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EC number: 223-095-2 | CAS number: 3734-33-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 421
- Principles of method if other than guideline:
- Reproduction/Developmental Toxicity Screening Test in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure of Denatonium Benzoate (CAS No 3734-33-6) over a relatively limited period of time.
- GLP compliance:
- yes
- Limit test:
- no
Test material
- 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:
- other: granules
- Details on test material:
- - Physical state: solid
- Appearance: white granules
- Storage condition of test material: room temperature until 22 February 1995 then room temperature in the dark
Constituent 1
- 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
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x wks; (F1) x wks: 9 to 10 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: Males: 240.11 g to 280.79 g
Females: 220.09 g to 253.90 g
- Fasting period before study:
- Housing:Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i. Pre mating
Two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period
- Water (e.g. ad libitum):Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes
- Acclimation period:nineteen days (including five days of acclimatizion) to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days)
ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.1 to 23.6oC
- Humidity (%):41 to 69%
- Air changes (per hr): air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour),
- Photoperiod (hrs dark / hrs light):12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES:
From: 20 October 2017
To:21 February 2018
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:Required quantity of test item was weighed into a clean glass beaker and there by the test item was dissolved by adding little volume of vehicle in to beaker, mixed well with clean glass rod and transferred to a measuring cylinder and the beaker was rinsed and the volume was transferred to measuring cylinder. The rinsing procedure was repeated until to ensure entire quantity of test item formulation was transferred to measuring cylinder. Finally, the volume was made up to the required quantity with the vehicle to get desired concentrations of 1.5, 3 and 6 mg/mL of test item for low, mid and high dose groups respectively.
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is clearly soluble in distilled water at the concentration of 6 mg/mL (highest dose concentration) based on the in-house solubility test results
- Concentration in vehicle: 0, 15, 30 and 60 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required):
- Purity: - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Homogeneity and stability of dose formulations was established by Analytical Department
- Details on mating procedure:
- - M/F ratio per cage:1:1 ratio
- Length of cohabitation: two weeks
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: The re-mating of females with proven males of the same group was considered for further one week in case of unsuccessful mating of females within two weeks.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: - Duration of treatment / exposure:
- Male 42 days
Female: 63 days - Frequency of treatment:
- Daily
- Duration of test:
- The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 42 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 15 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 60 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Total: 96
0 mg/kg bw/day: 12 male, 12 female
15 mg/kg bw/day: 12 male, 12 female
30 mg/kg bw/day: 12 male, 12 female
60 mg/kg bw/day: 12 male, 12 female - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
- Rationale for animal assignment (if not random):The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+15.49% and -12.75% for males and +7.14% and -8.23% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
- Other:
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included.: mortality and morbidity were examined.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
BODY WEIGHT: Yes
- Time schedule for examinations:The animals were weighed at receipt, on the first day of dosing, weekly thereafter (varied by -1 day) and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # : 14
- Organs examined:yes
OTHER: Organ Weights were examined. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: - Fetal examinations:
- The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined and subjected to gross pathological examination.
Hormone analysis:T4 (Thyroxine) andgross abnormalities were examined. - Statistics:
- The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group. - Indices:
- Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Gestation Index (%), Live Birth Index (%) per dam and Pup Survival index (%) on lactation day 4 were examined.
- Historical control data:
- not specified
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- When treated with 30 and 60 mg/kg bw, no clinical signs of toxicity up to 21 days of treatment period 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.
When treated with 15 mg/kg bw, no clinical signs of toxicity were observed in treated male and female rats as compared to control. - Dermal irritation (if dermal study):
- not specified
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality/morbidity observed at 15, 30 and 60 mg/kg bw treated animals of either sex during the experimental period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- When treated with 30 and 60 mg/kg bw, slight reduction in mean body weight and percent change in body weight gain was observed at mid dose group males (G3) and high dose group males (G4) 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.
When treated with 15 mg/kg bw,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.
Gestation Body Weight:
No related changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals.
Lactation Body Weight:
No changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- When treated with 30 and 60 mg/kg bw, 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.
When treated with 15 mg/kg bw, 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). - 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:
- no effects observed
- Description (incidence and severity):
- No treatment related changes observed in serum T4 levels in males when compared with vehicle control group.
