Denatonium benzoate

Endpoint summary

Description of key information

Acute toxicity: Oral

LD50 values were considered to be for all animals 749 mg/kg bw , Males: 841(707-1000) mg/kg bw and Females: 648 (506-832) mg/kg bw, When rat were treated with denatonium benzoate (3734-33-6) orally.  

Acute toxicity: Inhalation

The median lethal Concentration (LC50) and 95% confidence limits was considered to be0.20 mg/L (0.13 - 0.36) when the male and female Sprague-Dawley rats were treated with Denatonium benzoate ( Bitrex®) by inhalation route.

Acute toxicity: Dermal

LD50 was considered to be >2000mg/kg body weight. When rats were treated with denatonium benzoate (3734-33-6) by dermal application.

Based upon the study results and available information, the substance denatonium benzoate (CAS No 3734-33-6) is expected to show acute toxicity effect by the oral route for category IV as per CLP classification and for by the inhalation route category II and dermal route qualified as not classified as per CLP classification

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

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:

equivalent or similar to guideline

Guideline:

other: OECD environment monograph no. 45 (OCDE/ GD (92)32

Principles of method if other than guideline:

Acute oral toxicity study of denatonium benzoate was performed in rats.

GLP compliance:

yes (incl. QA statement)

Test type:

other: No data available

Limit test:

no

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
- purity:99.5%
- Storage conditions: room temperature, room temperature in the dark from 22 February, 1995

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Charles river (UK) Ltd, Margate Kent.
- Age at study initiation:8-12 weeks old
- Weight at study initiation: male : 200-247g and female 200-250g
- Fasting period before study: An overnight fast immediately before dosing and for approximately two hours after dosing
- Housing: The animals were housed in group of up to five by sex in solid floor polypropylene cages furnished with wood flakes.
- Diet (e.g. ad libitum): food (Rat and mouse expanded diet no. 1,Special diets services limited , witham , Essex, uk)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 - 22°C
- Humidity (%):46-56%
- Air changes (per hr):15 changes
- Photoperiod (hrs dark / hrs light):12 hours continuous light and 12 hours darkness

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Details on exposure
VEHICLE
- Concentration in vehicle:50.0,70.7,100.0 mg/kg
- Amount of vehicle (if gavage): 10mg/ml
- Justification for choice of vehicle: Test material dissolved in water
- Lot/batch no. (if required):No data available
- Purity: No data available
MAXIMUM DOSE VOLUME APPLIED: No data available
DOSAGE PREPARATION (if unusual):
The test material was freshly prepared, as required, as a solution at the appropriate concentration in distilled water.
CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Range finding study was performed

Doses:

500,707, 1000mg/kg

No. of animals per sex per dose:

Total :30
500mg/kg : 5male and 5 female
707mg/kg : 5male and 5 female
1000mg/kg : 5male and 5 female

Control animals:

not specified

Details on study design:

Details on study design
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:1/2, 1,2 and 4 hour after dosing and subsequently once daily for 14 days animals were observed for death or overt signs of toxicity.
Individual body weights were recorded prior to dosing on day 0 and on days 7 and 14 or at death.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathological examination were performed and the appearance of any macroscopic abnormalities was recorded

Statistics:

LD50 and 95% confidence limits were calculated using Thompson W.R ,

Preliminary study:

Range finding study was performed

Sex:

male/female

Dose descriptor:

LD50

Effect level:

749 mg/kg bw

Based on:

test mat.

95% CL:

664 - 845

Remarks on result:

other: 50% mortality was observed

Sex:

male

Dose descriptor:

LD50

Effect level:

841 mg/kg bw

Based on:

test mat.

95% CL:

707 - 1 000

Remarks on result:

other: 50% mortality was observed

Sex:

female

Dose descriptor:

LD50

Effect level:

648 mg/kg bw

Based on:

test mat.

95% CL:

506 - 832

Remarks on result:

other: 50% mortality was observed

Mortality:

Death were noted within 30 min of dosing or 2 to 4 hr after dosing

Clinical signs:

other: Hunched posture, lethargy and decreased respiratory rate were commonly noted on the day of dosing with additional signs of ataxia, labored respiration and noisy respiration. Clonic convulsions were noted in six animals prior to death. Incidents of hunched

Gross pathology:

Hemorrhagic lungs, dark liver, dark kidneys, pale gastric mucosa and haemorrhage or slight haemorrhage of the gastric mucosa were noted at necropsy of the animals that died during the study. No abnormalities were noted at necropsy of animals killed at the end of the study.

Other findings:

No data available

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

LD50 values and 95% confidence limits of the test material denatonium benzoate(3734-33-6) in the Sprague –Dawley strain were calculated by the method of Thompson W.R to be for all animals: 749 (664-845) mg/kg bw, Males: 841(707-1000) mg/kg bw and Females: 648 (506-832) mg/kg bw.

Executive summary:

