Dodecyltrimethylammonium bromide

Endpoint summary

Description of key information

Skin:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance showed skin irritating properties (UN GHS: Category 1 or Category 2) (reference 7.3.1-1).

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the test item showed no skin corrosive potential (UN GHS: No Category) (reference 7.3.1-2).

Based on the available data the test item is classified for skin irritation, Cat. 2 according to Regulation (EC) No 1272/2008 (CLP) and no further testing is necessary.

Eye

In an in vitro eye irritation study according to OECD Guideline 492, the eye hazard potential of the test item could not be predicted (reference 7.3.2-1). In order to further evaluate the eye hazard potential of the substance the Bovine Corneal Opacity and Permeability Test according to OECD Guideline 437 was performed. No prediction regarding the eye hazard potential can be made (reference 7.3.2-2). However, based on the available data the substance is classified for eye irritation, Cat. 2 according to Regulation (EC) No 1272/2008 (CLP) and no further testing is necessary.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

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 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

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

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

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

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.

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.

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).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-07-12 to 2018-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B. 40 bis. (in Vitro Skin Corrosion: Human skin Model Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTO X Protocol SkinEthic™ Skin Corrosivity Test

Version / remarks:

April 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The SkinEthic™ RHE-model RHE/S/17 obtained from Episkin/SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 18-RHE-064

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Gently rinsing with a minimum volume of 20 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 h ± 15 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
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.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 ± 3 mg

NEGATIVE CONTROL
- Amount(s) applied: 40 ± 3 µL

POSITIVE CONTROL
- Amount(s) applied: 40 ± 3 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; 3 min (mean value of tissue 1 and 2)

Value:

86.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1; 1 hour (mean value of tissue 1 and 2)

Value:

40.8

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: Yes

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

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	3 min	1.849	98.9%	1.891	101.1%	1.870	100.0%	1.6%
	1 hour	1.749	97.9%	1.822	102.0%	1.786	100.0%	2.9%
Positive Control	1 hour	0.013	0.7%	0.009	0.5%	0.011	0.6%	16.7%
Test item	3 min	1.595	85.3%	1.652	88.3%	1.624	86.8%	2.4%
	1 hour	0.666	37.3%	0.791	44.3%	0.729	40.8%	12.0%

Acceptability of the Test

Acceptability of the Negative Control: The negative control OD values were between 1.749 and 1.891, 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 0.6% after 1 hour exposure and, thus, lower than 15 %.

Test Substance Data Acceptance Criteria:The range between identically treated tissues was less than 30 % (3.5 % after 3 minutes exposure and 18.8% after 1 hour exposure).

The study met all acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated.

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 corrosion potential.

After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.6% and, thus, lower than the historically established threshold of 1.04%. After treatment with the negative control (deionised water) the mean ODs were 1.870 (3 minutes exposure) and 1.786 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.571 and 1.422, respectively. Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was >50% after 3 minutes exposure (mean viability: 86.8%) and >15% after 1 hour exposure (mean viability: 40.8%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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".

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.

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

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

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

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.

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

9 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: not specified, nine corneas
- Characteristics of donor animals: Age: 17-100 months (cornea diameter: 23 - 28 mm)
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: Not specified
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 150 mg
- Concentration: 150 mg/ 750 µL (20% w/v solution)

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9%
- Lot/batch no.: 17361013

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 min with fluorescein

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES : 3

SOLVENT/NEGATIVE CONTROL USED : 0.9 % sodium chloride solution

POSITIVE CONTROL USED : 20 % (w/v) Imidazole in 0.9 % sodium chloride solution

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) at 490 nm (OD490)
- Others: Each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:IVIS <= 3: No category, IVIS >3 - <= 55: no prediction, IVIS > 55: Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1; Tissue 1

Value:

33.935

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1; Tissue 2

Value:

30.935

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1; Tissue 3

Value:

33.73

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1; Mean

Value:

32.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD 1.7

Table 1: The IVIS obtained after treatment with dodecyltrimethylammonium bromide

		Opacity	Permeability	IVIS
				Per cornea	Per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	2.7	0.002	2.730	1.5	1.1
		0.8	0.001	0.815
		1.0	-0.001	0.985
Positive control	Imidazole (20%)	69.6	2.045	100.275	96.7	6.7
		66.4	2.302	100.930
		55.3	2.243	88.945
Test item	dodecyltrimethylammonium bromide	32.3	0.105	33.875	32.8	1.7
		24.8	0.409	30.935
		28.0	0.382	33.730

Study Acceptance Criteria

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 - 3.1). After treatment with the positive control (20% Imidazole) the calculated IVIS was 96.7 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.4 — 132.8). Therefore, the study fulfilled the acceptance criteria.

The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item Dodecyltrimethylammonium bromide cannot be predicted.

Executive summary:

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to dodecyltrimethylammonium bromide as a 20% (w/v) solution in a 0.9% sodium chloride solution according to OECD 437. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: -1.4 - 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 96.7 (study acceptance criteria range: 82.4 - 132.8). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 32.8 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:
The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (according to OECD guideline 431) and the Skin Irritation Test (according to OECD 439).

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the skin irritating properties of dodecyltrimethylammonium bromide was determined (reference 7.3.1-1).

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).

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated (reference 7.3.1-2).
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 corrosion potential.
After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.6% and, thus, lower than the historically established threshold of 1.04%. After treatment with the negative control (deionised water) the mean ODs were 1.870 (3 minutes exposure) and 1.786 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.571 and 1.422, respectively. Thus, the acceptance criteria were met.
Following treatment with the test item, the tissue viability was >50% after 3 minutes exposure (mean viability: 86.8%) and >15% after 1 hour exposure (mean viability: 40.8%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

According to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2 or Category 1). Further testing according to OECD 431 showed that the test item is not considered as corrosive to skin. Based on the results of both in vitro tests, the test item is considered to be irritating to the skin, cat. 2 (H315).

Eye irritation:

In an in vitro eye irritation test (RhCE) according to OECD Guideline 492, the potential of dodecyltrimethylaminonium bromideto induce eye irritation in an in vitro human cornea model was determined (reference 7.3.2-1).
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.

As no prediction can be made from the EpiOcular Test a second in vitro test was performed in a bottom-up approach as recommended by OECD (IATA for serious eye damage and eye irritation, 2017). The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item, dodecyltrimethylammonium bromide, as a 20% (w/v) solution in a 0.9% sodium chloride solution according to OECD 437 (reference 7.3.2-2). As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: -1.4 - 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 96.7 (study acceptance criteria range: 82.4 - 132.8). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 32.8 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

The test item was evaluated for eye irritation and serious eye effects in validated in vitro test methods using a Bottom-up approach. No prediction can be made based on both test systems therefore a weight of evidence approach is used to come to a conclusion. In the in vitro eye irritation tests adverse effects on cell viability and opacity of the cornea are observed but the substance is not identified as UN GHS Cat.1.

The mean IVIS in the Bovine Corneal Opacity and Permeability method (OECD TG 437) was 32.8 and thus below to the threshold of 55 for UN GHS Category 1. Furthermore, the mean cell viability obtained in the Eye Irritation Test (OECD TG 492) is 0.9 % and thus far below the threshold of 60 % which triggers “No Category”. In conclusion, the test item should be classified for eye irritation as UN GHS Cat.2. Therefore, no further testing is necessary.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for skin irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for skin irritation, cat. 2 (H315) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

The WoE evaluation of the consistency, quality and relevance of all available data allows a decision on the eye irritation/serious eye damage potential of the test item. The test item is classified for eye irritation cat. 2 (H319) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776. No further testing is necessary.