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No effects observed in absolute and relative organ weights at any of the tested dose group animals of either sex when compared with vehicle control group.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- 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 bwdose 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.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- 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. - Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- No changes were noted in pre-natal loss and post-natal loss at any of the tested dose group animals when compared with vehicle control group animals.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- No changes observed in number of pre and post-implantation loss in treated rats as compared to control.
- Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- No resorptions were noted at all the dose group animals observed during necropsy.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- No resorptions were noted at all the dose group animals observed during necropsy.
- Dead fetuses:
- not specified
- Changes in pregnancy duration:
- not specified
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified - Changes in number of pregnant:
- not specified
- Other effects:
- not specified
Effect levels (maternal animals)
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- clinical biochemistry
- early or late resorptions
- gross pathology
- histopathology: non-neoplastic
- mortality
- necropsy findings
- number of abortions
- organ weights and organ / body weight ratios
- pre and post implantation loss
- total litter losses by resorption
- Remarks on result:
- other: No effect observed
Maternal abnormalities
- Abnormalities:
- not specified
- Localisation:
- not specified
- Description (incidence and severity):
- not specified
Results (fetuses)
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There were no treatment related changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose group dams when compared with vehicle control group dams.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no treatment related changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose group dams when compared with vehicle control group dams. - Reduction in number of live offspring:
- not specified
- Changes in sex ratio:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant reduction in number of female pups at 15 mg/kg bw (G2) and 60 mg/kg bw (G4) was observed at lactation day 7 and 13. This change is not considered as treatment related as there were no effects noted in other parameters like, external observation of pups, pup weights and ano-genital distance of pups. This change is due to more number of male pups born at these tested groups when compared with vehicle control group.
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- No effect on the pup survival were observed as compared to control.
- External malformations:
- no effects observed
- Description (incidence and severity):
- No gross pathological changes (both external and internal) observed at any of the tested dose group pups of both the sex examined at termination.
- Skeletal malformations:
- not specified
- Visceral malformations:
- not specified
- Other effects:
- no effects observed
- Description (incidence and severity):
- Nipple Retention in Male Pups on Lactation Day 13
There were 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.
