Registration Dossier

Administrative data

Description of key information

Skin Irritation:

The dermal irritation potential of Tetraethylammonium chloride was estimated using OECD QSAR toolbox v3.4 with logPow as the primary descriptor.

Tetraethylammonium chloride was estimated to be irritating to the skin of rabbits.

Based on the estimated results, Tetraethylammonium chloride can be considered to be irritating to skin and can be classified under the category “Category 2” as per CLP regulation.

Eye Irritation:

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies.Based on the available studies,it can be concluded that the test chemical is unable to cause eye irritation and considered as irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2".

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
data is from OECD QSAR toolbox v3.4 and the QMRF report has been attached
Qualifier:
according to
Guideline:
other: estimated data
Principles of method if other than guideline:
Prediction was done using OECD QSAR toolbox v3.4
GLP compliance:
not specified
Specific details on test material used for the study:
Name of the test chemical: Tetraethylammonium chloride
Molecular formula: C8H20ClN
Molecular weight: 165.706 g/mol
Smiles Notation: [N+](CC)(CC)(CC)CC.[ClH-]
InChI: 1S/C8H20N.ClH/c1-5-9(6-2,7-3)8-4;/h5-8H2,1-4H3;1H/q+1;/p-1
Substance Type: Organic
Physical State: Hygroscopic white Powder
Species:
rabbit
Strain:
not specified
Details on test animals and environmental conditions:
no data available
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
0.5 ml
Duration of treatment / exposure:
24 hours
Observation period:
24,72 hours
Number of animals:
6
Details on study design:
no data available
Other effects / acceptance of results:
no data available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
72 h
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Irritation was observed

Estimation method: Takes mode value from the 5 nearest neighbours
Domain  logical expression:Result: In Domain

((((((("a" or "b" or "c" or "d" or "e" )  and "f" )  and ("g" and ( not "h") )  )  and ("i" and ( not "j") )  )  and "k" )  and "l" )  and ("m" and "n" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Cationic (quaternary ammonium) surfactants by US-EPA New Chemical Categories

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Ammonium salt by Organic Functional groups

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Ammonium salt AND Overlapping groups by Organic Functional groups (nested)

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] AND Aliphatic Carbon [-CH2-] AND Aliphatic Carbon [-CH3] AND Nitrogen, single bonds  [N{v+5}] by Organic functional groups (US EPA)

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Anion AND Cation AND Quaternary ammonium salt by Organic functional groups, Norbert Haider (checkmol)

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as Bioavailable by Lipinski Rule Oasis ONLY

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as Halogens AND Non-Metals by Groups of elements

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as Alkali Earth OR Transition Metals by Groups of elements

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] AND Aliphatic Carbon [-CH2-] AND Aliphatic Carbon [-CH3] AND Nitrogen, single bonds  [N{v+5}] by Organic functional groups (US EPA)

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as 1,2-Oxaza compounds [N-C-O-] OR Alcohol, olefinic attach [-OH] OR Aliphatic Nitrogen, one aromatic attach [-N] OR Aliphatic Nitrogen, two aromatic attach [-N-] OR Aliphatic-C=N-Aliphatic  OR Amide, aliphatic attach [-C(=O)N] OR Amino, aliphatic attach [-N<] OR Amino, aliphatic attach [-NH2] OR Amino-carbonyl compound [NCC(=O)-C] OR Aromatic Carbon [C] OR Aromatic Nitrogen OR Aromatic Nitrogen, [N{v+5}] OR Benzene to CCN  OR Carbonyl, aliphatic attach [-C(=O)-] OR Carbonyl, olefinic attach [-C(=O)-] OR Carbonyl, one aromatic attach [-C(=O)-] OR Chlorine, aliphatic attach [-Cl] OR Chlorine, aromatic attach [-Cl] OR Chlorine, olefinic attach [-Cl] OR Ester, aliphatic attach [-C(=O)O] OR Fluorine, aromatic attach [-F] OR Fluorine, olefinic attach [-F] OR Guanidine derivatives [NC(NH2)=N-aliphatic ring] OR Hydrazine [>N-N<] OR Hydrazo compound [-NH-NH-] OR Hydroxy, aliphatic attach [-OH] OR Hydroxy, aromatic attach [-OH] OR Hydroxy, phosphorus attach [-OH] OR Hydroxy, sulfur attach [-OH] OR Miscellaneous sulfide (=S) or oxide (=O) OR Nitro, aromatic attach [-NO2] OR Nitrogen {v+5}, nitrogen attach OR Nitrogen, hydrogen attach {v+5} OR Olefinic carbon [=CH- or =C<] OR Olefinic carbon [=CH2] OR Oxygen or Sulfur, nitrogen attach [-O- or -S-] OR Oxygen, aliphatic attach [-O-] OR Oxygen, nitrogen attach [-O-] OR Oxygen, one aromatic attach [-O-] OR Oxygen, two phosphorus attach [P-O-P] OR Phosphine oxide [O=P] OR Phosphite, aliphatic attach [-O-P] OR Pyridine, non fused rings  OR Suflur {v+4} or {v+6} OR Sulfate, linear [-O-SO2-O-] OR Sulfinic acid [-S(=O)OH] OR Sulfite, linear [-OS(=O)O-] OR Sulfonate, aromatic attach [-SO2-O] OR Sulfonic [SO2(-OH)-O] OR Sulfur, phosphorus attach [-S-] OR Sulphonate, aliphatic attach [-SO2-O] OR Tertiary Carbon OR Thiol, aliphatic attach [-SH] OR Thio-phosphorus [S=P] by Organic functional groups (US EPA)

