Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Vehicle:
water
Details on exposure:
The oral route is an accidental route of exposure in humans.
2- aminoethanol hydrobromide was completely soluble in analytical grade water hence analytical grade water was used as a vehicle for dosing formulation.
Details on mating procedure:
One male to one female (1 : 1) mating was used in this study. The female was placed with the same male until pregnancy occurs or one week has elapsed. Each morning the females were examined for the presence of sperm. Day 0 of pregnancy was defined as the day a sperm is found. In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered.
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
Details on study schedule:
Dosing of both sexes started two weeks prior to mating, after they had been acclimatized for at least five days and also females had been screened for normal oestrous cycles (in two-week pre-treatment period). The study was scheduled in such a way that oestrous cycle evaluation began soon after the animals had attained full sexual maturity. The day of birth (viz. when parturition was complete) was defined as day 0 post-partum. Females showing no-evidence of copulation were sacrificed 25 days after the last day of the mating period. Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum total dosing period of 28 days had been completed. They were then sacrificed. Daily dosing of the parental females was continued throughout pregnancy and up to, and including, day 13 post-partum (the day before sacrifice).
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

Parental animals: Observations and examinations:
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.

HAEMATOLOGY AND CLINICAL CHEMISTRY
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). 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.

DURATION OF GESTATION
The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed for signs suggestive of abortion, or of premature delivery.
Oestrous cyclicity (parental animals):
Females were screened for normal oestrous cycles (in a two-week pre-treatment period). Oestrous cycle was monitored before the start of treatment to select the females with regular cyclicity. Females that failed to exhibit typical 4 or 5-day cycles were not included in the study. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
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.
Litter observations:
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.

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
Postmortem examinations (parental animals):
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.

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.
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.

Microscopic examination was carried out on tissues as listed below:
On all listed organs and tissues (Ref. Appendix 16) 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.
Postmortem examinations (offspring):
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.
At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.
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: P0 (first parental generation)

General toxicity (P0)

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
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
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Exposure of female rats to 2- aminoethanol hydrobromide at and up to the dose levels of 300 mg/kg bw/d did not have any adverse effect on the length and frequency of their oestrous cycle. Total 60 females were screened for normal oestrous cycle for 2 weeks prior to the treatment period. All females exhibited oestrous cycle of 4-5 days. Stages of oestrous cycle were monitored daily in the females randomly assigned to four groups (15 females/group), from the beginning of the treatment period until evidence of mating.
Mean values of treatment groups did not differ from those of controls with statistical significance. The group mean values of the number of oestrous cycles during the two weeks before the mating period was 2.3, 2.1 and 2.2 among the females exposed to test item at the dose levels of 30 mg/kg bw/d, 100 mg/kg bw/d and 300 mg/kg bw/d respectively. These values were found to be comparable to that (2.3) of the control group.
The length of oestrus cycle was measured in days starting from detection of oestrous stage of the cycle till the oestrous stage of the subsequent cycle. The values of group mean length, in days, of each of the above estrous cycle occurring for two weeks before the mating period were 4.3, 4.2 and 4.6 among the females exposed to test item at the dose levels of 30 mg/kg bw/d, 100 mg/kg bw/d and 300 mg/kg bw/d respectively and were found to be comparable to that (4.6 days) of the control group. Thus the test item did not have any adverse effect on the oestrous cycle during the treatment period.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The pregnancy rate was 93.3% in control and 66.6%, 93.3%, 66.6% in 30, 100 and 300 mg/kg dose groups respectively. There was no treatment related adverse effect on pregnancy rate. A comparison of the incidence of various pregnancy related parameters of the control group rats and those treated with test item did not indicate any remarkable differences indicative of any adverse effects due to the treatment. These parameters included incidence of maternal deaths during pregnancy, pregnancy rate (%), mean gestational length and post implantation loss.

-Gestational Length:
The mean gestational length (duration of pregnancy in days) computed for groups G1 to G4 for all dams was 21.3 ± 1.1 days, 21.6 ± 0.7 days, 21.8 ± 0.6 days and 21.9 ± 0.6 days respectively. The values do not differ from each other significantly (p>0.05).

-Live births:
The mean live birth index was 90.9% in control whereas it was 100%, 99.1% and 90.9% in G2, G3 and G4 respectively. This index measures the female’s ability to maintain pregnancy, based on having delivered at least one live pup. The values do not differ from each other significantly (p>0.05). This indicated that the test item does not influence the mean live birth index.

-Implantations:
The number of uteral implantations in left and right arms of the uterus were counted on day 14 of lactation during terminal sacrifice of the females. There were no significant differences (p>0.05) in the group mean values of number of implantations, live, dead and total, observed in control group and groups treated with 2- aminoethanol hydrobromide. This indicated that the test item did not influence the implantation process.
Total Implants: The total number of implants in control group dams was 153 in control whereas it was 127, 150 and 131 in treated group G2, G3 and G4 respectively. The group mean values of the total number of implants per female were 10.9 (Control), 11.5 (G2), 10.7 (G3) and 11.9 (G4).

-Post-implantation Loss:
The group mean values of percent post implantation loss was 10.7 (Control), 0.6 (G2), 2.1 (G3) and 2.0 (G4).

Effect levels (P0)

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
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment of dams with 2- aminoethanol hydrobromide did not have any adverse effect on the offsprings body weight gain during the lactation period. The values of mean litter body weights and mean litter body weight gain for the offsprings of female rats treated with 2- aminoethanol hydrobromide did not differ significantly (p>0.05) from those of the concurrent control group females during the post-natal lactation period except in G2 and G4. However, at two time points on day 2 and day 13 post-natal lactation period, there was a significant lowering (p<0.05) of body weight gain. The same group pups had gained weight at all other time points up to 13 days which was comparable statistically to that of control animals at all time points and hence of no toxicological significance.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
The AGD (Anogenital distance) of each pup was measured daily from the postnatal day (PND) 0 to PND 4. The anogenital distance was recorded in millimeters. Treatment of dams with 2- aminoethanol hydrobromide did not have any adverse effect on the anogenital distance for male and female pups as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the postnatal day (PND) 0 to PND 4. Only significant lowering (p<0.05) of anogenital distance was observed in male and female pups from G2 which was considered as incidental and not to be treatment related.

The number of nipples / areolae in male pups was counted on PND 12 among all male pups from control and treated groups. One male each from control, G2 and G4 were observed with retention of nipple which was considered as incidental. Thus, the test item did not influence the nipple retention in male pups at post natal day 12.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive 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.