Effect levels (fetuses)
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- changes in sex ratio
- fetal/pup body weight changes
- changes in litter size and weights
- changes in postnatal survival
- external malformations
- other: Nipple Retention in Male Pups on Lactation Day 13
- Remarks on result:
- other: No effect observed
Fetal abnormalities
- Abnormalities:
- not specified
- Localisation:
- other: not specified
- Description (incidence and severity):
- not specified
Overall developmental toxicity
- Developmental effects observed:
- not specified
- Treatment related:
- not specified
- Relation to maternal toxicity:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
Any other information on results incl. tables
SUMMARY OF THE STUDY
Parameters ↓ |
Group & Dose (mg/kg body weight/day) |
||||
G1 & 0 |
G2 & 15 |
G3 & 30 |
G4 & 60 |
||
Reproductive Indices |
|||||
Mating Indices |
|||||
Pairs started (No.) |
12 |
12 |
12 |
12 |
|
Males showing evidence of mating (No.) within 14 days* |
11 |
11 |
10 |
11 |
|
Females showing evidence of copulation (No.) |
12 |
12 |
12 |
12 |
|
Male Mating Index (%) within 14 days* |
91.67 |
91.67 |
83.33 |
91.67 |
|
Female Mating Index (%) |
100.00 |
100.00 |
100.00 |
100.00 |
|
|
|||||
Fertility Indices |
|||||
Females achieving pregnancy (No.) |
11 |
11 |
12 |
12 |
|
Male Fertility Index (%)* |
91.67 |
91.67 |
83.33 |
91.67 |
|
Female Fertility Index (%) |
91.67 |
91.67 |
100.00 |
100.00 |
|
|
|||||
Copulatory Indices |
|||||
Conceiving days 1 to 5 (No.) |
6 |
11 |
7 |
9 |
|
Conceiving days >6 (No.) |
6 |
1 |
5 |
3 |
|
Mean Precoital Interval (Days) |
6.75 |
3.50 |
7.25 |
4.00 |
|
|
|||||
Gestation Indices |
|||||
Pregnancy≤21 days (No.) |
1 |
0 |
1 |
0 |
|
Pregnancy = 22 days (No.) |
8 |
6 |
10 |
7 |
|
Pregnancy≥23 days (No.) |
2 |
5 |
1 |
5 |
|
Mean Gestation length (Days) |
22.09 |
22.45 |
22.00 |
22.42 |
|
Gestation Index (%) |
100.00 |
100.00 |
100.00 |
100.00 |
|
|
|||||
Dams with live young born (No.) |
11 |
11 |
12 |
12 |
|
Dams with live young at day 4 post-partum (No.) |
11 |
11 |
12 |
12 |
|
Dams with live young at day 13 post-partum (No.) |
11 |
11 |
12 |
12 |
|
|
|||||
Implantation Index and Pre and Post implantation Losses |
|||||
Implants/dam (Mean) |
10.82 |
10.91 |
11.08 |
11.00 |
|
Corpora luetea/dam (Mean) |
11.00 |
11.18 |
11.33 |
11.33 |
|
Implantation Index (%) |
98.35 |
97.13 |
97.83 |
96.64 |
|
Pre-Implantation Loss (%) |
1.66 |
2.87 |
2.17 |
3.36 |
|
Post-Implantation Loss (%) |
0.00 |
4.17 |
1.45 |
0.00 |
|
|
|||||
Offspring Viability Indices |
|||||
Live Birth Indices and Sex Ratio at Birth |
|||||
Live pups/dam at birth (mean) |
10.82 |
10.55 |
10.92 |
11.00 |
|
Litter Size (Total No. of pups born/dam) at birth (mean) |
10.82 |
10.91 |
11.08 |
11.00 |
|
Mean Live Birth Index/dam (%) |
100.00 |
95.83 |
98.55 |
100.00 |
|
Male Live pups/dam at birth (mean) |
4.82 |
5.73 |
5.42 |
5.83 |
|
Female Live pups/dam at birth (mean) |
6.00 |
4.82 |
5.50 |
5.17 |
|
Sex Ratio (male/female) |
0.87 |
1.24 |
1.11 |
1.27 |
Parameters ↓ |
Group & Dose (mg/kg body weight/day) |
|||
G1 & 0 |
G2 & 15 |
G3 & 30 |
G4 & 60 |
|
Pup Survival Indices and Sex Ratio during lactation |
||||
Mean No. of Pups survived per dam ( LD1 to 4) |
10.82 |
10.55 |
10.90 |
11.00 |
Mean No. of Pups dead per dam ( LD1 to 4) |
0.00 |
0.00 |
0.00 |
0.00 |
Mean Pup Survival Index (%) per dam (LD1 to 4) |
100.00 |
100.00 |
100.00 |
100.00 |
Sex Ratio (male/female) per dam at LD 4 |
0.87 |
1.24 |
1.11 |
1.27 |
