Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 13.5.2019 to 16.8.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Purity: 93.7 %
- Storage conditions: Ambient (21°C to 29°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Housing: Maximumof twoanimals of same sex and group 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 feedand drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitumto the animals throughout the acclimatization and experimental period. The contaminant analysis test report of the feed is presented in Annexure 2.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-we ll water passed through Reverse Osmosis Unit was provided in plasticwater bottles with stainless steel sipper tubes
- Acclimation period: The animals were acclimatized for a period of five days to experimental room conditions prior to dosingand observed for clinical signs once daily. Veterinary examination and body weight recording of all the animalswasperformed on the day of receipt and on the day of grouping. Ophthalmoscopic examination of all the animals wasperformed on the day of grouping. All the data generated during acclimatization period isnot presented instudyreport but maintained with the study raw data file

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 23.2
- Humidity (%): 45 - 65
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/ 12 hours dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered through oral route using stainless steel (gavage) cannula because the peroral intake is the probable route in human.
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item formulations wereprepared daily before dose administration. Required quantity of test itemwasweighed into a clean glass beaker and there by adding little volume of vehicle in to beaker, mixed well with clean glass rod and transferred to a measuring cylinder. This procedure wasrepeated until to ensure entire quantity of test item formulation wastransferred to measuring cylinder. Finally, the volume wasadjusted to the required markin measuring cylinder with the vehicle to get desired concentrations of 5, 15 and 30 mg/mL of test item for low, mid and high dose groups respectively (Table 1 and Table 2).
- The quantity of the test item taken and the volume prepared was varieddepending on the requirement. The exact formulation preparation details wererecorded in the raw data.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily before dose administration
- Storage temperature of food: room temperature (up to 6 hours)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations was performed during week 1 and week 4 of the treatment. The samples werecollected in duplicates (5 mL × 2sets) from top, middle and bottom layer from low, mid and high dose concentrations andin duplicates from middle layer from vehicle control.
One set of aliquot of each formulation was analyzed. The second set of samples was discarded, as the analysis results of first set of samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is <15%
Duration of treatment / exposure:
The animals were subjected to detailed clinical examinations before initiation of the treatment and at weekly thereafter during the study. These observations were made outside the home cage and at the same time on the day of examination. Signs noted included, but were not limited to, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards).
Frequency of treatment:
The animals were dosed with vehicle and test item formulations once in a day for a period of 28 consecutive days.The test item formulations were stirred continuously by using a magnetic stirrer during sampling and during dose administration.Following the 28 days treatment period, the animals in the recovery groups (G1Rand G4R) was not given any treatment and maintained for 14 days post treatment period and observed for reversibilityor persistence of toxic effects.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 10 (5 Males + 5 females)
Recovety groups: 10 (5 Males + 10 Females)
Total of 35 Males and 35 Females were recieved.
Control animals:
yes
Details on study design:
The selected animals were assigned to vehicle control and different treatment groups as shown in the Table 1. Dose Selection and Justification for Selection The doses of 50, 150and 300 mg/kg body weight wasselected as low (G2),mid (G3) and high dose (G4/G4R) levels for test item 3 (isodecyloxy)propylammonium acetate based on the results obtained from the dose range finding study (Bioneeds Study Number: BIO-TX 3630)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during experiment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicicty and twice deaily for mortality and morbidity.

BODY WEIGHT: Individual animal body weights wererecorded on Day 1,before test item administration andat weekly thereafterduring the experiment. Fasting body weight of all surviving animals wererecorded at their scheduled terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during 4 weeks of treatment for G1 and G4 group animals; during 6 weeks for revocery group animals (G1R and R4R)
- Dose groups that were examined: high dose group during 4 weeks

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from all surviving animals from main groups on say 29 (from all surviving animals from recovery groups on day 43)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes (overnight before blood collection)
- How many animals: Not specified
- Parameters checked in table [No.3] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from all surviving animals from main groups on say 29 (from all surviving animals from recovery groups on day 43)
- Animals fasted: Yes
- How many animals: Not specified
- Parameters checked in table [No.4] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: from all surviving animals from main groups on say 29 (from all surviving animals from recovery groups on day 43)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight but water was provided ad libitum during the period
- Parameters checked in table [No.5] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 for all the main group animals (G1, G2, G3and G4), during last week (week 6) for recovery group animals (G1R and G4R).
- Dose groups that were examined: Not specified
- Battery of functions tested: home cage observation (convulsions, tremors, palpebral closure), handling observation (resistance, lacrimation, red and crusty deposits around eye, nose and mouth), open field observation (mobiity, gait, arousal, rearing, urination, defecation, stereotypes), sensory, neruromuscular, physiological (rectal temperature), grip strength, locomotor

