Denatonium benzoate

Acute Toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

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

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: granular

Details on test material:

- Name of test material: Denatonium benzoate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight : 446.58 g/mol
- Substance type: organic
- Physical state: Solid
- SMILES: CCN{+}(CC)(Cc1ccccc1)(CC(=O)Nc1c(C)cccc1C).O{-}C(=O)c1ccccc1

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

Test animals

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

Administration / exposure

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

Results and discussion

Preliminary study:

No data available

Effect levels

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

Any other information on results incl. tables

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)

 

Applicant's summary and conclusion

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.