Registration Dossier

Administrative data

Description of key information

WoE approach was used to assess the skin and eye hazards.

Ski Irritation

OECD 431 : non-corrosive

OECD 439 : not irritating (The result obtained in this study is close to the classification border (50% tissue viability)

Eye Irritation:

OECD 492 : conclusion cannot be made

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:
weight of evidence
Study period:
24.06.2020-17.07.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes
Specific details on test material used for the study:
Test Item Zinc chlorohydroxy sulphate and copper sulphide, precipitation Synonym: „Sulphide precipitate “
Lot No. 20190601
CAS No. Not provided
EINECS-No. 951-963-7
Molecular Formula Not provided
Molecular Weight Not provided
Purity 100 % (UVCB), Multi-constituent substance.
Main components
Zinc Chloride Sulphate Hydroxide Hydrate (Zn12(OH)15(SO4)3Cl3 x H2O)
Sodium Sulphate (Na2SO4)
Copper Sulfide (CuS)
Sodium Chloride (NaCl)
Appearance blackish-green, wet sticky solid
Solubility Water - poor
Homogeneity homogenous
Production Date Not known
Expiry Date 31st December 2025
Storage Room temperature 25±5°C
Test Item Handling and Storage Melting point: 120°C, Protect from light, protect from humidity
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The RhE-based skin corrosion test methods have shown to be predictive of in vivo skin corrosion effects assessed in rabbits according to the OECD guideline 404.
Vehicle:
unchanged (no vehicle)
Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™
- Tissue batch number(s): Lot No. 30880; strain 00267
- Production date: 5.7.2020
- Delivery date: 13.7.2020
- Date of initiation of testing: 13.7.2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3h
- Spectrophotometer: Spectrophotometer MRX Dynex, UK Spectra MRX / 1SPA0086
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A single testing run composed of at least two tissue replicates should be sufficient for a test chemical when the resulting classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements, a second run may be considered, as well as the third one in case of discordant results between the first two runs.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8 N KOH
Duration of treatment / exposure:
3-minute and 1-hour time points.
Number of replicates:
2
Amount / concentration applied:



- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
65
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
6.5
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-min exposure
Value:
93.2
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
62.4
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 6.8%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 16% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=50% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 12%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Results obtained with test item, PC and NC 

 

 

3 min endpoint

1-hour endpoint

 

Diff

[%]

Prediction

Viability [%]

Diff

[%]

CV

 [%]

Viability [%]

 

CV

 [%]

QC

NC

100.0

5.1

3.6

100.0

5.8

5.8

4.1

qualified

Not classified

PC

62.4

10.1

8.4

6.5

0.0

0.0

36.9

qualified

Cat 1B/1C

TA

93.2

7.4

7.7

65.0

3.4

3.4

0.0

qualified

Not classified

Diff – the difference between two identically treated tissues, CV – Coefficient of variation,QC – Quality criteria

NC – Negative control, PC – Positive Control, TA – Test article

Interpretation of results:
other: does not require classification regarding the skin corrosivity.
Conclusions:
Based on the experimental data conducted according to the OECD TG 431, it can be concluded, that test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” does not require classification regarding the skin corrosivity.
Executive summary:

The purpose of the study was to evaluate the skin corrosion potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Skin Corrosion Test utilising the Reconstructed Human Epidermis (RhE) Test Method (OECD TG 431).

The skin corrosion potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro skin corrosion test using the reconstructed human epidermal model EpiDermTM. Two tissue replicates were used for each treatment (exposure times 3 min and 60 minutes). Concurrent positive (8N KOH) and negative (UP H2O) controls were used to ensure the adequate performance of the experimental model.

 

The Optical densities (ODs) of Negative controlt reated tissues were 2,630 at three min and 2,417 at 1 hour. The viability of the tissues exposed for 1 hour to the positive control (8N KOH) was 6.5%.The Positive and Negative controls met the acceptance criteria set by the OECD TG 431.

