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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

One in-vitro key study is available

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed 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 VitroSkin 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 was106.9% after the 15 -Minute exposure period.

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

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

Eye Irritation:

BCOP Assay - In vitro

Introduction. A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”

Method. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS). 

Results. Thein vitroIrritancy scores are summarised as follows:

Treatment	In VitroIrritancy Score
Test Item	1.0
Negative Control	1.5
Positive Control	33.4

Conclusion. The test item was considered not to be an ocular corrosive or severe irritant. In Vivo Rabbit Assay

Introduction. The study was performed to assess the irritancy potential of the test item following a single application to the rabbit eye, after an in vitro Bovine Corneal Opacity and Permeability study showed the test item not to be corrosive or severely irritating to the eye.The method was designed to be compatible with the following;

OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002)

Method B5 Acute Toxicity (Eye Irritation) of Commissipn Regulation (EC) No. 440/2008

Result. A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 48 Hour observation and the other treated eye appeared normal at the 72 Hour observation.

Conclusion. The test item produced a maximum group mean score of9.0and was classified as amild irritant (Class4on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. It is not classified by the OECD GHS guidelines.

Conclusion for eye irritation

It may therefore be concluded that Isostearamide DEA is not an eye irritant based on the mean scores from a GLP, guideline in-vivo study in rabbits and an in vitro BCOP assay.

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
1 in vitro BCOP assay and 1 in vivo assay were both negative for eye irritation.

Justification for classification or non-classification

Skin irritation:

In accordance with EU CLP (Regulation (EC) No. 1272/2008), classification is not required for skin irritation based on the available data.

Eye irritation:

In accordance with EU CLP (Regulation (EC) No. 1272/2008), classification is not required for eye irritation based on the available data.