Reaction product of Saccharose, Glycerine, biodiesel propoxylated

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2013 - 12 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD testing guideline 439 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

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:

Purity: 100% UVCB substance
Homogeneity of the test substance was provided after shaking the test-substance container.
The stability under storage conditions over the study period was guaranteed by the sponsor.
pH of undiluted test substance: ca. 5

Test animals

Species:

other: 3D human epidermis model

Details on test animals or test system and environmental conditions:

The test is designed to predict skin irritation potential of a chemical by using the three dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the EpiDermTM 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 tissue 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 tissue treated with PBS and expressed as relative tissue viability.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 μL of the undiluted test substance

Duration of treatment / exposure:

25 minutes under the laminar flow hood at room temperature and 35 minutes in the incubator at 37°C.

Observation period:

ca. 42 hours

Details on study design:

MESH COMPATIBILITY PRETEST:
For liquid test substances a nylon mesh can be used as a spreading support. To exclude a reaction of the test substance with the mesh, the compatibility of the test substance with the nylon mesh was checked in a pretest. The test substance and the mesh are brought together on a slide and the reaction was observed after 60 minutes exposure, using a microscope. An interaction between test substance and the mesh was not noticed. However, it was judged that the use of a mesh was not necessary for the test substance.

DIRECT MTT REDUCTION:
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. 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 and visible residues of the test substance remained on the tissues after washing, subsequent testing of killed controls (one freeze-killed control tissue (KC)) was considered. Killed controls are treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure” (3.6),
additionally.

BASIC PROCEDURE:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 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 for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes
overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.
Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation 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. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTANCE CRITERIA:
- For negative control (NC): The absolute OD570 of the negative control tissues in the MTTtest 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 2.8.
- For positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- For tissue variability: For every treatment, 3 tissues are treated in parallel. The intertissue variability is considered to be acceptable if the SD of %- viability is ≤ 20.

EVALUATION OF RESULTS:
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability, presented as the quotient of the mean OD570 divided by the respective OD570 NC value in %

Value:

113

Remarks on result:

other:

Remarks:

Basis: mean. Reversibility: no data. Remarks: A chemical is considered as irritant if the mean relative tissue viability is <=50%.. (migrated information)

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 above, it was concluded, that Reaction product of Saccharose, Glycerine, biodiesel propoxylated does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.

Executive summary:

The potential of Reaction product of Saccharose, Glycerine, biodiesel propoxylated to cause dermal irritation was assessed by a single topical application of 30 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by a 42-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 both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results: The test substance is able to reduce MTT directly. However, subsequent testing of MTT reduction control was not performed, because no visible residues of the test substance remained on the tissues after washing. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 113%. 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 a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.