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Description of key information

Skin Irritation

Key study:

In a study performed to the standardised guideline OECD 439, under GLP conditions, the relative mean tissue viability for the test item was 86.1%, greater than 50%, and therefore, the test item was classified as non-irritant to skin.  The following classification criteria apply: The test item was not classified as a skin irritant according to the Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).

Eye Irritation

Key study:

In a study performed to the standardised guideline OECD 437, under GLP conditions, thein-vitro irritancy score of the test item was 1.9, based on this result, the test item in not an 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:
07 October 2017 to ****
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Purity: >93%
Physical state/Appearance: Dark amber viscous liquid
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis tissues
Cell source:
other: not specified
Details on test system:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker at room temperature for 15 minutes to homogenize the released mediators in the maintenance medium. Maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator IL-1α determination.

MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer and then refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Control samples:
other: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Amount/concentration applied:
10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Value:
86.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results.

 

Assessment of Color Interference with the MTT Endpoint

The test item did not change the color of the solution. However the test item was an amber viscous liquid which may have adhered to the tissue culture surface during the main test and thus cause color interference. Therefore, as a precaution, color correction tissues were incorporated into the test to correct for this possibility. However, the results obtained showed that negligible color interference occurred (Appendix 3). It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 86.1% (>50%) after a 15 Minute exposure period and 42 hour post exposure incubation period. Traces of residual test item remained on the test item treated tissue culture surfaces after rinsing due to viscosity.

 

Table 1: Mean Viabilities of the EPISKIN™ Tissues

Treatment

OD570
(Mean ± SD)

Relative Tissue Viability
(Mean ± SD)

Test Item

0.701 ± 0.044

86.1% ± 5.4%

DPBS (Negative Control)

0.814± 0.049

100%± 6.0%

0.5% SDS (Positive Control)

0.302 ± 0.044

37.1% ± 5.4%

 

Quality Criteria

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 37.1% relative to the negative control treated tissues and the standard deviation value of the viability was 5.4% (≤18%). The positive control acceptance criteria were therefore satisfied.

 

The mean OD570 for the negative control (DPBS) treated tissues was 0.814, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 6.0% (≤18%). The negative control acceptance criteria were therefore satisfied.

 

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.4% (≤18%). The test item acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).
Executive summary:

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model. Reconstructed human epidermal cultures were treated topically in triplicate with the test material for 15 minutes, rinsed, and then incubated post-exposure for 42 hours. Cell viability was measured by means of the colorimetric MTT reduction assay, in which viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan salt within the mitochondria of viable cells. 

The test item was found to directly reduce MTT; therefore additional non-viable tissues were incorporated for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint; therefore, as a precaution, additional tissues were incorporated to correct for this. A third set of controls was included, comprising non-viable tissues, to prevent a double correction for a colored test item that also reduces MTT.

At the end of the 42-hour post exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals. At the end of the formazan extraction period each tube was mixed thoroughly, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate, and the optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 86.1%, greater than 50%, and therefore, the test item was considered as non-irritant to skin and was not classified according to GHS. The quality criteria required for acceptance of results in the test were satisfied.

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:
09 October 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Physical state/Appearance: Dark amber viscous liquid
Purity: >93%
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Fresh eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics. They were transported to the test facility over ice packs on the day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the appropriate corneas
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
Preparation of Corneas
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling, and the iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red, plugged and incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects, only corneas free of damage were used.

Treatment of Corneas
The medium from both chambers of each holder was replaced with fresh complete EMEM and a pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the appropriate corneas and each holder was incubated at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The chamber was then refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated at 32 ± 1 ºC for 120 minutes. After incubation, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was removed and replaced with sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader and LT-com software.
Irritation parameter:
in vitro irritation score
Value:
1.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were clear post treatment and clear post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied

Test Item
The in vitro irritancy score of the test item was 1.9, based on this result, the test item in not an eye irritant.
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro irritancy score of the test item was 1.9. Based on this result, the test item in not an eye irritant.
Executive summary:

The Bovine Corneal Opacity and Permeability (BCOP) test was performed to assess the potential of the test material to induce serious eye damage and to identify those not requiring classification for eye irritation or serious eye damage.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) control items were tested concurrently. Quantitative measurements of changes in corneal opacity (assessed as decreased light transmission through the cornea) and permeability (assessed as increased passage of sodium fluorescein dye through the cornea) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The In Vitro irritancy scores are summarized as follows:

Test material: 1.9

Negative Control: 0.8

Positive Control: 43.1

The corneas treated with the test item were clear post treatment and clear post incubation with an IVIS of 1.9, which did not meet classification criteria (≤ 3; No Category; Not classified for irritation).

The corneas treated with the negative control item were clear post treatment and post incubation. The negative control gave opacity of ≤3.0, permeability of ≤0.077, and met acceptance criteria.

The corneas treated with the positive control item were cloudy post treatment and post incubation. The IVIS was within the historical range of 31.6 to 58.7 and satisfied the acceptance criterion.

Based on the results of the BCOP test, the test material is not classified for serious eye damage or eye irritation according to GHS criteria.

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

Additional information

Justification for classification or non-classification