Mean No. of Pups Sacrificed for Blood Collection on LD4 |
0.64 |
1.00 |
0.92 |
1.25 |
Mean No. of Pups survived per dam (LD4 to 7) |
10.18 |
9.55 |
10.00 |
9.75 |
Mean No. of Pups dead per dam (LD4 to 7) |
0.00 |
0.00 |
0.00 |
0.00 |
Mean Pup Survival Index (%) per dam (LD4 to 7) |
100.00 |
100.00 |
100.00 |
100.00 |
Sex Ratio (male/female) per dam at LD 7 |
0.98 |
1.56 |
1.75 |
2.14 |
Mean No. of Pups survived per dam (LD7 to 13) |
10.18 |
9.55 |
10.00 |
9.75 |
Mean No. of Pups dead per dam (LD7 to 13) |
0.00 |
0.00 |
0.00 |
0.00 |
Mean Pup Survival Index (%) per dam ( LD7 to 13) |
100.00 |
100.00 |
100.00 |
100.00 |
Sex Ratio (male/female) per dam at LD 13 |
0.98 |
1.56 |
1.75 |
2.14 |
|
||||
Pre and Postnatal loss |
||||
Mean Pre-natal (implantations minus live births) (No.) |
0.00 |
0.36 |
0.17 |
0.00 |
Females with 0 (No.) |
11 |
9 |
10 |
12 |
Females with 1 (No.) |
0 |
2 |
2 |
0 |
Post-natal (live births minus alive at post natal day 13) |
||||
Females with 0 (No.) |
11 |
11 |
12 |
12 |
Females with ≥ 1 (No.) |
0 |
0 |
0 |
0 |
|
||||
Litter Observations |
||||
Male Pup weight at birth (mean) in gram |
6.92 |
6.72 |
6.67 |
6.83 |
Female Pup weight at birth (mean) in gram |
6.19 |
6.09 |
6.17 |
6.11 |
Male Pup weight on LD4 (mean) in gram |
9.99 |
10.15 |
10.19 |
10.39 |
Female Pup weight on LD4 (mean) in gram |
9.36 |
9.28 |
9.49 |
9.10 |
Male Pup weight on LD7 (mean) in gram |
14.49 |
14.53 |
14.38 |
14.77 |
Female Pup weight on LD7 (mean) in gram |
13.46 |
13.73 |
13.58 |
13.25 |
Male Pup weight on LD13 (mean) in gram |
24.53 |
23.87 |
24.69 |
24.46 |
Female Pup weight on LD13 (mean) in gram |
23.43 |
22.85 |
23.24 |
22.89 |
SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD
Group, Sex & Dose (mg/kg body weight/day) |
No. of Animals |
Clinical Signs of Toxicity/ Detailed Clinical Examination (No. of animals revealed) |
Mortality (No. of Mortality / No. of Animals dosed) |
G1, M & 0 |
12 |
N (12) |
0/12 |
G2, M & 15 |
12 |
N (12) |
0/12 |
G3, M & 30 |
12 |
N (12), 25 (12), 26 (4) |
0/12 |
G4, M & 60 |
12 |
N (12), 25 (12), 26 (12), 81 (12) |
0/12 |
M: Male; N: Normal; 25: Aggression; 26: Excessive grooming; 81: Hair thinning at lower jaw regions
Group, Sex & Dose (mg/kg body weight/day) |
No. of Animals |
Clinical Signs of Toxicity/ Detailed Clinical Examination (No. of animals revealed) |
Mortality (No. of Mortality / No. of Animals dosed) |
G1, F & 0 |
12 |
N |
0/12 |
G2, F & 15 |
12 |
N |
0/12 |
G3, F & 30 |
12 |
N (12), 25 (12) |
0/12 |
G4, F & 60 |
12 |
N (12), 25 (12) |
0/12 |
F: Female; N: Normal; 25: Aggression
SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD
Group, Sex & Dose (mg/kg body weight/day) |
Epididymes |
Testes |
Prostate+Seminal vesicles with coagulating glands (PSC) |
Thyroid along with parathyroid# |
|
G1, M & 0 |
Mean |
1.4651 |
3.3673 |
3.3721 |
0.0298 |
±SD |
0.0929 |
0.2051 |
0.6067 |
0.0039 |
|
n |
12 |
12 |
12 |
12 |
|
G2, M & 15 |
Mean |
1.4296 |
3.2347 |
3.0935 |
0.0291 |
±SD |
0.2130 |
0.3679 |
0.8135 |
0.0054 |
|
n |
12 |
12 |
12 |
12 |
|
G3, M & 30 |
Mean |
1.3627 |
3.3504 |
3.0786 |
0.0281 |
±SD |
0.1850 |
0.3840 |
0.2723 |
0.0044 |
|
n |
12 |
12 |
12 |
12 |
|
G4, M & 60 |
Mean |
1.4151 |
3.4102 |
3.2846 |
0.0299 |
±SD |
0.2042 |
0.7029 |
0.7674 |
0.0048 |
|
n |
12 |
12 |
12 |
12 |
Group, Sex & Dose (mg/kg body weight/day) |
|
Thyroid along with parathyroid# |
G1, F & 0 |
Mean |
0.0281 |
±SD |
0.0029 |
|
n |
11 |
|
G2, F & 15 |
Mean |
0.0290 |
±SD |
0.0026 |
|
n |
11 |
|
G3, F & 30 |
Mean |
0.0285 |
±SD |
0.0027 |
|
n |
12 |
|
