Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 September 2012 to 04 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

EC Number:

309-629-8

EC Name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Cas Number:

100545-48-0

Molecular formula:

No discrete molecular formula available for this UVCB substance

IUPAC Name:

Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Species: human reconstructed epidermis (tissues).
- Supplier: SkinEthic Laboratories, Lyon, France.
- Selection: at receipt, the medium quality and temperature indicators were checked to ensure the good quality of the tissues before use.
- Storage conditions: the living tissues were kept at room temperature from receipt until the end of the in vitro phase.
- Description: the EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstituted from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

REMOVAL OF TEST SUBSTANCE
For all treated tissues (test item-treated, positive and negative controls), at the end of the designated incubation period, each tissue insert was removed from the well of the treatment plate and rinsed with Phosphate Buffered Saline Dulbeccos (PBS).
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL PBS to gently remove any residual dosage form. As the dose formulation adhered to the walls of the compartment, it was eliminated using a cotton bud.

POSITIVE CONTROL
Name: glacial acetic acid.

NEGATIVE CONTROL
Name: 0.9% NaCl.

SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm:
Relative mean viability (%) = 100 x mean OD(test item) / mean OD(negative control)

DECISION CRITERIA
see table 7.3.1/1 in Any Other Information on Materials and Methods

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg +/- 2 mg per tissue.
The test item was spread over the whole tissue surface without damaging the tissue sample.
As the test item was a solid, 100 µL of 0.9% NaCl were applied over the test item to ensure good contact with the epidermis.

Duration of treatment / exposure:

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes (duplicate tissues were treated with the positive
and negative control materials for an exposure period of 240 minutes).

Duration of post-treatment incubation (if applicable):

MTT-loading after a 3h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 3 minutes

Value:

86

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 60 minutes

Value:

79

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure duration: 240 minutes

Value:

104

Any other information on results incl. tables

Following the 3, 60 and 240 minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue. 

The relative mean tissue viability for the positive control treated tissues was 3% relative to the negative control treated tissues following the

240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

The mean OD value for the negative control was 0.232. The negative control acceptance criterion was therefore satisfied.

Table 7.3.1/2 Mean OD Values and viabilities for the negative and positive controls and the test item

Group	Exposure duration	OD570 nm		Viability (%)
		Mean	SD	Mean	SD
Negative control	240 min	0.232	0.000	100	0
Positive control	240 min	0.006	0.001	3	0
Test item	3 min	0.199	0.002	86	1
	60 min	0.184	0.010	79	4
	240 min	0.241	0.002	104	1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the in vitro study, the test substance was considered to be non-corrosive to the skin.

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test substance using the Episkin reconstituted skin model. This study was conducted in compliance with OECD Guideline No. 431 and the principles of Good Laboratory Practice.

A preliminary test was performed to detect the ability of the test item to directly reduce MTT. Following the preliminary test, the skin corrosion potential of the test item was tested in the main test. The test item, and both negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 240 minutes (± 5 minutes); test item for 3 minutes (± 5 seconds), 60 minutes (± 5 minutes) and 240 minutes (± 5 minutes). At the end of the designated incubation periods, each tissue was rinsed with phosphate Buffered Saline Dulbeccos. The cell viability was then assessed by means of the colourimetric MTT reduction assay and relative viability (%) calculated for each tissue relative to control tissues.

In the preliminary assay, the MTT solution colour did not turn blue/purple when compared to the negative control. The test item was presumed not to directly reduce MTT.

In the main test, the blue discolouration of the test item-treated tissues following the 3 , 60 and 240 minute exposure periods was representative of viable tissue. The relative mean viabilities of the test item-treated tissues were 86% , 79% and 104% of the negative control for the 3, 60 and 240 minutes exposure periods respectively.

The test item was considered to be non corrosive to the skin.