In acute oral toxicity study male and female Sprague –Dawley rats were treated withdenatonium benzoate(3734-33-6).The animals were housed in group of up to five by sex in solid floor polypropylene cages furnished with wood flakes provided with food (Rat and mouse expanded diet no. 1, Special diets services limited, witham , Essex, uk) and drinking water ad libitum. An overnight fast immediately before dosing and for approximately two hours after dosing were done. The test material was freshly prepared, as required, as a solution at the appropriate concentration in distilled water. The dose concentration 500,707, 1000mg/kg were administered to 5 male and 5 female per dose group by oral gavage route in 10mg/ml volume. The Range finding study was performed for selection of dose concentration. All the animals were observed for 1/2, 1,2 and 4 hour after dosing and subsequently once daily for 14 days animals were observed for death or overt signs of toxicity. Individual body weights were recorded prior to dosing on day 0 and on days 7 and 14 or at death. Gross pathological examination was performed and the appearance of any macroscopic abnormalities was recorded. Deaths were noted within 30 min of dosing 1 female in 500mg/kg dose group while 2 females in 707 mg/kg dose group and 5 females and 4 males in 1000mg/kg dose group in which 2 animals were found dead within 1 or2 minutes of dosing. All the animals were died in 1000mg/kg dose group at the end of 4hr. Clinical signs like Hunched posture, lethargy and decreased respiratory rate were commonly noted on the day of dosing with additional signs of ataxia, labored respiration and noisy respiration. Clonic convulsions were noted in six animals prior to death. Incidents of hunched posture, lethargy, decreased respiratory rate, noisy respiration or labored respiration persisted up to 2 days after dosing. Surviving animals appeared normal 1 to 3 days after dosing or throughout the study. Surviving animals showed an acceptable increase in bodyweight during the study. Hemorrhagic lungs, dark liver, dark kidneys, pale gastric mucosa and haemorrhage or slight haemorrhage of the gastric mucosa were noted at necropsy of the animals that died during the study. No abnormalities were noted at necropsy of animals killed at the end of the study. Hence LD50 values and 95% confidence limits of the test material denatonium benzoate(3734-33-6) in the Sprague –Dawley strain were calculated by the method of Thompson W.R to be forall animals: 749 (664-845) mg/kg bw, Males: 841(707-1000) mg/kg bw and Females: 648 (506-832) mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

749 mg/kg bw

Quality of whole database:

Data is Klimicsh 1 and from experimental study report

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

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:

OECD Guideline 403 (Acute Inhalation Toxicity)

Principles of method if other than guideline:

The acute inhalation toxicity study of Denatonium benzoate was performed in rats.

GLP compliance:

yes (incl. QA statement)

Test type:

other: No data available

Limit test:

no

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 granular solid
- batch no:22362
- Storage conditions: room temperature, room temperature in the dark

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Charles River (UK) ltd., Margate, Kent.
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: male: 200-237g and female 200-229g
- Fasting period before study:
- Housing: The animals were housed in group of five by sex in solid floor polypropylene cage with stainless steel lids, furnished with softwood flakes (Datesand Ltd ., Cheshire, UK).
- Diet (e.g. ad libitum): food ( Rat and mouse expanded diet no . 1 , special diets services limited , Witham , Essex, UK)ad libitum (with the exception of the exposure period)
- Water (e.g. ad libitum): ad libitum (with the exception of the exposure period)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 55± 15%
- Air changes (per hr): 15 changes per hr
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hr continuous light and twelve hr darkness

IN-LIFE DATES: From: To: No data available

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: wright dust feed
- Exposure chamber volume: 30 liters
- Method of holding animals in test chamber:Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by mean of a rubber o ring. Only the nose of each animal was exposed to the atmosphere.
- Source and rate of air:metered compressed air supply
- Method of conditioning air:No data available
- System of generating particulates/aerosols:No data available
- Method of particle size determination: Cascade impactor
- Treatment of exhaust air:No data available
- Temperature, humidity, pressure in air chamber: The temperatur and relative humidity inside the exposure chamber were measured by electronic thermometer/humidity meter located in a vacant port in the animals breathing zone of the chamber and recorded every thirty minutes throughout each exposure period .

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable):0.77, 0.36, 0.13 mg/ L
- Justification of choice of vehicle: No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

4 h

Concentrations:

0.77, 0.36, 0.13 mg/ L

No. of animals per sex per dose:

Total :30
0.77mg/L: 5 male and 5 female
0.36 mg/L: 5 male and 5 female
0.13 mg/l: 5 male and 5 female

Control animals:

not specified

Details on study design:

Details on study design
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs , at hourly intervals during the exposure, immediately on removal from the restraining tubes at the end of the exposure, one hour after termination of the exposure and subsequently once daily for 14days.
Individual bodyweights were recorded prior to treatment on the day of exposure and on days 7,14 or at death.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight and gross pathology were performed. Full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Statistics:

No data available

Preliminary study:

No data available

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.2 mg/L air

Based on:

test mat.

95% CL:

0.13 - 0.36

Exp. duration:

4 h

Remarks on result:

other: 50% mortality was observed

Mortality:

During exposure to 0.77 mg/l all animals died between 134 and 216 min after the start. During exposure to 0.36mg/l two males and three female died between 218 and230 min and the 5 remaining animals were killed in extremis approximately two hr after completion of exposure. No deaths occurred in a group of animals exposed to 0.13mg/l

Clinical signs:

other:

Body weight:

The animals exposed to 0.13 mg/L showed normal bodyweight gain during the study.

Gross pathology:

The animals exposed to 0.77 mg/L or 0.36 mg/L showed abnormally red lungs, several with dark patches. No abnormalities were detected at necropsy in the group animals exposed to 0.13 mg/L.

Other findings:

No data available

EXPOSURE CHAMBER ATMOSPHERE CONCENTRATIONS-DOSE GROUP 1

DURATION OF EXPOSURE (minutes)	NET WEIGHT OF SAMPLE (mg)	VOLUME OF AIR SAMPLED (l)	CHAMBER FLOW RATE  (l/min)	ATMOSPHERE CONCENTRATION (mg/l)
15	1.15	2	16	0.58
22	1.38	2	16	0.69
30	1.39	2	16	0.70
45	1.51	2	16	0.76
60	1.46	2	16	0.73
75	1.51	2	16	0.76
90	1.32	2	16	0.66
105	1.59	2	16	0.80
120	1.64	2	16	0.82
135	1.69	2	16	0.85
150	1.71	2	16	0.86
165	1.68	2	16	0.84
180	1.75	2	16	0.88
195	1.62	2	16	0.81
210	1.60	2-	16	0.80
216	-		16	-

 

