Reaction products resulting from esterification of sucrose with saturated C16-18 (even numbered) fatty acids

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Sept 2016 - 28 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products resulting from esterification of sucrose with saturated C16-18 (even numbered) fatty acids

EC Number:

947-474-3

Molecular formula:

not available

IUPAC Name:

Reaction products resulting from esterification of sucrose with saturated C16-18 (even numbered) fatty acids

Test material form:

solid

Details on test material:

- Name of test material: Reaction products resulting from esterification of sucrose with saturated C16-18 (even numbered) fatty acids
- Physical state: beige to brown solid
- Analytical purity: 83.1%
- Storage condition of test material: The sample of the test item was stored at ambient temperature without exposure to light in a tightly sealed container, according to recommendations of the sponsor.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0012661240
- Test-substance No.: 16/0236-1
- Purity test date: 84.1% UVCB, 13.2% Cetyl Palmitate
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

TISSUE MODEL
- Tissue model: OCL-200 (EpiOcularTM model)
- Tissue Lot Number: 21588 (test run 1) and 21593 (test run 2)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION) CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 5% humidity
- Spectrophotometer: SunriseTM Absorbance Reader for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiOcular™ OCL-200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Containing: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm² cultured in Millicells® (1 cm diameter).
- Assay medium: OCL-200-ASY assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

- Using a sharp spoon, a bulk volume of ca. 50 μL of the test material 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).

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

TISSUE PREPARATIONS
- 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 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
- 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.
- After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

DIRECT MTT REDUCTION
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) 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 3 hours. 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 where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.

BASIC PROCEDURE
- Several test substances were tested in parallel within the present tests using the same control tissues (NC and PC) within the respective test. Two tissues were treated with each, the test substance, the PC and the NC.
- There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical state of the test substance the protocol for solids 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 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

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.

POST TREATMENT
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 25 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.

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 ofthe 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.

METHODS FOR MEASURED ENDPOINTS
- 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.
- Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
- Barrier funciton and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 μL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline. Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
- Acceptance criteria 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 > 0.8. The mean OD570 of the NC should not exceed 2.5.
- Acceptance citeria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

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 60%.

A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests. See 'Any other information on materials and methods incl. tables'

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: % viability [% of NC]

Run / experiment:

1

Value:

106.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Irritation parameter:

other: % viability [% of NC]

Run / experiment:

2

Value:

100.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not eye irritating