Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an internationally recognized protocol and followed recognized good laboratory practice standards.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
In accordance with REACH section 1.4 Annex XI and in accordance with the IR CSA, Chapter R.7a Endpoint specific guidance and guidance on application of CLP criteria, an in vivo skin irritation study (required in section 8.1.1) is not considered necessary as a validated in vitro skin irritation study is available. The used assay, EpiSkin, has undergone formal ECVAM/ESAC validation, and was found reliable as test method of distinguishing non-irritants from irritants and may therefore fully replace the traditional skin irritation test (ECVAM/ESAC, 2008).
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 5-(sodiosulfo)isophthalic acid
- Physical state: white powder
- Analytical purity: 99.4%
- Lot/batch No.: 6F21174000
- Expiration date of the lot/batch: 2 December 2013
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark. The material is hygroscopic and must be stored in a well-sealed container.

Test animals

Species:
other: reconstituted human epidermis
Strain:
other: n/a
Details on test animals and environmental conditions:
The test system consisted of EPISKIN Small Model(TM) (EPISKIN-SM(TM), 0.38 cm^2, Batch no.: 12-EKIN-032).

This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a lighly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source

SkinEthic Laboratories, Lyon, France.

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: phosphate buffered saline (negative control); 5% sodium dodecyl sulfate (positive control)
Amount / concentration applied:
The solid test substance (11.6 to 12.2 mg) was added directly on top of the skin tissue and spread to match the size of the tissue.
Duration of treatment / exposure:
15 min
Observation period:
Not applicable
Number of animals:
Replicates of 3 for each test substance, positive or negative control.
Details on study design:
Preparation and preincubation

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 25 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions

All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 83 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Test for reduction of MTT by the test substance

5-(Sodiosulfo)isophthalic Acid was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 13.0 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours 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 together with negative and positive controls. 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 and the solid test substance (11.6 to 12.2 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (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 hours 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 (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 70 hours. The
amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan spectrum.

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.

Acceptability of the assay

The in vitro skin irritation test is considered acceptable if 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 hours 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 hours 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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean tissue viability (% of control)
Value:
96
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

5-(Sodiosulfo)isophthalic Acid was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that 5 - (Sodiosulfo)isophthalic Acid did not interact with MTT.

The positive control had a mean cell viability after 15 minutes exposure of 46% and did not meet the acceptability criteria. Approximately 1 hour after the performance of this test another in vitro skin irritation test was performed using the same batch of skin and the same batch of 5% (aq) SDS. The positive control had a mean cell viability of 5% and met the acceptability criteria. The absolute mean OD570 of the negative control tissues (in both in vitro skin irritation tests) were within the laboratory historical control data range (See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. It can be concluded that the deviation in the mean cell viability of the positive control in the first in vitro skin irritation test was caused by a technical error.

Mean tissue viability in the in vitro skin irritation test with 5 -(Sodiosulfo)isophthalic Acid (see table):

   Mean tissue viability(percentage of control)
Negative control   100
 test substance  96
 Positive control (note 1)  5

Note 1: Based on second in vitro skin irritation test

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: If the mean tissue viability after exposure for 15 min to the test substance is > 50%, the material is considered a non-irritant.
Conclusions:
It is concluded that this test is valid and that 5-(Sodiosulfo)isophthalic Acid is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.