9-Octadecenoic acid (Z)-, sulfonated, potassium 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 February 18, 2014 to February 24, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

EC Number:

271-843-1

EC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Cas Number:

68609-93-8

Molecular formula:

A generic formula cannot be provided for this UVCB substance. The alkyl chain length of the sulfonated fatty acids range from C12-C22, however the major alkyl chain is C18.

IUPAC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Test material form:

other: powder/flakes with lumps

Details on test material:

- Name of test material (as cited in study report): 9-Octadecenoic acid (Z)-, sulfonated, potassium salts
- Physical state: Beige-yellow powder/flakes with lumps (i.e., determined at WIL Research Europe B.V.)
- Analytical purity: 100%
- Water content: 0.5%
- pH: 6 at concentration of 1g/L
- Lot/batch No.: 7495382
- Expiration date of the lot/batch: December 31, 2015
- Storage condition of test material: At room temperature in the dark
- Stability in vehicle: Water: Yes
- Solubility in vehicle: Water: Completely miscible

Test animals

Species:

other: human three dimensional epidermal model (i.e., EPISKIN Small Model (EPISKIN-SMTM))

Test system

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 10.0 to 11.7 mg of solid test substance was applied directly on top of the skin tissue

Duration of treatment / exposure:

15 minutes

Observation period:

After a 42 h post-incubation period, determination of the cytotoxic (i.e., irritancy) effect was performed

Details on study design:

- Test for reduction of MTT by the test substance:
Test substance was checked for possible direct MTT reduction before the study was started. 11.9 mg of the test substance was added to 2 mL of MTT solution (i.e., 0.3 mg/mL in PBS). The mixture was incubated for 3 h at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

- Application/Treatment of the test substance:
The test was performed on a total of 3 tissues per test substance, negative and positive controls. 10.0 to 11.7 mg of solid test substance (i.e., with a small glass weight boat) was added into 12-well plates on top of the skin tissues. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue. Three tissues were treated with 25 μL PBS (i.e., negative control) and 3 tissues with 25 μL 5% sodium dodecyl sulphate (SDS) (i.e., positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 h at 37°C.

- Cell viability measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (i.e., 0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 h. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: Cell viability (%)

Run / experiment:

15 minutes treatment time followed by 42 h post-incubation period.

Value:

95

Negative controls validity:

valid

Remarks on result:

other: Basis: mean

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with test substance compared to the negative control tissues was 95%. Since the mean relative tissue viability for test substance was above 50%, the test substance was considered to be non-irritant.

Any other information on results incl. tables

The absorption at 570 nm measured after treatment with test substance and controls are presented below:

Table 1: Individual OD measurements at 570 nm.

Test system	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.402	1.004	1.152
OD570 measurement 2	1.311	1.037	1.160
Test substance
OD570 measurement 1	1.137	1.062	1.132
OD570 measurement 2	1.154	1.058	1.175
Positive control
OD570 measurement 1	0.196	0.441	0.226
OD570 measurement 2	0.234	0.452	0.229

Table 2: Mean absorption values of test substance, negative and positive controls.

 

 

Test System	A (OD570)	B (OD570)	C (OD570)	Mean±SD (OD570)
Negative control	1.315	0.979	1.114	1.136±0.169
Test substance	1.104	1.018	1.112	1.078±0.052
Positive control	0.173	0.404	0.186	0.254±0.130

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range.

Mean tissue viability obtained after 15 minutes treatment with test substance compared to the negative control tissues is given below:

Table 3: Mean tissue viability values of test substance, negative and positive controls.

 

Test System	Mean tissue viability (percentage of control)
Negative control	100
Test substance	95
Positive control	22

 

The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

Result of test for reduction of MTT by the test substance:

As no colour change was observed in the assay, it was concluded that test substance did not interact with MTT.

Acceptability of the assay:

 

The test is considered acceptable because it meets the following criteria:

a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.

b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.

c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures

A test substance is considered irritant in the skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 h of post incubation is ≤ 50% of the mean viability of the negative controls.

A test substance is considered non-irritant in the in vitro skin irritation test if:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 h of post incubation is >50% of the mean viability of the negative controls.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

Under the study conditions, the test substance was considered to be non-irritating to skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance in a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)) according to OECD Guideline 439, in compliance with GLP. Skin tissue was moistened with 5 μL of Milli-Q water and 10.0 to 11.7 mg of test substance was applied on top of the tissue for 15 minutes. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed using the MTT assay. The relative mean tissue viability, compared to the negative control, was 95%, therefore the substance was considered to be non-irritating. The positive control had a mean cell viability of 22% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. Under the study conditions, the test substance was considered to be non-irritating to skin (Verbaan, 2014).