Soybean oil, maleated, ester with triethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

EPISKIN TM reconstructed human epidermis model

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 21 September 2016 Experimental completion date 26 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, maleated, ester with triethanolamine

EC Number:

285-107-2

EC Name:

Soybean oil, maleated, ester with triethanolamine

Cas Number:

85029-82-9

Molecular formula:

C63H113NO12 to C67H123NO12

IUPAC Name:

Soybean oil, maleated, ester with triethanolamine

Test material form:

other: Clear amber viscous liquid

Details on test material:

100% product
Physical state/Appearance: Clear amber viscous liquid
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

Identification: Test item
Physical state/Appearance: Clear amber viscous liquid
Purity: 100% wt - (UVCB)
Expiry Date: 24 June 2018
Storage Conditions: Room temperature, in the dark

In vitro test system

Test system:

human skin model

Source species:

other: Reconstructed Human Epidermis

Cell type:

other: Reconstructed Human Epidermis

Cell source:

other: Reconstructed Human Epidermis

Source strain:

other: SkinEthic Laboratories, Lyon, France

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit

Supplier: SkinEthic Laboratories, Lyon, France
Date received: 20 September 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-038
Maintenance Medium lot number: 16-MAIN3-065
Assay Medium lot number: 16-ESSC-041

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was used as supplied.

Details on test system:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.
The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.

Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

Pre-incubation (Day 0: Tissue Arrival)

Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test

Application of Test Item and Rinsing (Day 1)

2 mL of maintenance medium, warmed to approximately 37 C, was pipetted into the second column of 3 wells of the 12-well plate.
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/cm2) 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. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). 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++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. 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 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 determination.
2 mL of a 0.3 mg/mL 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 using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of 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. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

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 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Duration of treatment / exposure:

6 days

Duration of post-treatment incubation (if applicable):

Overnight.

Number of replicates:

3

Test animals

Species:

other: Reconstructed Human Epidermis

Strain:

other: Reconstructed Human Epidermis

Test system

Type of coverage:

other: Reconstructed Human Epidermis

Preparation of test site:

other: Reconstructed Human Epidermis

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: cell viability (obtained from optical density)

Value:

85.8

Remarks on result:

other: The relative mean viability of the test item treated tissues was 85.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Other effects / acceptance of results:

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

The solution containing the test item was a very pale yellow color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

Any other information on results incl. tables

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the appendix. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in the appendix.

The relative mean viability of the test item treated tissues was 85.8% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1 Alpha analysis as the results of the MTT test were unequivocal.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2%. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 1.028 and the standard deviation value of the viability was 3.9%. 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 6.3%. The test item acceptance criterion was therefore satisfied.

Appendix 1     Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD562of tissues	Mean OD562of triplicate tissues	±SDofOD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.981	1.028	0.041	95.5	100*	3.9
	1.048			102.0
	1.054			102.6
Positive Control Item	0.083	0.088	0.022	8.1	8.5	2.2
	0.068			6.6
	0.112			10.9
Test Item	0.850	0.882	0.065	82.7	85.8	6.3
	0.840			81.7
	0.957			93.1

 

OD=     Optical Density SD=    Standard deviation

*=         The mean viability of the negative control tissues is set at100%

 

 

Appendix 2     Classification Criteria

 

Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42 ± 1 hour post-exposure incubation period according to the following table:

 

Criteria for in vitro interpretation	Prediction	EU CLP (Regulation (EC) No 1272/2008)	UN GHS
Relative mean tissue viability is ≤50%	Irritant	H315 Category 2	H315 Category 2
Relative mean tissue viability is >50%	Non-irritant	Not classified for irritation	Not classified or UN GHS Category 3 can not be determined

 

 

EU CLP/UN GHS:

 

Hazard - Code H315; Category 2; Statement “Causes Skin Irritation”

Applicant's summary and conclusion

Interpretation of results:

other: Not classified for skin irritation

Conclusions:

Relative to the concurrently treated negative control the viability of the test item treated tissues was 85.8%.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. 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 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 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 85.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied. Not classified for skin irritation

Conclusion

Relative to the concurrently treated negative control the viability of the test item treated tissues was 85.8%.