Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium 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:

From 27 April to 18 June, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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:

other: Commission regulation (EC) N. 761/2009 amending regulation (EC) N. 440/2008, B.46 "In vitro Skin Irritation: reconstructed Human Epidermis Model test"

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

EC Number:

290-844-8

EC Name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Cas Number:

90268-43-2

Molecular formula:

The substance is too complex, no molecular formula is available.

IUPAC Name:

Reaction product of lanolin with maleic anhydride and Sodium sulphate.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

In vitro test system

Test system:

human skin model

Remarks:

in vitro test EPISKIN (TM) - 0.38 cm2

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

not specified

Details on test system:

The test system in vitro test EPISKIN (TM) is a commercial reconstructed human epidermis (RhE) model that closely mimics the biochemical and physiological properties of the upper parts of the human skin, the epidermis.
MEDIA AND REAGENTS
SkinEthic Maintenance Medium;
SkinEthic Assay Medium;
Dulbecco’s Phosphate buffered saline (D-PBS);
MTT Reagent, stock solution, ready-to-use solution;
Acid Isopropanol (HCl in isopropanol)

TEST SYSTEM
The SkinEthic reconstructed human tissue model EPISKIN (TM) is an airlifted, living, multi-layered tissue constructed, produced in polycarbonate insert in serum-free and chemically defined medium, featuring normal ultra-structure and functionality equivalent to human tissue in vivo.
The principle of the RhE test method is based on thee premise that chemicals are able to penetrate the stratum corneum and irritant chemicals are cytotoxic to the cells in the underlying layers. Cell viability is measures by hydrogenase conversion of the vital dye MTT in to blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.

PREPARATION OF THE TEST SYSTEM
The plate of the test system was opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12 -well plate in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium (without bubbles).

Pre-treatment incubation period: culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

EXPERIMENTAL PROCEDURE- Preliminary test:
(Step 1)
Direct MTT ( [3 -(4,5 -Dimethylthiazol-2 -yl)-2,5 -diphenyltetrazolium bromide, thiazolyl blue, not specific reduction test
2 ml of MTT Ready-to-use Solution will be incubated with a 20 mg of the test item and incubated at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time implies the use of water killed epidermis as a control in the main assay.
(Step 2)
Colouring potential test
10 mg of sample have added to 0.09 ml of distilled water in a Eppendorf tube. The solution was mixed for approximately 15 minutes.
Colouring of the solution at the end of the incubation time implies the use of alive samples treated without MTT as a control in the main assay.
 
EXPERIMENTAL PROCEDURE- Main assay (test item, positive control with SDS 5% and negative controls)
Treatment procedure
Number of replicates: 3
20 mg of the test item were added to each single tissue spreading it to ensure contact with the whole tissue surface to allow a surface exposure of approximately 53 μl/cm2 or mg/cm2 being the treatment area of the commercial test system equal to 0.38 cm2.
An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature.
Each tissue was then be rinsed with approximately 25 ml of sterile D-PBS filling and empting the tissue insert removing the exceeding substance.
The sample was transferred in new wells pre-filled with 2 ml/well of pre-warmed maintenance medium and recovered for 42 ± 1 hours at incubation temperature of 37°C, 5% CO2 and saturated humidity.

MTT Assay
The plates were 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 was removed and stored at -20°C for possible future analysis.
The tissue inserts and controls were incubated with 2 ml/well of MTT ready to use solution and plates were incubated for 3 hours ± 5 minutes at 37°C, 5% CO2 and saturated humidity.
At the end of the incubation period, tissues will be placed on absorbent paper to dry. A total biopsy was carried out by means of a specific biopsy punch supplied by skinethic to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 0.5 mL of acidic isopropanol.
Tubes were 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 were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 0.2 mL from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded (blank: six aliquots 0.2 mL of acidic isopropanol).

WASHING
At the end of the exposure, the test item was mechanically removed from the epidermis surface and each tissue was rinsed three times with approximately 25 mL of sterile D-PBS filling and empting the tissue insert.
The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

Control samples:

no

Duration of treatment / exposure:

exposure time of 15 ± 0.5 minutes in a ventilated cabinet at room temperature.
Adding was carried out staggering samples of approximately 1 minute.

Positive control: an intermediate re-spreading was carried out approximately after 7 minutes of incubation.

Duration of post-treatment incubation (if applicable):

A 42 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

cell viability

Value:

ca. 71

Remarks on result:

other:

Remarks:

Basis: mean. Max. score: 100.0. Reversibility: no data.

Any other information on results incl. tables

Table.1 Direct MTT reduction test (Step 1)
Test item (mg)	MTT ready to use solution (mL)	Container	Incubation condition	Colour Observation
20	2.0	Ulrich Tube	3h at 37°C, 100% nominal humidity, 5% CO2	Yellowish and opaque (no interaction)

Table. 2 Colouring potential test (Step 2)
Test item (mg)	Water (mL)	Container	Incubation condition	Colour Observation
10	90	Eppendorf tube	15 minutes, ambient condition, in agitation	Colourless suspension

Table 3. Main test for lanolin alcohol solid.

BLANK
Percentage of viability (%)		5.9
NEGATIVE CONTROL
Percentage of viability (%)*		100.0
POSITIVE CONTROL
Percentage of viability (%)*		2.9
TEST ITEM
Percentage of viability (%)*		71.0
* refers to the negative control mean value

Applicant's summary and conclusion

Interpretation of results:

other: not classified as a skin irritant according to the CLP Regulation (EC) No.1272/2008

Conclusions:

Referring to the negative control, and according to the established criteria of the cell viability, the test item Solfosuccinate of lanolin alcohol solid is considered to have no effect on the test system, so is not considered to have irritant effect on the skin under the experimental conditions.

Executive summary:

The potential of the test item solid 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 assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of 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 20µL/epidermis unit each measuring 0.38 cm²(treatment level: 53 µL/cm²). 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 20mL/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. Therefore, the assay was regarded as valid.

The test item did not induce cell death, with a mean cell viability of 71.0% when compared to the negative control. Good homogeneity was observed between replicates.

The test item result was therefore accepted as valid.

According to the established criteria (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.