Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 22 August 2012 and 27 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 10-07-2012 Date of Signature: 28-12-2012

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: Liquid

Details on test material:

Sponsor's identification : Isostearamide DEA
Description : amber coloured liquid
Batch number : OE11124 (6/202593/00)
Purity : 100%
Date received : 14 May 2012
Expiry date : not supplied
Storage conditions : room temperature in the dark

Test animals

Species:

other: reconstructed human epidermis model

Strain:

other: reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: Not applicable

Controls:

no

Amount / concentration applied:

TEST ITEM

The test item was used as supplied.

Amount(s) applied (volume or weight with unit):
10 µl of the test item was applied to the epidermis surface.

Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
Area of exposure:
10 µl of the test item was applied to the epidermis surface.

PERCENTAGE COVERAGE:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

EXPOSURE TIME:
15 Minutes post exposure

TEST ITEM REMOVAL:
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.


SCORING SYSTEM
Quantitative MTT Assessment (percentage tissue viability):
For the test item the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test item / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

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

Mean tissue viability is ≤50% : Irritant

Mean tissue viability is >50% : Non-Irritant

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

106.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Direct MTT Reduction

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 no 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.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1. The relative mean viability of the test item treated tissues was 106.9% after a 15-Minute exposure period.

Quality Criteria

Positive Control: The relative mean tissue viability for the positive control treated tissues was 7.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.6%. The positive control acceptance criterion was therefore satisfied.

Negative Control: The mean OD₅₄₀ for the negative control treated tissues was 0.699 and the standard deviation value of the percentage viability was 5.0%. The negative control acceptance criterion was therefore satisfied.

Test Item: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.5%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD540 of tissues	Mean OD540 of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.659	0.699	0.035	94.3	100*	5.0
	0.719			102.9
	0.719			102.9
Positive Control Item	0.042	0.054	0.018	6.0	7.7	2.6
	0.075			10.7
	0.045			6.4
Test Item	0.690	0.747	0.052	98.7	106.9	7.5
	0.759			108.6
	0.792			113.3

SD=   Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test item was considered to be Non-Irritant.

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 colourimetric 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 is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: 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. 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-labelled 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-labelled 96-well plate. The optical density was measured at 540 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 106.9% after the 15 -Minute exposure period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test item was considered to be Non-Irritant.