Butyl hydrogen maleate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2012 - 08 June 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:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl hydrogen maleate

EC Number:

213-116-3

EC Name:

Butyl hydrogen maleate

Cas Number:

925-21-3

Molecular formula:

C8H12O4

IUPAC Name:

(2Z)-4-butoxy-4-oxobut-2-enoic acid

Test material form:

other: liquid

Details on test material:

Description, Identification and Storage Conditions
Sponsor's identification : Butyl Hydrogen Maleate
Description : pale yellow liquid
Lot number : 120224
Purity : ≥95%
Date received : 20 March 2012
Expiry date : 30 March 2013
Storage conditions : room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type lV collagen.

Details on animal used as source of test system:

Not applicable.

Justification for test system used:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type lV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by vialbe cells in the test item treated cultures relative to the negative control.

Vehicle:

unchanged (no vehicle)

Details on test system:

Method:
Pre-Test
Assessment of Direct Test ltem Reduction of MTT via MTT Dye Metabolism, Cell Viability Assay:
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 was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µl of the test item was added to 2.2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.

Pre-Incubation (Day 0: tissue arrival- Day 1)
2.2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37°C, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

Main Test
Application of Test Item and Rinsing (Day 2)
2.2 ml of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 50 pl of the test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 pl of 0.9% w/v sodium chloride solution served as negative controls.

Duplicate tissues, treated with 50 µl of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca“ and Mg”. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of12-well plate until all tissues were rinsed.

2.2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours :5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 ml micro tubes containing 850 pl of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT~loaded tissues.

Absorbance/Optical Density Measurements (Day 3)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS:
Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item was predicted from the relative mean tissue viabilites obtained after the 3, 60 and 240-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloried solution. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = mean OD540 of test item / mean OD540 of negative control x 100.

Quality Criteria:
The resuls iof the assay are considered acceptable if the following assay acceptance criteria are achieved.
Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is >=0.115 and <=0.400.
Positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240-minute exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µl of the test item was applied topically to be tissues.

Duration of treatment / exposure:

3, 60 and 240 minutes

Number of replicates:

Duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes

Value:

5.8

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

10.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

75.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 7.2% relatvie to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criteria was satisfied.
The mean OD540 for the negative control treated tissues was 0.207. The negative control acceptance criteria was satisfied.

Any other information on results incl. tables

Mean OD541, Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period (Minutes)	Mean OD540 of ducplicate tisssues	Relative mean viability (%)
Negetive Control Item	240	0.207	100*
Positive Contol Item	240	0.015	7.2
Test item	240	0.012	5.8
	60	0.022	10.6
	3	0.156	75.4

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

The test item was classified as Corrosive to the skin and classified as Skin Corrosive Cat. 1B according to CLP.
The test item was also classified as corrosive to the skin.

Executive summary:

Introduction:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the following:

-OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)

-Method B.40 of Commission Regulation (EC) No. 440/2008

The EPISKIN™ model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group ll & Ill) test items.

Methods:

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. 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 (OD) was measured at 540 nm (OD540).

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:

240 minutes exposure : 5.8%

60 minutes exposure 10.6%

3 minutes exposure 75.4%

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

Conclusion:

The test item was classified as Corrosive to the skin and classified as Skin Corrosive Cat. 1B according to CLP.

The test item was also classified as corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC. The symbol the indication of danger “Corrosive” and the risk phrase R 34 “CAUSES BURNS" are therefore required. The UN packing group ll is also required.