copper sulphide and zinc chloride sulphate hydroxide, recovery products from copper and zinc dusts

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Type:

Constituent

Test material form:

other: wet sticky solid

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

Test animals / tissue source

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

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 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%.

Results and discussion

In vitro

Results

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%

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 EpiOcular^TM. 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.