Tributylmethylammonium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28APR2015 - 01MAY2015

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26 September 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tributylmethylammonium chloride

EC Number:

260-135-8

EC Name:

Tributylmethylammonium chloride

Cas Number:

56375-79-2

Molecular formula:

C13H30N.Cl

IUPAC Name:

tributyl(methyl)azanium chloride

Test material form:

solid

Remarks:

White powder with lumps and flakes

Details on test material:

- Colour: White

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No.of test material: ZMG-187261
- Expiration date of the lot: 16 April 2016
- Purity test date: 31-07-2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: negative

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Normal, human-derived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure (3 minutes): room temperature
- Temperature used during exposure (1 hour) and during post-treatment incubation: 36.4 - 36.7°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: not described
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
- The test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

- 26.0 to 26.7 mg test substance (the skin was moistened with 25 μl Milli-Q water to ensure close contact)
- 50 μl Milli-Q water (negative control)
- 50 μl 8N KOH (positive control)

Duration of treatment / exposure:

3 minutes or 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2/ incubation time

Test animals

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.4 - 36.7
- Humidity (%): 75 - 87

Test system

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 26.0 to 26.7 mg (skin tissue was moistened with 25 μl Milli-Q water to ensure close contact)

VEHICLE
The test substance was \moistened with \ mL of the vehicle.

Duration of treatment / exposure:

3-minute and 1-hour exposure

Number of animals:

2 tissues/ exposure period

Details on study design:

TEST SITE
- EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N; obtained from MatTek Corporation, Ashland MA, U.S.A). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 3 minutes or one hour

POST INCUBATION PERIOD
- 3 hours

SCORING SYSTEM:
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

CONTROLS:
Negative control: 50μL Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: 50μL 8N KOH.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes incubation

Run / experiment:

1

Value:

86

Vehicle controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

14%

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour incubation

Run / experiment:

1

Value:

83

Vehicle controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7%

Other effects / acceptance of results:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MTBAC compared to the negative control tissues was 86% and 83% respectively. Because the mean relative tissue viability for MTBAC was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MTBAC is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 14% and 7% after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 22% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%. It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

MTBAC was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that MTBAC did not interfere with the MTT endpoint.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that MTBAC is not corrosive in the in vitro skin corrosion test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EpiDerm Skin Model), the influence of MTBAC on the viability of human skin was tested. The test substance was applied directly to 0.6 cm2 cultured skin (26.0 to 26.7 mg, in presence of 25 μl Milli-Q water). After 3 minutes or one hour, the substance was removed and cells were cultured for 3 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14/% following 3 minute exposure whereas the test substance showed cell viability of 86%. Exposure for 60 minutes resulted in a mean cell viability of 83% and 7% for resp. MTBAC and the positive control. Since the mean relative tissue viability after exposure to MTBAC was above 50% after 3 minute exposure and above 15% after 1 hour exposure, it can be concluded that MTBAC is not corrosive in the in vitro skin corrosion test.