Registration Dossier

Administrative data

Description of key information

Skin irritation:

The in vitro skin irritation/corrosion test in the EpiDerm model (combined OECD 431/439) (Orovecz, CiToxLAB Hungary 2019) with cadmium sulfate indicates that the test item is non-corrosive and non-irritant to the skin

Eye irritation:

Based on the in vitro eye irritation assay in the isolated chicken eyes test (OECD 438) (Orovecz, CiToxLAB Hungary 2019) , cadmium sulfate is not classified as a severe irritant and not classified as non-irritant.

Based on the in vitro eye irritation assay, in the in vitro EpiOcular model (OECD 492) (Orovecz, CiToxLAB Hungary 2019), cadmium sulfate is probably irritant to eyes. The mean viability was 2.1% compared to the negative control. This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium (RhCE).

Based on the in vitro eye irritation assay, Bovine Corneal Opacity and Permeability (BCOP) test method (OECD 437) (Aubert CitToxLAB France 2019), with

the mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas is 3. However, it has to be noted that the SD value of the mean IVIS is high (i.e. 2.5) and that one of the three test item treated corneas gave a discordant prediction from the mean of all three corneas and the discordant result (IVIS = 6, i.e. > 3) was > 10 IVIS units from the cut off threshold of 55. It was also noted that permeability values of the three corneas were higher than those obtained with the vehicle control, reflecting a loss of tight junction barrier function due to the test item. The slight increased permeability values lead us to consider that the test item may have slight irritant potential to the eye, confirmed by the opacity and the fluorescein fixation noted on the corneas.

Based on these overall results, the conclusion of the study could be considered as borderline between "no category" and "no prediction can be made".

The 3 in vitro tests were done consequently in a testing strategy to determine the classification

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
version 28 July 2015
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
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):19-EKIN-028
- Expiry Date: 15 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.6-25.0°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1washing step: rinsing thoroughly with PBS

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 (volume or weight with unit): 10 mg

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
Value:
94
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

0.674

0.626

100.9

Phosphate buffered saline

2

0.661

0.613

98.8

 

3

0.671

0.623

100.4

 

mean

--

0.621

100.0

Positive Control:

1

0.116

0.069

 

11.0

5% (w/v) SDS solution

 

 

2

0.116

0.069

  

11.0

 

3

0.114

 

0.067

  

10.7

 

mean

--

0.068

 

10.9

Test Item:

1

0.627

0.579

  

93.3

cadmium sulfate

2

0.639

0.591

 

95.2

 

3

0.628

0.581

93.5

 

mean

--

0.584

 

94.0

Notes:

1. Mean blank value was 0.048.

2. Optical density means the mean value of the duplicate wells for each sample

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with cadmium sulfate, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.
Executive summary:

An in vitro skin corrosivity and irritation test of cadmium sulfate was performed in a reconstructed human epidermis model.EPISKINTM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines. 

Disks of EPISKINTM(SM) were treated with the test item and incubated for 15 minutes (irritation testing) and 4 hours (corrosivity testing) 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,in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. Two additional disks were used to provide in each case 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. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes of 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.

Corrosivity testing:

Following exposure with Cadmium sulfate, the mean cell viability was 72.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Irritation testing:

Following exposure with Cadmium sulfate, the mean cell viability was 94.0 % 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 EPISKINTM(SM) model test with Cadmium sulfate, the results indicate that the test item is not corrosive and not irritant to the skin, UN GHS Classification: No Category.



 

Endpoint:
skin corrosion: 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
version 29 July 2016
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
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):19-EKIN-028
- Expiry Date: 15 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.6-25.0°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1washing step: rinsing thoroughly with PBS

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: 2

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 (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

NEGATIVE CONTROL: NaCI (9 g/l saline)
- Concentration (if solution): 50µl

POSITIVE CONTROL: glacial acetic acid
- Concentration (if solution): 50µl
Duration of treatment / exposure:
4h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Value:
72.4
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

0.745

0.697

100.2

Phosphate buffered saline

2

0.743

0.695

99.8

 

mean

--

0.696

100.0

Positive Control:

1

0.052

0.004

 

0.6

glacial acetic acid

 

 

2

0.053

0.005

 

0.6

 

mean

--

0.004

 

0.6

Test Item:

1

0.555

0.507

 

72.8

cadmium sulfate

2

0.549

0.501

 

72.0

 

mean

--

0.504

 

72.4

Notes:

1. Mean blank value was 0.048.

2. Optical density means the mean value of the duplicate wells for each sample

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with cadmium sulfate, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.
Executive summary:

An in vitro skin corrosivity and irritation test of cadmium sulfate was performed in a reconstructed human epidermis model.EPISKINTM(SM) is designed to predict and classify thecorrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines. 

Disks of EPISKINTM(SM) were treated with the test item and incubated for 15 minutes (irritation testing) and 4 hours (corrosivity testing) 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,in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. Two additional disks were used to provide in each case 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. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes of 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.