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the end of treatment (main groups) and recovery period (recovery groups), all surviving animalswerefasted overnight (water allowed) and on the day of necropsy (day 29 for main groups, day 43 for recovery groups), all surviving animals wereweighed and then subjected to necropsy. The animals wereeuthanized by using excess of carbon dioxide asphyxiation followed by exsanguination. The gross pathological examination wascarried out for each rat. Necropsy includedan examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. All the surviving animals weresacrificed in a serial block sequence and complete gross pathological examination wasperformed.Rats found dead and moribund were subjected to detailed necropsy.

HISTOPATHOLOGY: Yes
Initially, histopathological examination was carried out on the tissuesfrom the vehicle control (G1), high dose (G4) group animals and from dead or moribund sacrificed animals. The organs and tissue samples as defined in the study plan wereprocessed, embedded in paraffin, cut at a thickness4 to 5micrometers and stained with hematoxylin and eosin. The bone marrow smearswere fixed in methanol and stained with Giemsa Stain.Evaluation of spleen, thymus, stomach and skeletal muscle were extended to lower and recovery group animals.
Other examinations:
TISSUE COLLECTION:
The following organs and tissues were collected, trimmed off any adherent tissue, as appropriate. All the tissues mentioned belowfrom all the animalswere preserved in 10% v/v Neutral Buffered Formalin and Modified Davidson’s fixative for eyes and testes.
Eyes, Ovaries,Brain, Lungs, Spinal cord, Prostate gland, Stomach, Esophagus, Duodenum, Urinary bladder, Jejunum, Mesenteric Lymph nodes, Ileum with peyer’s patches,Mandibular Lymph nodes,Caecum,Sciatic nerve,Colon, Bone marrow smear, Rectum, Epididymis, Liver, Aorta, Kidneys, Skin (with Mammary glands in females), Adrenals, Sternum, Spleen, Skeletal muscle, Heart, Thyroid, Thymus, Parathyroid, Seminal vesicles with coagulating gland, Vagina, Pancreas, Trachea, Uterus and Cervix, Salivary glands, Testes, Gross lesion

BONE MARROW SMEAR EXAMINATION
Bone marrow smear (from one femur) was prepared for all the animals at the time of necropsy. Histological evaluation of hematopoietic system and complete blood countwere adequate the characterization of toxicity of hematopoietic system, hencecytological evaluation of the bone marrow not conducted

ORGAN WEIGHTS
The following organs from all animals at the scheduled sacrifices weretrimmed off any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying. List of the organs weighed: Kidneys*, Thymus Liver Spleen, Adrenals*, Brain, Testes*/Ovaries*, Uterus, Epididymis, Heart, Prostate + seminal vesicles with coagulating glands*: Paired organs were weighed together. Relative organ weights werecalculated against fasting body weight.
Statistics:
The computer printout of the data (in the form of appendix) was verified with the original raw data.After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight with respect to Day 1,feed consumption, FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, foot splay, grip strength andmotor activity), organ weights and ratios, hematological andclinical chemistry estimations andurine analysis parameters (pH, urobilinogen and specific gravity)were subjected to statistical analysis. Data of recovery groups during recovery period was subjected to ‘t’ test.All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts throughout the report as stated below: *: Statistically significant (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The detailed clinical examination of animals did not reveal any treatment related changes in low dose group animals (50 mg/kg) in either sex. All the animals from mid dose group (150 mg/kg) revealed treatment related clinical sign like slight salivation in either sex on Day 8, 15, 22 and 28 during detailed clinical examination. The detailed clinical examination of animals revealed treatment related changes like moderate salivation and brown staining around nose in all the surviving high dose group animals on Day 8; additionally wet perineum and dehydration was observed in
few of the high dose group animals on Day 8 and persisted up to the end of treatment period. The animals recovered and were found normal during recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Total mortalities observed at high dose (300 mg/kg/day) was 14 rats (11 rats were found dead and 3 rats were moribund sacrificed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment related statistical significant decrease in mean body weight was observed on Day 8 in G3, G4 males and on Day 15 in G3 males; treatment related statistical significant decrease in percent change in body weight with respect to Day 1 was noted during Day 1-8 in G3, G4 males and Day 1-15 in G3 males.

