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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 23, 2017 to March 24, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
CAS No.: 23054-60-6 + 23054-61-7; Batch No.: 7723329-R802; Purity: 100 %; Appearance: colourless/pale straw clear liquid (white solid was confirmed by visual inspection upon arrival at test facility at room temperature. Test substance was clear liquid after incubation at 37 °C).

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKINTM(SM)
Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-012, Expiry Date: 27 March 2017.
EPISKINTM(SM) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS).
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μl
Duration of treatment / exposure:
4 hours
Number of replicates:
Two replicates per test substance were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test substance was coloured, two additional test substance-treated tissues were used for the non specific OD evaluation.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
65.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the two negative control tissues was in the recommended range (0.915).
- The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
- The difference of viability between the two test substance-treated tissue samples in the MTT assay was 11.7 %.
- The difference of viability between the two negative control tissue samples in the MTT assay was 21.2 %.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated relative viability % of the samples:

Substance

Optcal density (OD)

Viability (%)

Measured

Blank corrected

Negative control

Physiological saline

(0.9% (w/v) NaCl)

 

1.058

1.011

110.6

0.864

0.818

89.4

.

mean: 0.915

100

Positive control

Glacial acetic acid

0.059

0.013

1.4

0.048

0.001

0.1

 

mean: 0.007

0.8

Test substance

0.681

0.634

69.4

0.611

0.564

61.7

 

mean: 0.599

65.5

Mean blank value was 0.046.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving %) of the additional control tissues:

Substance

Optcal density (OD)

NSCliving%

Measured

Blank corrected

Additional control

0.053

0.006

0.4

0.047

0.000

 

mean: 0.003

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the results of the in vitro EPISKIN™(SM) model test with the test substance indicate that the test substance is non corrosive to the skin.
Executive summary:

A study was conducted to determine the skin corrosion potential of the test substance using reconstructed human epidermis model EPISKINTM(SM) according to OECD Guideline 431 and EU-Method B.40-BIS, in compliance with GLP. Disks of EPISKINTM(SM) (two units) were treated with test substance and incubated for 4 hours at room temperature. Exposure of test substance was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test substance. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin. The experiment met the validity criteria, therefore the study was considered to be valid. Following exposure with the test substance the mean cell viability was 65.5 % compared to the negative control. This is above the threshold of 35%, therefore the test substance was considered as being non-corrosive. Under the study conditions, the results of the in vitro EPISKIN™(SM) model test with the test substance indicate that the test substance is non corrosive to the skin (Orovecz, 2017).