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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin 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:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD Draft Proposal for a New Guideline regarding the testing of chemicals (In Vitro Skin Irritation: Reconstructed Human Epidermis ( pertaining to the GHS classification system (Category 2 or "no-category"))
Deviations:
not specified
Principles of method if other than guideline:
The EpiDerm Skin Model was used to predict skin irritation potential of the test substance. Irritation potential was determined by measuring the relative conversion of MTT (3-[4,5 -dimethylthiazol-2-yl]-2,5 -diphenyltetrazolium bromide) in the test substance treated cultures after exposure to the test substance for a 60-minute exposure period, followed by a 42-hour post-exposure expression period.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Purity: not reported as such

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not reported
Source strain:
other: human cells
Details on test system:
The EpiDerm Skin Model was used to predict skin irritation potential of the test substance in the context of classification of skin irritation hazard according to the EU classification system (R38 or No label). Irritation potential was determined by measuring the relative conversion of MTT (3-[4,5 -dimethylthiazol-2-yl]-2,5 -diphenyltetrazolium bromide) in the test substance- treated cultures after exposure to the test substance for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. The test substance was administered to the test system without dilution. Since the test substance was a powder, a mesh was not applied after dosing onto the EpiDerm tissues. Therefore, a mesh compatibility test was not performed. The pH of the test substance was not determined because the test substance is a powder.
Assessment of Direct Test Substance Reduction of MTT: The test substance was added to a 1.0 mg/ mL MTT (Sigma) solution in warm Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM Lglutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 25 mg of the test substance were added to 1 mL of the MTT solution and the mixture was incubated in the dark at standard culture conditions for approximately one hour. A negative control, 30 μL of sterile, Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++ -Free DPBS) (Quality Biological), was tested concurrently. If the MTT solution color turned blue/purple, the test substance was presumed to have reduced the MTT. The test substance was not observed to directly reduce MTT in the absence of viable cells. The definitive assay included a negative control and a positive control. The negative control was 30 μL of sterile, Ca++Mg++ -Free DPBS and the positive control was 30 μL of 5% Sodium Dodecyl Sulfate (SDS). Both the positive and negative controls were tested in triplicate, and at the same exposure time as the test substances (60 ± 1 minutes).
The test substance was tested in one valid definitive trial. After the overnight incubation for 18 ± 3 hours, the 6-well plates containing the EpiDerm tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing. The EpiDerm cultures were tested in triplicate with the test substance for 60 ± 1 minutes. Since the test substance was a powder, immediately before application of the solid test substance, each tissue surface was moistened with 25 μL of sterile Ca++Mg++ -Free DPBS to improve contact of the tissue surface with the test substance. After adding the Ca++Mg ++ -Free DPBS, 25 mg of the test substance was added to each of three tissues at 1 minute intervals. After the three tissues were dosed with the test substance, the test substance was gently mixed and spread over the tissue surface. The EpiDerm cultures were similarly tested in triplicate with the positive or negative control. After 60 ± 1 minutes of test substance or control exposure, the tissues were rinsed with sterile, Ca++Mg++ -Free DPBS by filling and emptying the tissue insert 15 times. A stream of Ca++Mg++ -Free DPBS was directed onto the tissue surface. A sufficient stream may be applied directly onto the tissue surface to assure removal of the test substance or control. For the controls where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, the inserts were completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of Ca++Mg++-Free DPBS 3 times. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile Ca++Mg++ -Free DPBS from the wash bottle, and the excess Ca++Mg++ -Free DPBS was decanted. The bottom of the tissue insert was blotted on sterile paper towels. The tissue surface was visually observed for residual test substance or excess moisture and if present, removed. Once the rinsing was complete, the tissue inserts were transferred to new 6-well plates containing 0.9 mL of fresh assay medium. The cultures were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 24+2 hours. After the 24+2 hour post-treatment expression incubation, the 6-well plates were removed from the incubator and placed on a plate shaker. The plates were shaken at room temperature for at least 10 minutes. After the 10 minute shaking, the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh assay medium warmed to approximately 37°C. The tissues were placed back into the incubator for an additional 18+2 hours post-treatment incubation at standard culture conditions.
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/mL solution no more than two hours before use. Three hundred microliters of the MTT solution were added to each designated well of a pre-labeled 24-well plate. After the total 42 ± 2 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 mL of MTT solution. The 24-well MTT plates were incubated at standard culture conditions for 3 ±.1 hours. After the 3 ± 0.1 hour incubation, the EpiDerm cultures were submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of Ca++Mg++ -Free DPBS 3 times. The tissue was then blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plate was covered with parafilm and shaken for at least 2 hours at room temperature to extract the MTT. At the end of the extraction period, the insert was gently agitated up and down in its extractant well. The tissue was pierced with forceps to allow the extract to flow back into the well from which the insert was removed. The Millicell inserts were removed from the well from which the Millicell insert was taken. The extract solution was mixed (homogenized by pipetting up and down 3 times) and two x 200 μL aliquots were transferred to the appropriate wells of a 96-well plate. Two hundred μL of isopropanol was added to the wells designate as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg
NEGATIVE CONTROL
- Amount applied: 30 μL
POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% SDS
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: % Relative viability
Value:
96.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean tissue viability for the test substance was 96.9%. The mean OD570 of the negative control, sterile, Dulbecco's Phosphate Buffered Saline, was 2.334. The mean viability of the positive control, SDS, was 7.0%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 20 for the test substance, positive and negative control. Since all of the positive control results were viability ≤ 50% and the mean OD570 values of the negative controls were greater than 1.000 and less than 2.500, the assay results were considered valid. Based upon the results of this assay, the test substance was predicted to be non-skin irritating (mean tissue viability ≤ 50%).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this test using the EpiDerm Skin model, the test substance was predicted to be non-skin irritating.
Executive summary:

The Skin Irritation Test (SIT) using the EpiDerm Skin Model was used to predict the skin irritation potential of the test substance in the context of classification of skin irritation hazard according to the EU classification system (R38 or No Label). Irritation potential was determined by measuring the relative conversion of MTT (3 -[4,5 -dimethylthazol-2-yl]-2,5 -diphenyltetrazolium bromide) in the test substance- treated cultures after exposure to the test substance for a 60-minute exposure period, followed by a 42-hour post-exposure expression period. Skin irritation potential of the test substance was predicted if the relative viability was less than or equal to 50%.

The mean tissue viability for the test substance was 96.9%. The mean OD570 of the negative control, sterile, Dulbecco's Phosphate Buffered Saline, was 2.334. The mean viability of the positive control, SDS, was 7.0%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 20 for the test substance, positive and negative control. Since all of the positive control results were an R38 classification (i.e. viability ≤ 50%) and the mean OD570 values of the negative controls were greater than 1.000 and less than 2.500, the assay results were considered valid.

Based upon the results of this assay, the test substance was predicted to be non-skin irritating (mean tissue viability >50%).