Denatonium benzoate

Endpoint summary

Description of key information

Skin Sensitization:

Denatonium benzoate[BITREX] produced a 0% (0/20) sensitization rate and was considered to be non- sensitizing to guinea pig skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 81-6 (Skin Sensitisation)

Principles of method if other than guideline:

To assess the skin contact sensitization potential of the test material in the albino guinea pig

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

not specified

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:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Staffordshire
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]: no
- Microbiological status of animals, when known:no data available
- Age at study initiation:8-12 weeks old
- Weight at study initiation:336-449g
- Housing: the guinea pigs were housed in groups of upto two in solid floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Guinea pig FDI diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum acclimatisation period of 5 days for each animal
- Indication of any skin lesions: no data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22 deg C
- Humidity (%): 44-65%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.5 ml of 75%w/w in distilled water

Day(s)/duration:

3 , 6 hour exposures

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.5ml of the test material in 75% w/w in distilled water

Day(s)/duration:

6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

twenty test animals and 10 control animals

Details on study design:

RANGE FINDING TESTS:
The concentrations for the topical induction and topical challenge exposure were determined by the 'sighting tests' in which groups of guinea pigs were treated with various concentrations of the test chemical. The procedure followed is as follows:
a] Selection of concentration for topical induction exposure
2 untreated guinea pigs were treated with 0.5 ml of the four concentrations of test chemical in distiled water (75%, 50%, 25 and 10% w/w). The highest concentration causing mild to moderate irritation after 6 hour occlusive exposure was selected as the concentration for the topical induction.
b] Selection of concentration for the topical challenge exposure:
2 guinea pigs were treated with 0.5 ml of two concentrations of test chemical in distilled water (50,75% w/w).These animals had been treated identically to the control animals of the main study on days 0,7 and 14. The highest concentration of the test material which produced no evidence of irritation 24 or 48 hours after a 6 hour occlusive exposure were selected for the topical challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 - 6 hours exposure
- Exposure period: 6 hours
- Test groups: 20 guinea pigs
- Control group: 10 guinea pigs
- Site: left flank
- Frequency of applications: on day 0,7 and 14 - 6 hours exposure
- Duration: 6 hours exposure
- Concentrations: 0.5 ml of 75%w/w in distilled water

B. CHALLENGE EXPOSURE
- No. of exposures: single exposure
- Day(s) of challenge: day 28
- Exposure period: 24 hours
- Test groups: 20 guinea pigs
- Control group: 10 guinea pigs
- Site: right flank
- Concentrations: 0.5 ml of 75%, 50% w/w in distilled water
- Evaluation (hr after challenge): approximately 24 and 48 hours after removal, the degree of erythema and edema was quantified

OTHER: Classification of the Sensitization response
A comparison of the intensities and durations of the reactions at the test material challenge sites in both test and control animals permits the identification of sensitization reactions.
If the test material at the maximum non-irritant concentration produces reactions in test animals at 24 or 48 hour reading, these reactions are attributable to contact sensitization.If irritation is observed in the control animals, only reactions in the test animals that exceed the most severe reaction seen in the control animals are attributable to contact sensitization. The results are expressed in terms of incidence and severity of responses:
Severity score: sum of the skin responses at each observation divided by the number of animals in the group
Incidence score: total number of animals showing sensitization responses divided by the number of animals in the group

Challenge controls:

no data available

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole was used a control

Positive control results:

Contact sensitization reactions were elicited in 3 of the 20 previously induced test group animals in topical challenge.The known contact sensitizer,
2 -Mercaptobenzothiazole, therefore produced a 15% sensitization rate. This was considered to be a satisfactory sensitization response under the conditions of the test.

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75% w/w

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no dermal reactions apart from fur loss was reported at 24 and 48 hour observation period

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

other: vehicle control

Dose level:

0.5ml distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No dermal reactions were observed in the control animals

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% w/w in acetone

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

contact sensitization was elicited in 3 of 20 previously induced test animals in challenge exposure

Remarks on result:

positive indication of skin sensitisation

Table 1: Buehler delayed contact hypersensitivity study in the guinea pigs

Individual reactions in test animals during induction

Induction Concentration: 75%w/w, Vehicle: Distilled water

Animal number	Evaluation of induction sites on day
	1			8			15
	Erythema	Edema	Other	Erythema	Edema	Other	Erythema	Edema	Other
1	0	0	-	0	0	-	0	0	-
2	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-
4	0	0	-	0	0	-	0	0	-
5	0	0	-	0	0	-	0	0	-
6	0	0	-	0	0	-	0	0	-
7	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-
9	0	0	-	0	0	-	0	0	-
10	0	0	-	0	0	-	0	0	-
11	0	0	-	0	0	-	0	0	-
12	0	0	-	0	0	-	0	0	-
13	0	0	-	0	0	-	0	0	-
14	0	0	-	0	0	-	0	0	-
15	0	0	-	0	0	-	0	0	-
16	0	0	-	0	0	-	0	0	-
17	0	0	-	0	0	-	0	0	-
18	0	0	-	0	0	-	0	0	-
19	0	0	-	0	0	-	0	0	-
20	0	0	-	0	0	-	0	0	-

