Sodium dimethyl 5-sulphonatoisophthalate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-06 to 2012-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

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:

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:

Sodium dimethyl 5-sulphonatoisophthalate

EC Number:

223-578-8

EC Name:

Sodium dimethyl 5-sulphonatoisophthalate

Cas Number:

3965-55-7

Molecular formula:

C10H10O7S.Na

IUPAC Name:

sodium 3,5-bis(methoxycarbonyl)benzenesulfonate

Test material form:

other: solid

Details on test material:

Name: Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester)

Test animals

Species:

other: EpiSkin™ Small Model

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

The epidermal surface was first moistened with 10 μL deionised water (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly onto the skin.

Duration of treatment / exposure:

15 minutes at room temperature

Details on study design:

PERFORMANCE OF THE STUDY
Pre-incubation (day [-1])
The medium was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2.

APPLICATION (DAY 0)
Test Item
The epidermal surface was first moistened with 10 μL deionised water* (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

Positive and negative control
A volume of 20 μL positive control (SDS 5 % aq.) or negative control (1 x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

EXPOSURE (DAY 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

RINSING (DAY 0)
After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

POST-INCUBATION (DAY 0-2)
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “Maintenance Medium” (2 mL/well) below them and then incubated for 42 hours at 37 °C in an incubator with 5 % CO2.

MTT TEST (DAY 2)
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37 °C in an incubator with 5 % CO2 protected from light.

FORMAZAN EXTRATION (DAY 2)
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a Vortex Mixer to achieve a good contact of all of the material to the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY (DAY 2)
Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD’s of each well were recorded using a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at a wavelength of 570 nm while using an acidified isopropanol solution blank (6 × 200 μL).

VALIDITY OF THE TEST
– The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
– The acceptable mean percentage viability range for positive controls is 0-40 % and the standard deviation value (SD) of the % viability should be ≤ 18.
– For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

CRITERIA FOR IN VITRO INTERPRETATION
Mean tissue viability % is ≤ 50 % = Irritant
Mean tissue viability % is > 50 % = Non Irritant

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Duration of exposure: 15 min.

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The results of the optical density measured at 570 nm and the calculated % viability of the cells are presented below:

 

Substance	Mean Optical Density	Mean Viability (%)
Negative Control	0.816	100 (standard deviation: 2.18)
Positive Control	0.038	5 (standard deviation: 1.18)
Test Item	0.780	96 (standard deviation: 1.06)

 

Summary

All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item revealed no skin irritation in vitro (EPISKIN model).

Executive summary:

In a skin irritation study ( Toxi-Coop Zrt., 2012,EPISKIN model, OECD 439, GLP compliance) disks of epidermal units (three units / chemical) were treated with the test item Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester)and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. SIM-Ester did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin using the in vitro EPISKIN model.