beta-D-fructofuranosyl alpha-D-glucopyranoside octa(acetate and palmitate and stearate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

RhCE test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 21, 2017 - Nov. 6, 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:

adopted: July 28, 2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sucrose tetrastearate triacetate

IUPAC Name:

Sucrose tetrastearate triacetate

Test animals / tissue source

Species:

human

Strain:

other: Construct of normal human epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ human cell construct for eye irritation testing is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek Corporation). It models the corneal epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. In this assay, the test item is applied to the surface of the corneal epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Two construct tissues (replicates) are used for each test treatment and each control group; a 6-h exposure with an 18 hour incubation post-exposure. Relative tissue viability post-exposure is determined against the negative control-treated constructs by evaluating the reduction of MTT to a formazan product, determined spectrophotometrically (optical density). A concurrent positive control is used with each assay to determine validity of the test. Based on the "depth of injury model," the EpiOcular eye irritation test (EIT) is intended to differentiate those materials that are nonirritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as either GHS eye irritant category 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 20993) was obtained from MatTek Corporation, and tested by MatTek for potential bilogical contaminants, sterility, barrier function and tissue viability, results for which were within acceptable ranges and indicated appropriate formation of the mucosal barrier and a viable basal cell layer.

- Environmental conditions
Storage conditions: EpiOcular tissures and the medium were stored in 1 °C to 10 °C
Culture condition: In CO2 incubator (MCO-18AIC, SANYO Electirc); temperature 37 °C; under humid condition; 5% CO2 concentration

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied: 50 μL
- Lot/batch no.: K7B70

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Lot/batch no.: 090717ALA

Duration of treatment / exposure:

6 hours ± 15 minutes (applied in 1 min intervals)

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

Duplicate tissues for each treatment, positive and negative control groups, 2 absorbance measurements after MTT incubation were performed.

Details on study design:

- Details of the test procedure used : EpiOcular™ was transferred to plates containing assay medium and incubated for 60 ± 5 minutes, followed by transferring out the assay medium, adding fresh assay medium, and pre-incubating 16-24 hours at 37°C, in a humidified atmosphere of 5% CO2. After pre-incubation, the tissues were pre-treated by wetting with PBS and incubated at standard culture conditions for 30 ± 2 minutes. The negative and positive controls were tested by applying 50 μL topically on the tissues. The test material was tested by applying topically onto the tissue surface at 50 mg per tissue; two tissues (replicates) were used per treatment with test material, negative and positive controls. The cultures were incubated for 6 hours ± 15 minutes at 37°C, in a humidified atmosphere of 5% CO2. After the 6-hour exposure time, tissues were rinsed with PBS (three times) to remove any residual test material. After rinsing, the tissues were immediately transferred to and immersed in assay medium for a 25 ± 2 minutes immersion incubation (post-soak). The tissues were then returned to assay medium, and post-incubated for an additional 18 hours ± 15 minutes at 37°C, in a humidified atmosphere of 5% CO2. Then the cultures were transferred to 0.3 mL/well of MTT reagent and incubated for 180 ± 10 minutes. After incubation, the cultures were transferred to new tissue well-plates, and extracted in 2 mL of 2-propanol for 2 hours, with plate shaking. After 2 hours or more, the extracts were mixed to obtain homogeneous solutions, and duplicate volumes of 200 μL of each extraction solution were transferred to a 96-well plate and their optical density (ODs) were recorded, while 200 μL of 2-propanol was used as the blank.

- RhCE tissue construct used, including batch number : Epiocular™EIT (OCL-200); Test Kit name: OCL-200EIT kit (manufactured on 25 Sep 2017 by MatTek Corporation); Lot number: 20993; Receipt Date: 21 Sep 2017.

- Doses of test chemical and control substances used : test material: 50 mg of test substance; negative control: 50 μL of distilled water; positive control: 50 μL of Methyl acetate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure: 6 hours ± 15 minutes at 37°C in a humidified atmosphere of 5% CO2; Post-exposure immersion: 25 ± 2 minutes not further specified; Post-exposure incubation: 18 hours ± 15 minutes at 37°C in a humidified atmosphere of 5% CO2.

- Description of any modifications to the test procedure : None

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals : The test substance was tested for tissue-binding in assay medium without MTT, with resultant staining being below 60%. Therefore mean cell viability of the test substance group was reduced by the mean staining ratio. The test substance was tested for direct MTT-reduction and results were negative.

- Number of tissue replicates used per test chemical and controls: Two replicates per treatment: test substance, positive and negative controls

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : Wavelength of 570 nm, measured on a spectrophotometer

- Acceptable variability between tissue replicates for positive and negative controlss: OD in the negative control substance group is > 0.8 and < 2.5. The cell viability in the positive control substance group is < 50%.

- Acceptable variability between tissue replicates for the test chemical: Differences of two tissue cell viabilities in each treatment group (test material, positive control, negative control) are < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Run / experiment:

two tissue replicates per treatment

Value:

ca. 108.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in MTT medium did not led to a change in colour.
The mean staining ratio was below 60%, therefore the cell viability was corrected by the following formula: Corrected cell viability (%) = Mean cell viability of the test substance group (%) - Mean staining ratio (%).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.427, 1.411, 1.464 and 1.444. The mean OD is 1.437, thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the positive control reduced the cell viability at 38.8% and fulfilled the acceptance criteria.
- Acceptance criteria for variabilities: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 2.5%, 8.1% and 7.8%, respectively, and thus < 20%.

Any other information on results incl. tables

Group	Tissue	OD	Mean OD /Tissue	Mean OD of Tissue 1 and 2	Mean Cell Viability(%) /Tissue	Mean Cell Viability (%) of Tissue 1 and 2	Corrected Mean Cell Viability (%) *	Difference
Negative control	1	1.427	1.419	1.437	98.7	100	_	2.5
		1.411
	2	1.464	1.454		101.2
		1.444
Positive control	1	0.620	0.615	0.557	42.8	38.8	_	8.1
		0.610
	2	0.504	0.499		34.7
		0.494
Test substance	1	1.519	1.507	1.563	104.9	108.8	108.6	7.8
		1.495
	2	1.639	1.619		112.7
		1.596

* Correction of cell viability was conducted.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008.

Conclusions:

Under the conditions of the RhCE test method the test substance did not show irritant properties.
CLP: not classified