 

Table 2: Buehler delayed contact hypersensitivity study in the guinea pigs

Individual reactions in test animals during challenge

Induction Concentration: 75%, 50 w/w, Vehicle: Distilled water

Animal number	Skin reactions (Hours after removal of dressing)
	75% w/w						50% w/w
	24 hours			48 hours			24 hours			48 hours
	Erythema	Edema	Other reactions	Erythema	Edema	Other reactions	Erythema	Edema	Other reactions	Erythema	Edema	Other reactions
1	0	0	FL	0	0	FL	0	0	-	0	0	-
2	0	0	-	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-	0	0	-
4	0	0	-	0	0	-	0	0	-	0	0	-
5	0	0	-	0	0	-	0	0	-	0	0	-
6	0	0	-	0	0	-	0	0	-	0	0	-
7	0	0	-	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-	0	0	-
9	0	0	-	0	0	-	0	0	-	0	0	-
10	0	0	FL	0	0	FL	0	0	-	0	0	-
11	0	0	-	0	0	-	0	0	-	0	0	-
12	0	0	-	0	0	-	0	0	-	0	0	-
13	0	0	-	0	0	-	0	0	-	0	0	-
14	0	0	-	0	0	-	0	0	-	0	0	-
15	0	0	-	0	0	-	0	0	-	0	0	-
16	0	0	-	0	0	-	0	0	-	0	0	-
17	0	0	FL	0	0	FL	0	0	-	0	0	-
18	0	0	FL	0	0	FL	0	0	-	0	0	-
19	0	0	-	0	0	-	0	0	-	0	0	-
20	0	0	FL	0	0	FL	0	0	-	0	0	-
Sum of the ski n responses (A)	0+0			0+0			0+0			0+0
Severity (A/20)	0			0			0			0
Number showing sensitization response (N)	0			0			0			0
Incidence (N/20)	0						0

 FL - fur loss

Interpretation of results:

other: not sensitizing

Conclusions:

Denatonium benzoate[BITREX] produced a 0% (0/20) sensitization rate and was considered to be non- sensitizing to guinea pig skin.

Executive summary:

A study was performed to assess the dermal sensitization potential of Denatonium benzoate[BITREX] in guinea pigs. Buehler delayed contact hypersensitivity method following EPA Guidelines 81 -6 was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access). Twenty test and 10 control Dunkin Hartley male guinea pigs were used for the main study, 4 guinea pigs were used for the sight finding study.

The concentrations for the topical induction and topical challenge exposure were determined by the 'sighting tests' in which groups of guinea pigs were treated with various concentrations of the test  chemical. The procedure followed is as follows:

a] Selection of concentration for topical induction exposure

2 untreated guinea pigs were treated with 0.5 ml of the four concentrations of test chemical in distiled water (75%, 50%, 25 and 10% w/w). The highest concentration causing mild to moderate irritation after 6 hour occlusive exposure was selected as the concentration for the topical induction.

b] Selection of concentration for the topical challenge exposure:

2 guinea pigs were treated with 0.5 ml of two concentrations of test chemical in distilled water (50,75% w/w).These animals had been treated identically to the control animals of the main study on days 0,7 and 14. The highest concentration of the test material which produced no evidence of irritation 24 or 48 hours after a 6 hour occlusive exposure were selected for the topical challenge exposure.

The concentrations obtained from these studies for the topical induction and challenge are as follows:

Topical Induction: 75% w/w in distilled water

Topical Challenge: 50, 75% w/w in distilled water

During Induction phase, 0.5 ml of the test material at concentration of 75% w/w in distilled water was applied to the left flank of the test animals under an occlusive dressing for 6 hours. The induction exposure procedure was repeated on the same site on days 7, 14 for a total of 3 6 -hour exposures. Approximately 24 hours after each induction exposure (days 1, 8, 15), the degree of erythema and edema were quantified. The same procedure was followed for the control animals except that the vehicle alone was applied. On day 28, the right flank of the test animals was clipped free of hair and 0.5 ml of the test material at concentrations of 75%,50 w/w in distilled water was applied to right flank under occlusive conditions for 6 hours. After 6 hours, the patches were cut and the treatment sites were rinsed with distilled water and the test sites were marked with an indelible marker. Approximately 24, 48 hours after patch removal, the degree of erythema and edema were quantified. 2 -Mercaptobenzothiazole was used as a positive control. Contact sensitization reactions were elicited in 3 of the 20 previously induced test group animals in topical challenge. The known contact sensitizer, 2 -Mercaptobenzothiazole, therefore produced a 15% sensitization rate. This was considered to be a satisfactory sensitization response under the conditions of the test.

