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Description of key information

Based on results of this studies, the classification forCZ-HABI is as follows:

Globally Harmonized System of Classification and  Labeling of Chemicals: 

Not Skin Irritant

Not Eye Irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 11, 2018 - September 15, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 439
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermis (SkinEthicTM RHE)
Details on animal used as source of test system:
The test system used for the in vitro skin irritation test was reconstructed human epidermis (SkinEthicTM RHE) as recommended by the OECD 439 guideline. The SkinEthicTM RHE model consists of normal human keratinocytes cultured for 17-days on an insert of 0.5 cm2 polycarbonate filter at the air-liquid interface in a chemically defined growth medium. The cells form a multilayered, highly differentiated and stratified epidermis model of the human epidermis that consists of main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Reconstructed human epidermis tissues, SkinEthicTM RHE model, were procured from SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France; were used in the study (Lot N°18-RHE-111)
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 ± 2 mg of test item/0.5 cm2
Duration of treatment / exposure:
incubated at room temperature for 42 minute exposure
Duration of post-treatment incubation (if applicable):
at 37 ± 1°C in 5 ± 1% CO2 in a humidified incubator for 42 hours
Number of replicates:
Three tissues for each treatment (test item, Negative control and Positive control)
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 90.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, the classification for the tesi item is as mentioned below:
Globally Harmonized System of Classification and: No Category (Non Skin Irritant)
Executive summary:

This study was performed to evaluate the non-irritant and irritant potential of CZ-HABI using reconstructed human epidermis (RHE) tissue in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Tissues were exposed tothenegative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate,5% aqueous (SDS)) and the test ithem in triplicate for 42 minutes at room temperature.

The mean cell viability in tissues treated with the test item was 90.67% after 42 minutes exposure. A significant reduction in percent cell viability was not observed in treated tissues when compared with that of the concurrent negative control.

The Optical density (OD) values for the negative control replicates were between 1.274 and 1.305, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blankwasbetween0.042and0.045which met the guideline requirement of OD< 0.1.The positive control showed a 1.52% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, when compared with that of the concurrent negative control. Variation between tissue replicates (i.e., CV% value) was 0.18% for the test item group, 5.92% for positive control and 0.77% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTMRHE model. All criteria for a valid study were met as describe in the study plan.

Based on results of this study, the classification for the substance is as follows:

Globally Harmonized System of Classification and  Labeling of Chemicals:         No Category (Non Skin Irritant)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
December 14, 2018 - December 15, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 437
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra

Bovine Age: Between 1 to 5 years (the age of the animals was determined based on the teeth count and horn ring count in addition to the Horizontal Diameter of corneas and central corneal thickness)

Transportation Condition: Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]
Amount / concentration applied:
750 μL of the prepared test item [20% (w/v) concentration in corn oil]
Duration of treatment / exposure:
Corneas were exposed for 4 h at 32 ± 1 °C
Number of animals or in vitro replicates:
3 replicates for each conditions (Normal saline, Imidazole 20%, Corn Oil, the test item at 20% in corn Oil)
Details on study design:
This study was performed to evaluate the ocular irritancy of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea. Effects were measured by: 1) decreased light transmission through the cornea (opacity) and 2) increased passage of sodium fluorescein dye through the cornea (permeability). The opacity and permeability assessments of the cornea following exposure to a test item are considered individually and also combined to derive an In Vitro Irritancy Score, which is used to classify the irritancy level of the test item. This study was conducted in compliance with the OECD Principles of GLP (1998).

In this test, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability using an opacitometer and visible light (ELISA reader), respectively. These measurements are used to calculate an in vitro irritancy score (IVIS) to assign an in vitro irritancy hazard classification category of a test item.
Isolated corneas from eyes of freshly slaughtered cattle are used in this test. Corneal opacity is measured quantitatively, as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.
Irritation parameter:
cornea opacity score
Value:
ca. 1.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Value:
0.028
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
1.62
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The IVIS score, for the corneas treated with 750 μL test item (suspension) at 20% (w/v) concentration in corn oil, were found to be 1.62.
Based on results of this study, the classification for the test item is as follows:
Classification (OECD 437 UN GHS): No Category
Executive summary:

This study was conducted to evaluate theocular irritancy of test item,in compliance with the OECD437 for theBCOP test.

Four sets, each consisting of three corneas, were tested. The first set served as the control and was treated with750 μLnormal saline. The second set served as the positive control and was treated with750 μL20% (w/v) imidazole innormal saline. The third setwas treated with corn oil and served as vehicle control, and the fourth set was treated with750 μLtest item (suspension) at 20% (w/v) concentration in corn oil. Post application, corneas were incubated for approximately 4 h at32 ºC.At the end of incubation, opacity readings were taken.

The post opacity permeability reading was measured by applying1 mL of fluorescein sodium solution
(5 mg/mL)
on to the anterior surface of the cornea followed by incubation for approximately90 min at
32 ºC. At the end of incubation, the Optical Density (OD) was measured at 490 nm from fluid collected from the posterior chamber.

The mean in-vitro Irritancy Score (IVIS) of normal saline, corn oil and750 μL20% (w/v) imidazole innormal saline(positive control) treated corne as were found to be 1.54, 1.20 and 107.04,respectively. The IVIS score for the corneas treated with750 μLtest item (suspension) at 20% (w/v) concentration in corn oil was found to be 1.62.

Based on results of this study, the classification for the substance is as follows:

Classification (OECD 437 UN GHS):          No Category

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

Additional information

Justification for classification or non-classification