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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
28- days repeated oral toxicity study followed by 14 days recovery period
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
The study palnned to determined toxicological profile of test chemical to determine target organ of toxicity, its reversibility and No Observed Adverse Effect Level (NOAEL) in the rat after 28 repeated days by oral administration of test chemical.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrylammonium bromide
EC Number:
200-769-4
EC Name:
Tetrylammonium bromide
Cas Number:
71-91-0
Molecular formula:
C8H20N.Br
IUPAC Name:
N,N,N-triethylethanaminium bromide
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Tetrylammonium bromide (CAS No. 71-91-0)
- Molecular formula (if other than submission substance): C8H20N.Br
- Molecular weight (if other than submission substance): 210.157g/mol
- Substance type: Organic
- Physical state: Solid
- Impurities (identity and concentrations): No data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Justification for selection of the rat as the test system:
1) In order to meet the regulatory requirement for testing in a rodent species;
2) Widely used in as a species of choice for pre-clinical toxicological studies.
3) This strain is widely used throughout the industry in the non-clinical laboratory studies.
4)This study is intended to provide information on the health hazards likely to arise from exposure to the test item.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Male : Mean : 168.35 g (= 100 %)
Minimum : 160.5 g (- 4.66 %)
Maximum : 176.9 g (+ 5.08 %)

Female: Mean : 168.35 g (= 100 %)
Minimum : 160.5 g (- 4.66 %)
Maximum : 176.9 g (+ 5.08 %)

- Fasting period before study:
- Housing: The rats were housed in polycarbonate cages with paddy as bedding. After allocation to respective dose groups rats were housed 2/sex/cage.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was from a local source and passed through the reverse osmosis membrane before use provided ad libitum from individual bottles attached to the cages.
- Acclimation period: minimum of 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range, 20.4 °C to 21.7 °C)
- Humidity (%):30% to 70% (actual range, 55.8% to 58.9%).
- Air changes (per hr): ten air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light and dark cycle of 12 hours

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Justification for Route of Administration:
1) The dosage can be accurately administered;
2) It is the proposed route for clinical use

P
Vehicle:
water
Details on oral exposure:
REPARATION OF DOSING SOLUTIONS:
The test item was dissolved in distilled water for preparation of solution(s). The solution(s) of test chemical were made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test item was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days daily
Frequency of treatment:
Daily by oral gavage
Doses / concentrations
Remarks:
Group I- Vehicle- 0 mg/kg
Group II-Vehicle reversal- 0 mg/kg
Group III- 250 mg/kg
Group IV- 500 mg/kg
Group V- 1000 mg/kg
Group VI-Test chemical reversal- 1000mg mg/kg
No. of animals per sex per dose:
A total of 72 animals (36 males + 36 females)

Group I- Vehicle- 0 mg/kg (6 male and 6 female)

Group II-Vehicle reversal- 0 mg/kg (6 male and 6 female)

Group III- 250 mg/kg (6 male and 6 female)

Group IV- 500 mg/kg (6 male and 6 female)

Group V- 1000 mg/kg (6 male and 6 female)

Group VI-Test chemical reversal- 1000mg mg/kg (6 male and 6 female)
Control animals:
yes, concurrent vehicle
Details on study design:
Details on study design
- Dose selection rationale:
Salient features of the Dose Range Finding study were as follows :

The study was conducted at the dose levels of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.

Since all animals trested for 7 days servived throughout the dosing period, normal body weight gain at the end of teh study , daily clinical observations did not reveal any signs of toxicity, gross pathological examination did not reveal any abnormality attributable to the treatment. Hence dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment every day for a period of 28 days.
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: Rats were examined once daily for clinical signs.
Clinical observations were done for individual animal once before the start of dose administration and at least once a week thereafter until scheduled sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, food consumption recorded on commencement of treatment and weekly thereafter until scheduled sacrifice and the feed consumption per animal was calculated for each group.


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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice.

Eye examination was carried out by using a HEINE mini 2000 ophthalmoscope. The evaluation was carried after the induction of mydriasis with 1% solution of tropicamide sulfate.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood were withdrawn from the orbital sinus at the end of the study.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All treated and control male and female animals
- Parameters examined: Following haematological parameters were studied using Beckman Coulter haematology analyzer and coagulation analyzer (KC 1 Delta).

Hb : Hemoglobin (g/dL)
RBC : Red Blood Corpuscles (x 106 /µL)
HCT : Hematocrit (%)
MCV : Mean Corpuscular Volume (fL)
MCH : Mean Corpuscular Hemoglobin (pg)
MCHC : Mean Corpuscular Hemoglobin Concentration (g/dL)
Platelets: (x 103 /µL)
WBC : White Blood Corpuscles (x 103 /µL)
Following investigations were done manually:
Rt. : Reticulocytes (%)
N : Neutrophils (%)
L : Lymphocytes (%)
E : Eosinophils (%)
M : Monocytes (%)
B : Basophil (%)
Pt. : Prothrombin time (sec.)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of dosing period on day 29.
- Animals fasted: No data
- How many animals: All male and female rats.
- Parameters examined:
Following biochemical parameters were studied using Dimension XpandPlus and Acculyte 5P.