 

The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 93.2 % after 3 min and 65.0% after 60 min. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment

 

The Test Item is classified as non-corrosive on the basis of the 1-hour time-point.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24.06.2020-17.07.2020
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)
GLP compliance:
yes
Specific details on test material used for the study:
Test Item Zinc chlorohydroxy sulphate and copper sulphide, precipitation; Synonym: „Sulphide precipitate“
Lot No. 20190601
CAS No. Not provided
EINECS-No. 951-963-7
Molecular Formula Not provided
Molecular Weight Not provided
Composition Solid
Purity 100% (UVCB),
Main components
Zinc Chloride Sulphate Hydroxide Hydrate (Zn12(OH)15(SO4)3Cl3 x H2O)
Sodium Sulphate (Na2SO4)
Copper Sulfide (CuS)
Sodium Chloride (NaCl)
Appearance blackish-green, wet sticky solid
Solubility Water - poor
Homogeneity homogenous
Production Date Not known
Expiry Date 31st December 2025
Storage Room temperature 25±5°C
Test Item Handling and Storage Protect from light, protect from humidity
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The RhE-based skin irritation test methods have shown to be predictive of in vivo skin irritation effects assessed in rabbits according to the OECD guideline 404.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:The reconstructed human epidermal model EpiDerm™ (EPI-200)
- Tissue batch number(s): Lot No. 30880
- Production date: 5th July 2020
- Delivery date: 13th July 2020
- Date of initiation of testing: 14th July 2020

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline 15 times
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT solution
- Incubation time: 3h
- Spectrophotometer: Spectrophotometer MRX Dynex, UK Spectra MRX / 1SPA0086
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
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): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
60 minutes
Observation period:
42 hours
Number of animals:
not used, in vitro study
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
exposure 60 min, post exposure 42 h
Value:
55.2
Negative controls validity:
valid
Remarks:
100 % viability
Positive controls validity:
valid
Remarks:
5.8 % viability
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute optical density (OD) of negative control tissues (treated with sterile DPBS) in the MTT-test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is meeting the acceptance criterion of the OECD TG 439, if the mean OD of the NC tissues is ≥ 0.8 and ≤ 2.8.

- Acceptance criteria met for positive control: Mean viability of the tissue replicates exposed for 1 hour to the positive control (5% SDS), expressed as % of the negative control, should according to the OECD TG 439 be ≤ 20%.

- Acceptance criteria met for variability between replicate measurements: The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of three identically treated replicates is < 18%.

Results obtained with test item, PC and NC in the OECD Test Guideline No. 439.  

 

 

 

OD

SD

of OD

Viability [%]

SD

of viabilities

CV

 [%]

QC

Prediction

NC

2.010

0.118

100.0

5.9

5.9

qualified

Not classified

PC

0.117

0.010

5.8

0.5

8.2

qualified

Cat 2

TA

1.109

0.177

55.2

8.8

16.0

qualified

Not classified

OD – Optical Density, SD – standard deviation, CV – Coefficient of variation,QC – Quality criteria

NC – Negative control, PC – Positive Control, TA – Test article

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the experimental data conducted according to the OECD TG 439 and strictly adhering to the prediction model of the OECD TG 439, it can be concluded that the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation“ does not require classification concerning the skin irritation effects. However, since the result obtained in the current study is close to the classification border, a second confirmatory run is recommended.
Executive summary:

The purpose of the study was to evaluate the skin irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Skin Irritation Test utilising the Reconstructed Human Epidermis (RhE) Test Method (OECD TG 439).

The skin irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro skin irritation test using the reconstructed human epidermal model EpiDermTM. Three tissue replicates were used for each treatment (exposure time 60 minutes, postexposure time 42 hours). Concurrent positive (5% SDS) and negative (DPBS) controls were used to ensure the adequate performance of the experimental model.

 The Optical density (OD) of Negative control treated tissues was 2,010 and the viability of the tissues exposed for 1 hour to the positive control (5% SDS) was 5.8%. The Positive and Negative controls met the acceptance criteria set by the OECD TG 439.

 The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 55,2% with a SD of 8,8 and CV of 16%. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment.

