Reaction mass of 1,4:3,6-dianhydro-D-glucitol monooleate and 1,4:3,6-dianhydro-D-glucitol dioleate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP compliant near guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

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:

1,4:3,6-dianhydro-D-glucitol monooleate

EC Number:

249-031-3

EC Name:

1,4:3,6-dianhydro-D-glucitol monooleate

Cas Number:

28454-96-8

Molecular formula:

C24H42O5

IUPAC Name:

1,4:3,6-dianhydro-2-O-oleoyl-D-glucitol

Test animals / tissue source

Species:

other: EpiOcular™ OCL-200 kit

Details on test animals or tissues and environmental conditions:

TEST MODEL
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: freeze-killed control tissues

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- For better handling the solid test substance was heated shortly at ca. 50 °C. Before application it was cooled down to room temperature.

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Details on study design:

EXPERIMENTAL PROCEDURE
- Direct MTT reduction: To assess the ability of the test material to directly reduce MTT a pre-test was performed. 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 (deionized 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 where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control.
- Basic procedure: Several test substances were tested in parallel within the present test using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. In addition, two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and post-incubation 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 pre-conditioned in the incubator at 37 °C. After 1 hour the pre-incubation medium was replaced with fresh medium and pre-conditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
- Pre-treatment 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: For better handling the solid test substance was heated shortly at ca. 50 °C. Before application it was cooled down to room temperature. 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 deionized water (NC, killed tissue control, NC KC) 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 post-incubation 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 pre-warmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes (liquids) 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 (post-incubation 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 ratio 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 was an irritant.
- Calculation of individual and mean optical densities: The corrected measured OD570 value for each individual tissue was 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 was 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) was 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 was 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 was greater than 0.1 it was 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. In case the mean net OD570 KC was smaller than 0.1 the effect of direct MTT reduction was considered to be negligible and no subtraction followed.
- Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
- 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 < 60% 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 ≤ 50% of the NC.

Results and discussion

In vitro

Results

Irritation parameter:

other: viability [% of NC]

Run / experiment:

30 minutes

Value:

94

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks on result:

other: mean OD570: 1.817

Other effects / acceptance of results:

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that Isosorbid Monooleate does not show an eye irritation potential under the test conditions chosen.

Any other information on results incl. tables

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:

GHS criteria not met