Fatty acids, C18-unsatd., dimers, ethoxylated

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: Three-dimensional non-keratinized tissue: EpiOcularTM model (OCL-200)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-24 to 2012-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and reliable method compliant study.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

According to method 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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, ethoxylated

EC Number:

614-587-1

Cas Number:

68551-92-8

Molecular formula:

Unspecified

IUPAC Name:

Fatty acids, C18-unsatd., dimers, ethoxylated

Test material form:

other: liquid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

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.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μL
- Concentration: Undiluted

Duration of treatment / exposure:

The tissues were incubated at standard culture conditions for 30 minutes.

Observation period (in vivo):

Subsequently, the tissues were incubated at standard culture conditions for 2 hours (liquids) (postincubation period).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek Corp., Ashland MA, USA and Biochrom, Germany)
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.
- Time after start of exposure: 30 minutes

Controls
Negative control (NC): De-ionized water, sterile
Positive control (PC): Methyl acetate (98+%, CAS No.: 79-20-9, Merck KGaA, Germany)
MTT reduction control (KC): De-ionized water, sterile or test substance

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.

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.

Principle of Data Evaluation
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.

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

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Tissue Viability [% of NC]

Basis:

other: tissue 1

Time point:

other: 30 min

Score:

98.7

Remarks on result:

other: Inter-tissue variability 5.9%

Results 2

Irritation parameter:

other: Tissue Viability [% of NC]

Basis:

other: tissue 2

Time point:

other: 30 min

Score:

104.6

Remarks on result:

other: Inter-tissue variability 5.9%

Irritant / corrosive response data:

Negative Control Viability [% of NC]: 100
Positive Control Viability [% of NC]: 27

Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel. However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information