Corrosivity testing:

Following exposure with Cadmium sulfate, the mean cell viability was 72.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Irritation testing:

Following exposure with Cadmium sulfate, the mean cell viability was 94% compared to the negative control. This is above the threshold of 50%, thereforethe 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 EPISKINTM(SM)model test with Cadmium sulfate, the results indicate that the test item is not corrosive and not irritant to the skin, UN GHS Classification: No Category.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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:
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 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30mg


Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
not applicable
Details on study design:
treatments:
-test substance treated chicken eye: treated with 30 mg cadmium sulfate
-positive control chicken eye: treated with 30 mg imidazole
-negative control eye: treated with 30µL physiological saline (0.9% (w/v) NaCl solution

REMOVAL OF TEST SUBSTANCE
- Washing (if done): cornea surface was rinsed thoroughly with 20ml physiological saline
- Time after start of exposure: after 10'' of exposure

SCORING SYSTEM:
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable.

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.


TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope/fluorescein

Irritation parameter:
percent corneal swelling
Run / experiment:
up to 75 minutes
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 240 minutes
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
0.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
2.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no 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

0.0 %

I

Mean maximum corneal swelling at up to 240 min

0.0 %

I

Mean maximum corneal opacity

0.5

I

Mean fluorescein retention

2.67

IV

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Overall ICE Class

1xI 1xIV

Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium sulfate,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.

Positive Control

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.6 %

II

Mean maximum corneal swelling at up to 240 min

27.8 %

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 were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

The positive control Imidazole was 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 saline was classified as 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

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium sulfate, 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.
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 guideline (25 June 2018).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg test itemwas applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds exposure time, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL ofphysiological saline(0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in this experiment. Thus, the study was considered to be valid.

No cornea swelling was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on any eyes. Severe fluorescein retention change (severity 2 on one eye and severity 3 on two eyes) was noted.Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium sulfate, 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.


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:
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)
Version / remarks:
version 18 June 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
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): 50 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:
two replicates were used for the test item. Two negative controls and two positive controls
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 October 2017) 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 18 June 2019).
- RhCE tissue construct used, including batch number: The EpiOcular™ tissue constructs consisted of at least 3 viable layers of cells and a non-keratinized surface, showing a corneal-like structure analogous to that found in vivo. The EpiOcular™ human cell construct (MatTek Corporation) is a nonkeratinized epithelium prepared from normal human keratinocytes, grown in such a way as to produce a membrane similar to a cornea. The use of EpiOcular™ cultures offers features appropriate for a model of ocular irritation. First, the model is composed of stratified human keratinocytes in a three-dimensional structure. Next, test materials can be applied topically to the model, so that all substances including water insoluble materials can be tested.
Supplier: MatTek, Bratislava, Slovak Republic.
Batch No.: 30626
Expiry date: The living EpiOcular tissues were used within 72 hours after their production.

- Doses of test chemical and control substances used:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 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: T
The test was considered to be valid if:
the mean OD blank of each plate (i.e. extraction solvent) is <0.1,
the mean OD of the negative controls is between 0.8 and 2.5,
the relative mean viability of the positive control is <50% of the relative mean viability of the negative control,
the difference of viability between two tissue replicates 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:
2.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Additional controls

The test item did not react with MTT and therefore the use of additional NSMTT controls was not necessary.

The test item was white therefore additional colour controls were not necessary.

VIABILITY RESULTS

 

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table below. The mean OD values for the test item treated samples showed 2.1% relative viabilitycompared to the negative control.

Table : Optical Density (OD) and the calculated relative viability % of the samples

 

         Optical Density (OD)  Viability   
 Substance  Measured Blank corrected   % RV   
  Negative control: distilled water  1.573 1.527  0.0    
 2  1.787 1.740   100.0   
   mean  --  1.633  100.0   
 Positive control: methylacetate  1  0.593  0.546  33.4   
    2  0.473  0.426  26.1   
   mean  --  0.486  29.8   
 test item: cadmium sulfate  1  0.086  0.040  2.4   
   2  0.076  0.030  1.8   
   mean  --  0.035  2.1   

     

 

Interpretation of results:
other: test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. Further eye irrritation test done for conclusion on eye irritation classification
Conclusions:
Following exposure with Cadmium sulfate, the mean cell viability was 2.1% compared to the negative control. This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiOcular™ model test with Cadmium sulfate (Batch number: X09D060), the results indicate that the test item is probably irritant to eyes (UN GHS Classification: Category 2 or Category 1).
Executive summary:

The purpose of this study was to evaluate the eye hazard potential of the test item, Cadmium sulfate, based on its ability to induce cytotoxicity in the EpiOcularTMcornea epithelial model, as measured by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] assay.

Disks of EpiOcular™(two units) were treated with the test item 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 >95% humidified atmosphere. After the incubation, MTT solution was added to the units and incubated for a further 3 hours to determine cell viability. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.

Distilled water and Methyl Acetate treated Epithelium were used as negative and positive controls, respectively (two units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability is less or equal (≤) to 60% of the negative control, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium.

