Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium 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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods predicting corrosivity. In combination with the in vitro irritation test, it is considered relevant, adequate and reliable for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

90268-36-3

Test material form:

solid: particulate/powder

Remarks:

powder

Details on test material:

- Name of test material (as cited in study report): Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts
- Physical state: White powder
- Analytical purity: >95%
- Composition and impurities of test material, percentage of components: See confidential details
- Purity test date: 2012-07-03
- Lot/batch No.: 008249628
- Expiration date of the lot/batch: 2014-03-20
- Stability under test conditions: stable
- Storage condition of test material: At +10° to +25°C
- Other: Manufacturer/supplier: BASF Personal Care and Nutrition GmbH

In vitro test system

Test system:

human skin model

Remarks:

three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Skin model EST-1000 with BAtch no. EST-120611-001

Source strain:

not specified

Justification for test system used:

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.

Vehicle:

other: 50 µL Dulbecco's phosphate buffered saline (D-PBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST1000 model
- Tissue batch number(s): EST-120611-001
- Production date: 2012-08-21
- Shipping date: Not provided
- Delivery date: Not provided
- Date of initiation of testing (experimental phase): 2012-08-07

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not provided.
- Temperature of post-treatment incubation (if applicable): Not applicable.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing step; no data on volume used. 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).
- Observable damage in the tissue due to washing: Not provided.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Yes
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The magnitude of viability was quantified by using MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue ). In this case the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period. The stratum corneum has been shown to be sufficiently robust to resist the rapid penetration of certain cytotoxic maker chemicals (e.g. 1% Triton X-100). This property was estimated by the exposure time required to reduce cell viability by 50% (ET50) (for the EST 1000 model this is >2 hours). 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%).
- 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%). The barrier properties of the tissues were verified by the supplier. Barrier function result was 63.4% (>50%).
- Morphology: Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. Stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum would lead to poor modelling of the exposure to skin.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Moreover it has been shown to be capable of predicting the corrosive potential of the reference chemicals when used in the testing protocol selected.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MTT interference (Certificate of Analysis Epidermal Skin Test EST 1000)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 test sequence of the test, negative control and positive control group in triplicate at 3 minutes and 1 hour with duplicate OD measurements of the three tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the tissue viability after 3 minutes exposure was ≤ 50%, or if the viability after 3 minutes exposure was ≥ 50% and the viability after 1 hour exposure was < 15%.
- The test substance is considered to be non-corrosive for skin : if the tissue viability after 3 minutes exposure≥ 50% and the viability after 1 hour exposure was ≥ 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431: Not applicable.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 26.25 mg Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts were mixed with 50 µL Dulbecco's phosphate buffered saline (D-PBS) to a slurry to ensure good contact with the skin. 50 µL containing the 26.25 mg test substance were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface. Usually 25 mg are used, however a correction factor of 1.05 was used to correct for the purity of the test item. A minimum of 25 µL substance applied per cm2 is required by the guidelines.

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 µL Dulbecco's phosphate buffered saline (D-PBS)
- Lot/batch no. (if required): Batch no. 1080359

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Dulbecco's phosphate buffered saline (D-PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

Two exposure times of 3 minutes or 1 hour were employed.

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean test group versus negative control group

Run / experiment:

3 minutes

Value:

78.8

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

meann test group versus negative control group

Run / experiment:

1 hour

Value:

74.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean positive control group versus negative control group

Run / experiment:

3 minutes

Value:

0.5

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean positive control group versus negative control group

Run / experiment:

1 hour

Value:

< 0.1

Any other information on results incl. tables

The test item, Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts,was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control.8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure.The OD₅₄₀ values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively.Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and were below the cut-off values. Hence,8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Applicant's summary and conclusion

Interpretation of results:

other: predicted non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions the test itemapplied at two exposure times of 3 minutes or 1 hour was predicted non-corrosive 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 corrosion by test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The test item was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control.8 N KOHwas used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure.The OD₅₄₀ values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or<15% for a 3-minute or 1-hour treatment, respectively.Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin. The mean viability of cells treated with thepositive reference item 8 N KOH was 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Under the present test conditions the test item containing >95% active ingredient, tested at two exposure times of 3 minutes or 1 hour, was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.