 On the basis of the prediction model given by the OECD TG 439, the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” does not require classification concerning the skin irritation effects. However, since the result obtained in the current study is close to the classification border (50% tissue viability), a second confirmatory run is recommended.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.06.2020-17.07.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes
Specific details on test material used for the study:
Test Item Zinc chlorohydroxy sulphate and copper sulphide, precipitation Synonym: „Sulphide precipitate“
Lot No. 20190601
CAS No. Not provided
EINECS-No . 951-963-7
Molecular Formula Not provided
Molecular Weight Not provided
Composition Solid
Purity 100 % (UVCB), Multi-constituent substance.
Main components
Zinc Chloride Sulphate Hydroxide Hydrate (Zn12(OH)15(SO4)3Cl3 x H2O)
Sodium Sulphate (Na2SO4)
Copper Sulfide (CuS)
Sodium Chloride (NaCl)
Appearance blackish-green, wet sticky solid
Solubility Water - poor
Homogeneity homogenous
Production Date Not known
Expiry Date 31st December 2025
Storage Room temperature 25±5°C
Test Item Handling and Storage Protect from light, protect from humidity
Species:
other: in vitro study
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Protocol: In vitro EpiOcularTM eye irritation test (OCL-200-EIT)
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Cell line: The EpiOcular™ human cell construct
- Lot: 30667
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1°C
- Cell Culture Conditions: a humidified atmosphere of 5±0.5% CO2 in air
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
Duration of treatment / exposure:
6 ± 0.25 minutes
Duration of post- treatment incubation (in vitro):
post-exposure of 18±0.25 hours + tissues were incubated with MTT for 3 hours ± 5 min
Number of animals or in vitro replicates:
2
Details on study design:
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure lasted 6±0.25 hours (37±1ºC, 5±1% CO2 and 90±10% RH), followed by post-exposure of 18±0.25 hours (37±1ºC, 5±1% CO2 and 90±10% RH).

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
The test item may interfere with the MTT endpoint if it is coloured and/or able to directly reduce MTT and at the same time is present in the tissues when the MTT viability test is performed. Some non-coloured test items may change into coloured material in wet or aqueous conditions and thus stain tissues during the 60 min exposure.
To identify these possible interferences, a functional check was performed. The 50mg of a test item was added into 0.3 mL of purified water as well as isopropanol. The mixture was incubated in a glass test tube in the incubator at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 60 min. At the end of the exposure time, the presence of the staining was evaluated. To evaluate direct interference of the test item with the MTT, 50 mg of the test item was added to 1 mL of the MTT medium and incubated in the incubator (37±1°C in a humidified atmosphere of 5±1% CO2 in air) for 60 min. MTT medium was used as visual control.

- Description of the method used to quantify MTT formazan
After the completion of the post-exposure period, tissue were blot and moved into the 24-well plate prefilled with 300 µL of the MTT-media (c = 1mg/mL). Tissues were incubated with MTT for 3 hours ± 5 min at a humidified incubator (37±1ºC, 5±1% CO2 and 90±10% RH). After the MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted from the tissue with 2 mL/tissue of isopropanol. Extraction was performed overnight at room temperature. Plates were sealed with parafilm to prevent evaporation of isopropanol. On the following day, 200 μL of each extraction solution was transferred to a 96-well plate, and the optical density of extracted formazan was determined using a spectrophotometer at 570 nm. Isopropanol was used as blank.

-The assay was considered valid if the following criteria were met:

Negative Control (NC):
The absolute optical density (OD) of negative control tissues (treated with sterile DI H2O) in the MTT-test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the NC tissues is ≥ 0.8 and ≤ 2.8.

Positive Control (PC):
The assay meets the acceptance criterion if the mean viability of tissues treated with PC (Methyl acetate) and expressed as % of the negative control tissues is ≤ 50%.

Standard Deviation (SD or Diff):
Per OECD TG 492, Difference of viability between two tissue replicates should be less than 20% or the Standard deviation (SD) between three tissue replicates should not exceed 18%.

Irritation parameter:
in vitro irritation score
Run / experiment:
The mean tissue viability (%)
Value:
2.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
100 % viability
Positive controls validity:
valid
Remarks:
33.8 % viability
Remarks on result:
not determinable
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the Optical density (OD) of Negative control treated tissues was 2.011
- Acceptance criteria met for positive control: Yes, the viability of the tissues exposed for 6 hours to the positive control was 33.8%

Results obtained with the test item, PC and NC in the OECD Test Guideline No. 492.

 

 

OD

Diff

of OD

Viability [%]

Diff

[%]

QC

Prediction

NC

2.011

0.004

100.0

0.2

qualified

Not cat

PC

0.680

0.165

33.8

8.2

qualified

Positive

TA

0.059

0.006

2.9

0.3

qualified

Positive, no prediction can be made

OD – Optical density, Diff – Difference, QC – quality criteria

NC – Negative control, PC – Positive Control, TA – Test article

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the experimental data conducted according to the OECD TG 492 and strictly adhering to the prediction model of the guideline, it can be concluded that no prediction of eye irritating effect can be made for the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation”.
Executive summary:

The purpose of the study was to evaluate the eye irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Eye Irritation Test utilising the Reconstructed Human Cornea-like Epithelial (RhCE) model (OECD TG 492).

