Reaction products of fatty acids, tall oil and fatty acids, C18 unsaturated, trimers and fatty acids, C18 unsaturated, dimers with (9Z)-octadec-9-en-1-amine

Endpoint summary

Description of key information

Skin irritation/corrosion in vitro: irritating (BASF 2013, GLP, OECD 431 + 439)
Eye corrosion in vitro (BCOP): not corrosive (BASF 2013, GLP, OECD 437)
Eye irritation in vitro (EpiOcular): not irritating (BASF 2013, 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 to ensure homogenous distribution 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
- OD570 values for the test substance had to be corrected because of its ability to directly reduce MTT. For this, a freeze killed tissue sample each was treated with the test substance or the negative control in the same way as all other samples.

Irritation / corrosion parameter:

% tissue viability

Remarks:

irritation test

Run / experiment:

exposure period of 1 hour with about 42 hours post-incubation

Value:

6

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrosion test

Run / experiment:

exposure period of 3 minutes or 1 hour

Value:

94

Interpretation of results:

irritating

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
Since the test substance is able to reduce MTT directly, a freeze killed tissue sample each was treated with the test substance or the negative control in the same way as all other tissue samples. However, a difference below 0.1 in MTT reduction between test sample and negative control was obtained in this experiment. Thus, the results were not used for correction of the OD750 values obtained in the viable cultures.

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

Run / experiment:

mean (2.5 h)

Value:

83

Remarks on result:

other: not irritating

Remarks:

value: 83 %

Interpretation of results:

not irritating

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 the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine 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 2013). 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. Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes or 1 hour was 94%. 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 6%.

Based on the observed results the test substance shows a skin irritation, but no corrosion potential in the EpiDerm™ skin corrosion/irritation test.

Eye

To assess if the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine 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 0.5 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 the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine (EpiOcular, BASF 2013). 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%. Since the mean viability of tissue treated with the test substance was 83%, 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 the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine, 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 the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine 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.