Fatty acids, C14-18 and C16-18-unsatd., maleated, reaction products with oleylamine

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:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation: 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:

An error in the cited SOP was not adopted. Instead, correctly, 10 mg test substance + 90 μL water were used for checking the coloring potential of the test substance

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Test system

Amount / concentration applied:

Types of Treatment in the Main Test
- Negative control: Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium, dose volume 10 µl/tissue sample.
- Test Substance: WS400107, dose 10 µl/tissue sample.
- Positive control: 5% Sodium Dodecyl Sulphate (SDS) in distilled water, dose volume 10 µl/tissue sample.

Duration of treatment / exposure:

15 ± 0.5 minutes with the test substance, negative or positive control at room temperature

Details on study design:

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. 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 intrinsic colour and the colouring potential of the test substance, WS400107, in contact with water did not interfere with the assay. However, direct reduction of MTT by WS400107 itself without the involvement of mitochondrial dehydrogenase activity could not be ruled out, as there was a change of colour in a WS4000107/MTT solution during the pre-test. Therefore in the main test, water killed tissues (which have no metabolic activity but absorb and bind the test substance like viable tissues) were included in addition to the live tissues. In addition, the pH of the test substance was estimatedto be approximately 7.0 using pH indicator paper.

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 test substance treated and untreated water killed tissues, each in triplicate, were included in the main test and processed further in a similar manner as the live tissue samples.

Tissue Processing and Quantitative Determination of Cell Viability
Incubation of the tissues prior to treatment in maintenance medium:
- Live tissue samples: ≥ 24 h at 37 ± 2° C in humidified atmosphere of 5% CO2 in air
- Thawed, water killed tissues: ≥ 1 h at room temperature

Positive and negative control preparations and the test substance were dispensed over each assigned tissue sample using a positive displacement pipette and 7 minutes afterwards positive controls were re-spread with a curved flat spatula. Test material was spread over the tissue thus making contact with the whole surface of the tissue.

After the 15 ± 0.5 minutes treatment period, residual test substance or positive control substance was removed by rinsing each tissue with 25 mL sterile DPBS. Inserts were blotted on absorbent paper to remove remaining DPBS.

This was followed by incubation of each insert at 37 ± 2° C in humidified atmosphere of 5% CO2 first for 42 ± 1 h in wells each containing 2 mL maintenance medium and then for 3 hours ± 5 minutes in wells each containing 2 mL of 0.3 mg/mL MTT. Finally the tissue samples were processed further and formazan was extracted by vortexing and storage in acidic isopropanol, 500 µL/sample, at 2-8°C in the dark over a minimum of 70 hours. The absorbance was quantitatively determined at 540 nm with acidified isopropanol (0.04 N HCl final concentration) as a blank.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Negative Control Tissue viability as % of OD of negative controls

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and ≥70 h formazan extraction. Max. score: 100.0. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: WS400107 treated tissue viability as % of OD of negative controls

Value:

112.7

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and ≥70 h formazan extraction. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks and of water killed tissues has been accounted for by subtraction. Therefore, a maximum possible score is not applicable.. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Positive Control Tissue viability as % of OD of negative controls

Value:

12.8

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and ≥70 h formazan extraction. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. An accurate maximum possible score is not applicable.. (migrated information)

In vivo

Irritant / corrosive response data:

See Table 1

Other effects:

In a pre-test, direct reduction of MTT by WS400107 itself without the involvement of mitochondrial dehydrogenase activity was disclosed. This was accounted for in the main test by inclusion of water killed tissues (which have no metabolic activity but absorb and bind the test substance like viable tissues) in addition to the live tissues.

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	0.725	0.689	0.698	0.704 ± 0.019	100.0 ± 2.7
WS400107	0.701	0.756	0.740	0.732 ± 0.028	112.7 ± 4.0**
Positive Control	0.043	0.090	0.138	0.090 ± 0.047	12.8 ± 6.7

 

*  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.) = 0.138 ± 0.004

 

** Derived from “true MTT reduction” OD 540 values, thus accounting for possible direct reduction of MTT by the test substance

 

 

Hence assay validity was confirmed both, for the negative and the positive controls, and the test material, WS400107, proved to be not irritating.

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: criteria specified in the OECD 439 test guideline.