Mean achieved atmosphere concentration (mg/l) = 0.77

Standard Deviation = 0.80

-= not determined

Nominal concentration = Amount of material introduced (mg) / Volume of air passed (l)

                                         = 7600 / 3456

                                         = 2.20 mg/l

 

 

EXPOSURE CHAMBER ATMOSPHERE CONCENTRATIONS-DOSE GROUP 3

DURATION OF EXPOSURE (minutes)	NET WEIGHT OF SAMPLE (mg)	VOLUME OF AIR SAMPLED (l)	CHAMBER FLOW RATE  (l/min)	ATMOSPHERE CONCENTRATION (mg/l)
15	0.68	4	16	0.17
30	2.06	4	16	0.52
45	1.77	4	16	0.44
60	1.58	4	16	0.40
75	1.45	4	16	0.36
90	1.46	4	16	0.37
105	1.68	4	16	0.42
120	1.47	4	16	0.37
135	1.36	4	16	0.34
150	1.41	4	16	0.35
165	1.08	4	16	0.27
180	1.47	4	16	0.27
195	1.13	4	16	0.28
210	1.58	4	16	0.40
225	1.29	4	16	0.32
240	1.42	4	16	0.36
249	-	-	16	-

 

 

Mean achieved atmosphere concentration (mg/l) = 0.36

Standard Deviation = 0.08

-= not determined

Nominal concentration = Amount of material introduced (mg) / Volume of air passed (l)

                                         = 4900 / 3984

                                         = 1.23 mg/l

 

                                                                                                                                                     

EXPOSURE CHAMBER ATMOSPHERE CONCENTRATIONS-DOSE GROUP 2

DURATION OF EXPOSURE (minutes)	NET WEIGHT OF SAMPLE (mg)	VOLUME OF AIR SAMPLED (l)	CHAMBER FLOW RATE  (l/min)	ATMOSPHERE CONCENTRATION (mg/l)
15	1.04	10	16	0.10
45	1.20	10	16	0.12
60	1.28	10	16	0.13
75	1.26	10	16	0.13
90	1.42	10	16	0.14
105	1.47	10	16	0.15
120	1.32	10	16	0.13
150	1.52	10	16	0.15
165	1.44	10	16	0.14
180	1.43	10	16	0.14
195	1.44	10	16	0.14
210	1.34	10	16	0.13
240	1.28	10	16	0.13
249	-	-	16	-

                                                                                                                                                              

 

Mean achieved atmosphere concentration (mg/l) = 0.13

Standard Deviation = 0.01

-= not determined

Nominal concentration = Amount of material introduced (mg) / Volume of air passed (l)

                                         = 2100 / 3984

                                         = 0.53 mg/l

 

 

 PARTICLE SIZE DISTRIBUTION – DOSE GROUP 1

Impactor Stage	Cut off Diameter (µm)	Amount Collected (mg) During Sample number			Mean Amount in Sample (mg)	Mean Cumulative Percentage
		1	2	3
1#	0.5	0.13	0.13	0.06	0.11	2.66
2	0.9	0.25	0.23	0.09	0.19	7.40
3	1.6	0.67	0.65	0.69	0.67	24.13
4	3.5	1.00	0.89	0.95	0.95	47.75
5	6.0	1.40	1.37	1.53	1.43	83.53
6	10.0	0.65	0.64	0.69	0.66	100.00

 

 

PERDICTED DISTRIBUTION FROM MEAN DATA

Percentage of Aerosol	Predicted less than size (µm)	Particle Size (µm)	Predicted Percentage of Aerosol below this size
10	1.0	1	10.6
20	1.4	2	32.2
30	1.9	3	49.9
40	2.4	4	62.7
50	3.0	5	7.8
60	3.8	7	83.1
70	4.8	10	91.4
80	6.3	15	96.6
90	9.3	20	98.4

 

# values include amount collected on back up filter

Mean Mass Median Aerodynamic Diameter = 3.0 µm

Geometric Standard Deviation = 0.41 µm

Correlation coefficient = 0.9899 (p < 0.05)

 

 

 PARTICLE SIZE DISTRIBUTION – DOSE GROUP 3

Impactor Stage	Cut off Diameter (µm)	Amount Collected (mg) During Sample number			Mean Amount in Sample (mg)	Mean Cumulative Percentage
		1	2	3
1#	0.5	0.07	0.04	0.03	0.05	1.68
2	0.9	0.20	0.12	0.18	0.17	7.66
3	1.6	0.63	0.46	0.76	0.62	29.82
4	3.5	0.82	0.50	0.62	0.65	53.05
5	6.0	1.07	0.92	0.69	0.89	85.15
6	10.0	0.55	0.37	0.32	0.41	100.00

 

 

PERDICTED DISTRIBUTION FROM MEAN DATA

Percentage of Aerosol	Predicted less than size (µm)	Particle Size (µm)	Predicted Percentage of Aerosol below this size
10	1.0	1	10.3
20	1.4	2	34.2
30	1.8	3	53.6
40	2.3	4	67.2
50	2.8	5	76.5
60	3.4	7	87.2
70	4.3	10	94.2
80	5.5	15	98.1
90	7.9	20	99.2

 

# values include amount collected on back up filter

Mean Mass Median Aerodynamic Diameter = 2.8 µm

Geometric Standard Deviation = 0.44 µm

Correlation coefficient = 0.9839 (p < 0.05)

PARTICLE SIZE DISTRIBUTION – DOSE GROUP 2

Impactor Stage	Cut off Diameter (µm)	Amount Collected (mg) During Sample number			Mean Amount in Sample (mg)	Mean Cumulative Percentage
		1	2	3
1#	0.5	0.33	0.15	0.05	0.18	4.83
2	0.9	0.26	0.20	0.11	0.19	10.02
3	1.6	0.62	0.76	0.92	0.77	30.97
4	3.5	0.72	0.96	0.71	0.80	52.73
5	6.0	0.77	1.34	1.39	1.17	84.61
6	10.0	0.32	0.72	0.65	0.56	100.00

