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 hydroxide 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 2020), Cadmium Hydroxide is not classified as a severe irritant and not classified as non-irritant. It was concluded that further information was required for classification. Histopathological observations were therefore performed.


The histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed very slight erosion of the corneal epithelium in 6/6 sections. Very slight and/or slight vacuolation of the top epithelium were seen in 6/6 cases. 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, the criteria triggering serious eye damage were not met for Cadmium Hydroxide.


Based on the in vitro eye irritation assay, in the in vitro EpiOcular model (OECD 492) (Orovecz, CiToxLAB Hungary 2021), Cadmium Hydroxide is probably irritant to the eyes. The mean viability was 1% compared to the negative control. This is below the 60% threshold, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium (RhCE). To be noted: longer cell exposure than per the guideline (test item stuck to Epithelium surfaces for 27 hours instead of 6 hours.


  


The 2 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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
version 28 July 2015
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:
CP5275
- Expiration date of the lot/batch:
04 June 2020
- Purity test date:
98.92%
- 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:
93.1
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

1.108

1.061

108.2

Phosphate buffered saline

2

0.998

0.952

97.1

 

3

0.975

0.928

94.7

 

mean

--

0.980

100.0

Positive Control:

1

0.099

0.052

5.3

5% (w/v) SDS solution

 

 

2

0.207

0.160

16.3

 

3

0.091

0.044

 

4.5

 

mean

--

0.085

8.7

Test Item:

1

0.884

0.838

85.4

cadmium hydroxide

2

0.963

0.917

93.5

 

3

1.029

0.983

100.2

 

mean

--

0.912

93.1

Notes:

1. Mean blank value was 0.047.

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 hydroxide, 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 hydroxide 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 theOECD 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 hydroxide, the mean cell viability was 88.2% 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 hydroxide, the mean cell viability was 93.1% 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 thevalidity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM(SM) model test with Cadmium hydroxide, 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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
version 29 July 2016
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:
CP5275
- Expiration date of the lot/batch:
04 June 2020
- Purity test date:
98.92%
- 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:
88.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion

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

1.030

0.983

103.7

Phosphate buffered saline

2

0.961

0.913

96.3

 

mean

--

0.948

100.0

Positive Control:

1

0.057

0.010

1.0

glacial acetic acid

 

 

2

0.052

0.004

0.4

 

mean

--

0.007

0.7

Test Item:

1

0.917

0.869

91.7

cadmium hydroxide

2

0.851

0.803

84.7

 

mean

--

0.836

88.2

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 hydroxide, 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 hydroxide 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 theOECD 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 hydroxide, the mean cell viability was 88.2% 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 hydroxide, the mean cell viability was 93.1% 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 hydroxide, 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:
adverse effect observed (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:
key study
Study period:
05 Jul 2019 - 27 Nov 2020
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 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:
CP5275
- Expiration date of the lot/batch:
04 June 2020
- Purity test date:
98.92%
- 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:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary), commercial abattoir.
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): 7 wekks old (no info on sex or weight).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): ambient temperature, heads were wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft.. and processed within 2 hours of collection.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: not specified
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
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
No, however, test item remained stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.
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 eyeball 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 - yes

NUMBER OF REPLICATES - 7

NEGATIVE CONTROL USED - yes: physiological saline

POSITIVE CONTROL USED - yes: imidazole

APPLICATION DOSE AND EXPOSURE TIME - exposure time of 10s before rinsing
-test substance treated chicken eye: treated with 30 mg cadmium hydroxide
-positive control chicken eye: treated with 30 mg imidazole
-negative control eye: treated with 30µL physiological saline (0.9% (w/v) NaCl solution

OBSERVATION PERIOD - 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.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: cornea surface was rinsed thoroughly with 20ml physiological saline. Additional gentle rinsing with 3x20 mL (positive control treated eyes) or 5x20 mL (test item treated eyes) saline was performed at each time point when the test item and positive control material remaining on the cornea was observed.
- Time after start of exposure: after 10's of exposure
- Indicate any deviation from test procedure in the Guideline : none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit Bern 900 slit-lamp microscope
- Macroscopic morphological damage to the surface: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Others (e.g, histopathology): via microscopy

SCORING SYSTEM:
- Mean corneal swelling (%) : according to TG, measured at all time points.
- Mean maximum opacity score: according to TG, measured at all time points.
- Mean fluorescein retention score at 30 minutes post-treatment : according to TG, measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - yes
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 75 minutes
Value:
3.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
up to 240 minutes
Value:
4.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Irritation parameter:
fluorescein retention score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Physiological saline
Positive controls validity:
valid
Remarks:
Imidazole
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not observable (test item remained stuck on cornea despite washing

