Dodecyltrimethylammonium bromide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-17 to 2018-04-19

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

9 October 2017

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability : The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used: See "Any other information on materials and methods".

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg

POSITIVE CONTROL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

6 hours (± 15 minutes)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 minutes)

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used : The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential.
Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.
- RhCE tissue construct used, including batch number : EpiOcular™ Tissue (OCL-200, OCL-212), Lot No.: 27033, Keratinocyte strain: 4F1188, Supplier: MatTek In Vitro Life Science Laboratories
- Doses of test chemical and control substances used :
test substance 50 mg
pos./neg. Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 37 ± 1 °C and 5 % CO2
Post-exposure: room temperature
Post-exposure incubation: 37 °C and 5 % CO2
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
For correct interpretation of results, it is necessary to assess the ability of a test item to directly reduce MTT. For this purpose, a 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item were added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated at 37 ± 1°C and 5% CO2 and protected from light for 3 hours (± 5 minutes). Sterile deionised water (50 µL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Colored test items or test items which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties. The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1°C and 5% CO2 protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm (Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Description of the method used to quantify MTT formazan :
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 ml isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: % cell viability

Run / experiment:

1; Tissue 1

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: % cell viability

Run / experiment:

1; Tissue 2

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: % cell viability

Run / experiment:

1; Mean

Value:

0.9

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

Any other information on results incl. tables

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

Table 2: Results obtained after treatment of the reconstructed human cornea-like epithelium (RhCE) model with dodecyltrimethylammonium bromide

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.577	93.5%	1.795	106.5%	1.686	100.0%	9.19	13.0%
Positive Control	0.243	14.4%	0.358	21.2%	0.301	17.8%	4.81	6.8%
Test item	0.015	0.9%	0.8%	0.8%	0.015	0.9%	0.07	0.1%

Acceptability of the Test
1. The negative control OD is >0.8 and <2.5 (1.577 and 1.795).
2. The mean relative viability of the positive control is below 50% of the negative control viability (17.8%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.1% to 13.0%) in the same run (for positive and negative control tissues and tissues of single chemicals).
The study met all acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the present study, the eye hazard potential of dodecyltrimethylammonium bromide cannot be predicted.

Executive summary:

In an in vitro skin irritation test (RhCE) according to OECD Guideline 492, the potential of Dodecyltrimethylaminonium bromide to induce eye irritation in an in vitro human cornea model was determined.
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the mean OD was 1.686 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.8% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.
Following treatment with the test item, the tissue viability was 0.9% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.