Total Protein (g/dL)
Blood Urea Nitrogen (mg/dL)
Urea Nitrogen (mg/dL) Calculated
ALT: Alanine Aminotransferase (U/L)
AST: Aspartate Aminotransferase (U/L)
ALP: Alkaline Phosphatase (U/L)
GGT: Gamma Glutamyl Transferase (U/L)
Glucose (mg/dL)
Calcium (mmol/L)
Phosphorous (mg/dL)
Albumin (g/dL)
Total Bilirubin (mg/dL)
Creatinine (mmol/L)
Total Cholesterol (mg/dL)
Triglycerides (mg/dL)
Globulin (g/dL) Calculated
Sodium (mmol/L)
Potassium (mmol/L)
Chloride (mmol/L)
Bile acid (mmol/L).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine analyses was performed on all treated animals scheduled sacrificed during the last week of dosing period. The urine analysis on reversal group rats were performed at termination of recovery period on day 43.
- Metabolism cages used for collection of urine: Yes. Animals fasted: Yes / No / No data
- Parameters examined:
Physical Examination:
Volume
Appearance
Colour
Chemical Examination:
The following estimations were performed using Urine analyses strips as discussed below:
pH
Specific Gravity
Proteins
Glucose
Ketones
Bilirubin
Urobilinogen
Occult Blood
Nitrite


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: All dose group
- Battery of functions tested: sensory activity / grip strength / motor activity.

Sacrifice and pathology:
Rats were sacrificed by CO2 asphyxiation, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V) and rats from reversal groups were sacrificed on day 43 (Group II and VI). All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin.

GROSS PATHOLOGY: Yes All the rats surviving at the end of the treatment were sacrificed and gross lesions were noted.

HISTOPATHOLOGY: Yes, Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination.

Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Muscles - Skeletal muscle, Oesophagus, Ovaries, Pancreas, Pharyngeal Lymphnodes, Pituitary, Prostate, Rectum, Sciatic Nerve, Seminal Vesicles with coagulation gland, Skin with Mammary Gland, Spleen, Spinal Cord (Cervical, mid thoracic and lumbar), Sternum with bone marrow, Stomach, Testes, Thymus, Trachea, Thyroid, Urinary Bladder, Uterus, Vagina.
Other examinations:
Mortality: Twice daily observed in all treated and control animals.

Organ weight: Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together.

Clinical signs: Behavior, alterations, vocalization, respiration and palpebral closer were examined in all animals from control and treated rats
Statistics:
Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data does not meet the homogeneity of variance, Student’s t-test were performed to calculate significance.

Significance was calculated at 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed throughout the dosing period of 28 days and during the post-dosing recovery period of 14 days in control and 250, 500 and 1000mg/kg dose level treated rats.
Mortality:
no mortality observed
Description (incidence):
All animals from control and different dose groups at 250, 500 and 1000mg/kg survived throughout the dosing period of 28 days and the post-dosing recovery period of 14 days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All animals from control and 250, 500 and 1000mg/kg dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
During the post-dosing recovery period, male and female rats from 1000 mg/kg reversal group exhibited normal body weight gain with respect to control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
All male and female rats from control and 250, 500 and 1000mg/kg dose groups exhibited normal feed consumption at the end of the dosing period of 28 days and recovery period of 14 days.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were observed on ophthalmological examination in any control and treated rats during pre-exposure and at the end of the respective termination
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male and Female -
Haematological investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, some parameters revealed changes with respect to their controls however this changes were within limit and not considered to be dose dependent.

Neutrophils : Significantly increased in neutrophiles values were obtained for males from 500 mg/kg and 1000 mg/kg dose groups and for females from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05)

Lymphocytes: Decreased values were obtained for males from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical investigations investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, some parameters revealed changes with respect to their controls however this changes were within limit and not considered to be dose dependent.

Male and female:
Chloride : Elevated levels were observed in males and females from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),

Male :
Alanine Aminotransferase and Calcium : Decreased levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05) and
Sodium : Decreased levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05).

Female :
Blood Urea Nitrogen and Urea Nitrogen : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05).
Alanine Aminotransferase : Decreased levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05) and
Glucose: Decreased levels were observed in animals from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 25, 26, 27 and 43) in male and female animals from 250, 500 and 1000mg/kg dose groups as compared to control group animals
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory Reactivity Observations:
All animals from 250, 500 and 1000mg/kg showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response.

Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

Grip Strength:
No significant differences were seen in Grip strength values of control and 250, 500 and 1000mg/kg treated animals.