DEMONSTRATION OF TECHNICAL PROFICIENCY: not indicated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes - within historical data range, The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.
- Acceptance criteria met for positive control: yes - within historical data range, The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.
- Range of historical values if different from the ones specified in the test guideline:
For NEGATIVE control (physiological saline):
-Maximum corneal swelling at up to 75 min: Min. Value: -3.2 % ; Max. Value: 3.4 %
-Maximum corneal swelling at up to 240 min: Min.: -4.8 % ; Max.: 3.4 %
Maximum corneal opacity change: Min.: 0.00 ; Max.: 0.50
Fluorescein retention change: Min.: 0.00 ; Max.: 0.50
Number of eyes: 536
For POSITIVE control (Imidazole):
-Maximum corneal swelling at up to 75 min: Min. Value: -6.6 % ; Max. Value: 25.0 %
-Maximum corneal swelling at up to 240 min: Min.: -15.9 % ; Max.: 36.7 %
Maximum corneal opacity change: Min.: 3.50 ; Max.: 4.00
Fluorescein retention change: Min.: 2.00 ; Max.: 3.00
Number of eyes: 665

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

3.2 %

I

Mean maximum corneal swelling at up to 240 min

4.2 %

I

Mean maximum corneal opacity

1.00

II

Mean fluorescein retention

1.00

II

Other Observations

Test item 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

1xI 2xII

Based on the performed in vitro eye irritation assay in isolated chicken eyes with Cadmium hydroxide, 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.The results were very close to the borderline for non-irritant, but as it was just over the limit, the test item can not be classified. If a REACH classification is required, it is recommended that another in vitro test (for example the Epiocular test) is performed.

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.

Pathology report:

Semi-quantitative microscopic evaluation was performed on the cornea in the ICET. The classification of histopathology findings was performed based on the 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. The negative control, 30 μL of 0.9 % Sodium chloride (Salsol solution) cornea showed no abnormalities. Positive control, 30 mg Imidazole caused severe epithelial erosion of corneal epithelium in 3/3 cases and detachment form the basement membrane in 2/3 cases. No compromised Bowman’s or basement membrane as well as no endothelial changes were recorded.

Cadmium hydroxide produced very slight erosion of the corneal epithelium in 6/6 sections. Very slight and/or slight vacuolation of the top of the epithelium was seen in 6/6 sections. No stromal or endothelial changes were observed as well as no effects on integrity on basement, Bowman's and Descemet's membranes. Based on the published criteria for histopathological changes, no predictions can be made for Cadmium hydroxide.

Microscopic Findings:

Test Material

 

Eye number

 

Epithelium

Notes

Stroma

Endothelium

Erosion

Necrosis

Vacuolation

 

 

Disorder of fibers

 

 

Pyknotic nuclei

Necrosis

 

outer region (adjacent to epithelium)

inner region (adjacent to endothelium)

 

Top

Mid

Low

 

Test item

11A

1/2

-

-

-

1

-

-

-

-

-

 

11B

1/2

-

-

-

1

-

-

-

-

-

 

12A

1/2

-

-

-

½

-

-

-

-

-

 

12B

1/2

-

-

-

½

-

-

-

-

-

 

13A

1/2

-

-

-

-

-

-

-

-

-

 

13B

1/2

-

-

-

½

-

-

-

-

-

 

Imidazole

14

3

-

-

-

-

x

-

1

-

-

 

15

3

-

-

-

-

-

-

1

-

-

 

16

3

-

-

-

-

x

-

1

-

-

 

Physiological saline solution (Salsol solution,NaCl 0.9% (w/v)

Solution

17

-

-

-

-

-

-

-

-

-

-

 

- = not observed; P = present; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe;

x- detachment from basement membrane

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 hydroxide, 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. Further histopathological observations were performed by the study's pathologist and did not reveal evidence of severe irritant effects.
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 significant cornea swelling (mean swelling ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1) was observed on any eyes. Slight fluorescein retention change (severity 1) was observed on any eyes.Test item 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.

The results were very close to the borderline for non-irritant, but as it was just over the limit, the test item can not be classified. If a REACH classification is required, it is recommended that another in vitro test (for example the Epiocular test) is performed.

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

Histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed very slight erosion of the corneal epithelium in 6/6 sections. Very slight and/or slight vacuolation of the top epithelium were seen in 6/6 cases. 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, no prediction can be made for Cadmium hydroxide.

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)

 

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:

True

No severe corneal morphological changes:

True

Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:

False

 

Criteria for “Category 1” (one or more true)

 

2 or more endpoints classed as IV:

False

Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):

False

Corneal opacity = 4 at any time point (in at least 2 eyes):

False

Severe loosening of epithelium (in at least 1 eye):

False

 

Criteria for “No prediction can be made” (one or two true)

 

Based on the endpoints not classifiable for No Category, or for Category 1:

False

Particles of test item were stuck to the cornea and could not be washed off during the study:

True


Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
9 December 2019 - 26 January 2021
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)
Version / remarks:
version 18 June 2019
Deviations:
yes
Remarks:
EpiOcular™ tissues were pre-incubated for 13 hours 23 minutes instead of 16-24 hours. This fact had no impact on the study on the results or integrity of the study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material:
CP5275
- Expiration date of the lot/batch:
04 June 2020
- Purity test date:
98.92%
- 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
Observation period (in vivo):
not applicable
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 were also used.
Details on study design:
- Details of the test procedure used: Test design of this assay is 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), which is in agreement with OECD Guideline No. 492. (adopted 18 June 2019).
- 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. Its use for eye irritation testing involves a topical application of test items on the surface of the cornea epithelial construct.

