Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
other: EpiDerm™ 200 kit
Details on animal used as source of test system:
TEST SYSTEM:
- MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Vehicle:
unchanged (no vehicle)
Details on test system:
EXPERIMENTAL PROCEDURE
Mesh compatibility
For liquid test substances a nylon mesh can be used as a spreading support. However, it was judged that this was not necessary for the test substance. Thus, no pre-test for mesh compatibility was performed.

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pre-test 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 55 to 65 minutes. A negative control (deionized 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 one freeze-killed control tissue (KC) was treated with, each, the test article and the negative control.

Basic procedure
Several test substances were tested in parallel within the present test using the same control tissues (NC and PC). On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and pre-conditioned in the incubator at 37 °C. After 1 hour the pre-incubation medium was replaced with fresh medium and pre-conditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. In addition, one killed tissue was used for the test substance and NC, respectively, in order to detect direct MTT reduction. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. For better handling the solid test substance was heated shortly at ca. 50 °C. Before application it was cooled down to room temperature. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC, killed tissue control, NC KC) or with 30 μL of 5% SDS (positive control, PC) or test substance (killed tissue control, KC). A nylon mesh was placed carefully onto the tissue surface of the NC and PC controls 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 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. 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.

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 three tissues treated in the same way is calculated.
- Application of measurements using killed control tissues: Because the reduction of MTT may be influenced by the test substance, a killed control was tested concurrently. 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 test substance 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 OD570 KC of the NC from the OD570 KC of the test substance. In case the 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.

ACCEPTANCE CRITERIA
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control 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 2.8.
- Assay acceptance criterion for the 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.
- Assay acceptance criterion for tissue variability: For every treatment, except the killed controls, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.
- Assay acceptance criterion for killed controls (KC): The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: freeze-killed control tissue
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- For better handling the solid test substance was heated shortly at ca. 50 °C. Before application it was cooled down to room temperature.
Duration of treatment / exposure:
25 minutes at room temperature and for 35 minutes in the incubator
Duration of post-treatment incubation (if applicable):
After washing, 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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
% of NC
Run / experiment:
1 hour
Value:
115
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks on result:
other: mean OD570: 2.552
Other effects / acceptance of results:
Based on the observed results and applying the evaluation criteria it was concluded, that Isosorbid Monooleate does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.

Any other information on results incl. tables

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:
GHS criteria not met