BAP

Endpoint summary

Description of key information

Skin irritation

Key study

The study was performed to the standardised guidelines OECD 439 and EU Test Method B. 46., under GLP conditions. The relative mean tissue viability of tissues treated withfor the test mterial, was 112.4%, greater than 50%, and therefore, the test item was classified as non-irritant to skin. The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) (Envigo, 2018).

Eye irritation

Key study

In a study performed to the standardized guidelines OECD 437 and EU Test Method B.47., under GLP conditions, 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. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). Based on the results of the BCOP test, no prediction of eye irritation can be made for the test material

(Envigo, 2018).

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 February 2018 to 22 August 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Purity: >99.5% (nominal); This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Tan paste to semi-solid

Test system:

human skin model

Source species:

human

Cell type:

other: non-transformed keratinocytes

Cell source:

other: not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

The EPISKIN™ Kit consisted of one EPISKIN™ plate containing 12 reconstructed human epidermis tissues (0.38 cm^2), maintenance medium (used for tissue pre-conditioning and testing purposes) and assay medium (used for MTT assay). The maintenance medium and assay medium were refrigerated at 1 to 10 ºC.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm^2) of the test material 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. 1.6 mL of the 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, incubated for 3 hours at 37 °C, 5% CO2 in air, and then placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix, 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, mixed thoroughly on a vortex mixer, and then were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the LabTech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL (26.3 µL/cm^2) of the test material 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

Run / experiment:

15 minute exposure period and 42 hours post exposure (mean value)

Value:

ca. 112.4

Vehicle controls validity:

not applicable

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 no effects on OD570 values that indicated the test item was totally rinsed off with no residual test item remained on or in the tissues. 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 solution containing the test item was a brown color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. 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 112.4% (>50%) after a 15-minute exposure period and 42-hour post-exposure incubation period. It was considered unnecessary to perform the inflammatory mediator IL-1α analysis as the results of the MTT test were unequivocal. The maintenance medium that was retained for possible analysis was discarded without evaluation.

 

Quality Criteria

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6% (≤18%). The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control (DPBS) treated tissues was 0.736, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 1.2% (≤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 17.0% (≤18%). The test item acceptance criterion was therefore satisfied.

 

Individual and Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.743	0.736	0.009	100.9	100*	1.2
	0.726			98.6
	0.74			100.5
Positive Control Item	0.019	0.02	0.004	2.6	2.7	0.6
	0.016			2.2
	0.024			3.3
Test Item	0.707	0.828	0.125	96	112.4	17
	0.957			130
	0.819			111.2

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability of tissues treated withfor the test item, was 112.4%, greater than 50%, and therefore, the test item was classified as non-irritant to skin. The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The study was performed to the standardised guidelines OECD 439 and EU Test Method B. 46., under GLP conditions.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing 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 into the testing to correct for this. A third set of controls was included, comprising non-viable tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm (OD570)

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 112.4% after the 15-minute exposure period and 42-hours post-exposure incubation period.

 

The relative mean viability of the tissues treated with the test item was 112.4%, greater than 50%, and therefore, the test item was classified as non-irritant to skin. 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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2018 to 26 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Purity: >95%
- Physical state/Appearance: Brown colored paste

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same 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 material 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:

3

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.

Selection of Corneas and Opacity Reading
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.

Treatment of Corneas
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

Run / experiment:

10 minute exposure period and 120 minutes post exposure

Value:

4.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test material.

Executive summary:

In a study performed to the standardized guidelines OECD 437 and EU Test Method B.47., under GLP conditions, the undiluted test item 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. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available