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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.08.2018 - 01.11.2018
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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source: The Lab Animals Breeding Center, Branch of Institute of Bioorganic Chemistry RAS (www.spf-animals.ru)
Age: Approximately 10 weeks old on day of pre-mating oestrous cycle evaluation;
approximately 13 weeks old at the initiation of dose administration (day 1);
approximately 15 weeks old when paired on study day 14.
Body weight at first day of dosing: Males: 359 ± 22 g, N = 58, Females: 219 ± 14 g, N = 58
Number of males used: 58
Number of females used: 58

Animals will be acclimated to the facility for at least 14 days prior to the pre-dosing oestrus cycle evaluation in females.

Husbandry practice meets the standards defined by the Directive 2010/63/EU on the protection of animals used for scientific purposes.

Environment
Mean temperature ranged from 21 °C to 25 °C and mean relative humidity ranged from 32 % to 63 %. Temperature and humidity are constantly monitored in each room automatically by Eksis Visual Lab system (Practice-NC Ltd, Russia).

Cages
Following group forming and until mating, all F0 females and males were housed for two animals in solid bottom polycarbonate cages (Type-4) with bedding. Cages were equipped with steel lids, steel separators for the food and steel label holders. All cages were provided with environmental enrichment material Lignocel Nesting Ball (JRS Germany). For mating, males were housed alone and animals were paired for mating in the home cage of the male. After mating, dams were housed alone to deliver and their litters were housed in these cages until euthanasia on lactation day 13. Females with no evidence of mating or that failed to deliver were housed individually until 55 days of dosing. Males and females of satellite subgroup (not mated) were housed for 2-3 animals per cage.

Bedding
Commercial autoclaved woodchip bedding was used.

Diet
Animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaeten, GmbH) ad libitum.

Water
Tap filtered water will be provided ad libitum in standard water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Tap water filtrated with the MilliRO Millipore system
Details on oral exposure:
The vehicle and test item will be administered orally by gavage, via an appropriately sized stainless steel ball-tipped dosing cannula connected with syringe once daily. The dosage volume for all groups will be 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The validated method was used for detection of the test item concentration in vehicle formulations ranging from 10 to 90 mg/mL. The validation of the analytical method was performed before initiation of dosing where linearity, accuracy, precision/repeatability, specificity/selectivity, sensitivity (LLOQ), and sample stability was assessed.
Duration of treatment / exposure:
The males were dosed during study days 1-28 (14 days prior to pairing and continuing throughout the mating period) for a total of 28 doses. Males of satellite subgroup (not mated) were dosed for a total of 28 doses with following two weeks recovery period.

The females were dosed during study days 1 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 50-58 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia for a total of 55 doses. Females of satellite subgroup (not mated) were dosed for a total of 55 doses with following two weeks recovery period.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Satellite subgroup: 5 (from control and high dose group, not mated)
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
All rats were observed twice daily for morbidity and mortality. Each F0 male and female were also observed for signs of toxicity approximately 5-20 minutes following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.
Detailed physical examinations were recorded for all parental (F0) animals before first dosing and regularly on a weekly basis throughout the study.

Individual F0 male body weights were recorded during groups assignment, on the first day of dose administration, weekly thereafter throughout the study (at the same day as evaluation of food consumption), and prior to the scheduled euthanasia.
Individual F0 female body weights were recorded during group assignment, on the first day of dose administration, and weekly thereafter until evidence of copulation will be observed. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), and at the day 2 (for measurement of food consumption at lactation day 1-2), day 4 and day 13 post-partum.
Furthermore, body weights were also recorded during Functional Observation Battery (FOB) measurements (animals allocated to these components of the study only.

Food consumption was assessed quantitatively by weighing of feeder (cage lid) at the beginning of the day and 24 hours after.