Domain logical expression index: "k"

Similarity boundary:Target: CCN{+}(.Cl{-})(CC)(CC)CC
Threshold=30%,
Dice(Atom centered fragments)
Atom type; Count H attached; Hybridization

Domain logical expression index: "l"

Similarity boundary:Target: CCN{+}(.Cl{-})(CC)(CC)CC
Threshold=40%,
Dice(Atom centered fragments)
Atom type; Count H attached; Hybridization

Domain logical expression index: "m"

Parametric boundary:The target chemical should have a value of log Kow which is >= -3.4

Domain logical expression index: "n"

Parametric boundary:The target chemical should have a value of log Kow which is <= 3.19

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Tetraethylammonium chloride was estimated to be irritating to the skin of rabbits.
Executive summary:

The dermal irritation potential of Tetraethylammonium chloride was estimated using OECD QSAR toolbox v3.4 with logPow as the primary descriptor.

Tetraethylammonium chloride was estimated to be irritating to the skin of rabbits.

Based on the estimated results, Tetraethylammonium chloride can be considered to be irritating to skin and can be classified under the category “Category 2” as per CLP regulation

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
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)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Specific details on test material used for the study:

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST:solid
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien

The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek,In Vitro Life Science Lab. (Bratislava, Slovakia).The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of solid test chemical
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles, and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA)
for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles and controls at approximately 37°C, 5% CO2 in a humidified incubator.
After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls.Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.
- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette.2 tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid test articles and controls.

- Justification for the use of a different negative control than ultrapure H2O (Not applicable)
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
35.9
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of OD :0.760;irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
other: irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean of OD for test chemical was determined to be 0.760. The mean % tissue viability of test chemical was determined to be 35.9%. Thus, test chemical was considered to be irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.760.The mean % tissue viability of test chemical was determined to be 35.9%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

In different studies, Tetraethylammonium chloride has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits along with predicted data for target chemical and its functionally similar read across substances, Tetrabutylammonium bromide[CAS:1643-19-2] and N,N,N-trimethylhexadecan-1 -aminium chloride [CAS:112-02-7]. The predicted data using the OECD QSAR toolbox has also been compared with the experimental data.

In a prediction done by SSS (2017) using the OECD QSAR toolbox with log kow as the primary descriptor, the skin irritation potential was estimated forTetraethylammonium chloride. Tetraethylammonium chloride was estimated to be irritating to the skin of rabbits.

This result was supported by the study conductedin an OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access) for thefunctionally similar read across substance, Tetrabutylammonium bromide[CAS: 1643-19-2]. This study was performed as per OECD guideline No. 404.In the initial test one healthy rabbit of body weight 1.80kg±200gm selected for study after acclimatization. The test compound in the amount of 0.5 gm was applied at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm2) one day earlier before the treatment. The site of application was covered with impervious dressing secured in position with adhesive tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour. The test compound produced slight erythema at the site of application. Finally, the animal was observed for 14 days, for any irritation and corrosion. The test compound Tetrabutylammonium bromide produced slight erythema on skin at 24 hrs. Based on the above observations, a confirmatory test was performed using two additional animals following same procedure. In the confirmatory test; the test compound in the amount of 0.5 gm after moistened with water was applied on the shaven back skin of two animals, each with one patch, for an exposure period of four hours.After four hours the patch was removed and the skin reactions were graded according to Draize’s method. The test compound Tetrabutylammonium bromide applied on the shaven area of skin of New Zealand white rabbits in the amount of 0.5 gm showed slight skin erythema at the site of application. The skin irritation index of test compound Tetrabutylammonium bromide was calculated as 0.50.

