Germanium dioxide

Endpoint summary

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

Reference

Reference 1

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

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).

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 EPISKIN^TM(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 (NSC_living) 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.

 

 

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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.

Reference 2

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

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.

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 EpiOcular^TMcornea 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).

        

 

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.