The eye irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro eye irritation test using the reconstructed human epidermal model EpiOcularTM. Two tissue replicates were used for each treatment (exposure time 6 hours, postexposure time 18 hours). Concurrent positive (Methyl acetate) and negative (DI H2O) controls were used to ensure the adequate performance of the experimental model.

 

The Optical density (OD) of Negative control treated tissues was2.011 and the viability of the tissues exposed for 6 hours to the positive control was 33.8% (see Table 6 and Appendix 3). The Positive and Negative controls met the acceptance criteria set by the OECD TG 492.

 

The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 2.9 % with a Diff of 0.3 %. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment.

 

On the basis of the prediction model given by the OECD TG 492, for the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” no prediction could be made, and the test material requires further testing and clarification its eye irritating or corrosive effects. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion (OECD 431)

The purpose of the study was to evaluate the skin corrosion potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Skin Corrosion Test utilising the Reconstructed Human Epidermis (RhE) Test Method (OECD TG 431).

The skin corrosion potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro skin corrosion test using the reconstructed human epidermal model EpiDermTM. Two tissue replicates were used for each treatment (exposure times 3 min and 60 minutes). Concurrent positive (8N KOH) and negative (UP H2O) controls were used to ensure the adequate performance of the experimental model.

 

The Optical densities (ODs) of Negative controlt reated tissueswere 2,630 at three min and 2,417 at 1 hour. The viability of the tissues exposed for 1 hour to the positive control (8N KOH) was 6.5%.The Positive and Negative controls met the acceptance criteria set by the OECD TG 431.

 

The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 93.2 % after 3 min and 65.0% after 60 min. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment

 

The Test Item is classified as non-corrosive on the basis of the 1-hour time-point.

Skin Irritation (OECD 439)

The purpose of the study was to evaluate the skin irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Skin Irritation Test utilising the Reconstructed Human Epidermis (RhE) Test Method (OECD TG 439).

The skin irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro skin irritation test using the reconstructed human epidermal model EpiDermTM. Three tissue replicates were used for each treatment (exposure time 60 minutes, postexposure time 42 hours). Concurrent positive (5% SDS) and negative (DPBS) controls were used to ensure the adequate performance of the experimental model.

 The Optical density (OD) of Negative control treated tissues was 2,010 and the viability of the tissues exposed for 1 hour to the positive control (5% SDS) was 5.8%. The Positive and Negative controls met the acceptance criteria set by the OECD TG 439.

 The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 55,2% with a SD of 8,8 and CV of 16%. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment.

 On the basis of the prediction model given by the OECD TG 439, the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” does not require classification concerning the skin irritation effects. However, since the result obtained in the current study is close to the classification border (50% tissue viability), a second confirmatory run is recommended.

Eye Irritation (OECD 492)

The purpose of the study was to evaluate the eye irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” in In Vitro Eye Irritation Test utilising the Reconstructed Human Cornea-like Epithelial (RhCE) model (OECD TG 492).

The eye irritation potential of “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was examined in the in vitro eye irritation test using the reconstructed human epidermal model EpiOcularTM. Two tissue replicates were used for each treatment (exposure time 6 hours, postexposure time 18 hours). Concurrent positive (Methyl acetate) and negative (DI H2O) controls were used to ensure the adequate performance of the experimental model.

The Optical density (OD) of Negative control treated tissues was2.011 and the viability of the tissues exposed for 6 hours to the positive control was 33.8% (see Table 6 and Appendix 3). The Positive and Negative controls met the acceptance criteria set by the OECD TG 492.

The viability of cultures treated by “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” was 2.9 % with a Diff of 0.3 %. Since the material did not reduce MTT, nor it interacted with water, and was very easily removed from the tissue surface during the washing procedure, no additional steps were necessary to correct the result obtained on viable tissues in the main experiment.

On the basis of the prediction model given by the OECD TG 492, for the test item “Zinc chlorohydroxy sulphate and copper sulphide, precipitation” no prediction could be made, and the test material requires further testing and clarification its eye irritating or corrosive effects. 

Taken into account the worst case scenario based on the available in vitro data it is concluded that the substance need to be classified as irritation to skin and damaging to the eyes.

Justification for classification or non-classification

Based on the in vitro study results and application of the worst case scenario, sulphide precipitate has to be classified as Skin Irrit. 2 H315 and Eye Dam. 1 H318 according to CLP Regulation 1272/2008.