Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Second half of June 2012 to 6 months from despatch of the audited draft

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Glp compliant with international guidelines

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

EC Number:

270-307-4

EC Name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Cas Number:

68424-50-0

Molecular formula:

UVCB

IUPAC Name:

Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Test material form:

other: liquid

Details on test material:

- Name of test material: Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts
- Molecular formula: UVCB
- Physical state: liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Purity test date:
- Lot/batch No.:
- Expiration date of the lot/batch:
- Storage condition of test material:the storage conditions for the test item will be room temperature.

Test animals

Species:

human

Strain:

other: reconstructed human epidermis (RhE) model

Details on test animals or test system and environmental conditions:

The SkinEthic reconstructed human tissue model EPISKINTM consists of an airlifted, living, multi-layered tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionality equivalent to human tissue in vivo.

Test system

Type of coverage:

other: no coverage

Preparation of test site:

other:

Vehicle:

unchanged (no vehicle)

Controls:

not required

Number of animals:

no animals, 3 replicates

Details on study design:

-Media and reagents:
- SkinEthic Maintenance Medium Supplied with the EPISKINTM,to be stored at 2-8°C. To be used at room temperature (without pre-heating).
- SkinEthic Assay Medium Supplied with the EPISKINTM,to be stored at 2-8°C.
- MTT Reagent: Supplied by Sigma (cat. M2128 or equivalent).
- MTT Stock Solution: MTT Reagent 3 mg/mL in D-PBS.
- MTT Ready-to-use Solution: MTT Stock Solution diluted 1:10 (v/v) with pre-warmed SkinEthic Assay Medium (final concentration 0.3 mg/mL of MTT).
- Acidic Isopropanol: 0.04 N HCL in Isopropanol
- Water-killed epidermis: to be prepared only for MTT interacting reagents. Living epidermis is incubated with 2 mL of distilled water at 37°C, 5%CO2 and saturated humidity for 48±1 hours. The water is discarded and the samples frozen at -18 to -20°C for up to 6 months.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

> 61

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The plates will be shaked on a plate shaker for 15 ± 2 minutes at maximum speed (300 rpm/min). A volume of 1.6 mL of the medium used during the 42 h incubation period will be removed and stored at ‑20°C for possible future analysis .The tissue inserts and controls will be incubated with 2 mL / well of MTT ready to use solution with the exception of treated tissues without MTT which will be incubated with MTT ready to use solution. Plates will be incubated for 3 hours ± 5 minutes at 37°C , 5% CO₂and saturated humidity. At the end of the incubation period, tissues will be placed on absorbent paper to dry. A total biopsy will be carried out by means of a specific biopsy punch supplied by skinethic to allow biopsies of the same dimensions. The epidermis will be separated from the collagen matrix and will both be placed in a microtube prefilled with 500ml of acidic isopropanol. In the case of coloured collagen, unstained and untreated collagen matrices taken from killed epidermis may substitute the coloured collagen.Tubes will be mixed by vortexing and preserved for 4 hours at room temperature (vortexing after 2 hours, for analysis on the same day) or for 1-3 days at 4°C to allow formazan extraction.

At the end of the extraction period, debris will be eliminated by short centrifugation of the tubes (e.g. at 10000 -14000 rpm for 2 minutes). Aliquots of 200ml from each sample will be read in duplicate for their absorbance at 595 nm. Optical Density (OD) values will be recorded.

VALIDITY CRITERIA:

EPISKINTM batch acceptance:

Negative controls: OD values of the negative control samples > 0.600, CV% ≤ 18.

Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and CV% ≤ 18.

Test item data acceptance:

CV% ≤ 18.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the mean cell viability (61.1%) when compared to the negative control, the test item may be considered not irritant. However, since intra-replicate variability was higher than expected (CV% = 48.7% instead of < 18%), data are not considered reliable to classify the substance and a repetition of test should be carried out.
The negative and positive controls gave the expected results and the study was accepted as valid.
RTC

Executive summary:

The potential of the test item TALLOELFETTSAEURE-C15+-ALKYLESTER, SULFONIERT, NA-SALZ to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN^TM.

The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and colouring water per se.

No interaction was recorded between the test item and MTT in test conditions similar to those of the Main Assay. Moreover, no colouring potential of the test item in contact with water was recorded. Thus, no additional control was added in the main phase for the evaluation of non specific coloration which may influence evaluation of results.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 μL/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 μL/cm^2).

Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 L/epidermis unit.

The negative control gave the expected baseline value and variability, in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (2.9% of cell viability when compared to the negative control) and variability (CV% equal to 12.2).

Therefore, the assay was regarded as valid.

The test item induced cell death in the three replicates with a mean cell viability of 3.8% when compared to the negative control. Intra-replicate variability was higher than expected. This may be due to the nature of the substance (cream) and thus, to the fact that residues might be present on the surface even with a higher number of washings after treatment.

In these cases, according to the standard procedure defined by the Supplier, a repetition of test should be carried out.