Butanedioic acid, sulfo-, C-[2-[(1-oxododecyl)amino]ethyl] ester, monosodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

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:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted August 24, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

IUPAC Name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

Test material form:

other: liquid

In vitro test system

Test system:

human skin model

Remarks:

EST1000

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified: Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany

Justification for test system used:

Skin irritation by cytotoxic action of substances with direct human skin contact refers to the production of reversible damage to the skin following the application of a test item for up to 4 hours. This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach.
The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermis keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The Skin model EST1000 was used.
- Tissue batch number(s): Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
- Production date: August 13, 2012
- Shipping date: Not provided.
- Delivery date: Not provided.
- Date of initiation of testing:
Start of the experimental phase: July 18, 2012
Termination of the experimental phase: August 16, 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21°C
The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation: not specified
Viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the rinsed tissues in fresh medium of 42-hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours (37°C incubation temperature, 5% CO2, 95% humidity)
- Spectrophotometer: Yes, no further details given.
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability was the MTT. The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20-fold greater than the OD of the extraction solvent alone. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%; result 58.5%). The barrier properties of the tissues were verified by the supplier.
- Morphology: No. of cornified layers and No. of vital cell layers were in line with the target of 5 and 4 respectively.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1 ( at 20 min exposure + 42 h post-treatment incubation)
9 tests in total (Negative control, Test item and Positive control in triplicate at 1 timepoint)


PREDICTION MODEL / DECISION CRITERIA

- The test substance is considered to be irritant to skin with UN GHS category 2 if the tissue viability after exposure and post-treatment incubation was ≤ 50%
- The test substance is considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 75.3 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, were applied to the skin model with a surface of 0.6 cm2 to uniformly cover the skin surface. Usually 30 µL are used, however as the supplied test item contains only 39.8% active matter, a correction factor of 2.51 was used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 75.3 µL
- Concentration (if solution): water for injection Batch no. 120378141, B. Braun Melsungen AG, 34212 Melsungen, Germany

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS) Batch no. 15733; SERVA Electrophoresis GmbH, 69115 Heidelberg, Germany

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 replicates; % versus negative control group

Run / experiment:

test group

Value:

1.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Time point: 20 minutes, followed by 42h incubation.

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 replicates; % versus negative control group

Run / experiment:

positive control group

Value:

1.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Time point: 20 minutes, followed by 42h incubation.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No interactions of the test item with MTT were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
All quality criteria required were fulfilled.
No interactions of the test item with MTT were noted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (NC): For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries.
- Acceptance criteria met for positive control (PC): Tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method)
- Acceptance criteria met for variability between replicate measurements: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

Any other information on results incl. tables

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value.The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS,was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested at an exposure time of 20 minutes, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells, which might lead to irritation by Butandioic acid, 2(or 3)-sulfo, 4 - [2 - [(1 -oxo(C12 -C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl], esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST1000 model was employed. The test item containing 39.8% active ingredient was applied. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed, followed by refreshment of the medium and further 42 hours incubation. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.