 

 

PERDICTED DISTRIBUTION FROM MEAN DATA

Percentage of Aerosol	Predicted less than size (µm)	Particle Size (µm)	Predicted Percentage of Aerosol below this size
10	0.8	1	14.8
20	1.2	2	37.8
30	1.6	3	54.6
40	2.1	4	66.3
50	2.7	5	74.4
60	3.4	7	84.4
70	4.4	10	91.7
80	6.0	15	96.5
90	9.0	20	98.3

 

# values include amount collected on back up filter

Mean Mass Median Aerodynamic Diameter = 2.7 µm

Geometric Standard Deviation = 0.39 µm

Correlation coefficient = 0.9885 (p < 0.05)

 

Interpretation of results:

Category 2 based on GHS criteria

Conclusions:

The median lethal Concentration (LC50) and 95% confidence limits was considered to be 0.20 mg/L (0.13 - 0.36) when the male and female Sprague-Dawley rats were treated with Denatonium benzoate ( Bitrex®) by inhalation route.

Executive summary:

In acute inhalation toxicity study of Denatonium benzoate (3734-33-6) was performed in male and female Sprague-Dawley rats. The animals were housed in group of five by sex in solid floor polypropylene cage with stainless steel lids, furnished with softwood flakes (Datesand Ltd ., Cheshire, UK). Water andfood ( Rat and mouse expanded diet no . 1 , special diets services limited , Witham , Essex, UK)ad libitum (with the exception of the exposure period. Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by mean of a rubber o ring. Only the nose of each animal was exposed to the atmosphere for 4 hr. The dose concentration 0.77, 0.36, 0.13 mg/ L were given to 5 male and 5 female in each dose level.

 

All animals were observed for clinical signs , at hourly intervals during the exposure, immediately on removal from the restraining tubes at the end of the exposure, one hour after termination of the exposure and subsequently once daily for 14days.

Individual bodyweights were recorded prior to treatment on the day of exposure and on days 7,14 or at death. Gross pathology was performed. Full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.During exposure to 0.77 mg/l all animals died between 134 and 216 min after the start. During exposure to 0.36mg/l two males and three female died between 218 and230 min and the 5 remaining animals were killed in extremis approximately two hr after completion of exposure. No deaths occurred in a group of animals exposed to 0.13mg/l. During exposure wet fur and respiratory abnormalities including increased or decreased respiratory rate and labored respiration were commonly noted .one female exposed to 0.77 mg/l also showed pallor of the extremities. On removal from the chamber surviving animals showed additional signs of hunched posture, plo- erection, ptosis and pallor of the extremities. Lethargy, ataxia and an incident of noisy respiration were also noted in animals exposed to 0.36mg/l. one hr after completion of exposure surviving animals in this group continued to show sever signs of toxicity including an incident of gasping and noisy respiration . One hr after completion of exposure to 0.13mg/l the animal’s condition had improved; wet fur and pallor of the extremities were no longer evident although one male appeared lethargic. Increased respiratory rate, signs of hunched posture and pilo- erection and an incident of decreased respiratory rate were observed on day 1.Increased respiratory rate was persistently noted but animals in this group recovered to appear normal 2 to 4 days after exposure. The animals exposed to 0.13 mg/L showed normal bodyweight gain during the study. The animals exposed to 0.77 mg/L or 0.36 mg/L showed abnormally red lungs, several with dark patches. No abnormalities were detected at necropsy in the group animals exposed to 0.13 mg/L. Hencethe median lethal Concentration (LC50) and 95% confidence limits was considered to be0.20 mg/L (0.13 - 0.36) when the male and female Sprague-Dawley rats were treated withDenatonium benzoate (Bitrex®) by inhalation route.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

0.2 mg/m³ air

Quality of whole database:

Data is Klimicsh 1 and from experimental study report

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

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 81-2 (Acute Dermal Toxicity)

Principles of method if other than guideline:

Acute dermal toxicity study of denatonium benzoate (3734-33-6) was performed in rats.

GLP compliance:

yes (incl. QA statement)

Test type:

other: limit test

Limit test:

yes

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 granular solid
- batch no:22362
- Storage conditions: room temperature, room temperature in the dark

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Harlan U.K Ltd., Blackthron, Bicester, Oxon, U.K.
- Age at study initiation:10-14 weeks old
- Weight at study initiation: male 222-251g and female 202-232g
- Fasting period before study: Rats were not fasted prior to dosing
- Housing: The animals were housed in suspended polypropylene cage furnished with wood flakes. The animals were housed individually during the 24-hr exposure period and in group of 5 , by sex for reminder of the study.
- Diet (e.g. ad libitum): Fed ad libitum (Rat and mouse Expanded Diet no. 1, Special dies services Limited, Witham , Essex, U.k)
- Water (e.g. ad libitum):Drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-22°C
- Humidity (%):47-53%
- Air changes (per hr):15 changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give 12hr continuous light and 12hr darkness.
IN-LIFE DATES: From: To:

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

Details on exposure
VEHICLE:
- Concentration in vehicle:2000mg
- Amount of vehicle (if gavage): no data available
- Justification for choice of vehicle: no data available
- Lot/batch no. (if required): no data available
- Purity: no data available
MAXIMUM DOSE VOLUME APPLIED: no data available
DOSAGE PREPARATION (if unusual): no data available
CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:

Duration of exposure:

24hr

Doses:

2000mg/kg

No. of animals per sex per dose:

5 male and 5 female

Control animals:

not specified

Details on study design:

Details on study design
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for death or overt signs of toxicity ½, 1,2, and 4hr after dosing and subsequently once daily for 14 days .
Individual body weights were recorded prior to application of the test material on day 0 and on days 7 and 14
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathological examination was performed. The appearance of any macroscopic abnormalities was recorded.

