Germanium

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Germanium

EC Number:

231-164-3

EC Name:

Germanium

Cas Number:

7440-56-4

Molecular formula:

Ge

IUPAC Name:

germanium

Test material form:

solid: particulate/powder

Details on test material:

Batch number: Zwarte C-mal 229-2
Description: Grey Powder
Expiry date: 18 September 2018
Purity: 96.9%
Storage conditions: Controlled room temperature (15-25°C, below 70% RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test system

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): yes
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 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

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Run / experiment:

1

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

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, and after subtraction of the blank OD, the OD of the test item solution was > 0.08 (i.e. 0.424 and 0.456). Therefore, the test item was considered as probably interacting with the MTT viability test. As a result, additional controls were performed on viable tissues in parallel to the main test for the evaluation of the non‑specific OD.

 

 

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 (incubated in MTT) 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.

The difference of viability within each tissue duplicate was calculated. As the difference was ≤20%, the test was considered as valid.

 

The calculated value of the mean non-specific color NSC_viablewas 0%.

As the mean NSC_viableis<50% relative to the negative control OD, the test item was considered compatible with the test. Therefore, a Colorant Control (CC) corrected viability of the tissues treated with the test item was calculated.

Individual and mean corrected OD values and tissue viabilities for the test item, the  negative and positive controls

Group	Exposure duration	Tissue   No.	OD570nm measurements		Mean blank	cOD570nm measurements		MeancOD570nm	Viability (%)
			1"	2nd		1''	t'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	1.839	1.840	0.040	1.799	1.800	1.799	101
		2	1.863	1.875		1.823	1.835	1.829	103
Test item treated (viable tissue) not incubated with MTT	6h	1	0.042	0.041	0.040	0.002	0.001	0.001	0
		2	0.040	0.041		0.000	0.001	0.000	0

OD= optical density

cOD=blank corrected optical density

MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

Mean tissue viability and standard deviations for the test item, the negative and positive controls

Mean tissue viability and standard deviations for the test item, the negative and positive controls

Group	Exposure duration	cOD570nm		Viability(%)			NSC (%)
		Mean	so	Mean	so	Dfference(%)	Mean
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
Test item treated (viable tissue) not incubated with MTT	6h	0.001	0.001	0	0	0	0
Test item (true MTT metabolic conversion)	6h	1.813		102

cOD=blank corrected optical density

SO= standard deviation

NSC= non specific colour

MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

The relative corrected mean viability of the tissues treated with the test item was 102%.

As the mean viability was > 60% after the MTT reduction,the results met the criteria for a non-irritant response.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, GERMANIUM, 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, by measurement of its cytotoxic effect on the EpiOcular^TMcornea epithelial model.

The studywas 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 to have colouring potential but no direct MTT reducing properties.As a result, additional controls were added to the main test (i.e.viable tissues treated with the test item but not incubated with MTT) for the evaluation of the non-specific OD.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

The calculated value of the mean non-specific color NSC_viablewas 0%.

As the mean NSC_viableis<50% relative to the negative control OD, the test item was considered compatible with the test.Therefore, a Colorant Control (CC) corrected viability of the tissues treated with the test item was calculated.

 

The relative corrected mean viability of the tissues treated with the test item was 102%.

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, 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).