Denatonium benzoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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

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:

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

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

Sex:

male/female

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

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Distilled 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:

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: No Data Available

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

Duration of treatment / exposure:

Male 42 days
Female: 63 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

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:

Positive control:

No Data Available

Examinations

Parental animals: Observations and 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:

OTHER: Organ Weights were observed

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.

Sperm parameters (parental animals):

Spermatogenesis and interstitial testicular cell structure, spermatogenic cycle and presence of all germ layers (cells) were examined.

Litter observations:

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.

Postmortem examinations (parental animals):

Hormone analysis:T4 (Thyroxine), Gross Pathology and Histopathology were examined.

Postmortem examinations (offspring):

gross 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.

Reproductive 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.

Offspring viability indices:

Yes, live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.
No treatment related changes observed in the copulatory interval and number of conceiving days at any of the tested dose group animals when compared with vehicle control group animals.
No changes observed in number of corpora lutea, number of implantations and no changes were noted in pre and post-implantation loss, pre-natal loss, post-natal loss at any of the tested dose group animals when compared with vehicle control group animals. No resorptions were noted at all the dose group animals observed during necropsy.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effect on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells) were observed in all male rats as compared to control.

Reproductive performance:

no effects observed

Description (incidence and severity):

No related changes observed in the 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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: No effect observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in any of the pups of tested dose group animals during lactation period.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on the pup survival were observed as compared to control.

Body weight and 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.

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:

no effects observed

Description (incidence and severity):

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 dependant. Hence, the variation observed in the T4 levels is considered to be incidental.

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

No changes observed in ano-genital distance ratio of both male and female pups recorded on lactation day 4 at any of the tested dose group litters when compared with vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

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.

Histopathological findings:

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Remarks on result:

other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

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 (mg/kg body weight/day)	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 in reproductive parameters such as 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 changes observed in the 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.