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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and justification for the selection of the species: Rat (Rattus norvegicus); The regulatory guidelines for this test has preferred rat among the species of rodents.
Strain and justification for the selection of the strain: Wistar; This strain has been used for non-clinical safety studies, recommended by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
Age at start of study: 11 to 12 weeks (Healthy virgin animals)

Accommodation:
Animals were housed maximum three per sex / cage in solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. The cages were suspended on stainless steel racks. Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period. Animals were housed in groups or singly, as below.
Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex.
During mating (co-habitation) : one male : one female, per cage.
Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dam were housed individually.

Environmental Conditions: Air conditioned rooms with 10 to 15 air changes per hour, temperature between 19 to 25 °C, relative humidity 30 to 70%, and illumination cycle set to 12 hours light and 12 hours dark.

Diet: 'Altromin' brand extruded pelleted rat feed was provided ad libitum. The diet has been tested and certified to be free from undesired levels of environmental contaminants.

Water: Potable water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles.

Acclimatisation: The animals were acclimatised for a period of at least five days.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route is an accidental route of exposure in humans.
Vehicle:
water
Details on oral exposure:
2- aminoethanol hydrobromide was completely soluble in analytical grade water hence analytical grade water was used as a vehicle for dosing formulation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle for maximum possible highest and minimum possible lowest concentrations were determined at Analytical Chemistry Section of test facility, in a separate study. Homogeneity analysis of formulation was not performed as test item was completely soluble in analytical grade water.
Test item formulations (for all concentrations) were subject for verification of concentration twice (i.e. in the first week after initiation of treatment and in the last week at termination of treatment) during the study.

Formulation analysis was performed at Analytical Chemistry Section of test facility.
Duration of treatment / exposure:
Males were dosed for a minimum of four weeks, up to and including the day before scheduled sacrifice (this included a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post mating).
Females were dosed throughout the study (50 to 60 days). This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 15 females/dose (total 40 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
The test item 2- aminoethanol hydrobromide was administered daily by oral gavage at three graduated doses and a dose volume of 5 mI/kg body weight to 10 male and 15 female rats per dose group. A total of 25 animals (10 males / 15 females) received solely the vehicle i.e. Analytical grade water as control item via the same route and at same dose volume. The individual dose volumes were calculated from the latest actual body weight data. The first day of dosing was considered as day 1.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS AND MORTALITY

-General Clinical Signs and Mortality:
All signs of illness, together with any behavioural changes or reaction to treatment were observed (cage side observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets. Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.

-Detailed Clinical Examinations:
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subject to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period. These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and 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, difficult or prolonged parturition or bizarre behavior were also recorded.

FUNCTIONAL OBSERVATION BATTERY
Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. These included the functional observational battery. In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry. In females these functional tests were made during the last week of lactation (e.g., LD 6-13), shortly before scheduled sacrifice.

BODY WEIGHT
Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum. These observations were reported individually for each adult animal.

FOOD CONSUMPTION
During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation days 0, 7, 14 and 20 and during lactation on days 4 and 13. Food consumption during mating period was not measured. Food consumption was computed as the amount of food consumed in grams per animal per day.

DURATION OF GESTATION AND LITTER EXAMINATION
The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, still births, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 and day 13 post-partum. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

-Anogenital Distance:
The AGD (Anogenital distance) of each pup was measured daily on the postnatal day (PND) 0 to PND 4. Pup body weight was recorded daily on PND 0 to PND 4 and at termination on day 13. The AGD was measured by using Dial caliper (Mitutoyo dial caliper, Japan). The arms of the Dial caliper were aligned as follows: for males, the anogenital distance was measured from the cranial (or anterior) edge of the anus to the base (or posterior edge) of the anogenital aperture; and for females, the anogenital distance was measured from the cranial edge of the anus to the base of the urinary aperture (not the base of the vulva). The anogenital distance was recorded in millimeters.

-Nipple Retention:
The numbers of nipples / areolae in male pups were counted on PND 12.

-Dams with Signs of Abortion or Premature Delivery:
Dams were observed for signs suggestive of abortion, or of premature delivery.

-Dams with No Evidence of Copulation / Mating and those Found Non Pregnant:
Females showing no-evidence of copulation / mating were sacrificed 25 days after last day of the mating period. Dams found sperm positive and failed to deliver by gestation day 24 were weighed and sacrificed by
CO2 asphyxiation on their gestation day 25. The dams were subject to a necropsy examination to observe any gross pathological changes, and the findings were recorded.
Sacrifice and pathology:
HAEMATOLOGY AND CLINICAL CHEMISTRY
Animals were fasted overnight prior to blood sampling but had access to water ad libitum. Blood sampling was performed, under light CO2 anaesthesia, through the orbital sinus of all males and females sacrificed at termination (day 29 for males and day 14 of lactation for females). Samples were collected separately in tubes containing EDTA (dipotassium salt), for haematology, and Heparin, for clinical chemistry, as anticoagulants. For hormone analysis (serum total T4), blood samples were collected separately in plain tubes to yield serum. Blood samples from the pups were collected by cardiac puncture. Pups were anesthetized by barbiturate anaesthetic (Sodium Thiopentone) agent prior to blood collection.
Baseline Sampling: The base-line values for haematological and clinical chemistry parameters including the hormone estimation (Total T4) were obtained by randomly selected five males and five females. Sampling was performed before start of treatment.

