Denatonium benzoate

Endpoint summary

Description of key information

Skin irritation

The dermal irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies conducted for test chemical.Based on the available key data and supporting studies,it can be concluded that the testchemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye irritation

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies conducted for test chemical.Based on the available key data and supporting study, it can be concluded that the test chemical is able to cause sever irreversible eye damage and hence considered as corrosive. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 1 (irreversible effects on the eye) ''.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

data is from experimental reports following standard procedures

Qualifier:

according to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

Principles of method if other than guideline:

To assess the irritancy potential of the test material to the skin of New Zealand White rabbits

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate
-Common name: Denatonium benzoate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight: 446.58 g/mol
- Substance type: organic
- Physical state: Solid
- Form: white granules
- Batch Number: 22362
- Storage conditions: room temperature, room temperature in the dark from 22 February, 1995

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: Male and female
- Source: David Percival Ltd, Cheshire, U.K
- Age at study initiation: 12-20 weeks old
- Weight at study initiation: 2.23 -2.71 kg
- Housing: individually housed in separate suspended metal cages
- Diet (e.g. ad libitum): STANRAB SQC Rabbit diet, ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: minimum acclimatisation period of 5 days to each animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21 deg C
- Humidity (%): 43-58%
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped intact skin

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

0.5 g of the test material moistened with 0.5 ml distilled water

Duration of treatment / exposure:

4 hours

Observation period:

one hour following the removal of patches and 24, 48 and 72 hours after patch removal

Number of animals:

6(3/sex)

Details on study design:

TEST SITE
- Area of exposure: dorsal/ flank area
- % coverage: 2.5 * 2.5 cm gauze patch
- Type of wrap if used: the patch was secured with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5*2.5 cm)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: four hours after exposure, any residual material was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated spirits

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : one hour following the removal of patches and 24, 48 and 72 hours after patch removal

SCORING SYSTEM:
- Method of calculation: The test sites were scored and evaluated according to ECHA guidance on the application of CLP criteria v5.0 (July 2017)

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24 and 72 hours

Score:

2.8

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of moderate irritation

Irritant / corrosive response data:

Very slight to well defined erythema was noted at the all test sites 24 and 28 hour observation period and at five treatment sites at 72 hour observation. Very slight to moderate edema was noted at the test sites. Desquamation was also noted at five treatment sites 7 days after treatment.This reaction was considered to be reversible.

Other effects:

No corrosive effects were observed

Table 1: Bitrex Acute dermal irritation test in the rabbit: Individual skin reactions

 

Skin reaction	Observation time	Individual scores: Rabbit number and sex (Bodyweight kg)
		177 Female (2.23)	178 Male (2.63)	188 Male (2.71)	185 Male (2.68)	192 Female (2.52)	197 Female (2.44)	Total
Erythema/ Eschar formation	1 hour	1	0	1	1	1	1	(5)
	24 hours	1	2	2	2	2	2	11
	48 hours	1	1	2	2	2	1	(9)
	72 hours	0	1	2	2	2	1	8
	7 days	0	0D	0D	0D	0D	0D	0
Edema formation	1 hour	1	0	2	2	1	0	(6)
	24 hours	0	2	3	2	2	1	10
	48 hours	0	1	2	2	2	1	(8)
	72 hours	0	0	1	2	1	1	5
	7 days	0	0	0	0	0	0	0
Sum of 24 and 72 hours readings [S]	34
Primary Irritation Index [S/12]	34/12 = 2.8
Classification	Moderate irritant

 

Where

() = values not required for calculation of Primary Irritation Index

 

D = desquamation

2.

Animal No.	OBSERAVTION TIME												Average Score (ERY)	Average Score (OED)
	Erthyema (ERY)						Oedema (OED)
	1hr	24hr	48hr	72hr	7days	14days	1hr	24hr	48hr	72hr	7days	14days
177	1	1	1	0	0	NA	1	0	0	0	0	NA	0.66	0
178	0	2	1	1	OD*	NA	0	2	1	0	0	NA	1.33	1
188	1	2	2	2	OD*	NA	2	3	2	1	0	NA	2	2
185	1	2	2	2	OD*	NA	2	2	2	2	0	NA	2	2
192	1	2	2	2	OD*	NA	1	2	2	1	0	NA	2	1.66
197	1	2	1	1	OD*	NA	0	1	1	1	0	NA	1.33	1
Evaluation is made based on the averge score per animal for 24/48/72 hours.													0	0
Table based on ECHA Guidence on the application of CLP criteria V5.0,July 2017. Examples 3 from section 3.2.5.1.3 on page 298 “test carried out”.													0	0
O.D.: Response of Desuamation judege to be reverbile.													0	0

