - Irritation parameter:
- other: % tissue viability
- Run / experiment:
- 1
- Value:
- 97
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the experimental conditions of this study, the test item, GERMANIUM DIOXIDE, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be:
No Category (GHS 2015 and Regulation (EC) No. 1272/2008). - Executive summary:
The purpose of this study was to predict the acute eye irritation potential of the test item,GERMANIUM DIOXIDE, by measurement of its cytotoxic effect on the EpiOcularTMcornea epithelial model.
The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.
Methods
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.
Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
Results
Preliminary tests
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.
Main test
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
The relative mean viability of the tissues treated with the test item was 97% with a difference of 5% between duplicate tissues.As the mean viability was > 60% after the MTT reduction,the results met the criteria for a non-irritant response.
Conclusion
Under the experimental conditions of this study, the test item,GERMANIUM DIOXIDE, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
According to the results of this study, the classification of the test item should be: No Category (GHS 2015 and Regulation (EC) No. 1272/2008).
Referenceopen allclose all
The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.
Observation |
Value |
ICE Class |
Mean maximum corneal swelling at up to 75 min |
1.7 % |
I |
Mean maximum corneal swelling at up to 240 min |
2.2 % |
I |
Mean maximum corneal opacity |
1.00 |
II |
Mean fluorescein retention |
1.33 |
II |
Other Observations |
None |
|
Overall ICE Class |
1xI 2xII |
Based on this in vitro eye irritation in the isolated chicken eyes test with GERMANIUM DIOXIDE,the test item is not classified as a severe irritant and not classified as non-irritant.
Positive Control
Observation |
Value |
ICE Class |
Mean maximum corneal swelling at up to 75 min |
8.5 % |
II |
Mean maximum corneal swelling at up to 240 min |
25.5 % |
III |
Mean maximum corneal opacity |
4.00 |
IV |
Mean fluorescein retention |
3.00 |
IV |
Other Observations |
Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. |
|
Overall ICE Class |
1xIII 2xIV |
The positive control Imidazolewas classified as severely irritating, UN GHS Classification: Category 1.
NEGATIVE Control
Observation |
Value |
ICE Class |
Mean maximum corneal swelling at up to 75 min |
0.0 % |
I |
Mean maximum corneal swelling at up to 240 min |
0.0 % |
I |
Mean maximum corneal opacity |
0.00 |
I |
Mean fluorescein retention |
0.00 |
I |
Other Observations |
None |
|
Overall ICE Class |
3xI |
The negative control Physiological salinewasclassifiedas non-irritating, UN GHS Classification:No Category.
Table:Assessment of the general IN VITRO eye irritancy and regulatory GHS classification.
UN GHS Classification |
Combinations of the three ICE Classes |
No Category |
3×I 2×I, 1×II |
No prediction can be made |
Other combinations |
Category 1
|
3×IV 2×IV, 1×III 2×IV, 1×II* 2×IV, 1×I* Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) Corneal opacity = 4 at any time point (in at least 2 eyes) Severe loosening of epithelium (in at least 1 eye) |
Remark:*:combinations of categories less likely to occur
Histopathology:
GERMANIUM DIOXIDE produced slight erosion of the corneal epithelium in 5/6 sections and very slight severity in one case. At the top epithelial layer, necrosis with very slight degree in 3/6 sections, moderate in 1/6 section and severe in 2/6 sections was seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, GERMANIUM DIOXIDE was classified as Category 1.
AISE ICE (Isolated Chicken Eye) method: Proposed histopathology criteria
Tissue layer |
Effects triggering serious eye damage (Eye Cat 1) identification |
Epithelium |
- erosion ≥ moderate in at least 2 out of 3 eyes - and/or, any vacuolation (≥ very slight) observed only in the mid and lower parts in at least 2 out of 3 eyes - or, if erosion ≥ moderate in 1 out of 3 eyes + vacuolation ≥ very slight (mid/low part) is observed in at least another eye out of the 3 eyes - and/or, necrosis ≥ moderate observed in at least 2 out of 3 eyes |
Stroma |
and/or, pyknotic nuclei ≥ slight in at least 2 out of 3 eyes |
Endothelium |
and/or, any effect observed in at least 2 out of 3 eyes |
1.1 PRELIMINARY TESTS
1.1.1 Test for direct MTT reduction with the test item
The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
1.1.2 Test for the detection of the colouring potential of the test item
During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.