Statistics:

No data available

Preliminary study:

No data available

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

There were no death during study

Clinical signs:

other: No signs of systemic toxicity were noted during the study. No signs of skin irritation were noted during the study in nine animals. Necrosis, well- defined erythema, very slight oedema and a hardened dark brown/ black colored scab or sunken scab resemblin

Gross pathology:

No abnormalities were noted at necropsy

Other findings:

No data available

Interpretation of results:

other: Not classified

Conclusions:

LD50 was considered to be >2000mg/kg body weight. When rats were treated with denatonium benzoate (3734-33-6) by dermal application.

Executive summary:

In acute dermal toxicity study male and female Sprague – Dawley rats were treated withdenatonium benzoate(3734-33-6).The animals were housed in suspended polypropylene cage furnished with wood flakes. The animals were housed individually during the 24-hr exposure period and in group of 5 , by sex for reminder of the study. Fed ad libitum (Rat and mouse Expanded Diet no. 1, Special dies services Limited, Witham , Essex, U.k)and Drinking water ad libitum. Acclimation period was 5 days , Rats were not fasted prior to dosing. On the day before treatment the back and flanks of each animals were clipped free of hair using veterinary clippers to expose a skin area of approximately 5cmx 7cm.A group of 5 male and 5 female rats were treated with the test material at dose concentration of 2000mg/kg. The appropriate amount of the test material, as received, pre- weighed into a plastic tube, was applied uniformly to an area of shorn skin approximating to 10% of the total body surface area which had previously been moistened with distilled water .A piece of surgical gauze measuring 7 cm x4 cm was placed over the treatment area and semi occluded with a piece of self-adhesive bandage. The bandage was further secured with a piece of BLENDERM wrapped around each end. The animals were caged individually for the 24hr exposure period. Shortly after dosing the dressing were examined to ensure that they were securely in place. The animals were observed for death or overt signs of toxicity ½, 1,2, and 4hr after dosing and subsequently once daily for 14 days .Individual body weights were recorded prior to application of the test material on day 0 and on days 7 and 14 gross pathological examination was performed. The appearance of any macroscopic abnormalities was recorded.

There was no death during study.No signs of systemic toxicity were noted during the study. No signs of skin irritation were noted during the study in nine animals. Necrosis, well- defined erythema, very slight oedema and a hardened dark brown/ black colored scab or sunken scab resembling a crater were noted at the remaining test site during the study. Scab lifting to reveal dried blood and scab lifting to reveal further bleeding were also noted and an accurate evolution of the degree of erythema and/or oedema was not possible from day 5 onwards at this treatment site.All animals showed expected gain in bodyweight. No abnormalities were noted at necropsy. HenceLD50 was considered to be >2000mg/kg body weight. When rats were treated with denatonium benzoate(3734-33-6)by dermal application.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimicsh 1 and from experimental study report

Additional information

Acute oral toxicity

In different studies,denatonium benzoate(3734-33-6)has been investigated for acute oral toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments and estimated data in rodents, i.e. most commonly in rats fordenatonium benzoate(3734-33-6).

The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) In acute oral toxicity study male and femaleSprague –Dawley rats were treated withdenatonium benzoate(3734-33-6).The animals were housed in group of up to five by sex in solid floor polypropylene cages furnished with wood flakes provided with food (Rat and mouse expanded diet no. 1, Special diets services limited, witham , Essex, uk) and drinking water ad libitum. An overnight fast immediately before dosing and for approximately two hours after dosing were done. The test material was freshly prepared, as required, as a solution at the appropriate concentration in distilled water. The dose concentration 500,707, 1000mg/kg were administered to 5 male and 5 female per dose group by oral gavage route in 10mg/ml volume. The Range finding study was performed for selection of dose concentration. All the animals were observed for 1/2, 1,2 and 4 hour after dosing and subsequently once daily for 14 days animals were observed for death or overt signs of toxicity. Individual body weights were recorded prior to dosing on day 0 and on days 7 and 14 or at death. Gross pathological examination was performed and the appearance of any macroscopic abnormalities was recorded.

Deaths were noted within 30 min of dosing 1 female in 500mg/kg dose group while 2 females in 707 mg/kg dose group and 5 females and 4 males in 1000mg/kg dose group in which 2 animals were found dead within 1 or2 minutes of dosing. All the animals were died in 1000mg/kg dose group at the end of 4hr. Clinical signs like Hunched posture, lethargy and decreased respiratory rate were commonly noted on the day of dosing with additional signs of ataxia, labored respiration and noisy respiration. Clonic convulsions were noted in six animals prior to death. Incidents of hunched posture, lethargy, decreased respiratory rate, noisy respiration or labored respiration persisted up to 2 days after dosing. Surviving animals appeared normal 1 to 3 days after dosing or throughout the study. Surviving animals showed an acceptable increase in bodyweight during the study. Hemorrhagic lungs, dark liver, dark kidneys, pale gastric mucosa and haemorrhage or slight haemorrhage of the gastric mucosa were noted at necropsy of the animals that died during the study. No abnormalities were noted at necropsy of animals killed at the end of the study. Hence LD50 values and 95% confidence limits of the test materialdenatonium benzoate(3734-33-6) in the Sprague –Dawley strain were calculated by the method of Thompson W.R to be forall animals: 749 (664-845) mg/kg bw, Males: 841(707-1000) mg/kg bw and Females: 648 (506-832) mg/kg bw.

