Denatonium benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer-reviewed journal

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Final Report of the Safety Assessment of Alcohol Denat.,IncludingSDAlcohol3-A,SDAlcohol30,SDAlcohol39,SDAlcohol39-B,SDAlcohol 39-C,SDAlcohol 40,SDAlcohol 40-B,SDAlcohol 40-C,and the Denaturants,Quassin,Brucine Sulfate/Brucine,and Denatonium Benzoate1

Author:

Cosmetic Ingredient Review Expert Panel

Year:

2008

Bibliographic source:

International Journal of Toxicology, 27(Suppl. 1):1–43, 2008

Reference 2

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Chronic toxicity study of denatonium benzoate in cynomolgus monkeys

GLP compliance:

not specified

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

Details on test material:

- Name of test material (as cited in study report): N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate
-Common name: Denatonium benzoate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight: 446.58 g/mol
- Substance type: organic
- Physical state: Solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): Denatonium benzoate
- Molecular formula (if other than submission substance): C28H34N2O3
- Molecular weight (if other than submission substance): 446.58 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data available
- Age at study initiation: No data Available
- Weight at study initiation:
Males: 1.60 to 3.45 Kg
Females: 1.40 to 2.90 Kg
- Fasting period before study: No data available
- Housing: The monkeys were housed individually in hanging wire mesh “squeeze type” cages
- Diet (e.g. ad libitum): Purina Monkey Chow was fed twice daily and fresh apples were fed three times a week
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled environment
- Humidity (%):Controlled environment
- Air changes (per hr): Controlled environment
- Photoperiod (hrs dark / hrs light): Controlled environment

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Denatonium benzoate was dissolved in distilled water so that a volume of 2 ml/kg/day was administered at all dosage levels. Individual daily doses were based upon the body weights obtained twice a week or weekly.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water was used as a vehicle.
- Concentration in vehicle: 0, 1.6, 8.0 and 16.0 mg/kg body weight /day
- Amount of vehicle (if gavage): 2 ml/kg/day
- Lot/batch no. (if required): : No data available
- Purity: : No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 year

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 1.6, 8 and 16 mg/kg/day

No. of animals per sex per dose:

Total: 66
0 mg/kg bw/day: 8 male, 9 female
1.6 mg/kg bw/day: 8 male, 8 female
8.0 mg/kg bw/day: 9 male, 8 female
0 mg/kg bw/day: 8 male, 8 female

Control animals:

yes, concurrent vehicle

Details on study design:

Eight male and 8 female monkeys were initiated at each dosage level and also as a control group. Two monkeys dying during the first 6 weeks of study were replaced.

Positive control:

not specified

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly for first 5 weeks and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once for control animals and at 3, 6 and 12 months of study for test animals
- Dose groups that were examined: All 66 animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Once for control animals and at 3, 6 and 12 months of study for test animals
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all dose group animals
- Parameters checked: Hematological studies included hemoglobin , hematocrit , total erythrocyte count , total and differential leucocyte counts,
clotting time and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once for control animals and at 3, 6 and 12 months of study for test animals
- Animals fasted: No data
- How many animals: all dose group animals
- Parameters checked: Biochemical studies included fasting glucose, urea nitrogen , serum glutamic pyruvic and oxalacetic transaminase activities, serum alkaline phosphatase, carbon dioxide, serum total protein, serum electrophoresis, albumin, bilirubin, serum sodium, potassium, chloride, calcium and prothrombin time.

URINALYSIS: Yes
- Time schedule for collection of urine: Once for control animals and at 3, 6 and 12 months of study for test animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: Urinalysis included measurement of volume, pH and specific gravity; description of color and appearance; qualitative tests for albumin, glucose, ketones, bilirubin and occult blood; and microscopic examination of the sediment

NEUROBEHAVIOURAL EXAMINATION: No data - Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Heart function was examined by using electrocardiograms
- Time schedule to obtained electrocardiograms: Once in quarantine period and at 3, 6 and 12 months of study.
- How many animals: All 66 animals were examined.
- Parameters checked in table [No.?] were examined: Variations or abnormalities were examined.

Organs weight :
Spleen, liver, adrenals, kidney, ovaries, liver, lung, thyroid, brine and pituitary were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At 3 months of study, 2 male and 2 female monkeys from the control and 1.6 mg/kg/day group, 2 males and 1 female from the 8 mg/kg/day group and 2 males fro mthe 16 mg/kg/day group.
At 3 months of study , 2 male and 2 female monkeys from the control group, 2 males and 1 female from 1.6 mg/kg/day group and 1 male and 2 females from each of the 8 mg/kg/day and 16 mg/kg/day groups were sacrificed
At the end of 12 months of study, all remaining monkeys were sacrificed and necropsied.
At necropsy selected organs were weighed and representative tissues from each monkey were collected in buffered neutral 10% formalin.

HISTOPATHOLOGY: Yes
Hematoxylin and eosin stained paraffin sections of tissues were prepared and examined.
Oil red 0 stained frozen sections of livers, from selected monkeys sacrificed at 3 months and, from those that died, were prepared and examined.
Organs examined.
Brain (3 levels),spinal cord (thoracic), sciatic nerve, eye, pituitary, thyroid/parathyroid, adrenals, trachea, lung, heart, spleen, lymph node (mesenteric), bone marrow (rib, junction), esophagus, stomach, small intestine (3 levels), large intestine, pancreas, liver, kidneys, urinary bladder, testes, epididymus/ovaries, prostate/uterus, seminal vesicles, skeletal muscle, skin (mammary gland) and any other tissue with gross lesions were examined.

Other examinations:

not specified

Statistics:

not specified

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No effect was observed on treated monkeys as compared to control.

Mortality:

mortality observed, treatment-related

Description (incidence):

When treated with 16 mg/kg bw/day, 2 male and 2 female died on 9th, 11th, 17th and 31st week of study.
When treated with 8 mg/kg bw/day 2 male and 1 female died on 3rd, 10th and 18th week of study.
When treated with 1.6 mg/kg bw/day, 1 female died on 21 weeks of study and one animal died in control group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treated male monkeys gained less body weight as compared to control monkeys. Female monkeys, control and treated, generally maintained or showed slight gains in body weight.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmoscopic changes were observed in treated monkeys as compared to control.

Haematological findings:

no effects observed

Description (incidence and severity):

When treated with 1.6 mg/kg bw/day, marked decreases in hemoglobin, hematocrit and total erythrocyte count for one female monkey were observed at 3 months of study. Changes were considered to be incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes were observed in clinical chemistry of treated monkeys as compared to control.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes in urinalysis were observed in treated monkeys as compared to control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No behaviour changes were observed in treated monkeys as compared to control.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No absolute and relative changes in organ weight were observed in treated monkeys.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound related gross pathologic lesions were observed in any of the monkeys which were sacrificed at 3 months, 6 months, or 1 year, or which died during the course of study.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic lesions were observed in tissues from monkeys sacrificed at 3 months from 8 or 16 mg/kg bw /day group or those sacrificed at 6 months or 1 year at the 16 mg/kg/day group or in monkeys which died during the course of the study.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

No effect on electrocardiograms were observed in treated monkeys.

Effect levels

Dose descriptor:

LOAEL

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other: Effect on survival of monkeys

Target system / organ toxicity

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

LOEAL was considered to be 8 mg/kg bw when Macaca fascicularis (cynomolgus) male and female monkeys were treated with denatonium benzoate orally by gavage for 2 years.

Executive summary:

A Chronic toxicity study, Macaca fascicularis (cynomolgus) male and female monkeys were treated with denatonium benzoate n the concentration of 0, 1.6, 8 and16 mg/kg/day orally by gavage. 2 male and 2 female died on 9^th, 11^th, 17^thand 31^st week of study at 16 mg/kg bw/day, 2 male and 1 female died on 3^rd, 10^thand 18^thweek of study at 8 mg/kg bw/day and 1 female died on 21 weeks of study and one animal died in control group at 1.6 mg/kg bw/day. No effect on clinical sign and behaviour changes were observed on treated monkeys as compared to control. Treated male monkeys gained less body weight as compared to control monkeys. Female monkeys, control and treated, generally maintained or showed slight gains in body weight. No ophthalmoscopic changes were observed in treated monkeys as compared to control. Marked decreases in hemoglobin, hematocrit and total erythrocyte count for one female monkey were observed at 3 months of study at 1.6 mg/kg bw/day. Changes were considered to be incidental. No changes were observed in clinical chemistry, urinalysis and electrocardiograms of treated monkeys as compared to control. No absolute and relative changes in organ weight and no compound related gross pathologic lesions were observed in any of the monkeys which were sacrificed at 3 months, 6 months, or 1 year, or which died during the course of study. No microscopic lesions were observed in tissues from monkeys sacrificed at 3 months from 8 or 16 mg/kg bw /day group or those sacrificed at 6 months or 1 year at the 16 mg/kg/day group or in monkeys which died during the course of the study. Therefore, LOEAL was considered to be 8 mg/kg bw when Macaca fascicularis (cynomolgus) male and female monkeys were treated with denatonium benzoate orally by gavage for 2 years.