Denatonium benzoate

Skin irritation / corrosion

Administrative data

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

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 81-5 (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)

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, room temperature in the dark from 22 February, 1995

Test animals

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

Test system

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)

Results and discussion

In vivo

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.