The results obtained from present study concluded that Tetrabutylammonium bromide was slightly irritating to skin of New Zealand white rabbits.

These results are also supported by the experimental study summarized in INVENTORY MULTITIERED ASSESSMENT AND PRIORITISATION (IMAP) – NICNAS, 7/3/2017; for the functionally similar read across substance, N,N,N-trimethylhexadecan-1-aminium chloride [CAS:112-02-7]. 24-26% of test chemical was applied to the intact skin of 3 New Zealand White rabbits for 4 hours exposure period and observed for 14 days. The mean score values at the 24, 48 and 72hours post application readings were 3 for erythema and 1.9 for oedema. At days seven and 14, grade 2–3 erythema and grade 1–2 oedema were found. At day 14, oedema was observed only in one animal.

Based on the observations and scores, Trimethylhexadecylammonium chloride was considered to be moderately irritating to the skin of New Zealand White rabbits.

Based on the available data for the target and read across substances and applying the weight of evidence approach, Tetraethylammonium chloride can be considered to be irritating to skin.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”.

Eye Irritation:

In different studies, the test chemical has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in- vitro and in-vivo experimental conducted in rabbits conducted which have been summarized as below:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.760.The mean % tissue viability of test chemical was determined to be 35.9%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

This result was supported by the study conducted in an OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access) for thefunctionally similar read across substance, Tetrabutylammonium bromide(CAS: 1643-19-2). This study was performed as per OECD guideline No. 405. Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mg of test item (pulverised) was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour and at day 7 after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg (0.1 gm) of test item (pulverized)was applied into the conjunctival sac of the left eye of animal no.1 whereas the right eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at till 72 hours observation which recovered on day 7 hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item(pulverised) was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 till 72 hours observation which was recovered on day 7 observations. Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours.

The following grading scores were observed in treated eye of tested rabbits. Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was seen in animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2; Area of Opacity-Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2;Iris:Normal in all the animals. Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible in animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3;Chemosis:Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3. Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity-Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3; Iris:Normal in all the animals. Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% damage in animal no. 1, 2 and 3 respectively. Observation at 48 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity-One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3;Chemosis:Obvious swelling with partial eversion of lids was seen in all 3 animals. Observation at 72 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity-One quarter (or less) but not zero was seen in all 3 animals; Iris:Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2. Observation on day 7 after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae -Blood vessels normal was seen in all 3 animals; Chemosis: No swelling (Normal) was seen in all the 3 animals.

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were: 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively.

Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1, 2 and 3 was observed till 72 hours which were recovered on day 7.

Hence under the experimental test conditions, “Tetrabutylammonium bromide(CAS No. - 1643-19-2) is “Mildly Irritating” to New Zealand White female rabbit eyes and is being classified as an eye irritant in 'category 2' as per the CLP regulation.

 

These results are further supported by the experimental study summarized in International Journal of Toxicology, Vol 16, Issue 3, pp. 195 – 220, 1997; for the functionally similar read across substance, N,N,N-trimethylhexadecan-1-aminium chloride (CAS:112-02-7). 0.2 ml of10% cetrimonium chloride solution was instilled in the eyes of 2 rabbits and observed for signs of irritation. The reactions were observed and scored after 1, 24 and 48 hours instillation of test chemical. The maximum possible score for irritation was 110.The overall score for 10% cetrimonium chloride solution was 47.5/110.

Based on the scores, cetrimonium chloride can be considered to be moderately irritating to rabbit eyes.

All these studies lead to a conclusion that Test chemical is indeed not irritating to eye. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “category 2 - irritant”.

Justification for classification or non-classification

Based on the available information, Test chemical is likely to cause irritation to eyes and skin. Hence,Test chemical can be classified under the category “Category 2” as per CLP regulation.