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Administrative data

skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test substance contained 64% water and was not taken into account for setting the limit dose

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction product of tin, citric acid and nitric acid
EC Number:
Molecular formula:
not available
Reaction product of tin, citric acid and nitric acid
Test material form:

In vitro test system

Test system:
artificial membrane barrier model
unchanged (no vehicle)
Details on test system:

Corrositex® kit:
InVitro International, Irvine CA, USA, containing: reagents required for qualification and categorization screen,
biobarrier matrix powder and diluent, membrane discs and vials containing the Chemical Detection System.

Negative control (NC): 10% citric acid
Positive control (PC): Sodium hydroxide (solid)

The Corrositex® assay is a standardized in vitro corrosion test. The Corrositex® assay kit is commercially available from InVitro International. The Corrositex® Biobarrier Membrane is a test system consisting of a reconstituted collagen matrix. The assay is based on the time that is required for the test substance to penetrate through the Corrositex® Biobarrier Membrane and produce a change in the Chemical Detection System (CDS).
The Corrositex® assay is used to determine the corrosive potential of test substances. The assay is limited to testing those materials which cause detectable pH changes in the CDS.

The experimenal design of this study consisted of a qualification screen with the CDS (to determine if a color change can be detected) and a categorization screen (to categorize weak acids/bases and strong acids/bases), which were performed as a pretest, and a definitive Corrositex® assay.
The Corrositex® assay was evaluated on the basis of the color change of the CDS. The time that a color change was observed was recorded manually and the breakthrough times of the four replicates was used to determine the corrosive potential of the test substance.

Qualification screen:
For the qualification screen, 150 µL of the test substance was added to the CDS screening tube. If the test substance failed to produce a color change in the CDS within one minute, the test substance could not be analyzed in this system, and no further testing was required.

Categorization screen:
The categorization screen was used to assess the appropriate scoring scale for the test substance. It was performed by adding 150 µL of test substance to each tube A and B. Each tube was mixed and the resulting color observed. If required, 2 drops of the "confirm" reagent were added to tube B, the tube mixed, and the resulting color observed. The categorization kit and color chart provided by InVitro International were used to determine the category. The test substance was scored as category 1 (high acid/alkaline reserve) or category 2 (low acid/alkaline reserve) as described under "Evaluation of results".

Biobarrier preparation:
The vial containing the biobarrier matrix powder was placed in a water bath at 64 – 68ºC. The entire contents of the biobarrier diluent vial was added slowly to the matrix powder. The stir bar rotated slowly enough to avoid foaming of the solution. 200 μL of the solubilized matrix was pipetted into each of the membrane discs. The membrane discs were then refrigerated for at least 2 hours at 2 – 8ºC. The biobarriers were wrapped and stored at 2 – 8ºC for a maximum of 7 days. Any remaining matrix solution was stored at 2 – 8ºC for up to 30 days in order to prepare additional biobarrier membrane discs.

Corrositex® assay:
Following the acceptance of the positive control the Corrositex® assay was performed for the test substance. Four vials containing the CDS were used for the test substance. In addition, one vial was used for the PC, NC and for the color (blank) control, each. A membrane disc coated with the biobarrier matrix was placed into one vial containing the CDS and 500 µL of the undiluted test substance was added onto the membrane disc. An electronic time clock was started with the application. The vial was observed for three minutes for any change in the CDS.
If no color change was observed within three minutes, the remaining membranes were treated with the test substance. An electronic time clock was started with each application. The vials were observed continuously for the first ten minutes. Thereafter the vials were observed for approximately ten minutes around the time points relevant for evaluation or until breakthrough of the test substance occurred. The elapsed time between test substance application and the first change in the indicator solution (i.e. barrier penetration) was recorded.
The positive control vial was prepared as described above and received one pellet of sodium hydroxide on top of the membrane disc. This vial was monitored continuously until breakthrough had occurred.
The negative control vial was prepared as described above and received 500 μL of 10% citric acid. This vial was observed for 60 minutes and was evaluated as “non-corrosive” if no reaction had been observed.

The Corrositex® assay was accepted if the breakthrough time for the positive control substance was in the historic control range (mean ± 2-3 x standard deviations). To demonstrate the functional integrity of the membrane barrier, the acceptance criterium for the negative control was not to induce membrance breakthrough within a 60 min observation period.
Control samples:
yes, concurrent negative control
yes, concurrent positive control

Results and discussion

In vitro

Irritation / corrosion parameter:
other: mean breakthrough time through Corrositex® Biobarrier Membrane in minutes
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Remarks on result:
The mean breakthrough time of the test substance in 4 vials was 4 minutes 50 seconds

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Migrated information Criteria used for interpretation of results: EU

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