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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:
2015-05-05 to 2015-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study with detailed documentation, in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ dated 31. May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
MAEEBP
IUPAC Name:
MAEEBP
Details on test material:
- Name of test material (as cited in study report): MAEEBP- Physical state: colourless liquid- Composition of test material, percentage of components: 4-methacryloyl diethoxy benzophenone 85 area%, impurities 4-hydroxybenzophenone <5%, 4-methacryloyl benzophenone <5%, polymethacrylate <5%- Lot/batch No.: 150301- Expiration date of the lot/batch: Sep. 2015- Storage condition of test material: The test item was stored in the test facility in a closed vessel in the refrigerator (2-8°C) (exception: 22.- 28. Apr. 2015 storage at room temperature).

Test animals

Species:
human
Details on test animals or test system and environmental conditions:
IN VITRO TEST SYSTEM- Source: Commercially available EpiDermTM (EPI-200), consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. - Origin: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Test system

Type of coverage:
other: topically applied to tissue of EpiDermTM
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
50 μL of the liquid test item
Duration of treatment / exposure:
exposure with test item for 3 min and 1 h
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
-Negative controls: demineralised water-Positive controls: KOH (CAS No. 1310-58-3) solution in demineralised water containing 8.0 mol/L Two tissues of the human skin model EpiDermTM were treated with MAEEBP for 3 min and 1 h, respectively. 50 μL of the liquid test item were applied to each tissue and spread to cover the tissue size.After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.DESCRIPTION OF THE METHODFour 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containingthe tissues were transferred to the wells using sterile forceps and the 6-well-plateswere set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 h (pre-incubation).For each experiment (“3 min” and “1 h”), one 24-well-plate was prepared as holding plate.12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μLMTT reagent. One additional plate was left empty. The plates were stored in the incubator.For each experiment (“3 min” and “1 h”), two 6-well-plates were used. After pre-incubation,the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL H2O demin., two wells as positive controls with50 μL potassium hydroxide solution and two other wells for testing the test item.The liquid test item was applied without preparation (50 μL).At the start of each experiment (application of negative controls), a stop watch was started.After the respective incubation time (3 min and 1 h), the inserts were removed from theplates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted withsterile cellulose tissue and set into the respective holding plate, using the wells containingassay medium. After transfer of all inserts, they were immediately moved to the wells containingMTT reagent, blotting the bottom with cellulose tissue again before setting the insertinto the MTT well. The tissues were incubated with MTT reagent for 3 h at 37 ± 1°Cand 5.0 ± 0.5% CO2. After this time, the MTT reagent was aspirated and replaced byDPBS buffer. DPBS is aspirated subsequently and replaced 3 times.At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of theinsert. The plate was then covered with Parafilm® and left to stand overnight at room temperature.On the next day, the inserts in which formazan had been produced overnight were piercedwith an injection needle, taking care that all colour was extracted. The inserts were thendiscarded and the content of each well was thoroughly mixed in order to achieve homogenisation.From each well, 3 replicates with 200 μL solution (each) were pipetted into a 96-well-platewhich was read in a plate spectral photometer at 570 nm.EVALUATIONThe values of the 96-plate-reader were transferred into a spreadsheet (Microsoft Excel®).The photometric absorbance of the negative controls was considered as 100 %. For themean of the 3 replicates of test item and positive control, formazan production was calculatedas % photometric absorbance compared with the negative control.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: relative viability (%) compared to negative control (100%)
Basis:
mean
Time point:
other: after 3 min (experiment 1)
Score:
70.5
Reversibility:
no data
Irritation parameter:
other: relative viability (%) compared to negative control (100%)
Basis:
mean
Time point:
other: after 1 h (experiment 1)
Score:
107
Reversibility:
no data
Irritation parameter:
other: relative viability (%) compared to negative control (100%)
Basis:
mean
Time point:
other: after 3 min (experiment 2)
Score:
123.5
Reversibility:
no data
Irritation parameter:
other: relative viability (%) compared to negative control (100%)
Basis:
mean
Time point:
other: after 1 h (experiment 2)
Score:
132.8
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item MAEEBP the relative absorbance values are above the thresholds for corrosion potential (50% after 3 min treatment and 15% after 1 h treatment). Therefore, the test item ist considered as "non-corrosive to skin" as stated in OECD guideline 431.

Any other information on results incl. tables

RESULTS CONTROLS:

In the first experiment, after treatment with the negative control, the absorbance value for the 3 min treatment interval was not within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8, OD was 3.1. For the 1 h treatment interval the absorbance value was within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8, OD was 2.0.

The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 12.3 % for the 1 h treatment.

A second experiment was performed, because in the first experiment the validity criterion for the 3 min treatment for the negative control was not fulfilled (mean OD ≥ 0.8 and ≤ 2.8), OD was 3.1.

In the second experiment, after treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. OD was 1.7 (3 min treatment) and 1.3 (1 h treatment).

The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 10.0 % for the 1 h treatment.

RESULTS TEST ITEM:

In the first experiment, after 3 min treatment with the test item, the relative absorbance values were reduced to 70.5 %. This value is well above the threshold for corrosion potential (50 %). After 1 h treatment, relative absorbance values were increased to 107.0 %.

This value, too, is well above the threshold for corrosion potential (15 %).

In the second experiment, after 3 min treatment with the test item, the relative absorbance values were increased to 123.5 %. This value is well above the threshold for corrosion potential (50 %). After 1 h treatment, relative absorbance values were increased to 132.8 %.

This value, too, is well above the threshold for corrosion potential (15 %).

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive to skin
Conclusions:
MAEEBP is considered as non- corrosive to skin in the Human Skin Model Test.
Executive summary:

In the first experiment, after 3 min treatment with the test item, the relative absorbance values were reduced to 70.5 %. This value is well above the threshold for corrosion potential (50 %). After 1 h treatment, relative absorbance values were increased to 107.0 %.

This value, too, is well above the threshold for corrosion potential (15 %).

In the second experiment, after 3 min treatment with the test item, the relative absorbance values were increased to 123.5 %. This value is well above the threshold for corrosion potential (50 %). After 1 h treatment, relative absorbance values were increased to 132.8 %. This value, too, is well above the threshold for corrosion potential (15 %).