Reaction mass of docosyl acrylate and octadecyl acrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-18 to 2011-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

April 24, 2002

Deviations:

no

Principles of method if other than guideline:

no official national or international guidelines for the EpiOcularTM test:
- 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)

Remarks:

BASF SE

Test material

Test material information

Constituent 1

Reference substance name:

Behenylacrylate (Acrylate 22 45%)

IUPAC Name:

Behenylacrylate (Acrylate 22 45%)

Constituent 2

Reference substance name:

2-Propenoic acid, C18-26-alkyl esters

EC Number:

285-348-3

EC Name:

2-Propenoic acid, C18-26-alkyl esters

Cas Number:

85085-17-2

Test material form:

other: solid

Test animals / tissue source

Species:

human

Strain:

other: Three dimensional human cornea model

Details on test animals or tissues and environmental conditions:

Test system: 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)

Controls:

other: Negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

90 min

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- 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 (highly 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.

- 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 solids was applied.

- Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value.
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.

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test-substance treated tissues was 96%. Behenylacrylate (Acrylate 22 45%) does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Test substance		Tissue 1	Tissue 2	Mean KC	mean	Inter-tissue variability [%]
NC	Mean OD570	1.334	1.373	0.025	1.353
	Viability [% of NC]	98.6	101.4	-	100	2.9
11/0546-1	Mean OD570	1.299	1.299	0.029	1.299
	Viability [% of NC]	96.0	96.0	-	96	0.0
PC	Mean OD570	0.135	0.118	-	0.126
	Viability [% of NC]	10.0	8.7	-	9	1.3

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for eye irritation according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritatio potential in the EpiOcular eye irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of the test substance to a reconstructed three dimensional human cornea model (EpiOcular).

Because the waxy test substance could not be applied with a pipette, a metal pin was covered with about 50 mg of the undiluted test substance.

Two EpiOcular tissue samples were incubated with the test substance for 90 minutes followed by a 18 -hours post-incubation period.

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 test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular eye irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test substance treated tissues was 96 %.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular eye irritation test under the test conditions chosen.