Motor Activity:
Motor activity values observed in male and female animals for control and different dose groups were not significant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male -
At the end of dosing on day 29, organ weight data of animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed decreased relative weights of brain and kidneys (p<0.05) with respect to control animals. In addition, decreased relative weights were observed for liver (250 mg/kg and 1000 mg/kg), testes (250 mg/kg) and Prostate + Seminal Vesicle with Coagulation gland as whole (1000 mg/kg) (p<0.05).
At the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group was observed to be comparable with respect to control animals.

Female -
At the end of dosing on day 29, organ weight data of animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver (p<0.05) in comparision to control animals. In addition, increased relative weights were observed for kidneys (500 mg/kg and 1000 mg/kg) and uterus (500 mg/kg) (p<0.05).
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group revealed increased relative weights of liver (p<0.05).

Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examination on male and female animals from control and 250, 500 and 1000mg/kg dose groups did not reveal any abnormality.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological changes were observed in male and female animals from control and 1000mg/kg dose groups.

Incidental, congenital and physiological histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, focal to multifocal periportal mononuclear cells infiltration in liver; minimal, focal mononuclear cells infiltration and/or tubular vacuolation and/or mineralization in the kidneys; minimal, diffuse dilatation of zona reticularis in the adrenals; minimal, diffuse luminal dilatation in the uterus; presence of Ectopic thymus in thyroid; minimal, multifocal glandular dilatation in stomach; minimal, multifocal vacuolation of acinar cells of pancreas; presence of Rathke’s pouch in pituitary in male and female animals from control and 1000mg/kg dose group.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Clinical signs:
Before commencement of treatment:
In home cage observation, rat from 250, 500 and 1000mg/kg dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field for motor activity, rat did not reveal any abnormality from different dose groups and control group.


During treatment:
In home cage observation, revealed normal behavior, alterations, vocalization, respiration and palpebral closer in all rats from control and 250, 500 and 1000mg/kg dose groups.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation for motor activity, rat did not reveal any abnormality from different dose groups and control group.

Detailed clinical observation did not reveal any abnormality in treated groups at 250, 500 and 1000mg/kg during the dosing period of 28 days and during the post-dosing recovery period.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
other: Clinical signs
Remarks on result:
other: No toxic effect were observed

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the observations of experimental study of repeated Dose 28-day toxicity by daily gavage followed by a 2 week recovery period, the NOAEL for the test chemical was considered to be 1000mg/kg in the male and female Sprague Dawley rats
Executive summary:

The study aimed to determined toxicological profile of test chemical to determine target organ of toxicity and its reversibility in Sprague Dawley rats. In the present study the rats administered orally to different dose levels at 250, 500 and 1000mg/kg for 28 days followed by 14 days of recovery period in reversal groups (Group II-Vehicle reversal- 0 mg/kg and Group VI-Test chemical reversal- 1000mg mg/kg). The control and treated animals observed formortaliy, Clinical signs, clinical observation, body weight, food consumption, Opthalmoscopic examination, haematology, neurobehavuioral examination, urinalysis, organ weight, gross examination and histopathological study. The results of the study revealed no mortality in treated animals at250, 500 and 1000mg/kg dose group throughout the dosing period of 28 days and the post-dosing recovery period of 14 days. No clinical signs of toxicity were observed throughout the dosing period of 28 days and during the post-dosing recovery period of 14 days in control and 250, 500 and 1000mg/kg dose level treated rats. All animals from control and 250, 500 and 1000mg/kg dose groups exhibited normal body weight gain throughout the dosing period of 28 days. However, In the recovery period, male and female rats from 1000 mg/kg reversal group exhibited normal body weight gain with respect to control animals. The food consumption in control and 250, 500 and 1000mg/kg dose groups obse4rved normal with respect to control animlas at the end of the dosing period of 28 days and recovery period of 14 days. Ophthalmological examination in any control and treated rats during pre-exposure and at the end of the respective termination, No ocular abnormalities were observed. The haematological investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, some parameters revealed changes (Neutrophiles and lymphocytes) with respect to their controls however these changes were within limit and not considered to be dose dependent.  Biochemical investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, some parameters revealed changes with respect to their controls however this changes were within limit and not considered to be dose dependent. The urinalysis relealed No statistically significant variation conducted at the end of the dosing period in week 4 and 6 (on day 25, 26, 27 and 43) in male and female animals at 250, 500 and 1000mg/kg dose groups as compared to control group animals. No significant differences were seen in Grip strength and motor activity values of control and 250, 500 and 1000mg/kg treated animals. All animals from250, 500 and 1000mg/kgshowed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

 Although significant changes in the values of organ weight were observed in male and female animals from 250, 500 and 1000mg/kg dose groups, since no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Gross pathological examination on male and female rats from control and 250, 500 and 1000mg/kg dose groups did not reveal any abnormalities. Thehistopathological examination of the organs revealed No treatment related changes in male and female animals from control and 1000mg/kg dose groups.

From the observations of experimental study of repeated Dose 28-day toxicity by daily gavage followed by a 2 week recovery period, the NOAEL for the test chemical was considered to be 1000mg/kgin the male and female Sprague Dawley rats

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