Oligomerisation products of sucrose with ethylene oxide and methyloxirane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study following the testing strategy for determination of eye irritation/corrosion as given in the following OECD TG 405 (GLP).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure, the test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values from tissues and expressed as relative tissue viability.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation products of sucrose with ethylene oxide and methyloxirane

EC Number:

701-303-7

Molecular formula:

C12 H14O11. ( C3 H6 O )(b+d+f+h+j+l+n+p).(C2 H4 O)(a+c+e+g+i+k+m+o) ; 1 < a+c+e+g+i+k+m+o < 16.5; 1 < b+d+f+h+j+l+n+p < 16.5

IUPAC Name:

Oligomerisation products of sucrose with ethylene oxide and methyloxirane

Details on test material:

- Name of test material (as cited in study report): Sucrose, ethoxylated and propoxylated (>1 < 16.5 mol EO and >1 < 16.5 mol PO)
-Test item number: 11/0043-1
- Physical state: Liquid, viscous / colorless, clear
- Analytical purity: >99%
- Lot/batch No.: T35/035/10
- Stability under test conditions: guaranteed
- Storage condition of test material: room temperature, protect against humidity
- pH-value of the undiluted test substance: 5

Test animals / tissue source

Species:

other: in vitro study

Strain:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- The test system (target tissue): three dimensional human cornea model.
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.
- Supplier: MatTek Corp., Ashland MA, USA.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control tissues were concurrently applied with 50 μL of highly de-ionized water (NC)

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 min

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Each treatment group (test substance, NC and PC) consisted of 2 tissues.

Details on study design:

TEST PROCEDURE
- Direct MTT reduction: the test substance was added to the MTT solution, and 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 direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, in the same way as described below, additionally.

BASIC PROCEDURE:
Two tissues were treated with the test substance, the PC and NC, respectively.
-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 standard culture conditions for 16 – 24 hours (pre-incubation).
-Pretreatment of the tissues: after the pre-incubation the tissues were pre-treated with 20μL 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: 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 highly de-ionized water (NC) or with 50 μL of methyl acetate (PC). 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 postincubation 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 prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 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. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation 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 value determined for each tissue was taken as a measure of its 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.
-Tissue viability: the quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

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 (1) the mean OD570 of the NC is ≥ 1.0 and (2) the mean OD570 of the NC ≤ 2.5.
-Acceptance criteria for the PC: methyl acetate used as PC usually leads to a tissue viability of approx. 30%. 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%.

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 50%. At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

HISTORICAL CONTROL VALUES:
Historical control values of negative and positive controls, gathered over an appropriate time period, were available. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Value:

95

In vivo

Irritant / corrosive response data:

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

Any other information on results incl. tables

Table 1: Findings of the eye irritation test.

Test substance		Tissue 1	Tissue 2	mean         SD
NC	mean OD570	1.4788	1.4973	1.4880
	viability [% of NC]	99.4	100.6	100             1.2
11/0043-1	mean OD570	1.4743	1.3458	1.4100
	viability [% of NC]	99.1	90.4	95               8.6
PC	mean OD570	0.3028	0.3353	0.3190
	viability [% of NC]	20.3	22.5	21               2.2

 

Table 2: Historical control values of the negative control (NC), the positive control (PC), and the viability. (SD = standard deviation)

Historical range of NC (OD570) Historical period: Apr 2010 - Apr 2011
Mean OD	SD	Mean+2 SD	Mean–2 SD
1.487	0.099	1.69	1.29
Historical range of NC (OD570) Historical period: Apr 2010 - Apr 2011
Mean OD	SD	Mean+2 SD	Mean–2 SD
0.372	0.097	0.57	0.18
Viability (%); Historical period: Apr 2010 - Apr 2011
Mean %	SD	Mean+2 SD	Mean–2 SD
25.18	5.72	36.62	13.73

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information