Interpretation of results:

other: Not irritating

Conclusions:

A study was performed to assess the irritancy potential of Denatonium benzoate (BITREX) to the skin of New Zealand White rabbits according to EPA 81 -5 Guidelines.
Based on the observed scores in the study (the original 1995 Skin Irritation Toxicology Report), documented in table x, and when using the calculation method from ECHA Guidance on the application of the CLP criteria v 5.0 (July 2017), none of the average scores per animal (at 24, 48 and 72 hours), reached the cut off value of 2.3. So, no animals are positive for skin irritancy. Therefore, in conclusion Denatonium Benzoate (BITREX) is not classified as Skin Irritant under CLP.

Executive summary:

A study was performed to assess the irritancy potential of Denatonium benzoate (BITREX) to the skin of New Zealand White rabbits. The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) according to EPA 81 -5 Guidelines and 6 New Zealand White rabbits (3/sex) were used for the study.

One day before the test, the rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. On the day of the test, a suitable test site was selected on the back of each rabbits. 0.5 g of the test material moistened with 0.5 ml distilled water was applied under semi -occlusive conditions ( 2.5 * 2.5 cm gauze patch) on the shorn skin of rabbits. The patch was secured with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5*2.5 cm). To prevent the animals from interfering with the patches, the trunk of each rabbit was wrapped in elastic corset. and the rabbits were returned to their respective cages. After 4 hours of exposure, the patches and corset were removed. Also, any residual material was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated spirits. The test sites were observed and scored according to Draize one hour following the removal of patches and 24, 48 and 72 hours after patch removal. The scores of erythema and edema at 24 and 72 hours readings were totaled for the six test animals(24 values) and divided by 12 to give the primary irritation index of the test material. The test material was classified according to the Draize classification criteria. Very slight to well defined erythema was noted at the all test sites 24 and 28 hour observation period and at five treatment sites at 72 hour observation. Very slight to moderate edema was noted at the test sites. Desquamation was also noted at five treatment sites 7 days after treatment. This reaction was considered to be reversible. No corrosive effects were observed.

Based on the observed scores in the study (the original 1995 Skin Irritation Toxicology Report), documented in table x, and when using the calculation method from ECHA Guidance on the application of the CLP criteria v 5.0 (July 2017), none of the average scores per animal (at 24, 48 and 72 hours), reached the cut off value of 2.3. So, no animals are positive for skin irritancy. Therefore, in conclusion Denatonium Benzoate (BITREX) is not classified as Skin Irritant under CLP.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 17, 2017

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

Guideline:

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

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material:- Expiration date of the lot/batch:- Purity test date:RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature or 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 availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Prior to the main test, the test articles are tested for their ability to reduce/interact with MTT and their ability to stain the tissues itself. All tests are performed according to the by MatTek provided test protocol. - 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 availableFORM AS APPLIED IN THE TEST: SolidOTHER SPECIFICS: No data available

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Source strain:

other: Not applicable

Details on animal used as source of test system:

- 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 epidermis. 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 IdentificationAll of the EpiDerm™ 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 are included in this report. Tissue plates were appropriately labeled with study information.

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. 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). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ 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 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 3 hours with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability is 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 EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 25 mg of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (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. 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 mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)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 mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit):25 mg- Concentration (if solution): neat VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL sterile DPBS- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate

Duration of treatment / exposure:

The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

For a total of an approximately 42 hour post-exposure incubation.

Number of replicates:

3 tissues were used for test compound and control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

6

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean of OD:0.130; irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Interpretation of results:

other: irritating

Conclusions:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The mean of OD for test chemical was determined to be 0.130 .The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 6.0%. Thus, test chemical was considered to be irritating to the human skin.

Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article bydetermining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met and passed the acceptance of criteria.The mean of OD for test chemical was determined to be 0.130. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 6.0%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritant to human skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

data is from experimental reports following standard procedures

Qualifier:

according to guideline

Guideline:

EPA OPP 81-4 (Acute Eye Irritation)

Principles of method if other than guideline:

To assess the irritancy potential of the test material to the eyes of New Zealand White rabbits

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate
-Common name: Denatonium benzoate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight: 446.58 g/mol
- Substance type: organic
- Physical state: Solid
- Form: white granules
- Batch Number: 22362
- Storage conditions: room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Sex: male
- Source: David Percival Ltd, Cheshire, U.K
- Age at study initiation: 12-20 weeks old
- Weight at study initiation: 2.84 kg
- Housing: individually housed in separate suspended metal cages
- Diet (e.g. ad libitum): STANRAB SQC Rabbit diet, ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: minimum acclimatisation period of 5 days to each animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-22 deg C
- Humidity (%): 48-66%
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml volume (approximately 40 mg)

Duration of treatment / exposure:

1 second

Observation period (in vivo):

approximately 1, 24,48 and 72 hours following treatment. Additional observations were made on day 7,14 and 21 to check the reversibility of effects.

