Sulfonic acids, C16-alkane hydroxy and C16-alkene, 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:

From 30th April 2013 to 7th June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts

EC Number:

294-430-8

EC Name:

Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts

Cas Number:

91722-28-0

IUPAC Name:

Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts

Test material form:

liquid

Details on test material:

Identification: Internal Olefin Sulfonate
Description: pale yellow liquid
Storage conditions: approximately 4°C in the dark
The test item was heated to approximately 30°C and homogenized prior to use.

Specific details on test material used for the study:

Expiry/retest date: 30 June 2013

In vitro test system

Test system:

other: EpiSkin Kit, Lot No.: 13-EKIN-021

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Vehicle:

other: Deionised water

Details on test system:

-Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on June 04, 2013. After receipt EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.

-Dose Selection
Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test item in deionised water were applied to each of triplicate tissues for 15 minutes. The purity of the test item was taken into consideration for dose calculation.
The test was performed two times. In the first experiment, an incorrect dilution ratio for the test item (w/v instead of v/v) was used. The raw data of both experiments are archived, but only the results of the second experiment are reported in the present document.

-Test for Direct MTT Reduction
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 µL of the 20% (v/v) dilution of the test item in deionised water were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

-Pre-warming of EpiSkin™ Tissues
After incubation period of about 2 hours of the EpiSkin™ tissues were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium under sterile conditions using sterile forceps, the inserts.

-Treatment
The negative and the positive control, and the two different dilutions of the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were placed into the incubator for 15 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

-MTT Assay
The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (MatTek, USA) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 21.5 hours in the refrigerator.
Per each tissue sample 2  200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the medium beneath the tissues was replaced before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". MTT reducing capability of the test item was tested as described in section “9.5 Test for Direct MTT Reduction”.

-Evaluation of Results
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (OD test item / OD mean of negative control) x 100

For the test item and the positive control the mean relative viability  standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

-Acceptability of the Assay
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is  0.6 till ≤ 1.5.
The relative standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS).
The historical data (means, standard deviation, and ranges) of the positive control as well as for the negative control obtained with the EpiSkin™ model at Harlan CCR are mentioned in this report.

- Negative Control
Deionised water (produced in-house) was used as negative control. 10 µL were applied to each of triplicate tissues for 15 minutes.

- Positive Control
A 5% SLS (Fluka, Sigma-Aldrich) solution in deionised water, prepared freshly prior to the performance of the experiment, was used as positive control. 10 µL were applied to each of triplicate tissues for 15 minutes.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test substance

Duration of treatment / exposure:

15 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2% (v/v) dilution of the test substance, mean of 3 replicates

Value:

95.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

20% (v/v) dilution of the test substance, mean of 3 replicates

Value:

4.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≧ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 19.2% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls were below 13% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table. Results after treatment with Internal olefin sulfonate (IOS) and comtrols

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	1.030	1.165	1.199	1.131	91.0, 102.9, 106.0	7.9	100.0
Positive Control	15 min	0.236	0.209	0.207	0.217	20.9, 18.4, 18.3	7.5	19.2
Test Item2% (v/v)	15 min	1.098	1.086	1.043	1.076	97.1, 96.0, 92.2	2.7	95.1
Test Item20% (v/v)	15 min	0.051	0.053	0.064	0.056	4.5, 4.7, 5.6	12.4	4.9

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]: 100×(absorbancetissue)/(mean absorbancenegative control)

*** relative absorbance per treatment group [rounded values]: 100×(mean absorbancetset item)/(mean absorbancenegative control)

 

 

Table. HISTORICAL DATA

Positive Control		Negative Control
Mean Viability	19.2%	Mean OD	1.004
Standard Deviation	10.0%	Standard Deviation	0.219
Range of Viabilities	1.7% - 35.4%	Range of ODs	0.607 – 1.521

Data of 206 studies performed from October 2007 until March 2013

Applicant's summary and conclusion

Interpretation of results:

other: Non irritant if used as 2% (v/v) dilution in deionised water, and irritant to skin if used as 20% (v/v) dilution in deionised water

Conclusions:

In this study and under the experimental conditions reported, the 2% (v/v) dilution of Internal olefin sulfonate (IOS) in deionised water is not irritant to skin, whereas the 20% (v/v) dilution possesses a clear irritating potential according to UN GHS and EU CLP regulation. The purity of the test item was taken into consideration for dose calculation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Internal olefin sulfonate (IOS) by means of the Human Skin Model Test according to OECD TG 439. Three tissues of the human skin model EpiSkin™ were treated with two different dilutions of the test item, the negative or the positive control for 15 minutes. Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test item in deionised water, of the positive and the negative control were applied to each tissue, spread to match the surface of the tissue.

 

After treatment with the negative control (deionised water) the absorbance values were well within the required acceptability criterion of mean OD ≧ 0.6 till ≦ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control (5% SDS in deionised water) induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

 

After exposure of the 2% (v/v) test item solution the mean relative absorbance value decreased to 95.1%. This value is well above the threshold for irritancy of ≤ 50%. With the 20% (v/v) solution the mean relative absorbance value was reduced to 4.9%.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, Internal olefin sulfonate (IOS) is irritant to skin if used as 20% (v/v) dilution in deionised water, and is non irritant if used as 2% (v/v) dilution in deionised water according to UN GHS and EU CLP regulation.