 

 It is supported by experimental study given by E. W. Schafer, Jr. and W. A. Bowles, Jr. (Arch. Environ. Contain. Toxicol. 14, 111-129)In a acute oral toxicity study, Deer mouse were treated with denatonium benzoate orally by gavage in geometrically spaced dosage level using water, corn oil, or 1.0% carbopol as solvent and observed for 3 days. No more than 50% of the mice were killed at 1225 mg/kg bw. Therefore, LD50 value was considered to be ≥1225 mg/kg bw when Deer mouse were treated with denatonium benzoate (CAS No 3734-33-6) orally by gavage.

 It is supported by experimental study given by Cosmetic Ingredient Review Expert Panel. (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008) In a acute oral toxicity study, Charles River CD male and female rat were treated with denatonium benzoate in the concentration of 127.1, 201.7, 320.2, 508.4, 807 and 1281 mg/kg in distilled water orally by gavage. No mortality was observed in treated male and female rats at 127.1, 201.7, 320.2 mg/kg bw, one animals were died at 508.4 mg/kg bw and all the animals died at 807 and 1281 mg/kg bw. Lungs congested and pale yellow fluid in stomach in one female rat at 508 mg/kg bw, pinpoint red foci in lungs and severe hemorrhage and glandular were in two male and female rat and stomach mucosa in male rat at 807 mg/kg bw and no gross lesions were observed in male and femlae rat at 1281 mg/kg bw. Therefore, LD50 was considered to be 612 mg/kg bw (558-671) for male adnd female rat, 640 mg/kg bw (554-740) for male rat and 584 mg/kg bw (485-702) for female rat when treated with denatonium benzoate orally by gavage.

 It is supported by experimental study given by Cosmetic Ingredient Review Expert Panel. (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008)Acute oral toxicity (LD50) studies were conducted with Denatonium Benzoate in neonatal albino rats. The neonatal rats were derived from pregnant Charles River CD female albino rats. As required, all litters were reduced in number to 10 pups.  Each of the neonatal rats used were administered the test compound within the first 24 hours following parturition. Dosing was accomplished by means of a polyethylene catheter attached to a syringe. The test compound was dissolved in distilled water and administered orally at dosage levels of 7.9, 12.5, 19.8, 31.5, 50.0, 79.4, 125 and 315 mg/kg. Ten neonatal rats were used at each dosage level. Volumes of 10 ml/kg of body weight were administered" at all dosage levels. An additional ten neonatal rats were employed as a control group and were administered distilled water. Volumes of 10 ml/kg of body weight were also used in the control group.The neonatal rats were observed for pharmacodynamics signs and mortality during the first 5 hours following oral intubation, at 24 hours and daily thereafter for a total of 14 days. Gross necropsy examinations were conducted on animals which died during the study period. Individual body weights were obtained just prior to compound administration and again 14 days after compound administration. After the 14 day observation period, all surviving neonatal rats were sacrificed and discarded. No effect on survival of treated neonatal rats at 7.9 and 12.5 mg/kg bw,3 rat dead within 60 minutes following oral dosing at 19.8 mg/kg bw, 3 rat dead within 5 hours and two additional rats at 24 hours at 31.5 mg/kg bw, all neonatal rats were dead within 24 hours at 50 mg/kg bw and all neonatal rats were dead wlthin 4 hours at 79.4, 125 and 315 mg/kg. cyanosis was observed in one animal in the first 60 minutes at 19.8 mg/kg bw, cyanosis in one rat pup from 0 to 2 hours and cyanosis in several pups and hypothermia in one pup from 3 to 4 hours were observed at 31.5 mg/kg bw and decreased motor activity, cyanosis and hypothermia were observed in treated neonetal rats at 50, 79.4, 125 and 315 mg/kg bw. All surviving neonatal rats, in both the control and treated groups, exhibited normal body weight gains during the study period, with the exception of the one rat surviving at the 31.5 mg/kg dosage level. This one rat exhibited a lesser body weight gain than did surviving animals in the other treated groups or in the Control group. In addition, milk curd in stomach (10/10 )and small intestine (1/10) were observed in treated rat at 315 mg/kg bw. milk curd in stomach (10/10), distended urinary bladder (1/10) and pale foci in liver (2/10) at 125 mg/kg bw. milk curd in stomach at 79.4 mg/kg (10/10), 50 mg/kg (7/7) and 31.5 mg/kg (7/7) and yellow fluid/powder in stomach (313), congested lung (1/3) and distended urinary bladder (1/3) were observed in treated rat at 19.8 mg/kg bw.Based upon the results obtained, an acute oral toxicity (LD50) for Denatonium Benzoate in neonatal rats was calculated to be 23 mg/kg with confidence limits of 19-27mg/kg.

 It is supported by experimental study given by Cosmetic Ingredient Review Expert Panel. (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008) in a acute oral toxicity study, New Zealand White male and female rabbits were with denatonium benzoate in the concentration of 201.7, 320.2, 508.4, 807.1 and 1281 mg/kg orally by gavage. All male and female rabbits were dead within 5 hours at 807.1 and 1281 mg/kg bw. Decreased cardiac rate, decreased respiratory rate, cyanosis, decreased activity, ataxia, prostration, tremors, fasciculations, clonic convulsions, mydriasis (2/2) and pupillary reflex absent, corneal reflex absent (1/2) were observed male and femlae rabbits at 1281 mg/kg, Cyanosis, hypothermia, decreased activity, flaccidity, ataxia, prostration, mydriasis, corneal refle:· absent, pupi.llary reflex absent1/2 tremors, clonic convulsi.ons (2/2) were observed male and female rabbits at 807.1 mg/kg. Ataxia, tremors, fasciculations clonic convulsions (1/2), increased respiratory rate, soft stool, nasal discharge, ataxia, tremors, fasciculations, clonic convulsions (1/2) and soft stool, nasal (clear) discharge and lacrimation (1/2) were observed male and female rabbits at 508.4 mg/kg, Diarrhea (1/2) in male and clear nasal discharge (1/2) in female rabbits were observed at 320.2 mg/kg and no adverse findings were observed in treated rabbits at 201.7 mg/kg. No adverse effects observed in treated male and female rabbits. In addition, thymus hemorrhagic, thymus congested, lungs congested, ungs edematous, hemorrhagic trachea congested, blood fille and trachea hemorrhagic and froth filled were observed in treated male and female rabbits at 1281 mg/kg bw, subcutaneous edema, neck, thymus hemorrhagic, lungs congested, lungs emphysematous, trachea congested and blood in trachea were observed in treated male and female rabbits at 807.1 mg/kg bw and lungs congested, edematous, serosanguineous fluid, thoracic cavity and proximal colon and cecum wall, edematous in treated male and female rabbits at 320.2 mg/kg bw. Therefore, LD50 was considered to be 593 mg/kg bw (483-728) for male and female rabbits, 508 mg/kg bw (202-1281) for male rabbits and 640 mg/kg bw (508-807) for female rabbits when treated with denatonium benzoate orally by gavage.

