Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-26 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
BADGE MXDA (#42) [Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7)] is a clear colorless viscous liquid. The test material lot number was ZH1650NZ08. The test material was protected from exposure to light.

Test animals

Species:
human
Strain:
other: EpiDerm Skin Model Bioassay Kit (MatTek Corporation).
Details on test animals and environmental conditions:
Not applicable.

Test system

Type of coverage:
other: Not applicable.
Preparation of test site:
other: Not applicable.
Vehicle:
unchanged (no vehicle)
Controls:
other: deionized water was used as control sample
Amount / concentration applied:
50 ul which covered the entire dermal surface.
Duration of treatment / exposure:
3 minutes and 60 minutes
Observation period:
See below.
Number of animals:
Not applicable.
Details on study design:
Assessment of Direct Test Substance Reduction of MTT
The test substance was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 50 μL of the test substance were added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for approximately one hour. A negative control, 50 μL of sterile, deionized water (Quality Biological), was tested concurrently. If the MTT solution color turned blue/purple, the test substance was presumed to have reduced the MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface between the test substance and the medium.

In cases where the test substance was shown to reduce MTT, only those test substances that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, could present a problem. To evaluate whether residual test substance was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed as described in the section labeled “Killed Controls (KC)”.

The test substance was observed to directly reduce MTT in the absence of viable cells. The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed control experiment was performed concurrently in the definitive assay to determine the extent of the direct MTT reduction (if any) by the test substance and the positive control in non-viable freeze-killed tissues.

Preparation and Delivery of Test Substance
The test substance was tested neat. Prior to dosing, the test substance was aliquotted in a glass vial. The vial was sealed with parafilm and was incubated for at least 30 minutes in a water bath at 37C. Fifty μL of the test substance were applied directly on the tissue so as to cover the upper surface.

The negative control was treated with 50 μL of sterile, deionized water. The positive control, 8N KOH (Sigma), was tested using the same method.

pH Determination
The pH of the test substance was measured using pH paper (EMD Chemicals Inc.). Initially, the test substance was added to pH paper with a 0-14 pH range in 1.0 pH unit increments to approximate a narrow pH range. Next, the test substance was added to pH paper with a narrower range of 0-6 pH units with 0.5 pH unit increments, to obtain a more accurate pH value.

Corrosivity Assay
The test and control substances were tested by treating four EpiDerm™ tissues per material. Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. Fifty microliters of liquid test and control substances were applied topically on the EpiDerm™ tissue. The negative and positive controls were treated as described above. The three-minute exposure time began as soon as the material was spread onto the tissue. This short exposure time precluded treating more than a small number of tissues at once. The tissues exposed for 3 minutes were held at room temperature during dosing, while the tissues exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

A 1.0 mg/mL solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium was decanted. Three-tenths mL of MTT reagent were added to designated wells in a prelabeled 24-well plate. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 180±3 minutes.

After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with parafilm and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for at least two hours at room temperature.

At the end of the extraction period, the liquid within the tissue inserts was decanted into the well from which the tissue insert was taken. The extract solution was mixed and 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred μL of isopropanol were placed in the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

Killed Controls (KC)
To evaluate whether residual test substance was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed. Freeze-killed tissues were received from MatTek Corporation, and were stored in the freezer until use.

For the test substance and the positive control, 8N KOH, single killed tissues were treated with the test substance and positive control in the normal fashion for 3 and 60 minutes. The rinsing, MTT exposure, and solvent extraction procedures were performed exactly as described for the viable tissues. Single killed-control tissues were treated with the negative control for 3 and 60 minutes. A small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue. This background reduction of MTT will be compared to the MTT reduction observed in the test substance-treated killed-control tissues.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: tissue viability
Value:
34.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: tissue viability
Value:
21
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
The test substance was observed to directly reduce MTT in the absence of viable cells. The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed-control experiment was performed. The results of the killed control experiment showed that there was significant direct MTT reduction in the test substance-treated and positive control-treated killed controls. Additional calculations were performed to correct for the amount of MTT reduced directly by the positive control and test substance residues as described in the Presentation of Data section.

The test substance was tested in EpiDerm™ Corrosivity Assay. Two tissues were used to assess viability after a 3-minute exposure, and two tissues were used to assess viability after a 60-minute exposure. Negative and positive controls were tested in parallel. Table 1 summarizes the mean % viability results for the test substance and the positive control.
Other effects:
The test substance was observed to directly reduce MTT in the absence of viable cells. The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed-control experiment was performed. The results of the killed control experiment showed that there was significant direct MTT reduction in the test substance-treated and positive control-treated killed controls. Additional calculations were performed to correct for the amount of MTT reduced directly by the positive control and test substance residues as described in the Presentation of Data section.

Any other information on results incl. tables

Table 1

Test Substance   Name  Conc.  Exposure Time  % Viability  Corrosive?*  pH
 Test Substance  BADGE MXDA  Neat  3 minutes  34.1 Yes   5.5
 Test Substance  BADGE MXDA  Neat  60 minutes  21.0    
 Positive Control  8N KOH  NA  3 minutes  30.0  Yes  NA
 Positive Control  8N KOH  NA  60 minutes  15.5    

NA – Not Applicable

* - A material is considered corrosive if the percent viability is less than 50% at the 3-minute exposure, or less than 15% at the 60-minute exposure

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Based upon the results of this assay, the test substance, BADGE MXDA (#42) Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7), was predicted to be corrosive to skin according to the prediction model validated by ECVAM. Accordingly, the test substance, BADGE MXDA (#42) Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7), would be labeled corrosive to skin within the Globally Harmonized System.
Executive summary:

The MatTek Corporation’s EpiDermTM reconstituted human epidermis model was used to assess the potential skin corrosivity of the test substance, BADGE MXDA (#42) Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7), Lot No. ZH1650NZ08. The skin corrosivity potential of the test substance was evaluated by measuring the relative cell viability in treated tissues after a 3- and 60-minute exposure to the test substance, following the protocol that is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”1. Based upon the results of this study, the test substance, BADGE MXDA (#42) Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7), is predicted to be a skin corrosive. Accordingly, the test substance, BADGE MXDA (#42) Reaction product of m-phenylenebis(methylamine) with oligomerisation products of 4,4’-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane (IUPAC name, ECnr. 500-302-7), would be labeled corrosive to skin within the Globally Harmonized System.