Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

There are no official national or international guidelines for the EpiOcularTM test yet; however, 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)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with N-(2-aminoethyl)ethane-1,2- diamine

IUPAC Name:

Oleic acid, compound with N-(2-aminoethyl)ethane-1,2- diamine

Details on test material:

- Name of test substance: Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine
- Test-substance No.: 11/0603-1
- Batch identification: 11000829U0
- Purity: Reaction product of Oleic acid and N-(2-aminoethyl)ethane-1,2-diamine:
Elemental-analysis yielded 100.4 g/100g
"Triamide"-content: 5.2 g/100g (HPLC analysis)
(for details see analytical report No. 11L00415)
- Homogeneity: The test substance was homogeneous by visual inspection. In addition the homogeneity was provided after shaking the test substance container.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- pH-value: Ca. 5 (undiluted test substance)
- Physical state: Liquid, viscous

Test animals / tissue source

Species:

other: EpiOcular™ OCL-200 kit

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek Corp., Ashland MA, USA

Three dimensional human cornea model
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: historical control values

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the undiluted liquid test substance

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

2 hours

Details on study design:

EXPERIMENTAL PROCEDURE
Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure”, additionally.

Basic procedure:
Two tissues were treated with the test substance, the PC and NC, respectively. In addition two killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for liquids was applied.

Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 – 24 hours (pre-incubation).

Pretreatment of the tissues:
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance:
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC) or test substance (killed tissue control, KC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).

MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation:
Principle:
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities:
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.

Application of measurements using killed control tissues:
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the mean OD570 KC of the NC from the mean OD570 KC of the test substance. In case the mean net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

Tissue viability:
The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA:
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.

Assay acceptance criterion for the NC:
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Acceptance criteria for the PC:
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

Assay acceptance criterion for tissue variability:
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

Acceptance criteria for the KC:
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT-reduction of a test substance should be ≤ 30% of the NC.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

Mean tissue viability
(% of negative control) Prediction
≤ 50 irritant
> 50 ≤ 60 no prediction
> 60 non-irritant

Results and discussion

In vivo

Results

Irritation parameter:

other: viability (% of Negative Control)

Basis:

mean

Score:

38

Remarks on result:

other: Mean OD570: 0.854; Mean tissue viability (% of negative control) Prediction: ≤ 50 irritant; > 50 ≤ 60 no prediction; > 60 non-irritant

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU