Morpholinium sulphamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-22 to 2011-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 439, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test, adopted 23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Morpholinium sulphamate

EC Number:

258-061-6

EC Name:

Morpholinium sulphamate

Cas Number:

52636-67-6

Molecular formula:

C4H9NO.H3NO3S

IUPAC Name:

morpholin-4-ium sulfamate

Test material form:

solid: crystalline

In vitro test system

Test system:

human skin model

Cell type:

other: highly differentiated and stratified epidermis model

Cell source:

other: EpiSkinTMSM, EPISKIN Laboratories Lyon, France

Source strain:

other: not applicable

Details on test system:

The EPISKIN Model (EPISKIN SNC Lyon, France, Supplier: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69366 – LYON, France) is a three-dimensional human epidermis model. Adult human-derived 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 a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and
a functional stratum corneum. 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.

Details on Study design

Pre-incubation (day -1):
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2.

Application (day 0):
Epidermal surface was first moistened with 10 µL deionised water (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

A volume of 20 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Rinsing (day 0):
After the incubation time the EPISKIN units were removed and rinsed thoroughly with PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

Post-incubation (day 0-2):
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2):
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37°C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material to the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The optical densities of each well were recorded using a 96-well plate spectrophotometer at a wavelength of 570 nm while using an acidified isopropanol solution blank (6×200 µL).

Control samples:

yes, concurrent no treatment

Amount/concentration applied:

The test item was applied in its original form, no formulation was required. 10 mg of the test item was applied to each EpiSkin epidermis units.

Duration of treatment / exposure:

15 min

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

In conclusion, the results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions

Executive summary:

In this in vitro skin irritation test using the EPISKIN model, the test item Morpholinium Sulphamate did not show a significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits, thus the experiment was considered to be valid. The results indicated that the test item Morpholinium Sulphamate revealed no skin irritantion potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Morpholinium Sulphamate is considered a non-irritant to skin and is therefore not classified.