Thus, based on the above studies and predictions ondenatonium benzoate (3734-33-6)it can be concluded that LD50 value is749mg/kg bw. Thus, comparing this value with the criteria of CLPdenatonium benzoate (3734-33-6)can be classified as “Category IV” for acute oral toxicity.

Acute inhalation toxicity

In different studies,denatonium benzoate(3734-33-6)has been investigated for acute inhalation toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments and estimated data in rodents, i.e. most commonly in rats fordenatonium benzoate (3734-33-6).

The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) In acute inhalation toxicity study of Denatonium benzoate (3734-33-6) was performed in male and female Sprague-Dawley rats. The animals were housed in group of five by sex in solid floor polypropylene cage with stainless steel lids, furnished with softwood flakes (Datesand Ltd ., Cheshire, UK). Water and food ( Rat and mouse expanded diet no . 1 , special diets services limited , Witham , Essex, UK)ad libitum (with the exception of the exposure period. Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by mean of a rubber o ring. Only the nose of each animal was exposed to the atmosphere for 4 hr. The dose concentration 0.77, 0.36, 0.13 mg/ L were given to 5 male and 5 female in each dose level.

All animals were observed for clinical signs , at hourly intervals during the exposure, immediately on removal from the restraining tubes at the end of the exposure, one hour after termination of the exposure and subsequently once daily for 14days.Individual bodyweights were recorded prior to treatment on the day of exposure and on days 7,14 or at death. Gross pathology was performed. Full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity. During exposure to 0.77 mg/l all animals died between 134 and 216 min after the start. During exposure to 0.36mg/l two males and three female died between 218 and230 min and the 5 remaining animals were killed in extremis approximately two hr after completion of exposure. No deaths occurred in a group of animals exposed to 0.13mg/l. During exposure wet fur and respiratory abnormalities including increased or decreased respiratory rate and labored respiration were commonly noted .one female exposed to 0.77 mg/l also showed pallor of the extremities. On removal from the chamber surviving animals showed additional signs of hunched posture, plo- erection, ptosis and pallor of the extremities. Lethargy, ataxia and an incident of noisy respiration were also noted in animals exposed to 0.36mg/l. one hr after completion of exposure surviving animals in this group continued to show sever signs of toxicity including an incident of gasping and noisy respiration . One hr after completion of exposure to 0.13mg/l the animal’s condition had improved; wet fur and pallor of the extremities were no longer evident although one male appeared lethargic. Increased respiratory rate, signs of hunched posture and pilo- erection and an incident of decreased respiratory rate were observed on day 1.Increased respiratory rate was persistently noted but animals in this group recovered to appear normal 2 to 4 days after exposure. The animals exposed to 0.13 mg/L showed normal bodyweight gain during the study. The animals exposed to 0.77 mg/L or 0.36 mg/L showed abnormally red lungs, several with dark patches. No abnormalities were detected at necropsy in the group animals exposed to 0.13 mg/L. Hence the median lethal Concentration (LC50) and 95% confidence limits was considered to be 0.20 mg/L (0.13 - 0.36) when the male and female Sprague-Dawley rats were treated with Denatonium benzoate (Bitrex®) by inhalation route.

It is supported by experimental study given by Cosmetic Ingredient Review Expert Panel. (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008)Acute inhalation toxicity study was studied in a group of 5 males and 5 females Sprague-Dawley rats exposed by inhalation-"snout only" for a period of 4 hours, to Bitrex, formulated as 0.1% (w/v) solution in water and generated as a liquid aerosol at a mean gravimetric concentration of 2.32 mg/L. A further group of 5 males and females was exposed to the vehicle (water) only and acted as controls.The rats were checked for clinical signs, body weight changes and gross pathology examinations were conducted. No mortality observed in exposed rats. Similarly, No clinical signs were observed in treated rats at 2.32 mg/kg. Satisfactory weight gain was observed in treated and control group animals and No abnormalities were observed in treated rats. Therefore, LC0 was considered to be 2.32 mg/L when Sprague-Dawley male and female rats were exposed with denatonium benzoate by nose only inhalation for 4 hours.

Thus, based on the above studies and predictions ondenatonium benzoate (3734-33-6)it can be concluded that LC50 value is 0.20 mg/L (0.13 - 0.36) .Thus, comparing this value with the criteria of CLPdenatonium benzoate (3734-33-6)can be classified as “Category II” for acute inhalation toxicity.

In addition to the above, it has to be noted that the highly controlled and standardized laboratory animal inhalation test conditions specified for hazard identification in international guidelines (in particular, the employment of homogenous aerosols of small particle diameter) may not be representative of the workplace or real-life conditions. Based on the particle size distribution of the substance , <<10% would be inhalable, and hence an additional classification of Category IV for inhalation tpxicity is applied since the above category is overly conservative.

