Fatty acids, C18-unsatd., dimers, compds. with coco alkylamines

Endpoint summary

Description of key information

Skin irritation/corrosion in vitro: irritating (BASF 2012, GLP, OECD 431 + 439)
Eye corrosion in vitro (BCOP): not corrosive (BASF 2012, GLP, OECD 437)
Eye irritation in vitro (EpiOcular): not irritating (BASF 2012, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 2004

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2010

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

adopted May 2008

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2009

GLP compliance:

yes (incl. QA statement)

Species:

other: human-derived keratinocyte cell line

Details on test animals or test system and environmental conditions:

Three dimensional human epidermis model Epi-200
- Source: MatTek in vitro Life Science Laboratories, Bratislava, Slovakia
- Culture Medium: DMEM
- Cell death detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma,Germany)
- duplicate cultures (corrosion test), triplicate cultures (irritation test)

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative controls: De-ionized water (corrosion test), PBS (irritation test); Positive controls: 8-n potassium hydroxide solution (corrosion test), 5% (w/v) sodium dodecyl sulfate (irritation test)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µl (corrosion test), 30µl (irritation test)
- A nylon mesh was used to ensure homogenous distribution of positive and negative control substances, but was not required for the test substance

Duration of treatment / exposure:

Corrosion test: 3min (room temperature), 1h (incubator)
Irritation test: 25 min (room temperature) + 35 min (incubator) = 1h overall exposure

Details on study design:

Washing
- with PBS at the end of exposure

Post-incubation period: app. 42h, only irritation test

MTT incubation
- Time: after removal of the test substance (corrosion test) or after the post-incubation period (irritation test) for 3h
- Formazan detection: extraction using isopropanol, measurement of optical density at 570nm

Irritation parameter:

other: cell survival

Time point:

other: 3min

Remarks on result:

other: 86% (not corrosive)

Irritation parameter:

other: cell survival

Time point:

other: 1h

Remarks on result:

other: 88% (not corrosive)

Irritation parameter:

other: cell survival irritation

Basis:

mean

Time point:

other: 1h

Remarks on result:

other: 5% (irritating)

The test substance is not able to reduce MTT directly.

Interpretation of results:

irritating

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye 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: GLP study meeting generally accepted scientific standards and documented in detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The present test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. After application of 50µl of the test material to the surface of the three dimensional human cornea model "EpiOcular" for 30minutes, the test substance is removed. After a 2h post-exposure period, the induced cytotoxicity (= loss of viability) is measured as the reduction of mitochondrial dehydrogenase activity using MTT. A chemical is considered as irritant, if the mean viability of smaller or equal 50%, and as not-irritant, if the mean viability is above 60%. In the range between 50% and 60% no prediction can be made.

Though there are no official national or international guidelines for the EpiOcularTM test yet, the study was performed according to the methods described in the following publications:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.
In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, April 24, 2002 (“Acute Eye Irritation/Corrosion”)

GLP compliance:

yes (incl. QA statement)

Species:

other: human cornea model

Details on test animals or tissues and environmental conditions:

Three dimensional human cornea model EpiOcular OCL-200
- Source: MatTek corp., Ashland MA, USA
- Culture Medium: DMEM
- Cell death detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek corp., Ashland MA, USA / Sigma,Germany)
- duplicate cultures
The test substance is not able to reduce MTT directly.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

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

Duration of treatment / exposure:

30min

Details on study design:

Negative control: De-ionized water
Positive control: Methyl acetate (98%+)

Incubation times:
Pre-incubation in culture medium: 16-24h
Pre-treatment with PBS to wet the tissue surfact for 30min
Exposure time: 30min
Post-incubation period: 2h (after washing)
MTT incubation: 3h

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with PBS and medium subsequently at the end of the exposure time. After soaking for 12minutes in medium, the tissues were dried on absorbant paper.

Irritation parameter:

other: cell viability

Basis:

mean

Remarks on result:

other: 65% (not irritating)

Interpretation of results:

not irritating

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The potential of Fatty acids, C18-unsatd., dimers, compds. with coco alkylamines to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™) (BASF 2012). Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 86%, and it was 88% after an exposure period of 1 hour. Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 5%.

Based on the observed results, Fatty acids, C18-unsatd., dimers, compds. with coco alkylamines shows a skin irritation, but no skin corrosion potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Eye

To assess if Fatty acids, C18-unsatd., dimers, compds. with coco alkylamines causes damage to the eyes, 750µL of the undiluted test substance were applied to the epithelial surface of three isolated bovine corneas per group and incubated for 10 minutes, followed by a 2h post-incubation period. Corneal opacity was measured quantitatively as the amount of light transmission through the

cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance relative to the control corneas. An IVIS value of 6.9 was observed, which is well below the threshold for corrosivity (55), thus it is concluded, that the test substance does not cause damage to the eyes.

Since the evaluation of the BCOP cannot differentiate between irritant and non-irritant substances, a second in vitro test was performed to dermine the eye irritation potential of Fatty acids, C18 -unsatd., dimers, compds. with coco alkylamines (EpiOcular, BASF 2012). 50µl of the undiluted test substance were applied to duplicate tissue samples of a reconstructed three dimensional human cornea model, which were incubated for 30min, followed by a 2h post-incubation period. Cell viability was determined as reduction in mitochondrial dehydrogenase activity using MTT. A substance is considered irritating to the eye, if the relative viability compared to vehicle controls is below 50%. The mean viability of tissue treated with the test substance was 65%, thus the substance is not considered to be irritating to the eye.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

The in vitro test for skin irritation /corrosion showed an irritating potential of Fatty acids, C18-unsatd., dimers, compds. with coco alkylamines, but excluded corrosive properties. Both in vitro assays regarding eye corrosion or irritation led to negative results, i.e., the test substance is not irritant to the eye.

Based on these results fatty acids, C18 -unsatd., dimers, compds. with coco alkylamines is not required to be classified for eye irritation/corrosion, but requires classification with R38 according to 67/548/EEC and as a skin irritant cat.2 according to CLP/EU-GHS requirements.