Trimethyloctadecylammonium bromide

Endpoint summary

Description of key information

EYE IRRITATION/CORROSION

The test substance causes serious eye damage.

SKIN IRRITATION/CORROSION

Skin irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 05 to 08, 2016

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)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B40bis, 2008

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek
- Tissue batch number(s): 23344
- Delivery date: 05 July 2016
- Date of initiation of testing: 05 July 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/ml
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

DECISION CRITERIA
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD of test item/mean OD of negative control ) x 100

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes incubation

Value:

ca. 96.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes incubation

Value:

ca. 99.9

Negative controls validity:

valid

Positive controls validity:

valid

Direct MTT Reduction

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

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a white color. This was attributed to the intrinsic color of the test item and was considered not to have the potential to cause color interference.

Positive Control Item and Negative Control Item

The relative mean viabilities for each treatment group were as follows:

Percentage Viability:

Negative Control

3 minute exposure period = 100

60 minute exposure period = 100

Positive Control

3 minute exposure period = 3.0

60 minute exposure period = 3.0

Quality Criteria

The mean OD562 for the negative control treated tissues was 2.289 for the 3-Minute exposure period and 2.263 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 3.0 % relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

other: The substance is considered to be non-corrosive according to the OECD guideline 431

Conclusions:

Non-corrosive

Executive summary:

Method

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes, according to the OECD guideline 431.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µl samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The percentage of tissue Viability , after incubation of the test substance, was:

3 minute exposure period = 96.9

60 minute exposure period = 99.6

Conclusion

The test item was considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 28 to August 11, 2016

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:

2013

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:

2008

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: local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): cattle typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS). They were transported to the test facility over ice packs on the same day of slaughter.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item: 0.75 ml (20 % w/v solution in 0.9 % w/v sodium chloride solution).
Negative control item: 0.75 ml (0.9 % w/v sodium chloride solution).
Positive control item: 0.75 ml (20 % w/v solution in 0.9 % w/v sodium chloride solution).

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: the EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE: Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: visually observed
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of the Anthos 2001 microplate reader (OD492)
- Histopathology: the corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG.

Irritation parameter:

in vitro irritation score

Value:

ca. 65.9

Negative controls validity:

valid

Positive controls validity:

valid

Corneal Epithelium Condition

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	65.9
Negative Control	1.2
Positive Control	87.5

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

other: Category 1 according to the OECD 437 criteria

Conclusions:

The test substance causes serious eye damage.

Executive summary:

Method

The potential eye irritation/corrosion of the test substance has been evaluated using the BCOP test ( “Bovine Corneal Opacity and Permeability Assay”) according to the OECD guideline No. 437 (updated 26 July 2013).

Observations

The IVIS score recorded for the test Item is 65.9

Conclusion

According to the classification reported in the OECD Guideline, if the IVIS Scor recorded is > 55, the test item shell be classified as Category 1. UN GHS or EU CLP Causes serious eye damage.

The test substance causes serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION/CORROSION

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes, according to the OECD guideline 431.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µl samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The percentage of tissue Viability , after incubation of the test substance, was:

3 minute exposure period = 96.9

60 minute exposure period = 99.6

EYE IRRITATION/CORROSION

The potential eye irritation/corrosion of the test substance has been evaluated using the BCOP test ( “Bovine Corneal Opacity and Permeability Assay”) according to the OECD guideline No. 437 (updated 26 July 2013).

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The IVIS score recorded for the test Item is 65.9

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

As reported in the OECD guideline 431, " Skin corrosion refers to the production of irreversible damage to the skin manifested as visible necrosis through the epidermis and into the dermis, following the application of a test chemical [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)] (1). This updated Test Guideline 431 provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS (1). It also allows a partial sub-categorisation of corrosives".

The prediction models for the EpiDermTM SCT, associated with the UN GHS (1) classification system, are shown in Table below:

Viability measured after exposure time points (t=3 and 60 minutes)	Prediction to be considered
STEP 1 for EpiDerm^TMSCT
< 50% after 3 min exposure	Corrosive (Category 1A)
≥ 50% after 3 min exposure AND < 15% after 60 min exposure	Corrosive (Category 1B or 1C)
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure	Non-corrosive
STEP 2 for EpiDerm^TMSCT for substances/mixtures identified as Corrosive in STEP 1
< 25% after 3 min exposure	Optional Sub-category 1A *
≥ 25% after 3 min exposure	A combination of optional Sub-categories 1B-and-1C

The value of tissue viability calculated for the test substance using the EpiDermTM SCT method, is 96.9 % in the 3 minute exposure period and 99.9 % in the 60 minutes exposure period. Therefore , according to the the classification criteria reported in the OECD guideline 431 , the substance should be considered as Non Corrosive.

Due to the result obtained in the Eye Irritation/Corrosion test (serious eye damage - Category 1) and using a conservative approach, it is possible to suppose that the test substance would show a skin effect, therefore it has been classified as Skin irritant Category 2, according to the CLP Regulation EC 1272/2008.

EYE IRRITATION/CORROSION

As reported in the OECD guideline 437: "It is currently generally accepted that, in the foreseeable future, no single in vitro eye irritation test will be able to replace the in vivo Draize eye test to predict across the full range of irritation for different chemical classes. However, strategic combinations of several alternative test methods within a (tiered) testing strategy may be able to replace the Draize eye test.... The BCOP test method is an in vitro test method that can be used under certain circumstances and with specific limitations for eye hazard classification and labeling of chemicals".

Using this test the criteria of classification is based on the "in vitro irritancy score (IVIS)". The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤ 55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

The value of IVIS calculated for the test item is 65.9. This value is greater than 55, so it shall be concluded that the test substance causes serious eye damage (Category 1), according to the CLP Regulation (EC 1272/2008).