Acute dermal toxicity

In different studies,denatonium benzoate(3734-33-6)has been investigated for acute dermal toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments and estimated data in rodents, i.e. most commonly in rats fordenatonium benzoate (3734-33-6).

The study was conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) In acute dermal toxicity study male and female Sprague – Dawley rats were treated withdenatonium benzoate(3734-33-6).The animals were housed in suspended polypropylene cage furnished with wood flakes. The animals were housed individually during the 24-hr exposure period and in group of 5 , by sex for reminder of the study. Fed ad libitum (Rat and mouse Expanded Diet no. 1, Special dies services Limited, Witham , Essex, U.k)and Drinking water ad libitum. Acclimation period was 5 days , Rats were not fasted prior to dosing. On the day before treatment the back and flanks of each animals were clipped free of hair using veterinary clippers to expose a skin area of approximately 5cmx 7cm.A group of 5 male and 5 female rats were treated with the test material at dose concentration of 2000mg/kg. The appropriate amount of the test material, as received, pre- weighed into a plastic tube, was applied uniformly to an area of shorn skin approximating to 10% of the total body surface area which had previously been moistened with distilled water .A piece of surgical gauze measuring 7 cm x4 cm was placed over the treatment area and semi occluded with a piece of self-adhesive bandage. The bandage was further secured with a piece of BLENDERM wrapped around each end. The animals were caged individually for the 24hr exposure period. Shortly after dosing the dressing were examined to ensure that they were securely in place. The animals were observed for death or overt signs of toxicity ½, 1,2, and 4hr after dosing and subsequently once daily for 14 days .Individual body weights were recorded prior to application of the test material on day 0 and on days 7 and 14 gross pathological examination was performed. The appearance of any macroscopic abnormalities was recorded.There was no death during study. No signs of systemic toxicity were noted during the study. No signs of skin irritation were noted during the study in nine animals. Necrosis, well- defined erythema, very slight oedema and a hardened dark brown/ black colored scab or sunken scab resembling a crater were noted at the remaining test site during the study. Scab lifting to reveal dried blood and scab lifting to reveal further bleeding were also noted and an accurate evolution of the degree of erythema and/or oedema was not possible from day 5 onwards at this treatment site. All animals showed expected gain in bodyweight. No abnormalities were noted at necropsy. HenceLD50 was considered to be >2000mg/kg body weight. When rats were treated with denatonium benzoate(3734-33-6)by dermal application.

 

It is supported by experimental studywas conducted in a GLP certified laboratory (Sustainability Support Services (Europe) AB has letter of access) In acute dermal toxicity study male and femaleSPF Wistar rats (MoI:WIST) were treated with denatonium benzoate(3734-33-6).The rats were individually ear-tagged and kept in Macrolone cage TypeIII(42x26x15 cm) 2or 3 to a cage , male and female separated. The bedding was pinewood sawdust Spanvall Special White from Spanvall A/S, Jorlose, Dk- 4490 Jerslev. Fed ad libitum a complete rodent diet Altromin 1314 frm Chr Petersen A/S , DK-4100 Ringsted and Drinking water acidified with hydrochloric acid to pH 2.5.Acclimation period was 2 days. The test article was pulverized in mortar and mixed with white petrolatum in the ratio 1:3 (w/w) in dose concentration 2000mg/kg bw .5 male and 5 female were used in study. The rats were not fasted prior to dosing. The day before application of the test substance the hair was removed from the back and flanks with an electric clipper. The day after the test substance was applied onto an area of 6x8 cm skin, Which then was covered with a 4 layer gauze pack. The gauze packs were fixed with scanpor tape (705 cm wide) wound round the trunk. The application took place from 10 to 11 hr. The rats were caged individually for the 24hr of exposure time after which the dressings were removed. The skin and surrounding hair were sponged with soap and lukewarm water. The animals were then caged in group of 2 or3 during the 14- days observation period. Each rats was observed 1,3 and 6hr after application and then daily over a period of 14 consecutive days

Body weight was recorded on day 0,7and 14.No rats died from the treatment. Clinical sign like Sedation were observed in all rats at the observation on the day of treatment. This is a common finding among partly immobilized rats. Sedation was also observed on day 1and 2, probably due to the skin irritating properties of test article. After removal of the dressing the skin of all animals showed a moderate erythema but no oedema. Four animals had small wounds which, during the 14- days observation period, gradually healed up. From day three and throughout the rest of the observation period the rats showed normal behavior. All rats had a normal body weight gain throughout the experiment. Autopsy of the rats after two weeks observation period revealed no gross pathological organ changes, which could be related to the treatment, with the exception of a yellow discoloration of the skin on the back of two animals. HenceLD50 was considered to be >2000mg/kg body weight. When rats were treated with denatonium benzoate(3734-33-6)by dermal application.

Thus, based on the above studies and predictions ondenatonium benzoate (3734-33-6) it can be concluded that LD50 value is > 2000 mg/kg bw .Thus, comparing this value with the criteria of CLPdenatonium benzoate (3734-33-6)can be not classified for acute dermal toxicity.

 

 

Justification for classification or non-classification

Based upon the study results and available information, the substance denatonium benzoate (CAS No 3734-33-6) is expected to show acute toxicity effect by the oral route for category IV as per CLP classification and for by the inhalation route category II and dermal route qualified as not classified as per CLP classification.

In addition to the above, it has to be noted that the highly controlled and standardized laboratory animal inhalation test conditions specified for hazard identification in international guidelines (in particular, the employment of homogenous aerosols of small particle diameter) may not be representative of the workplace or real-life conditions. Based on the particle size distribution of the substance , <<10% would be inhalable, and hence an additional classification of Category IV for inhalation tpxicity is applied since the above category is overly conservative.