Following exposure withCadmium sulfate, the mean cell viability was 2.1% compared to the negative control. This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiOcular™ model test with Cadmium sulfate (Batch number:X09D060), the results indicate that the test item is probably irritant to eyes (UN GHS Classification: Category 2 or Category 1).

 

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 November 2019 (experimental period was within a single day)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
adopted 09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:X09D060
- Expiration date of the lot/batch: 04 June 2020
- Purity test date:99.99%
- Description:White solid (in the powder form)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity).
- Safety precautions: Enhanced safety precautions (half mask at least with P3 filter cartridge, nitrile gloves, lab coat) were applied considering the supplied safety datasheet to assure personnel health and safety.
Species:
other: bovine cattle (Bos taurus)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: bovine eyes will be obtained from freshly slaughtered cattle at the abattoir (SOCAVIA, Cany Barville – France, SOCAVIA, Beuvillers – France or EVA, Saint-Pierre-sur-Dives, France).
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle will be up to 12 months old (typically, 5 to 8 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport from supplier to Citoxlab France: the eyes will be immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Citoxlab France. Containers with smooth internal surfaces will be used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
- Time interval prior to initiating testing: Upon arrival at Citoxlab France, the selection and preparation of corneas will be performed as soon as possible.
- indication of any existing defects or lesions in ocular tissue samples: : a careful macroscopic examination will be performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects will be discarded.



Controls:
yes, concurrent positive control
yes, concurrent negative control
Number of animals or in vitro replicates:

3 corneas per substance (test item, positive control, negative control)
Irritation parameter:
in vitro irritation score
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
the SD value of the mean IVIS is high (i.e. 2.5) and one of the three test item treated corneas gave a discordant prediction from the mean of all three corneas and the discordant result (IVIS = 6, i.e. > 3) was > 10 IVIS units from the cut off threshold of 55. Also permeability values of the three corneas were higher than those obtained with the vehicle control, reflecting a loss of tight junction barrier function due to the test item.
Interpretation of results:
GHS criteria not met
Remarks:
Based on these overall results, the conclusion of the study could be considered as borderline between "no category" and "no prediction can be made".
Conclusions:
The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas is 3. Based on the OECD guideline and on its strict classification, as the mean IVIS is ≤ 3, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). However, it has to be noted that the SD value of the mean IVIS is high (i.e. 2.5) and that one of the three test item treated corneas gave a discordant prediction from the mean of all three corneas and the discordant result (IVIS = 6, i.e. > 3) was > 10 IVIS units from the cut off threshold of 55. It was also noted that permeability values of the three corneas were higher than those obtained with the vehicle control, reflecting a loss of tight junction barrier function due to the test item. The slight increased permeability values lead us to consider that the test item may have slight irritant potential to the eye, confirmed by the opacity and the fluorescein fixation noted on the corneas.

Based on these overall results, the conclusion of the study could be considered as borderline between "no category" and "no prediction can be made".
Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, Cadmium sulfate, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method (OECD Guideline 437) can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas is 3. Based on the OECD guideline and on its strict classification, as the mean IVIS is ≤ 3, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). However, it has to be noted that the SD value of the mean IVIS is high (i.e. 2.5) and that one of the three test item treated corneas gave a discordant prediction from the mean of all three corneas and the discordant result (IVIS = 6, i.e. > 3) was > 10 IVIS units from the cut off threshold of 55. It was also noted that permeability values of the three corneas were higher than those obtained with the vehicle control, reflecting a loss of tight junction barrier function due to the test item. The slight increased permeability values lead us to consider that the test item may have slight irritant potential to the eye, confirmed by the opacity and the fluorescein fixation noted on the corneas.

Based on these overall results, the conclusion of the study could be considered as borderline between "no category" and "no prediction can be made".

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation:

Based upon OECD 431/439 in vitro skin irritation/corrosion data, cadmium sulfate does not require classification as a skin irritant

Eye irritation:

Based upon the testing strategy with 3 performed in vitro tests (ICE test OECD 438, RhCE test OECD 492 and the BCOP test OECD 437), it can be concluded in a weight of evidence approach that cadmium sulfate requires 'category 2' classification for eye irritation.

The OECD 438 ICE test concluded the test item (cadmium sulfate) is not classified as a severe irritant and not classified as non-irritant. It concluded that further information was required for classification.

Therefore, the RhCE test (OECD 492) was performed to provide information whether the test item does not require any classification for eye damage/eye irritation.

Following exposure with cadmium sulfate, the mean cell viability was 2.1% compared to the negative control. This is below the threshold of 60%, and therefore cadmium sulfate is considered as being irritant to Reconstructed human Cornea-like Epithelium.

Given the OECD 438 and OECD 492 give a conclusion that cadmium sulfate is an eye irritant, the BCOP (OECD 437) was performed to determine the clasification. The outcome of the BCOP being borderline between no classification and no conclusion but no category 1 together with the test data from the ICE and RhCE (EpiOcular test) test concludes that cadmium sulfate requires eye irritation, category 2; H319 classifcation according to the EC criteria