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Description of key information

4-Chlor-PMA HCl was corrosive in a human 3D-Skin model.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
other information
Study period:
Mar 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 431 guideline draft
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: reconstructed human skin (RHS)
Strain:
other: not applicable
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control: 0.9 % NaCl
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg per insert
- Concentration (if solution): 100 %
Duration of treatment / exposure:
Incubation period: 3 and 60 minutes
Details on study design:
RECONSTRUCTED TISSUES:
The experiment was carried out on a reconstructed human epidermis epiCS (CeIlSystems, Troisdorf, Germany). The tissue equivalents were shipped in 24 well cell culture plates on Agarose supplemented with maintenance medium (Kit contents epiCS; CellSystems, Cat.NO. CS-1001). Inserts were of 0.6 cm2 size. All tests were performed in triplicates for each concentration and each time point (3 min or 60 min).

ADAPTATION TO CELL CULTURE CONDITIONS:
Inserts with epiCS reconstructed human epidermis (0.6 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5 % CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1ml new maintenance medium (37 °C) for each well.

ENVIRONMENTAL CONDITIONS:
The environmental conditions in the incubator were standardised as folIows:
Incubator temperature: 37 ± 2 °C, CO2 gas concentration: 5 %, Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).

APPLICATION OF THE TEST MATERIAL AND INCUBATION:
For testing of chemical induced corrosivity the epiCS inserts were exposed to 25 mg of the test item (plus 50 µl 0.9 % NaCl to moisten and ensure good contact with the skin) for 3 min (RT) and 60 min in the incubator (3 inserts per period of incubation time), respectively. 0.9 % NaCI (50 µl) treated epidermal models were used as negative controls (determination in triplicates).

DETERMINATION OF CELL VIABILITY (MTT):
After the incubation period the inserts were washed carefully in PBS and MTT reduction was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl of MTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems). The tissues were incubated for about 3 hours under cell culture conditions (5 % CO2, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 2 ml/insert) on a vertical shaker (at least 60 min). For deterrnination of cell viability the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatie reader (EL808, Bio-Tek; 96 well format, 200 µl). Data acquisition and evaluation were done with "Gen5" (software by Bio-Tek). The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Sigma, Deisenhofen, Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

RELIABILITY CHECK:
Reliability of the test was confirmed before by interlaboratory validation.

Table 1: Summary of results from in vitro corrosion test with 4-Chlor-PMA HCl

 Compound  Cell viability after 3 min. [%]  Cell viability after 60 min. [%]  Classification
4-Chlor-PMA HCl 63.07  6.04

  corrosive

 Negative control 100.00 100.00

  non-corrosive

4-Chlor-PMA HCl was characterised by a significant impact on cell viability after the 60 min. period. Thus, the substance is to be labeled as corrosive to skin.

Interpretation of results:
corrosive
Remarks:
Migrated information
Executive summary:

A study for predicting non-specific, corrosive potential of 4-Chlor-PMA HCl by using reconstructed human skin (OECD TG 431) was performed (Leidenfrost, 2014). For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 63.07 % viability; 60 min.: 6.04 % viability) showed, that the test item has a corrosive property under the conditions of the assay used.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study for predicting non-specific, corrosive potential of 4-Chlor-PMA HCl by using reconstructed human skin (OECD TG 431) was performed (Leidenfrost, 2014). For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 63.07 % viability; 60 min.: 6.04 % viability) showed, that the test item has a corrosive property under the conditions of the assay used.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available

Effects on skin irritation/corrosion: corrosive

Justification for classification or non-classification

According to OECD TG 431 the measured viabilities after 3 and 60 minutes lead to a combination of the sub-categories 1B and 1C. However, taking into account the low pH value (< 2) of the test substance, a classification to sub-category 1B is recommended. Therefore based on the study results (corrosive in a human 3D-Skin model), a classification with R34 (causes burns) according to Directive 67/548/EEC or with Skin Corr. Cat. 1B (H314: causes severe skin burns and eye damage) according to Regulation (EC) No. 1272/2008 (CLP) is required.