Treatment related statistical significant decrease in mean body weight was observed on Day 8 in G3, G4 females and from Day 15 to 28 in G2, G3 and G4 females and treatment related statistical significant decrease in percent change in body weight with respect to Day 1 was noted during Day 1-8 to Day 1- 22 in G2, G3 and G4 females and during Day 1-28 in G3 and G4 females.

Treatment related statistical significant decrease in mean body weight was observed from Day 8 to 28 in G4R males during treatment period and continued up to the end of recovery period on Day 35 and 42 in G4R males and treatment related decrease in percent change in body weight with respect to Day 1 was noted during Day 1-8 to Day 1-42 in G4R males.

Treatment related statistical significant decrease in mean body weight and percent change in body weight with respect to Day 1 was observed on Day 8 and during Day 1-8 in G4R females.

All these changes observed in lower body weight in G3 and G4 groups were associated with lower feed consumption and considered as treatment related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No treatment related changes in feed consumption were noted in low dose group animals in either sex. However, treatment related statistical significant decrease in feed consumption was observed in G3 and G4 group males during week 1, G3 and G4 females during week 1 and 2 and only in G4 females during week 3 and week 4 of the treatment period. In recovery group animals, treatment related decrease in feed consumption was observed in G4R males during week 1 to week 6 and in G4R females during week 1. The variations observed are considered treatment related as the same was associated with lower body weights.
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 ophthalmological abnormalities were noted during Week 4 in main group and Week 6 in recovery group animals.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related changes in hematology parameters were noted.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant variations noted in clinical chemistry parameters at all the tested dose groups. However, the following statistically significant variations were noted.
- In main group males, increase in total protein (G3), ALP (G3), cholinesterase (G2, G3), globulin (G3) was noted. In main group females, increase in triglycerides (G4), ALP (G3), potassium (G3), decrease in creatinine (G3) was noted. All the observed variations noted are considered as incidental in the absence of dose responsiveness and the variations could be due to random biological variation.
- In recovery males, increase in triglycerides (G4R), phosphorous (G4R), decrease in total bilirubin (G4R) was noted. The changes observed are considered incidental as the same variations were not observed in other sex.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related changes in urinary parameters were noted. However, statistical significant increase in urobilinogen (G2M) noted was considered incidental in the absence of dose responsiveness.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No treatment related neurological/functional abnormalities was observed during neurological/functional examinationinlow dose group animals; slight salivation was noted in all mid dose and high group animals.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in fasting body weight was noted in all test item treated main group females and only in recovery males. The following statistically significant changes were noted in organ weights.
Absolute organ weights: In main group females, decrease in thymus (G3), spleen (G4), uterus (G4), heart (G4), decrease in adrenals (G3, G4) was noted.
Relative organ weights: In main group females, increase in kidneys (G4), brain (G4), liver (G4) was noted. In recovery males, increase in adrenals (G4R), spleen (G4R), epididymis (G4R), heart (G4R), kidneys (G4R), liver (G4R), prostate+ seminal vesicles and coagulating glands (G4R) was noted.
Animals from G4 and G4R group showed atrophy of thymus and spleen, erosion/ulcer of non-glandular stomach and skeletal muscle necrosis which are considered as treatment related. All other differences were not associated with microscopic changes in high dose main group animals at the end of treatment period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment related gross pathological lesions were noted in stomach, thymus and spleen during necropsy at high dose group animals.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals from G4 and G4R group showed atrophy of thymus and spleen, erosion/ulcer of non-glandular stomach and skeletal muscle necrosis. These lesions considered as treatment related.
- One male animal from vehicle control group showed mononuclear cells in prostate gland. This lesion considered to be spontaneous.
- A presence of ultimobranchial cyst and ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
3-(isodecyloxy)propylammnoium acetate
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
behaviour (functional findings)
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
other: body weight
Organ:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
spleen
stomach
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
musculoskeletal system
Organ:
other: skeletal muscle
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item 3-(isodecyloxy)propylammonium acetate is 50 mg/kg body weight/day when administered for a period of 28 consecutive days by oral (gavage) route to Sprague Dawley rats under the test conditions employed.