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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015 to 2015-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Deviations:
yes
Remarks:
Different batches of the negative and positive control substances then indicated in the protocol were used. The fact that different batches were used (but with the same purity) has no effect on the study results.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
Identification Maleic acid
Appearance White solid
Purity/composition correction factor No correction factor required
Test substance handling Use amber glassware or wrap container in aluminum-foil
Stability at higher temperatures available
Chemical name (IUPAC), synonym or trade name 2-Butenedioic acid (Z)-
CAS Number 110-16-7
Molecular formula C4H4O4
Molecular weight 116.07

Test animals

Species:
other: in vitro: Corrositex® test system

Test system

Amount / concentration applied:
Maleic acid was applied (approximately 500 mg), directly on top of the bio-barrier. During weighing was the test substance protected from light by wrapping the glassware in tin foil.
Duration of treatment / exposure:
Corrositex® penetration response time (minutes)

Timescale Category 1: 0 to 3 min. (Cat 1A); > 3 to 60 min.(Cat 1B); > 60 to 240 min. (Cat 1C); > 240 min. (Non-corrosive)
Timescale Category 2: 0 to 3 min. (Cat 1A); > 3 to 30 min. (CAT 1B); > 30 to 60 min. (CAT 1C); > 60 min. (Non-corrosive)

Details on study design:
Test system
Corrositex® is a test system that is composed of two components, a hydrated collagen matrix (bio-barrier) on a supporting filter membrane and CDS, an underlying aqueous solution of two pH indicator dyes.

The Corrositex® kit contained:
- Bio-barrier matrix.
- Bio-barrier diluent.
- Vials with Chemical Detection System (= CDS).
- Membrane discs.
- Compatibility test tubes.
- Timescale categorize test tubes with buffer A and B.
- Confirm reagent.

The batch number of the kit used for the experiment was CT051214
Rationale
Recommended test system in international guideline (OECD 435).

Source
InVitro International Inc., Irvin CA, USA.
The bio-barrier diluent (10 ml) was added to the bio-barrier matrix powder (1 gram) which contained a micro stir bar. This was stirred and heated slowly to 68°C in a water bath container. After preparation the matrix was stored refrigerated.

The day before testing the matrix was solubilized by heating it for 3-4 minutes at 60 °C. The membrane discs (at room temperature) were filled with 200 µl solubilised bio-barrier matrix. The entire membrane was covered with matrix and care was taken to avoid air bubbles.

The bio-barrier was prepared the day before testing and stored in the refrigerator until use.

All tests were performed at room temperature. The samples were at room temperature when applied.

Prior to performing the membrane barrier penetration study, Maleic acid was evaluated for the compatibility with the CDS. Maleic acid (102 mg) was added to the compatibility test tubes filled with CDS fluid. The tube was shaken to dissolve solids. In case the test substance was immiscible, the vial was shaken and the colour change was read at the interface after 1 minute.

If the CDS solution detected a colour or consistency change within 5 minutes, the test substance was tested for corrosivity using the Corrositex® kit. In case the CDS solution detected no colour or consistency change the membrane barrier penetration study was not performed with Corrositex®.
Maleic acid was classified into one of the two timescale categories.

This category determined how the penetration response time (if one occurs) was interpreted. The two different penetration response timescales were based on the acid or alkali reserve of the chemical. Maleic acid, 109 and 114 mg, was added to buffers A and B, respectively, shaken vigorously for
10 seconds and after 1 minute the colour change was read on the chart. In case the test substance was immiscible, the colour change was read at the interface after another minute. Buffer A detected weak or strong acids and buffer B detected strong or weak bases. In case no colour change was observed in both buffers a conformation test was performed. Two drops of confirm reagent were added to buffer B. The tube was shaken vigorously for 5 seconds and the colour of the solution was read on the chart confirming that the substance is a timescale category 2 substance.

The test was performed on a total of 4 membrane discs with bio-barrier matrix together with a negative and positive control. A disc was placed on a vial with CDS fluid. The test substance (526, 503, 518 and 504 mg) was applied on top of the matrix within two minutes after the disc was placed on the CDS fluid. One disc was exposed to 500 µl citric acid (10%, negative control) and one disc was exposed to 510 mg of the positive control sodium hydroxide. The test substance and controls were evenly distributed. Each vial was at least monitored for the first 5 minutes and 5 minutes before and after each packing cut-off time. The time of a change in the CDS fluid was recorded. Changes in the CDS may be colour changes (red, orange or lightening) and flaking or precipitation. The elapsed time between application and penetration of the membrane was determined.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
mean
Value:
25
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Maleic acid was evaluated for the compatibility with the CDS. Because a colour change (to pink/red) was observed after 1-minute, Maleic acid was compatible with the Corrositex® test system.
Maleic acid was classified into one of the two timescale categories. The timescale category test showed that Maleic acid is a timescale 1 substance (after one-minute tube A showed a colour change into pink). After 5 minutes the colour was pink-red. Since there appeared a colour change after 1 minute, the addition of the confirm reagent to tube B was not performed.
The membrane barrier test was performed and the elapsed time between the application of Maleic acid and controls and penetration through the membrane was recorded. The individual penetration times are presented in APPENDIX 2.
Since Maleic acid was a timescale 1 test substance and showed a mean penetration time of 25 minutes, based on this test Maleic acid would be classified as Skin Corrosive 1B (CLP) and UN packing group II (= GHS 1B). The negative control citric acid (10%) showed a mean penetration time of >60 minutes (actual penetration time 95 minutes) and was therefore non-corrosive. The positive control sodium hydroxide (as it is) showed a mean penetration time of 22 minutes and was therefore classified as UN packing group II (= GSH 1B). It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
It is concluded that this test is valid and that Maleic acid would be classified as Skin Corrosive 1B (CLP) and UN packing group II (= GHS 1B) in the Corrositex® assay under the experimental conditions described in this report.
Executive summary:

This report describes the ability of Maleic acid to pass through a bio-barrier and to elicit a colour change in the underlying liquid chemical detection system. The study procedures described in this report were based on the most recent OECD guideline OECD 435. Maleic acid was a white solid with a purity of 100.1%. The test substance compatibility test showed that Maleic acid was compatible with the chemical detection system. Based on the acid reserve, Maleic acid was classified as a timescale 1 compound. Maleic acid was applied directly (approximately 500 mg) on top of the bio-barrier matrix. The elapsed time between application and penetration was determined by monitoring changes in the chemical detection system.  The penetration time of Maleic acid was 25 minutes. Since Maleic acid was a timescale 1 test substance and showed a mean penetration time of 25 minutes, based on this test Maleic acid would be classified as Skin Corrosive 1B (CLP) and UN packing group II (= GHS 1B). The negative control citric acid (10%) showed a mean penetration time of >60 minutes (actual penetration time 95 minutes) and was therefore non-corrosive. The positive control sodium hydroxide (as it is) showed a mean penetration time of 22 minutes and was therefore classified as UN packing group II (=GSH 1B). It was therefore concluded that the test system functioned properly. Finally, it is concluded that this test is valid and that Maleic acid would be classified as Skin Corrosive 1B (CLP) and UN packing group II (= GHS 1B) in the Corrositex® assay under the experimental conditions described in this report.