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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Aug 2012 - 10 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Constituent 1
Reference substance name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
EC Number:
263-174-9
EC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
Cas Number:
61791-42-2
Molecular formula:
UVCB
IUPAC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
Details on test material:
- Name of test material (as cited in study report): trade name given; Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
- Physical state: white powder
- Analytical purity: > 90%
- Lot/batch No.: 524038
- Expiration date of the lot/batch: 10 May 2013
- Storage condition of test material: at room temperature in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: EPISKIN™ model kit 0.38 cm²; reconstructed three-dimensional human epidermis
Details on animal used as source of test system:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France
- Description: The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
2.2 mL of maintenance medium, warmed to appr. 37 °C was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for the test item, control and time point item. The tissues were incubated at 37 °C, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, each tissue was removed from the well and rinsed with phosphate buffered saline (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
- Time after start of exposure: 3, 60 and 240 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, 2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 h at room temperature in a biological safety cabinet ensuring that the plates were protected form light. At the end of the 3-hour incubation period, each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using a punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containg 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues. The optical density was measured at 540 nm wave length in a plate spectrophotometer.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 20 mg (100 µL of 0.9% w/v sodium chloride was added for wetting of the test item)

NEGATIVE CONTROL
- Negative control: 0.9% w/v sodium chloride solution
- Amount(s) applied: 50 µL

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: glacial acetic acid (50 µL)
Duration of treatment / exposure:
3, 60 and 240 min
(EPISKIN cultures exposed to the control compounds for 240 min serve as the controls for all three exposure periods)
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Duplicates for each treatment and control group.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
99.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
95.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
240 min exposure
Value:
97.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 2: Mean OD540 values and viabilities of the negative control item, positive control item and test item

Item

Exposure period

Mean OD540 of duplicate tissues

Relative mean viability (%)

Negative control

240 min

0.156

100*

Positive control

240 min

0.020

12.8

Test substance

240 min

0.152

97.4

 

60 min

0.149

95.5

 

3 min

0.155

99.4

*: The mean viability of the negative control tissues is set at 100%.

EPISKIN cultures exposed to the control compounds for 240 min serve as the controls for all three exposure periods.

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive (in vitro)
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.