Fatty acids, C12-14, .alpha.-sulfo, disodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Nov 2015 to 25 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Official Journal of the European Union, No. L 142
30 May 2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate

Cas Number:

2156594-77-1

Molecular formula:

Unspecified

IUPAC Name:

tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm(TM) model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Vehicle:

unchanged (no vehicle)

Details on test system:

MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5%humidity
- Spectrophotometer: SunriseTM Absorbance Reader For the determination of the optical density of colored extracts.Measurement using a filter wavelength 570 nm without reference filter
- EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

TEST SYSTEM
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23305
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DIRECT MTT REDUCTION
- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently.
- If the color of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, two freeze-killed control tissue (KC) per exposure time were treated with, the test article and the negative control.

BASIC PROCEDURE
Several test substances were tested in parallel within the present test using the same control tissues (NC and PC).
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTTtest is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): Potassium hydroxide as 8.0 normal ready-made solution is used as positive reference. A tissue viability of ≥ 30%
is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.
- Acceptance criteria for the variability of the tissues: For every treatment two tissues are treated in parallel. In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%.

EVALUATION OF RESULTS
- Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off values cited above, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
25 μL de-ionized water was applied first (vehicle). Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

CONTROLS
Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).

Duration of treatment / exposure:

3 min and 1 hour

Duration of post-treatment incubation (if applicable):

42 hour post-incubation period

Number of replicates:

2 replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 3 min exposure

Value:

97.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 1 hour exposure

Value:

73.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

In combination with the irritation results (OECD 439)