Denatonium benzoate

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

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

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

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:8-12 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

Administration / exposure

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 ,

Results and discussion

Preliminary study:

Range finding study was performed

Effect levels

Effect levels 1

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

Effect levels 2

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

Effect levels 3

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

Applicant's summary and conclusion

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.