Reaction product of Saccharose, Glycerine, biodiesel propoxylated

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: Study conducted according to internationally accepted experimental procedure and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

no guideline available

Guideline:

other: EpiOcularTM test

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of Saccharose, Glycerine, biodiesel propoxylated

EC Number:

940-005-3

Molecular formula:

Unspecified

IUPAC Name:

Reaction product of Saccharose, Glycerine, biodiesel propoxylated

Test material form:

other: liquid

Details on test material:

100% UVCB substance
pH ca. 5 (undiluted test substance)
The test substance was homogeneous by visual inspection. In addition the homogeneity of the test substance was provided after shaking the test-substance container.

Test animals / tissue source

Species:

other: 3D human cornea model

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

50 μL

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

2 hours post-incubation

Details on study design:

TEST SYSTEM:
The EpiOcularTM assay is designed to predict an eye irritation potential of a chemical based on the observation that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short-term topical exposure. Cytotoxicity is expressed as a reductionof the activity of mitochondrial dehydrogenase, which reduces the yellow water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolim bromide (MTT) to the insoluble blue formazan in the test medium. Thus,the loss of viability of the tissues is measured by a colorimetric assay after administration of the substance.
For the EpiOcularTM assay, the OCL-200 model supplied by MatTEK Corp. (Ashland MA, USA) was used. Methyl acetate was used as a positive control.

DIRECT MTT REDUCTION PRETEST:
To assess the ability of the test material to directly reduce MTT a pretest was performed as described in the following. 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 (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 that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, additionally.

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.

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 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:
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 de-ionized water (NC) 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 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 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 testsubstance 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.

EVALUATION CRITERIA:
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.

Results and discussion

Any other information on results incl. tables

Table 1. Mean OD570 values and tissues viabilities of test item, negative control (NC) and positive control (PC).

Test substance		tissue 1	tissue 2	mean KC	inter-tissue mean variability [%]
NC	mean OD570	1.960	1.845	0.030	1.903
	viability [% of NC]	103.0	97.0	-	100	6.0
Reaction product of Saccharose, Glycerine, biodiesel propoxylated	mean OD570	1.648	1.633	0.030	1.640
	viability [% of NC]	86.6	85.8	-	86	0.8
PC	mean OD570	0.523	0.555	-	0.539
	viability [% of NC]	27.5	29.2	-	28	1.7

KC...killed control (MTT reduction control). 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:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the observed results and applying the evaluation criteria cited in chapter 3.8 it was concluded, that Reaction product of Saccharose, Glycerine, biodiesel propoxylated does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Executive summary:

The potential of Reaction product of Saccharose, Glycerine, biodiesel propoxylated to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-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 testsubstance 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 86%.