G4, F & 60 |
Mean |
0.0283 |
±SD |
0.0026 |
|
n |
12 |
SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD
Group, Sex & Dose (mg/kg body weight/day) |
Fasting Body Weight (g) |
Epididymes |
Testes |
Prostate+Seminal vesicles with coagulating glands (PSC) |
Thyroid along with parathyroid |
|
G1, M & 0 |
Mean |
393.47 |
0.3749 |
0.8618 |
0.8608 |
0.0076 |
±SD |
37.99 |
0.0375 |
0.0857 |
0.1576 |
0.0014 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G2, M & 15 |
Mean |
399.09 |
0.3601 |
0.8168 |
0.7781 |
0.0073 |
±SD |
51.00 |
0.0448 |
0.0957 |
0.1835 |
0.0014 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G3, M & 30 |
Mean |
378.11 |
0.3619 |
0.8933 |
0.8213 |
0.0075 |
±SD |
42.57 |
0.0429 |
0.1156 |
0.0957 |
0.0013 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G4, M & 60 |
Mean |
372.82 |
0.3803 |
0.9172 |
0.8848 |
0.0080 |
±SD |
31.86 |
0.0512 |
0.1859 |
0.2208 |
0.0012 |
|
n |
12 |
12 |
12 |
12 |
12 |
Group, Sex & Dose (mg/kg body weight/day) |
|
Fasting Body Weight (g) |
Thyroid along with parathyroid |
G1, F & 0 |
Mean |
294.69 |
0.0095 |
±SD |
20.97 |
0.0004 |
|
n |
11 |
11 |
|
G2, F & 15 |
Mean |
301.92 |
0.0096 |
±SD |
13.91 |
0.0010 |
|
n |
11 |
11 |
|
G3, F & 30 |
Mean |
306.55 |
0.0093 |
±SD |
25.38 |
0.0005 |
|
n |
12 |
12 |
|
G4, F & 60 |
Mean |
294.37 |
0.0096 |
±SD |
21.51 |
0.0005 |
|
n |
12 |
12 |
SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY
Group & Dose |
No. of Females |
No. of Females with Regular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14 |
No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14 |
G1 & 0 |
12 |
12 |
0 |
G2 & 15 |
12 |
12 |
0 |
G3 & 30 |
12 |
12 |
0 |
G4 & 60 |
12 |
12 |
0 |
SUMMARY RECORD OF ANO-GENITAL DISTANC RATIO ON LACTATION DAY 4
Group & Dose (mg/kg body weight/day) |
Mean Male AGD Ratio |
Mean Female AGD Ratio |
||
G1 & 0 |
Mean |
2.63 |
1.17 |
|
±SD |
0.11 |
0.05 |
||
n |
11 |
11 |
||
G2 & 15 |
Mean |
2.56 |
1.21 |
|
±SD |
0.08 |
0.08 |
||
n |
11 |
11 |
||
G3 & 30 |
Mean |
2.56 |
1.13 |
|
±SD |
0.10 |
0.06 |
||
n |
12 |
12 |
||
G4 & 60 |
Mean |
2.58 |
1.19 |
|
±SD |
0.06 |
0.08 |
||
n |
12 |
12 |
SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - MALES
Group, Sex & Dose (mg/kg body weight/day) |
Serum T4 Levels (ng/mL) |
||
G1, M & 0 |
Mean |
101.421 |
|
±SD |
8.369 |
||
n |
12 |
||
G2, M & 15 |
Mean |
104.912 |
|
±SD |
14.992 |
||
n |
12 |
||
G3, M & 30 |
Mean |
92.769 |
|
±SD |
16.056 |
||
n |
12 |
||
G4, M & 60 |
Mean |
93.937 |
|
±SD |
10.089 |
||
n |
12 |
SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 13PUPS
Group & Dose (mg/kg body weight/day) |
Serum T4 Levels (ng/mL) |
||
G1 & 0 |
Mean |
85.910 |
|
±SD |
12.782 |
||
n |
11 |
||
G2 & 15 |
Mean |
75.686 |
|
±SD |
5.761 |
||
n |
11 |
||
G3 & 30 |
Mean |
72.360* |
|
±SD |
6.839 |
||
n |
12 |
||
G4 & 60 |
Mean |
78.787 |
|
±SD |
12.236 |
||
n |
12 |
Applicant's summary and conclusion
- Conclusions:
- NOAEL was considered to be 60 mg/kg bw when Sprague-Dawley male and female rats were treated with Denatonium Benzoate orally by gavage in water for Male 42 days and 63 days female rats.
- Executive summary:
In a Reproduction/Developmental Toxicity Screening Test, 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 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, developmental effects were observed in treated rats and pups such as 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 60 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.
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