Tetrabutyltin

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th to 30th April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutyltin

EC Number:

215-960-8

EC Name:

Tetrabutyltin

Cas Number:

1461-25-2

Molecular formula:

C16H36Sn

IUPAC Name:

tetrabutylstannane

Test material form:

liquid

Details on test material:

Sponsor's identification: CAS No 1461-25-2
Description: clear colourless liquid
Date received: 06 April 2010
Expiry date: 28 March 2012
Storage conditions: approximately 4 °C in the dark

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstituted Human Epidermis (RHE)

Details on animal used as source of test system:

The EPISKIN model is a three-dimensional reconstituted 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 IV 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.

EPISKIN Model Kit 0.38 cm2
Supplier: SkinEthic Laboratories, Nice, France
Date received: 27 April 2010

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST
ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
- 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-diphenyl-tetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material 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 by the procedure described below.

- Test for Direct MTT Reduction:
As specified, a test material 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 material was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test material 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.
If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
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 material, control and time point. The tissues were incubated at 37 °C, 5 % CO2 in air for at least 24 hours. After 24 hours the medium underneath the tissues was refreshed and tissues were returned to the incubator for a further 24 hours.

MAIN TEST
APPLICATION OF TEST MATERIAL 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 material for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control materials for an exposure period of 240 minutes. 50 µL of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 µL 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 the 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 material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.2 mL of 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. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability). Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was taken using the EPISKIN™ 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 µL 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of the test material was applied

Duration of treatment / exposure:

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes.

Duration of post-treatment incubation (if applicable):

3, 60 and 240 minutes.

Number of replicates:

Two per time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 3 minutes

Value:

99

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 60 minutes

Value:

108.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 240 minutes

Value:

92.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

TEST MATERIAL, POSITIVE CONTROL MATERIAL AND NEGATIVE CONTROL MATERIAL:
Mean OD540 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean viability of the test material treated tissues was as follows:
240 minutes of exposure: 92.8 %
60 minutes of exposure: 108.7 %
3 minutes of exposure: 99.0 %

The qualitative evaluation of tissue viability is given in Table 2.
Following the 3, 60 and 240-minute exposure periods, the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

QUALITY CRITERIA
The relative mean tissue viability for the positive control treated tissues was 4.8 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table1: Mean OD540 Values and Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	Exposure Period (minutes)	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative Control Material	240	0.207	100*
Positive Control Material	240	0.010	4.8
Test Material	240	0.192	92.8
	60	0.225	108.7
	3	0.205	99.0

*The mean viability of the negative control tissues is set at 100 %

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Exposure Period (minutes)	Tissue 1	Tissue 2
Negative Control Material	240	-	-
Positive Control Material	240	++	++
Test Material	240	-	-
	60	-	-
	3	-	-

MTT Visual Scoring Scheme of EpiSkin Tissues

-     =  Blue tissue (viable)

+    =  Blue/white tissue (semi-viable)

++  =  Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of the test material using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

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 II & III) chemicals.

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test material 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 was measured at 540 nm.

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

The relative mean viability of the test material treated tissues were as follows:

240 minutes of exposure : 92.8 %

60 minutes of exposure : 108.7 %

3 minutes of exposure : 99.0 %

The quality criteria required for acceptance of results in the test were satisfied. The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.