-Haematology:
After completion of premating period (day 14 from start of treatment), haematological examination was made in five males and five females randomly selected from each group. The following haematological parameters were estimated on blood samples: haemoglobin, haematocrit, total erythrocyte count, reticulocytes, total leukocyte count, total platelet count, prothrombin time as a measure of blood clotting, differential leukocyte count, general blood picture.

-Clinical Chemistry:
At termination, clinical chemistry examinations were made in five males and five females randomly selected from each group. The clinical chemistry parameters that were measured were: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bile acids, total thyroid hormone, urea nitrogen. Plasma samples were analysed individually for determination of clinical chemistry parameters.

-Hormone Analysis:
Blood (Serum) samples were taken based on the following schedule:
1) two of the surplus pups, pooled, and used for determination of serum total T4 levels on day 4 after birth -
2) at least two pups per litter at termination on day Lactation day 13 - serum total T4
3) from all adult males at termination (day 29) - serum total T4
4) from all dams at termination on lactation day 14 - serum total T4 (dams were fasted over night on lactation day 13)
Blood samples of pups (on day 4 after birth or at lactation day 13) and adults (at day 29 for the males and at lactation day 13 for the females) were assessed for serum levels of thyroid hormone (T4). The hormone analysis was employed using commercially available ELISA kits.

GROSS NECROPSY
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
All the tissues listed in Appendix 16, from all adult males and females were preserved in 10% neutral buffered formalin. Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative. Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle. Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

ORGAN WEIGHTS
The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14. From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

HISTOPATHOLOGY
Microscopic examination was carried out on tissues as listed below:
On all listed organs and tissues of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination. All tissues were fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 μm thickness and stained with haematoxylin and eosin, for microscopic examination. These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group. In absence of any microscopic alteration in the thyroid glands of adult male and female rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.
Statistics:
Statistical analysis was performed using IBM SPSS Statistical Software (version 23).
For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters. The results of these statistical analysis were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

One-way ANOVA followed by Dunnett's test was used for the following parameters:
- Premating Body weight and body weight gain
- Premating Food Consumption
- Gestational and lactational body weights and body weight gain
- Gestational and lactational food consumption
- Organ weight (Absolute and relative)
- Live and dead foetuses
- Foetal Body weight
- Foetal Anogenital distance (AGD)
- Nipple retentions

Kruskal-Wallis followed by Mann-Whitney test was used for the following parameters:
- Number of male and female pups
- Sex ratio
- Number of live and dead foetuses

Chi-Square / fisher test was used for the following parameters:
- Number of pregnant / non pregnant females
- Number of live / dead females

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
During Premating Period:
After completion of premating treatment period (day 14 from start of treatment), haematological examinations were made on five males and five females randomly selected from each group. There were no treatment related changes in the above list of haematological parameters as the mean values were comparable to the same from concurrent control animals. Few statistical significant changes were observed in haematocrit, MCH, MCV, reticulocyte in male rats whereas eosinophils in female rats. These changes were not dose dependant and hence considered as incidental and not to be treatment related. General blood picture evaluation performed on stained blood smear slides revealed no morphological abnormalities and immature cells in red blood cells, white blood cells and platelets in any of the treated rats including the control animals.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
During Termination Period:
At termination of treatment, above listed clinical chemistry parameters were determined in all males (on day 29) and all females (on lactation day 14). The test item, at and up to the dose level of 300 mg/kg of body weight/d, did not induce any change in clinical chemistry parameters. Few statistical significant changes were observed in total bile acid (TBA) and albumin in male rats whereas alanine aminotransferase in female rats. These changes were considered as incidental as there were no dose dependant changes. The TBA values in male rats were within the historical control data of INTOX and hence not considered to be treatment related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernous muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed at termination of treatment (on day 29). While in all female rats, ovaries and uterus with cervix were weighed at termination on lactation day 14. In addition, from five randomly selected adult male and female rats from each group, the liver, kidneys, adrenals, thymus, spleen, brain, heart and thyroid glands were trimmed off any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.
The values of absolute and relative organ weights were found to be comparable between the treated and control group of male rats except for following few changes. A few instances of statistically significant (p<0.05) changes when compared to that of control organ weights were noticed and were considered incidental as there was no dose dependency and values were well within historical control ranges for these parameters.
The values of absolute and relative organ weights of treated group female rats were found to be comparable to those of the control group female rats which were terminated on lactation day 14. A few instances of statistically significant (p<0.05) changes when compared to that of control organ weights were noticed and were considered incidental as there was no dose dependency and values were well within historical control ranges for these parameters.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Systemic effect:
Histopathological examinations of the tissues from five adult male and female rats randomly selected from control group and high dose treated groups did not reveal any significant treatment related histopathological alterations. The various histopathological changes noticed in few organs have been presented in individual animal pathology findings. The summary of these changes has been tabulated in the summary table.
Some incidental and spontaneous lesions observed in animals from the control and high dose group (300 mg/kg bw/d) included perivascular lymphocytic aggregation, peribronchial lymphoid tissue hyperplasia, foam cells in lungs, cortical vacuolation in adrenal, dilated glands in stomach, submucosal lymphoid hyperplasia in colon and lymphocytic infiltration in prostrate. All these changes were of minimal severity and are commonly observed in rats of this age and were not considered to be treatment related. Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of 2- aminoethanol hydrobromide in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity and for reproductive and developmental toxicity was found to be 300 mg/kg body weight/d.