Denatonium benzoate

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Denatonium benzoate

EC Number:

223-095-2

EC Name:

Denatonium benzoate

Cas Number:

3734-33-6

Molecular formula:

C21H29N2O.C7H5O2

IUPAC Name:

N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate

Test material form:

solid

Details on test material:

- 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

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

Test animals / tissue source

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

Test system

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.

Results and discussion

In vivo

Results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.