Denatonium benzoate

Endpoint summary

Description of key information

NOAEL was considered to be 5359.05mg/kg for mice when exposed to denatonium orally for 3 weeks.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting 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:

The toxicity of intake of 6 mM denatonium in B6 mice was assessed over a 3-week experiment.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Denatonium
- 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

Species:

mouse

Strain:

other: C57BL/6J (B6)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories; Bar Harbor, ME, USA
- Age at study initiation: 7 weeks old
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: housed in standard tub cages
- Diet (e.g. ad libitum): laboratory chow ,ad libitum
- Water (e.g. ad libitum): The control mice were offered water ad libitum over the 3-week experiment, whereas the experimental mice were offered 6 mM denatonium ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

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

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Denatonium was given as a solution in the concentration of 6 mM.

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): No data available
- Concentration in vehicle: 6 mM denatonium
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3 weeks (21 days)

Frequency of treatment:

Daily, ad libitum

Remarks:

6 mM (535.896 mg/kg)

No. of animals per sex per dose:

Total: 48
0 mM: 24 male
6 mM: 24 male

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on prior work with mice and guinea pigs. Mice would adapt to the aversive taste of the denatonium solution, and thereby obtain sufficient water. So 6mM of dose were selected for the study.
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: : Daily
- Cage side observations included: Decreased activity, ataxia, ocular porphyrin discharge, diarrhea, corneal opacity, tremors, hypothermia, hypersalivation and respiratory congestion

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily during the initial week and weekly thereafter.

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

FOOD EFFICIENCY: No data available
- 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: No data available

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

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: No data available
- Time schedule for collection of blood: No data available
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals:
- Parameters checked in table [No.?] were examined. No data available

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

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

OTHER: No data available

Sacrifice and pathology:

No data available

Statistics:

Statistical analysis was performed by using two-way ANOVA, with time (i.e., day 1, 7, 14 and 21) as a within factor and fluid treatment (i.e., water or denatonium) as a between factor for body weight changes over the 3-week exposure period.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical sign of toxicity od test substence were observed in treated mice as compared to control.

Mortality:

not specified

Description (incidence):

No data available

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No change was observed in treated mice as compared o control. Body weight gain: Weight gain was observed in treated mice as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Effect level:

535.896 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effect on clinical signs and body weight.

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

Conclusions:

NOAEL was considered to be 6 mM (5359.05mg/kg) when B6 male mice were treated with denatonium orally.

Executive summary:

In a subacute repeated dose toxicity study, male B6 mice were exposed to denatonium in the concentration of 6 mM for 21 days. No clinical sign of toxicity and change in body weight were observed in treated mice as compared to control. However, an increased body weight gain were observed in treated mice. Therefore, NOAEL was considered to be 6 mM (5359.05mg/kg) for mice when exposed to denatonium orally for 3 weeks.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

535.896 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

K2 level data has been obtained from peer reviewed journal

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Repeated dose toxicity: Oral:

In different studies, denatonium benzoate has been investigated for repeated dose toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents for denatonium benzoate and summarized as below.

In a experimental study summarized by Cosmetic Ingredient Review Expert Panel (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008) and rapporteur Member State Portugal (Draft Assessment Report (DAR), Volume 3, Annex B, part 2, B.6, part 1, August 2008), 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.

In another subacute repeated dose toxicity study conducted by Glendinning et al (Physiology & Behavior, Volume 93, Issues 4–5, 18 March 2008, Pages 757–765), male B6 mice were exposed to denatonium in the concentration of 5359.05mg/kg for 21 days. No clinical sign of toxicity and change in body weight were observed in treated mice as compared to control. However, an increased body weight gain were observed in treated mice. Therefore, NOAEL was considered to be 5359.05mg/kg for mice when exposed to denatonium orally for 3 weeks.

This is further supported by experimental study summarized by Cosmetic Ingredient Review Expert Panel (International Journal of Toxicology, 27(Suppl. 1):1–43, 2008) and Rapporteur Member State Portugal (Draft Assessment Report (DAR), Volume 3, Annex B, part 2, B.6, part 1, August 2008),) A chronic study was conducted to evaluate the toxic effects of repeated administration of Denatonium benzoateto in male and female rats by gavage. Denatonium benzoate was administered to 65 animals/sex/species in distilled water at dosages of 0, 1.6, 8 or 16 mg/kg/day for 2 years. 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. 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 indicate towards a NOAEL for repeated dose toxicity study was considered to be 16 mg/kg/day in male and female rats when exposed to Denatonium benzoate by oral route for 2 years.

