Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 19 December 2012 and 24 December 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

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:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:
- OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation”
- Method B.46 of Commission Regulation (EC) No. 440/2008/EC
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : Sodium Glucoheptonate (EC 250-480-2)
Description : dark brown liquid
Lot number : 921000100
Purity : 99%
Date received : 09 February 2012
Expiry date : 09 February 2015
Storage conditions : room temperature in the dark

Test animals

Species:
other: not applicable in vitro study
Strain:
other: not applicable in vitro study
Details on test animals and environmental conditions:
Animals were not used as this is an In Vitro study, however the tissues were prepared as followed:

Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12-well plate containing 2.2 ml of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO 2 in air for 48 ±1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.2 ml of maintenance medium for approximately 1 hour at room temperature.

Test system

Type of coverage:
not specified
Preparation of test site:
other: not applicable as in vitro study
Vehicle:
unchanged (no vehicle)
Controls:
not required
Duration of treatment / exposure:
15 minutes
Observation period:
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 hours.
Number of animals:
not applicable as in vitro study, however the test was done in triplicate.
Details on study design:
NEGATIVE AND POSITIVE CONTROL ITEMS
Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. The negative control item was used as supplied.
Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control. The positive control item was prepared as a 5% w/v aqueous dilution.

Pre test:
Assessment of Direct Test Item Reduction of MTT:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:
10 μl of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT. The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.

In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

Pre-Incubation (Day 0: tissue arrival):
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: overall irritation score
Value:
98.3
Remarks on result:
other:
Remarks:
Basis: mean relative mean viability. Time point: 15 minutes. Reversibility: other: not classified for irritation. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 98.3% after a 15-Minute exposure period.

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1. Table 1 has been attached within the attached background information section.
Other effects:
Direct MTT Reduction:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Any other information on results incl. tables

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.926 and the standard deviation value of the percentage viability was 6.8%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 5.8%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be Non-Irritant (NI).
Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. This method was designed to be compatible with the following:

- OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

- Method B.46 of Commission Regulation (EC) No. 440/2008/EC.

Method:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results:

The relative mean viability of the test item treated tissues was 98.3% after the 15-Minute exposure period.

Quality criteria:

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:

The test item was considered to be Non-Irritant (NI).