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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-02-26 to 2014-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity: >98.5%
Physical form: White solid powder

Test animals

Species:
other: in vitro
Details on test animals and environmental conditions:
The following Reconstructed Human Epidermis Model was used:
EpiDermTM (EPI-200-SCT, Lot no. 19622) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

Test system

Vehicle:
water
Remarks:
sterile deionised water
Amount / concentration applied:
test item: 25 mg test item moistered with µl sterile deionised water were applied to the skin model with a surface area of 0.63 cm2.
positive and negative control: 50 µl
Duration of treatment / exposure:
3 minutes and 1 hour
Details on study design:
TEST FOR INTERFERENCE OF TEST ITEM WITH MTT REDUCTION ASSAY
- To identify the potential of the test item to interfere with the MTT assay, the following checks were performed:
25 mg test item was dissolved in 300 μL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% humidity for 60 minutes.
No discoloration of the test item was noted.
- A further check was performed with two tissue replicates, which underwent the whole skin corrosion test but were incubated with medium instead
of MTT solution during the MTT incubation step. No discoloration of the test item was noted. Hence, no possible interacting with the MTT
measurement had to be considered and no additional test had to be performed.

ADMINISTRATION
- EpiDerm tissues were conditioned by pre-incubation (1 hour) for release of transport stress related compounds and debris in the incubator
(37°C, 5% CO2, 95% humidity)
- After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with test item and controls
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface.
- Test item was moistened with 25 μL sterile deionised water to ensure good contact with the skin.
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- Three replicate tissues for each treatment (3 minutes, 1 hour, positive and negative control) were employed.
- At the end of the exposure period, tissues were rinsed (with Dulbecco's phosphate buffered saline (D-PBS)), blotted and assay medium is
replaced by MTT assay medium (final concentration: 1 mg MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide,
Thiazolyl blue)/mL medium).
- After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS), blotted and the blue formazan salt was extracted
with Isopropanol
- Optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as
% of the mean of the negative control tissues
- Skin corrosivity potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure
with the test chemical


Results and discussion

In vivo

Results
Remarks on result:
other: Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificialthree-dimensional model of human skin (see Executive summary below).
Other effects:
no other effects

Any other information on results incl. tables

Assay acceptability criteria

Assay acceptance criterion 1: Negative control

The absolute OD of the negative control (NC) tissues (treated withsterile deionised water) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.

The assay meets the acceptance criterion if the mean OD540of the NC tissues is ≥ 0.8 and ≤ 2.8.

Assay acceptance criterion 2: Positive control

A8N KOHwas used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.

Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1hour exposure: < 15%).

Assay acceptance criterion 3:variability between tissue replicates

Associated and appropriate measures of variability between tissue replicatesshould not exceed 30% (in the range of 20 – 100% viability).

Interpretation of results

The mean OD values obtained for the test item and the positive control were used to calculate percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items was used to evaluate the results and identify corrosive materials and shown to be appropriate. The prediction of corrosivity associated with theEpiDermTMmodel is:

The test item is considered to be corrosive to skin:

If the viability after 3 minutes exposure was < 50%, or
if the viability after 3 minutes exposure was ≥ 50% and the viability after 1hour exposure was < 15%.

The test item is considered to be non-corrosive to skin:

If the viability after 3 minutes exposure was ≥ 50% and the viability after 1hour exposure was ≥ 15%.

Applicant's summary and conclusion

Conclusions:
Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificialthree-dimensional model of human skin.
Executive summary:

The purpose of this study was to assess the corrosive properties of the test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EpiDermTMmodel was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

The test item was applied topically as solid test item to the model skin surface, which was moistened with sterile deionised water. Sterile deionised water was used as the negative control.8 N KOH was used as the positive reference item.The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 97.7% after a 3-minute exposure period and 117.0% after a 1-hour exposure.The 3-minute and the 1-hour exposure values were above the cut-off percentage cellviability values distinguishing corrosive from non-corrosive test items of >50% and >15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 3 tissues was 1.602 (3-minute exposure) or 1.433 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8 N KOH were 2.4% (3-minute exposure) and 3.9% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-h exposure. The standard deviation of all triplicates determined (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

 

Conclusion

Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificialthree-dimensional model of human skin.