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

1 male rabbit

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not washed
- Time after start of exposure: no data available

SCORING SYSTEM: Draize method

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: One day prior to the test, both the eyes of the test rabbit were examined under ultra-violet light, after treatment with Sodium Fluorescien BP. The cornea, iris and conjunctivae were examined for lesions. Immediately before treatment, the rabbit eyes were again examined with the aid of a light source from a standard ophthalmoscope. For scoring of ocular lesions, the examination of the eye was facilitated by the use of a standard ophthalmoscope.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

7 d

Score:

18

Max. score:

110

Reversibility:

not fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Single application of test material produced areas of translucent opacity, iridial inflammation and moderate conjuncitval irritation. Vascularisation of the cornea was present on day 7 and was still apparent on day 21. The ocular reactions were therefore considered to be irreversible.

Table 1:Bitrex Acute eye irritation test in the rabbit: Individual reactions

Rabbit number and sex (bodyweight kg)	IPR =3
	27 Male (2.84)
	1 hours	24 hours	48 hours	72 hours	7 days	14 days	21 days
Cornea
E –degree of opacity	2SI	2SI	1	2	1V	0V	0V
F –Area of opacity	4	4	4	2	2	0	0
Score – E*F*5	40	40	20	20	10	0	0
Iris
D	1	1	1	1	0	0	0
Score –D*5	5	5	5	5	0	0	0
Conjunctivae
A -redness	2	2	2W	2W	1W	0	0
B – chemosis	2	2	2	2	1	0	0
C –discharge	3	3	3	3	2	0	0
Score (A+B+C)*2	14	14	14	14	8	0	0
Total score	59	59	39	39	18	0	0

 

IPR – initial pain reaction, W- white area over the nictitating membrane, approximate size 3 mm-*3mm, V – Vascularistion along the bottom edge of the cornea, approximately 4mm in length and invading up to 2mm onto the cornea, Sl –Sloughing of the cornea

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Single application of test material produced areas of translucent opacity, iridial inflammation and moderate conjuncitval irritation. Vascularisation of the cornea was present on day 7 and was still apparent on day 21. The ocular reactions were therefore considered to be irreversible.
Hence, Denatonium benzoate (BITREX) was considered to be corrosive to rabbit eyes.

Executive summary:

A study was performed to assess the irritancy potential of Denatonium benzoate (BITREX) to the eyes of New Zealand White rabbits. The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) according to EPA 81 -4 Guidelines.

Initially one male New Zealand white rabbit was used for the study. After consideration of ocular responses in the first rabbit, no additional rabbits were treated.

For the purpose of this study, the test material was ground to a fine powder. One day prior to the test, both the eyes of the test rabbit were examined under ultra-violet light, after treatment with Sodium Fluorescien BP. The cornea, iris and conjunctivae were examined for lesions. Immediately before treatment, the rabbit eyes were again examined with the aid of a light source from a standard ophthalmoscope. 0.1 ml volume (approximately 40 mg) of the finely ground powder was instilled in to the right conjunctival sac of the rabbit. The upper and lower eyelids were held together for about one second immediately after application to prevent loss of the test material and then released. The left eye remained untreated and served as control. The assessment and scoring of ocular reactions was done approximately 1, 24, 48 and 72 hours following treatment. Additional observations were made on day 7,14 and 21 to check the reversibility of effects. For scoring of ocular lesions, the examination of the eye was facilitated by the use of a standard ophthalmoscope. The ocular reactions were scored according to the Draize method. Single application of test material produced areas of translucent opacity, iridial inflammation and moderate conjunctival irritation. Vascularisation of the cornea was present on day 7 and was still apparent on day 21. The ocular reactions were therefore considered to be irreversible. Hence, Denatonium benzoate (BITREX) was considered to be corrosive to rabbit eyes.

Reference 2

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

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:

3.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean of OD:0.064; irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).

