Reaction mass of sodium 1-methoxy-1-oxohexadecane-2-sulphonate and sodium methyl 2-sulphooctadecanoate

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 March - 02 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro Skin Corrosion: Human Skin Model Test (13 April 2004)

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)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-methoxy-1-oxohexadecane-2-sulphonate

EC Number:

223-676-0

EC Name:

Sodium 1-methoxy-1-oxohexadecane-2-sulphonate

Cas Number:

4016-24-4

Molecular formula:

There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C17H34O5S.Na C17H33NaO5S C14H29CH(CO2CH3)SO3Na CH3(CH2)13CH(SO3Na)COOCH3

IUPAC Name:

sodium 1-methoxy-1-oxohexadecane-2-sulfonate

Constituent 2

Reference substance name:

Sodium methyl 2-sulphooctadecanoate

EC Number:

223-770-1

EC Name:

Sodium methyl 2-sulphooctadecanoate

Cas Number:

4062-78-6

Molecular formula:

There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C19H38O5S.Na or C19H37O5S.Na CH3(CH2)15CH(SO3Na)COOCH3

IUPAC Name:

sodium methyl 2-sulphooctadecanoate

Test material form:

solid: flakes

Details on test material:

- Physical state: pale yellow flakes
- Storage condition of test material: In refrigerator (2-8 ºC) in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: human skin model
- % coverage: 0.6 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 3 minutes and 1 hour

SCORING SYSTEM: percentage viability
Cell viability measurement:
The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the Multiskan Spectrum.

Control samples:

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, crushed and ground in a mortar with pestle to improve the consistency

Duration of treatment / exposure:

3 minutes and 1 hour exposure times

Duration of post-treatment incubation (if applicable):

After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Number of replicates:

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to MIZULAN FL-80 and two for a 1-hour exposure.

Test animals

Species:

other: human-derived epidermal keratinocytes

Details on test animals or test system and environmental conditions:

EpiDerm Skin Model (EPI-200, Lot no.: 12961 kit I). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those foundin vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 91 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.1 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EpiDerm Skin Model (EPI-200, Lot no.: 12961 kit I). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those foundin vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 91 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.1 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MIZULAN FL-80 compared to the negative control tissues was 100% and 88% respectively. Because the mean relative tissue viability for MIZULAN FL-80 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MIZULAN FL-80 is considered to be not corrosive.

MIZULAN FL-80 was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that MIZULAN FL-80 did not interact with MTT.

Any other information on results incl. tables

The absolute mean OD₅₄₀(optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 10%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 26% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

MIZULAN FL-80 is not corrosive in the in vitro skin corrosion test.