Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

Skin irritation / corrosion

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

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

Test material information

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

Results

Results 1

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

Results 2

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

Results 3

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.