Supplier: MatTek, Bratislava, Slovak Republic.
Batch No.: 30638
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 >95% humidified atmosphere.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed Epithelium units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled), were used
- 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 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 2.8,
the relative mean viability of the positive control (after 6 hours exposure time) 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
Overall, as all the validity criteria were met. However the fact that the test item could not be washed off of the tissue samples, caused a prolonged exposure to test item, and the extraction-related procedures could have caused physical damage to the epithelium.
Irritation parameter:
other: % tissue viability
Run / experiment:
1
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
test item could not be removed during the study, despite best efforts to wash it off.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not determinable because the test material remained stuck to the cornea surface.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: The negative controls OD were higher than Historical Control range but these data were between the OECD Guideline range.

Additional controls

Two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm)of tissues was 0.003,Non-Specific Colour % was calculated as 0.1% (see Table 5).

 

Table 5: Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%)
of the Additional Control Tissues

Additional control

Optical Density (OD)

NSC %

 

measured

Blank corrected

(living)

Treated with

1

0.051

0.003

0.1

Cadmium hydroxide

2

0.051

0.003

 

mean

--

0.003

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).

As colour change (blue) was observed after three hours of incubation of the test item in MTT working solution, the potential for the test material to interact with MTT was identified. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed Epithelium are shown in Table 6. The non-specific MTT reduction (NSMTT) was calculated to be -0.040 and the calculated NSMTT% was -1.7%. Due to negative value the correction with NSMTT was not necessary.

Table 6: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues

Additional control

Optical Density (OD)

NSMIT

NSMIT %

 

measured

Blank corrected

Treated with

1

0.073

0.025

-0.038

- 1.7

Cadmium hydroxide

2

0.069

0.021

-0.042

 

mean

--

0.023

-0.040

Treated with

1

0.105

0.057

--

Distilled water

2

0.117

0.069

 

mean

--

0.063

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).

 

As the test item was shown being an MTT-interacting substance and the NSClivingcontrol was used, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.003, Non-Specific Colour % (NSCkilled%) was calculated as 0.1% (see Table 7).

 

Table 7:Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the Additional Control Tissues

Additional control

Optical Density (OD)

NSC %

(killed epidermis)

 

measured

Blank corrected

(killed)

Treated with

1

0.053

0.005

0.1

Cadmium hydroxide

2

0.049

0.001

 

mean

--

0.003

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).

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  2.390 2.342  102.7
 2  2.268 2.220  97.3
   mean  --  2.281  100.0   
 Positive control: methylacetate  1  0.357  0.309  13.5
    2  0.593  0.545  23.9
   mean  --  0.427  18.7
 test item: cadmium hydroxide  1  0.071  0.023  1.0
   2  0.069  0.021  0.9
   mean  --  0.022  1.0

     

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Following exposure with Cadmium hydroxide, the mean cell viability was 1.0% compared to the negative control (after adjustment for non-specific colour controls). This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium but the test item was stuck on all Epithelium surfaces after the treatment and the test item could not be removed during the study. The cell exposure period was longer than per the guideline (the test item was on Epithelium surfaces approximately 27 hours instead of 6 hours) and the extraction-related procedures could have caused physical damage to the epithelium.

In conclusion, in this in vitro EpiOcular™ model test with Cadmium hydroxide, the test item was not compatible with the test system. It is concluded that further information is required for classification.
Executive summary:

The purpose of this study was to evaluate the eye hazard potential of the test item, Cadmium hydroxide, 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, 5% CO2 in a >95% humidified atmospherefor 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 isopropanol and quantified spectrophotometrically at 570 nm.

 

Distilled waterand Methyl acetate treated epithelium tissues were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed Epithelium units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled), were used. 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 with Cadmium hydroxide, themean cell viability was 1.0% compared to the negative control (after adjustment for non-specific colour controls). This is below the threshold of 60%, therefore, theoretically,the test item would be considered as being irritant to Reconstructed human Cornea-like Epithelium. However, the test item was stuck on all Epithelium surfaces after the treatment and the test item could not be removed during the study, despite best efforts to wash it off. The cell exposure period was longer than per the guideline (the test item was on Epithelium surfaces approximately 27 hours instead of 6 hours) and the extraction-related procedures could have caused physical damage to the epithelium.

In conclusion, in this in vitro EpiOcular™ model test with Cadmium hydroxide, the test item was not compatible with the test system. It is concluded that further information is required for classification.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

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

Additional information

Justification for classification or non-classification

Skin irritation:

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

Eye irritation:

Based upon the testing strategy with 2 performed in vitro tests (ICE test OECD 438 - to include histopathological observations and RhCE test OECD 492), it can be concluded in a weight of evidence approach that Cadmium Hydroxide requires 'category 2' classification for eye irritation.