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 0.064 .The mean % tissue viability of test chemical was determined to be 3.4 %. 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 0.064.The mean % tissue viability of test chemical was determined to be 3.4%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritant to the human eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

Various studieshas been investigated for the test chemical to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in-vitro and in-vivo experiments conducted for target chemicalwhich have beensummarized as below;

 

A study was performed to assess the irritancy potential of Denatonium benzoate (BITREX) to the skin of New Zealand White rabbits. The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) according to EPA 81 -5 Guidelines and 6 New Zealand White rabbits (3/sex) were used for the study. One day before the test, the rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. On the day of the test, a suitable test site was selected on the back of each rabbits. 0.5 g of the test material moistened with 0.5 ml distilled water was applied under semi -occlusive conditions ( 2.5 * 2.5 cm gauze patch) on the shorn skin of rabbits. The patch was secured with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5*2.5 cm). To prevent the animals from interfering with the patches, the trunk of each rabbit was wrapped in elastic corset. and the rabbits were returned to their respective cages. After 4 hours of exposure, the patches and corset were removed. Also, any residual material was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated spirits. The test sites were observed and scored according to Draize one hour following the removal of patches and 24, 48 and 72 hours after patch removal. The scores of erythema and edema at 24 and 72 hours readings were totaled for the six test animals(24 values) and divided by 12 to give the primary irritation index of the test material. The test material was classified according to the Draize classification criteria. Very slight to well defined erythema was noted at the all test sites 24 and 28 hour observation period and at five treatment sites at 72 hour observation. Very slight to moderate edema was noted at the test sites. Desquamation was also noted at five treatment sites 7 days after treatment. This reaction was considered to be reversible. No corrosive effects were observed.Based on the observed scores in the study (the original 1995 Skin Irritation Toxicology Report), documented in table, and when using the calculation method from ECHA Guidance on the application of the CLP criteria v 5.0 (July 2017), none of the average scores per animal (at 24, 48 and 72 hours), reached the cut off value of 2.3. So, no animals are positive for skin irritancy. Therefore, in conclusion Denatonium Benzoate (BITREX) is not classified as Skin Irritant under CLP.

 

The above in-vivo result is further supported by the in-vitro dermal irritation study carried out for test chemical to assess the dermal irritation potential of test article according to the OECD 439 test guideline. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The mean of OD for test chemical was determined to be 0.130. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 6.0%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritant to human skin. Comparing the above annotations with the criteria of CLP regulation, the test chemical can be classified under the category “non-irritant” based on the in-vivo study result.

 

Thus based on the available key data and supporting study,it can be concluded that the testchemical is unable to cause skin irritation and hence considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

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:

 

A study was performed to assess the irritancy potential of Denatonium benzoate (BITREX) to the eyes of New Zealand White rabbits. The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) according to EPA 81 -4 Guidelines.

Initially one male New Zealand white rabbit was used for the study. After consideration of ocular responses in the first rabbit, no additional rabbits were treated.

For the purpose of this study, the test material was ground to a fine powder. One day prior to the test, both the eyes of the test rabbit were examined under ultra-violet light, after treatment with Sodium Fluorescien BP. The cornea, iris and conjunctivae were examined for lesions. Immediately before treatment, the rabbit eyes were again examined with the aid of a light source from a standard ophthalmoscope. 0.1 ml volume (approximately 40 mg) of the finely ground powder was instilled in to the right conjunctival sac of the rabbit. The upper and lower eyelids were held together for about one second immediately after application to prevent loss of the test material and then released. The left eye remained untreated and served as control. The assessment and scoring of ocular reactions was done approximately 1, 24, 48 and 72 hours following treatment. Additional observations were made on day 7,14 and 21 to check the reversibility of effects. For scoring of ocular lesions, the examination of the eye was facilitated by the use of a standard ophthalmoscope. The ocular reactions were scored according to the Draize method. Single application of test material produced areas of translucent opacity, iridial inflammation and moderate conjunctival irritation. Vascularisation of the cornea was present on day 7 and was still apparent on day 21. The ocular reactions were therefore considered to be irreversible. Hence, Denatonium benzoate (BITREX) was considered to be corrosive to rabbit eyes.

The above in-vivo result is further supported by the in-vitro eye irritation study carried out for test chemical to assess 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 0.064.The mean % tissue viability of test chemical was determined to be 3.4%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritant to the human eyes.

 

Thus based on the available key data and supporting study,it can be concluded that the testchemical is able to cause severe irreversible eye damage and hence considered as corrosive to the eyes. Comparing the above annotations with the criteria of CLP regulation, the test chemical can be classified under the category “Category 1 (irreversible effects on the eye)”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical were observed in various studies. The results obtained from these studies indicate that the chemical is not likely to cause skin irritation but can cause severe irreversible eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and “Category 1 (irreversible effects on the eye)” for eye as per CLP.