Again supported by experimental study conducted by Sustainability Support Services (Europe) AB (OECD Lab, 2018), Sprague-Dawley male and female rats were treated withDenatonium Benzoate in the concentration of 0, 15, 30 and 60 mg/kg bw orally by gavage in water. No clinical signs of toxicity up to 21 days of treatment period at 0 and 60 mg/kg bw and started to reveal aggressiveness approximately from day 22, excessive grooming and hair thinning at lower jaw region along with aggressiveness from day 28 onwards and continued till the end of treatment period. In females no any clinical signs of toxicity up to 22 days of treatment period and started to reveal aggressiveness approximately from day 23 to day 53 and later became normal at the end of treatment period. These observations are due to bitter nature of the test item and biting/ scratching of animals due to the bitter or irritant nature of the test item. No clinical signs of toxicity were observed in treated male and female rats at 15 mg/kg bw as compared to control. No mortality/morbidity observed at 15, 30 and 60 mg/kg bw treated animals of either sex during the experimental period. Slight reduction in mean body weight and percent change in body weight gain was observed in males (G3) and (G4) at 30 and 60 mg/kg bw during the experimental period when compared with vehicle control group. This reduction can be considered as treatment related as the change is consistent throughout the experimental period. No treatment related changes observed in mean body weight and percent change in body weight with respect to day of either sex during the experimental period at 15 mg/kg bw. No related changes observed in the gestation and lactation body weight and percent change in gestation and lactation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals. Similarly, statistical significant reduction in feed consumption was observed at G3 (30 mg/kg bw) and G4 (60 mg/kg bw) group males during post-mating period (treatment day 30 to 35 and 35 to 42) when compared with vehicle control group. This change can be considered as treatment related as the mean body weight and percent change in body weight gain of these dose group males (G3 and G4) during this duration were found to be reduced which were biologically significant. No treatment related changes observed in feed consumption at any of the tested dose group animals of either sex during the pre-mating treatment period of the experiment (i.e. first two weeks of the treatment period) at 15 mg/kg bw. No treatment related changes observed on serum T4 levels in male rats, absolute and relative organ weights at any of the tested dose group animals of either sex when compared with vehicle control group. No gross pathological changes (both external and internal) observed at 15, 30 and 60 mg/kg bw tested dose group adult females and at 15 mg/kg bw dose group adult males. All the adult males from 30 and 60 mg/kg bw revealed hair thinning at lower jaw region during gross pathological examination. This finding is due to the biting/ scratching of animals because of the bitter or irritant nature of the test item. Pustule formation in stratum corneum was observed in seven and eleven males at 30 and 60 mg/kg bw dose groups respectively. The severity of the lesion in most of the animal was minimal except one animal showed mild grade lesion. These pustules may arise from skin injury that may be by biting or scratching of animals. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, epididymis and ovaries did not show any pathological findings/lesions. In addition, no effect on oestrus cyclicity, copulatory interval and number of conceiving days at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14, number of corpora lutea, number of implantations and pre and post-implantation loss, pre-natal loss, post-natal loss, resorptions, gestation length, number of pups delivered, sex ratio and live birth index of each litter at any of the tested dose group animals when compared with vehicle control group animals. No clinical signs and pup survival were observed in any of the pups of tested dose group animals during lactation period. Statistically significant decrease in T4 levels at 30 mg/kg bw dose group lactation day 13 pups was noted when compared with vehicle control group. This significant variation is not considered as treatment related as the values obtained were within historical control range only and the change is not dose dependent. Hence, the variation observed in the T4 levels is considered to be incidental. No changes observed in ano-genital distance ratio and no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13. No gross pathological changes (both external and internal) observed at any of the tested dose group pups of both the sex examined at termination. Therefore, NOAEL was considered to be 15 mg/kg bw whenSprague-Dawley male and female rats were treated withDenatonium Benzoate orally by gavage in water for Male 42 days and 63 days female rats.

Thus, based on the above studies on denatonium benzoate, it can be concluded that NOAEL is 16 and 5359.05mg/kg in rat and mice and LOAEL value is 8 mg/kg bw for monkye . Thus, comparing NOAEL value with the criteria of CLP regulation, denatonium benzoate can be Not classified for repeated dose oral toxicity.

Repated dose toxicity: dermal

Given the low vapour pressure, exposure by the inhalation route is not likely and hence this end point was considered for waiver.

Repated dose toxicity: inhalation

Considering the use of the chemical, repeated exposure to denatonium benzoate by the dermal route is not very likely and hence this end point has been considered for waiver

Justification for classification or non-classification

Based upon the available data,the chemical denatonium benzoate is not likely to be toxic via repeated dose by oral route since the higher ranking Klimisch data indicates a NOAEL of 535.896 mg/kg bw/day and inhalation and dermal toxicity is waived.