Dodecyltrimethylammonium bromide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-11 to 2018-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dodecyltrimethylammonium bromide

EC Number:

214-290-3

EC Name:

Dodecyltrimethylammonium bromide

Cas Number:

1119-94-4

Molecular formula:

C15H34N.Br

IUPAC Name:

dodecyltrimethylazanium bromide

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 18-RHE-037
- Date of initiation of testing: 2018-04-11

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: ELx800 (BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the tissue viability after exposure and post-treatment incubation is greater than 50%
- The test substance is considered to be irritating to the skin (Category 1 or 2) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50%.
- Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL

Duration of treatment / exposure:

42 minutes (± 1 minute)

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; Tissue 1

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; Tissue 2

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; Tissue 3

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; Mean

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: No. The standard deviation between the three tissues replicates treated with the test item was 27.3% and, thus, >18%. Due to the very irritant potential of the test item very low viabilities were obtained. Little differences between very low viabilities resulted in a high standard deviation >18%. However, this does not influence the outcome or validity of the study.

Any other information on results incl. tables

Table 1: Results abtained after treatment of the reconstructed human epidermis model with the test item

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.507	90.8%	1.756	105.8%	1.716	103.4%	1.660	100.0%	8.1%
Positive Control	0.024	1.4%	0.022	1.3%	0.019	1.1%	0.022	1.3%	15.4%
Test item	0.020	1.2%	0.022	1.3%	0.014	0.8%	0.019	1.1%	27.3%

Acceptability of the Test

Acceptability of the Negative Control: The negative control OD values were between 1.507 and 1.756, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive Control: After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 1.3% and thus lower than 40 %.

The standard deviations between the three tissue replicates of the negative control and the positive control were 8.1% and 15.4%, respectively, and, thus, <18%.

Test Substance Data Acceptance Criteria: The standard deviation between the three tissues replicates treated with the test item was 27.3% and, thus, >18%. Due to the very irritant potential of the test item very low viabilities were obtained. Little differences between very low viabilities resulted in a high standard deviation >18%. However, this does not influence the outcome or validity of the study.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the present study, dodecyltrimethylammonium bromide is considered to possess an irritant potential to skin (UN GHS: Category 2 or Category 1).

Executive summary:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the skin irritating properties of dodecyltrimethylammonium bromide was determined.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, dodecyltrimethylammonium bromide, the tissue viability was 1.1% and, thus, lower than 50%, i.e. according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2 or Category 1).