Addition reaction products of conjugated sunflower-oil fatty acids and tall-oil fatty acids with maleic anhydride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Reconstructed Human Epidermis Test Method of 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test 15min - 42 hours, Standard Operating Procedure: February 2009 Version 1.8.

Deviations:

yes

Remarks:

Dose volume per tissue sample was at least 50 µL (132 µL/cm2) instead of 10 µL to adequately cover the entire epidermis surface

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

In vitro test system

Test system:

human skin model

Remarks:

Episkin

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System
EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes and forming a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum (matrix: collagen type 1 coated with type IV collagen).

Principle of the Test – Main Test
Irritant substances are sufficiently cytotoxic to cause cell deaths in the cell layers. Therefore, cell viability of the multilayers was determined by measurement of mitochondrial dehydrogenase activity assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, blue coloured, formazan salt. The degree of formazan salt formation (positively correlated with the degree of cell viability) was measured photometrically (i.e. determination of the optical density of formazan extracts from tissue at 540 nm).

Depending on the percentage of tissue viability attained (compared to negative control viability) a test substance is classified as skin irritating or not skin irritating.

Pre-Tests – Checking for Interference of the Test Substance with the Assay
It was demonstrated, that the test material WS400104 itself did not interact with MTT, i.e. possible false estimation of tissue viability caused by direct interaction of WS400104 with MTT could be ruled out. However, intrinsic colour of WS400104 was evident. Therefore, non specific colour % (NSC%) was determined using one additional yyyyyliveyyyyy control tissue (treated and processed as test material treated tissue but incubated with fresh assay medium instead of MTT) and accounted for when calculating the tissue viability of the test item treated tissue of the main test.

Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 live (viable) tissue samples placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. In addition the above oneyyyyyliveyyyyy control tissue was included in the main test for determination of non specific colour % (NSC%) induced by the test material.
Incubation of these tissues before treatment in maintenance medium: ≥ 24 h at 37°C, 5% CO2 in air, in humidified atmosphere (>95% r.h.), in wells
each containing 2 mL fresh pre-warmed maintenance medium.
Test material administration: Spreading of thin even layer over the epidermal surface.
Termination of 15 ± 0.5 minute exposure period: Removal of residual test material or positive control substance by thorough
rinsing of each epidermis unit with phosphate buffered saline 1x solution (0.9%)
Removal of remaining PBS by use of a Pasteur pipette linked to a vacuum.
Posttreatment incubation (all tissue samples) : 42 ± 1 h at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL fresh pre-warmed maintenance medium
Then MTT incubation (all except NSC sample): 3 hours (± 5 minutes) at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL of 0.3 mg/mL MTT.
NSC sample: 3 hours (± 5 minutes) at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL fresh pre-warmed maintenance medium.
Formazan extraction (all tissue samples) : Further processing of tissue samples & formazan extraction by vortexing in
acidic isopropanol, 500 µL/sample and then storing the vortexed samples with
gentle agitation at room temperature in the dark for ca. 3 hours.
Qantitative determination of optical density: At 540 nm with acidified isopropanol (0.04 N HCl final concentration,
6 x 200 µL) as blanks.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

14

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

0.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

2.3

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

  

Tabelle 1:  Results of in vitro EpiSkin (TM) Skin Irritation Test
Exposure Period: 15 ± 0.5 minutes
	OD 540* Tissue 1	OD 540* Tissue 2	OD 540* Tissue 3	OD 540 Mean of Tissue 1, 2 + 3	Tissue Viability [% of Negative Control ± s.d.]
Negative Control	1.048	0.902	1.014	0.988	100 ± 7.94
WS400104	0.331	0.191	0.212	0.056**	5.5 ± 7.44**
Positive Control	0.226	0.171	0.130	0.176	18 ± 5.03

 

*  OD 540 of individual tissues = Mean Optical Density [wavelength 540 nm] of 2 measurements minus Mean OD of six blanks

   Mean OD of six blanks ± standard deviation (s.d.) = yyyy ± yyyyy

 

** Derived from “true” OD 540 values, the direct colouring potential of WS400104 has already been accounted for.

  OD of Non Specific Colour (NSC) tissue sample = 0.189, NSC% = 19%

Hence assay validity was confirmed both, for the negative and the positive controls, and the test material, WS400104, was irritating.

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant)