Denatonium benzoate

Carcinogenicity

Description of key information

No Observed Adverse Effect Level (NOAEL) (relating systemic chronic effects and carcinogenicity) was considered to be 16 mg/kg per day. When  rats were treated with Denatonium benzoate (37.34-33-6)orally for 2 year.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: 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

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Chronic toxicity study of Denatonium benzoate in rats.

GLP compliance:

not specified

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): C21H29N2O.C7H5O2
- Molecular weight (if other than submission substance): 446.588 g/mole
- Substance type: Organic
- Physical state: Solid

Species:

rat

Strain:

not specified

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data available

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Denatonium benzoate was dissolved in distilled water at varying concentrations so that a volume of 10 ml/kg/day was administered at all dosage levels.

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): Distilled water
- Concentration in vehicle: 0, 1.6, 8 or 16 mg/kg/day.
- Amount of vehicle (if gavage): 10 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 years

Frequency of treatment:

Daily

Post exposure period:

No data

Remarks:

0, 1.6, 8 or 16 mg/kg per day

No. of animals per sex per dose:

Total: 520
Control (vehicle): 65 males, 65 femles
1.6 mg/kg per day: 65 males, 65 females
8 mg/kg per day: 65 males, 65 females
16 mg/kg per day: 65 males, 65 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations: Toxicity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): No data
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Daily during the first 5 weeks of study and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once in the control period and at 3, 6, 12, 18 and 24 months of study
- Dose groups that were examined: All rats were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24 months of study
- Anaesthetic used for blood collection: No data
- Animals fasted: : No data
- How many animals: 5 male and 5 female rats from each group
- Parameters checked: Haemoglobin, hematocrit, total erythrocyte count, total and differential leucocyte counts, clotting time and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24 months of study
- Animals fasted: Yes
- How many animals: 5 male and 5 female rats from each group
- Parameters checked: biochemical studies included fasting glucose, blood urea nitrogen, serum glutamic oxalacetic and pyruvic transaminase activities, serum alkaline phosphatise carbon dioxide, serum total protein, albumin, bilirubin, serum sodium, potassium, chloride and calcium and prothrombin time. Serum electrophoresis was also conducted.

URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6, 12, 18 and 24 months of study
- 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, bilirubins and occult blood; and microscopic examination of the sediment.

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

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After compound administration and at different times during the treatment schedule, 5 male and 5 female rats from the control and treatment groups were sacrificed by decapitation and necropsied. At necropsy, selected organs were weighed and representative tissues from each rat were collected. All rats which died or were sacrificed in extremis during the course of study were necropsied.

HISTOPATHOLOGY: Yes
Sections of the brain, spinal cord, peripheral nerve, eye, pituitary, thyroid, parathyroid, adrenals, mesenteric lymph node, trachea, esophagus, heart, lungs, liver, spleen, kidneys, urinary bladder, stomach, pancreas, small intestines (3 levels) large intestine, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, mammary gland, skin, skeletal muscle, bone marrow and bone (rib junction) were prepared and examined from male and female rats for the control and the high dosage level groups.

Other examinations:

No data available

Statistics:

No data available

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in general behaviour and appearance were observed.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in treated rats.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Increases in body weight were similar for control and treated rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption & compound intake was similar in control and treated rats

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No compound related effects were observed in treated rats as compared to control.

Haematological findings:

no effects observed

Description (incidence and severity):

No changes considered to be related to compound were observed in the haematological parameter.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes were observed in clinical chemistery of treated rats.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No compound-related organ weight variations were observed in any rats necropsied at scheduled sacrifices. The only statistically significant organ weights variations occurred in terminally sacrificed rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No pathological lesions which were considered compound-related were seen at necropsy in any rats from the experimental groups which were sacrificed or died during the study.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of tissues from terminally sacrificed rats and deaths or sacrifices in the extremis did not reveal any compound related lesions.

Adenomas, Pituitary carcinomas, C-cell adenomas, Thyroid follicular cell adenomas, pheochromocytomas, adrenal cortical cell carcinoma, alveolar cell adenoma, hepatocellular adenoma and carcinoma in the liver, hemangioma in the liver, embryonal nephroma in the kidney, testicular interstitial cell tumors, prostatic adenocarcinoma with metastasis to the lungs, granulosa cell tumors in the ovaries, ovarian thecal cell tumor, uterine endometrial polyps, polyploidy leiomyoma in the cervix, mammary gland fibroadenomas, mammary gland adenoma, mammary gland cystadenomas, intraductalpapillomas, mammary gland adenocarcinomas, squamous papillomas, sebaceous gland adenocarcinoma in the skin, osteosarcoma arising from a rib with metastasis to the lungs, hematopoetic neoplasms including malignant lymphoma in the lungs, mesentery, lymphnodes, gastrointestinal tract and pancreas, granulocytic sarcomas identified in the lungs, liver, spleen, lymphnodes, heart, kidneys, bone marrow, malignant fibrohystiocytomas in the lungs, liver, kidneys, pancreas, omentum, spleen, kidneys, subcutis, lymphnodes, jejunum and small intestine, subcutaneous neoplasms including lipomas, hemangioma and fibrosarcoma, fibromas on the ear, fibrosarcomas in the lip region and on the tail, osteosarcoma on the nose, squamous cell carcinomas in the head region, lipoma in the abdominal cavity, and preputial gland intraductal squamous pappillomas were noted in either male or female or both sex rats in either of the groups ranging from I-IV.

The types and incidences of neoplasms identified in this study did not indicate any tumorigenic effect related to compound administration.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic changes were observed in treated rats as compared to control.

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: Tumorigenic but not carcinogenic and not related to the test chemical

Remarks on result:

other: No effect observed

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

Mortality:

Survival after 2 years of compound administration was as follows:

Dosage level (mg/Kg/day)	Group	No. surviving/ No. initiate less Interim sacrifice
		Male (50)	Female (50)
Control	I	24	24
1.6	II	22	23
8	III	21	22
16	IV	19	26

Organ Weights:

Organ	Dosage level (mg/Kg/ day)	Sex	Weight		Change	p<
Liver	8	F		Relative	Decrease	0.05
	16	M	Absolute		Increase	0.05
Heart	16	F	Absolute		Increase	0.05
Brain	1.6	M		Relative	Decrease	0.05
	8	M		Relative	Decrease	0.01
Adrenals	16	M		Relative	Decrease	0.05
Pituitary	1.6	M		Relative	Decrease	0.05, 0.05
	16		Absolute		Decrease	0.05

The biological significance of these variations is unknown

Conclusions:

No Observed Adverse Effect Level (NOAEL) (relating systemic chronic effects and carcinogenicity) was considered to be 16 mg/kg per day. When rats were treated with Denatonium benzoate (37.34-33-6)orally.

Executive summary:

In a Chronic toxicity study, Denatonium benzoate, dissolved in distilled water, was administered by gavage at dosage levels of 1.6, 8 or 16 mg/kg/day with a total administration volume of 10 ml/kg/day. The rats were observed for overt signs of toxicity, mortality, body weight, food consumption and efficiency. Ophthalmoscopic, haematological, biochemistry, urine analysis, gross and histopathological examinations were also investigated. No mortalities occurred that could be directly attributed to treatment. Similarly, no changes were observed in body weight, food consumption, food efficiency, opthalmoscopic, hematology or clinical chemistry. In addition, no change were observed in organ weight, gross pathology and histopathology of treated male and female rat when compared with control. Some changes were observed on terminal sacrifice but the effect is not regarded to be substance-related. Since no alterations were observed in the test groups from the control groups the effects observed were not considered to be compound related. The tumors observed were not regarded to be the result of a tumorigenic effect of the test material. Despite the fact that no statistical calculations could be performed due to the low numbers of animals tested, and since no compound related effects were observed, the results still indicates towards a No Observed Adverse Effect Level (NOAEL) (relating systemic chronic effects and carcinogenicity) of 16 mg/kg per day for the given test chemical.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

16 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

K2 data as the information is from a peer reviewed journal

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation, Denatonium benzoate (3734-33-6) can be not classified for Carcinogenicity in rats by oral route exposed for 2 years.

Additional information

Carcinogenicity by oral route

In given study, Denatonium benzoate (3734-33-6) has been investigated for Carcinogenicity to a greater or lesser extent. Often the studies based on in vivo experiments in rodents, i.e. most commonly in rats Denatonium benzoate (3734-33-6)

In experimental study given by Cosmetic Ingredient Review Expert Panel (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008) In a Chronic toxicity study, Denatonium benzoate, dissolved in distilled water, was administered by gavage at dosage levels of 1.6, 8 or 16 mg/kg/day with a total administration volume of 10 ml/kg/day. The rats were observed for overt signs of toxicity, mortality, body weight, food consumption and efficiency. Ophthalmoscopic, haematological, biochemistry, urine analysis, gross and histopathological examinations were also investigated. No mortalities occurred that could be directly attributed to treatment. Similarly, no changes were observed in body weight, food consumption, food efficiency, opthalmoscopic, hematology or clinical chemistry. In addition, no change were observed in organ weight, gross pathology and histopathology of treated male and female rat when compared with control. Some changes were observed on terminal sacrifice but the effect is not regarded to be substance-related. Since no alterations were observed in the test groups from the control groups the effects observed were not considered to be compound related. The tumors observed were not regarded to be the result of a tumorigenic effect of the test material. Despite the fact that no statistical calculations could be performed due to the low numbers of animals tested, and since no compound related effects were observed, the results still indicates towards a No Observed Adverse Effect Level (NOAEL) (relating systemic chronic effects and carcinogenicity) of 16 mg/kg per day for the given test chemical.

Thus, comparing this value with the criteria of CLP regulation, Denatonium benzoate (3734-33-6) can be not classified for Carcinogenicity in rats by oral route exposed for 2 years.