1.2 MAIN TEST
1.2.1 Evaluation of the colouration of tissues at the end of the MTT incubation period
The qualitative evaluation of the MTT staining was performed with the naked eye
All test item-treated tissues appeared blue which was considered indicative for viable tissues.
1.2.2 Evaluation of the MTT results
The individual and mean OD values, standard deviations and viabilities for the test item, negative control and positive control tissues are presented in Table below
All of the acceptance criteria for the negative and positive controls were fulfilled (see table below), therefore the study was considered to be valid.
Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls
Group |
Exposureduration |
Tissue
No. |
OD570nmmeasurements |
Mean blank |
cOD570nmmeasurements |
MeancOD570nm |
Viability (%) |
||
1ol |
2'd |
1ot |
2'd |
||||||
Negative control |
6h |
1 |
1.875 |
1.872 |
0.040 |
1.835 |
1.832 |
1.833 |
103 |
2 |
1.774 |
1.776 |
1.734 |
1.736 |
1.735 |
97 |
|||
Positive control |
6h |
1 |
0.433 |
0.430 |
0.040 |
0.393 |
0.390 |
0.391 |
22 |
2 |
0.399 |
0.398 |
0.359 |
0.358 |
0.358 |
20 |
|||
Test item |
6h |
1 |
1.816 |
1.816 |
0.041 |
1.775 |
1.775 |
1.775 |
100 |
2 |
1.720 |
1.725 |
1.679 |
1.684 |
1.682 |
94 |
OD=optical density; cOD=blank corrected optical density
Mean tissue viability and standard deviations for the test item, the negative and positive controls
Group |
Exposureduration |
cOD570nm |
Viability(%) |
|||
Mean |
so |
Mean |
so |
Dfference(%) |
||
Negative control |
6h |
1.784 |
0.070 |
100 |
4 |
6 |
Positive control |
6h |
0.375 |
0.023 |
21 |
1 |
2 |
Test item |
6h |
1.729 |
0.066 |
97 |
4 |
5 |
cOD=blank corrected optical density
SO= standard deviation
The relative mean viability of the tissues treated with the test item was 97% with a difference of 5% between duplicate tissues.
As the mean viability was > 60% after the MTT reduction,the results met the criteria for a non-irritant response.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation/corrosion:
The in vitro skin irritation test in the EPISKIN model with germanium dioxide (Orovecz, CiToxLAB Hungary 2016) indicate that the test item is Non Irritant (NI).
Eye irritation:
Based on the in vitro eye irritation assay in the isolated chicken eyes test with germanium dioxide (Orovecz, CiToxLAB Hungary 2017) , the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.
To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed slight erosion of the corneal epithelium in 5/6 sections and very slight severity in one case. At the top epithelial layer, necrosis with very slight degree in 3/6 sections, moderate in 1/6 section and severe in 2/6 sections was seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, germanium dioxide was classified as Category 1 (classification of UN GHS) according to criteria published by Prinsen et al in 2011 and Cazelle et al in 2014.
Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.
In addition to the result of the ICE test alone and the result of the supplemental histopathology data, the RhCE test was performed to provide information whether the substance does not require any classification for serious eye damage/eye irritation. Based on the in vitro eye irritation assay, Reconstructed Human Cornea-like Epithelium (RhCE) Test method (OECD 492) (Richez, CiToxLAB France 2017), germanium dioxide is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
Justification for classification or non-classification
Based upon OECD 439 in vitro skin irritation data, germanium dioxide does not require classification as a skin irritant.
Based upon OECD 438 ICE test alone and the result of the supplemental histopathology data a definitive conclusion on the classification cannot be made. Therefore, the RhCE test (OECD 492) was performed to provide information whether the substance does not require any classification for serious eye damage/eye irritation. In a weight of evidence approach and based on the in vitro eye irritation assay, RhCE test, germanium dioxide is considered to be non-irritant to Reconstructed human Cornea-like Epithelium and should not be classified as eye irritant.
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