Denatonium benzoate[BITREX] produced a 0% (0/20) sensitization rate and was considered to be non- sensitizing to guinea pig skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensitization:

Denatonium benzoatehas been investigated in various studies for potential for dermal sensitization to a greater or lesser extent. The studies are based on in vivo experiments in humans and guinea pigs.

A study was performed to assess the dermal sensitization potential of Denatonium benzoate[BITREX] in guinea pigs. Buehler delayed contact hypersensitivity method following EPA Guidelines 81 -6 was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access). Twenty test and 10 control Dunkin Hartley male guinea pigs were used for the main study, 4 guinea pigs were used for the sight finding study.

The concentrations for the topical induction and topical challenge exposure were determined by the 'sighting tests' in which groups of guinea pigs were treated with various concentrations of the test  chemical. The procedure followed is as follows:

a] Selection of concentration for topical induction exposure

2 untreated guinea pigs were treated with 0.5 ml of the four concentrations of test chemical in distiled water (75%, 50%, 25 and 10% w/w). The highest concentration causing mild to moderate irritation after 6 hour occlusive exposure was selected as the concentration for the topical induction.

b] Selection of concentration for the topical challenge exposure:

2 guinea pigs were treated with 0.5 ml of two concentrations of test chemical in distilled water (50,75% w/w).These animals had been treated identically to the control animals of the main study on days 0,7 and 14. The highest concentration of the test material which produced no evidence of irritation 24 or 48 hours after a 6 hour occlusive exposure were selected for the topical challenge exposure.

The concentrations obtained from these studies for the topical induction and challenge are as follows:

Topical Induction: 75% w/w in distilled water

Topical Challenge: 50, 75% w/w in distilled water

During Induction phase, 0.5 ml of the test material at concentration of 75% w/w in distilled water was applied to the left flank of the test animals under an occlusive dressing for 6 hours. The induction exposure procedure was repeated on the same site on days 7, 14 for a total of 3 6 -hour exposures. Approximately 24 hours after each induction exposure (days 1, 8, 15), the degree of erythema and edema were quantified. The same procedure was followed for the control animals except that the vehicle alone was applied. On day 28, the right flank of the test animals was clipped free of hair and 0.5 ml of the test material at concentrations of 75%,50 w/w in distilled water was applied to right flank under occlusive conditions for 6 hours. After 6 hours, the patches were cut and the treatment sites were rinsed with distilled water and the test sites were marked with an indelible marker. Approximately 24, 48 hours after patch removal, the degree of erythema and edema were quantified. 2 -Mercaptobenzothiazole was used as a positive control. Contact sensitization reactions were elicited in 3 of the 20 previously induced test group animals in topical challenge. The known contact sensitizer, 2 -Mercaptobenzothiazole, therefore produced a 15% sensitization rate. This was considered to be a satisfactory sensitization response under the conditions of the test.

Denatonium benzoate[BITREX] produced a 0% (0/20) sensitization rate and was considered to be non- sensitizing to guinea pig skin.

This is supported by the experimental study performed by Bert Bjorkner (Contact Dermatitis, 1980: 6: 466-471) to assess the sensitization potential of Denatonium benzoate in human volunteers. A 30-year-old male developed asthma and pruritus after using an insecticidal spray (Pyrex®). The same symptoms appeared with denatonium benzoate (Bitrex®).

For all the above reasons, Open patch test was performed on the volunteer to assess the sensitizing potential of insecticidal spray (Pyrex®) and denatonium benzoate (Bitrex®).

Denatonium benzoate was tested at a concentration from 2 X 10-2 mg/l in water and diluted till 2x10-7mg/l. Water was used as a control. 0.1 ml of Denatonium benzoate was spread with a glass rod on normal skin on the volar aspect of the forearms. The test reactions were read after 20 and 30 min. Ten control subjects were tested with the same chemicals and in the same way.

Denatonium benzoate was sensitizing to the skin at a concentration of 2 X 10-2 mg/L, 2 X 10-3 mg/L, 2 X 10-4 mg/L, 2 X 10-5 mg/L, 2 X 10-6 mg/L. No sensitizing reaction was observed when the test solution was diluted to 2x10-7 mg/l.

Hence, Denatonium benzoate can be considered to be not sensitizing to human skin when exposed to concentrations below 2x10-7mg/l.

Based on the available data, Denatonium benzoate was considered to be not sensitizing to skin.Comparing the above annotations with the criteria of CLP regulation, test chemical can be classified under the category “Not Classified”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Available studies indicate a strong possibility of Denatonium benzoate being not sensitizing to skin. Hence, it can be classified under the Category "Not Classified" as per CLP regulation.