FOB assessments, locomotor activity, and grip strength were recorded for six F0 males and six F0 females, randomly selected from each group on the day 28 of dose administration for males, and on the last day before euthanasia for females. Satellite subgroup animals were tested before euthanasia after 2 weeks post-treatment. Animals were tested at the same time on each day following approximately 30-40 minutes of dosing in a randomized order.
Locomotor activity was measured after FOB handling observation automatically using a personal computer-controlled system OPTO-VARIMEX with Auto-Trek software v. 4.2 (Columbus Instruments) utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity.
Sacrifice and pathology:
A complete necropsy were conducted on all F0 animals died spontaneously, euthanized in extremis or at scheduled termination. Necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera. Organ weights were collected (at scheduled necropsy) and tissues were preserved.

Tissues (excluding thyroids) from six randomly selected adult males and females in the control and high-dose groups (half of the animals in the group euthanized at the scheduled necropsy) and from all animals that die or are euthanized in extremis will be trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
Liver, spleen, thymus, heart, lungs, kidneys, stomach, ovaries, testes, and mammary gland (males) were examined in the low- and mid-dose groups as well as in satellite subgroup animals.
Microscopic examination of thyroid glands was done for all adult males and females in all groups including satellite subgroups.

Six randomly selected animals/sex from all F0 groups have been chosen for bone marrow smear. Bone marrow was collected by aspiration from the right femur into a centrifuge tube and smear was prepared for hematological analysis.

For F0 females, the number of former implantation sites was recorded. Uteri, which appear non-gravid by macroscopic examination, were opened and placed in 2 % sodium hydroxide solution for detection of early implantation loss.
Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

The F0 females that delivered were euthanized on lactation day 14. Females with total litter loss were euthanized on the day of litter loss.
The F0 females, which have not delivered were euthanized after 55 days of dosing.
F0 males were euthanized after completion of the mating period and 28 days of dose administration.
Not mated males and females of satellite subgroup were subjected to euthanasia at the end of two weeks post-treatment period.
Other examinations:
Urine samples were collected in all F0 males before scheduled euthanasia (day 28-29, overnight) and in satellite subgroup males after 2 weeks post-treatment before scheduled euthanasia (day 42-43, overnight) using metabolic cages (Tecniplast s.p.a): Diuresis, Appearance, Specific gravity, pH, Protein, Glucose, Blood cells, Ketones, Volume.

Blood samples for clinical pathology evaluations (hematology, coagulation and serum chemistry, T4) were collected from all surviving F0 animals at the scheduled necropsies.
Statistics:
All statistical tests were performed separately for each sex using Microsoft Excel (descriptive statistics) and statistical software Statistica for Window v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean, standard deviation (S.D.), and N) are presented.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For scheduled euthanized animals, the treatment-related rales were noted during daily clinical examination in the 450 mg/kg bw/day group for four males starting from the first week of dosing, and four females starting from the 4-6 weeks of dosing. The total number of males with rales was six (including dead males), which is significantly different from the control group.

The main clinical finding related to the administration of the test item was hyperemia of auricles observed 5-10 minutes after dosing in the 150 mg/kg bw/day males and females starting from the third week of dosing, and in the 450 mg/kg bw/day males and females starting from the first week of dosing. The frequency of hyperemia incidents was significantly higher compared to the control group in 150 and 450 mg/kg bw/day male and female groups.

The reactivity to handling with vocalization was noted in three males (including dead male No.33) with clinical finding of rales in the 450 mg/kg bw/day dose group and in one male (No. 66) in the 150 mg/kg bw/day group.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 450 mg/kg bw/day group, three males (No. 33, No. 71, and No. 72) were found dead on day 17, day 14 and day 2, respectively. Male No. 71 had marked hyperemia of right pinna, focal opacity in the right eye, and red-brown nasal discharge as a postmortem observation associated with dark-red total discoloration of lungs revealed during necropsy. A day earlier, weak rales were noted. In the male No. 72, the same discoloration of lungs was associated with rales after the first dosing, which developed in respiratory failure followed by death. Male No. 33 was found dead after rales, and the trauma of esophagus was revealed during necropsy. The lungs of this male were dark-red discolorated as for other males discussed.
The death of these males can be associated with cardiovascular failure and is considered to be treatment-related.

Female No. 90 (50 mg/kg bw/day dose group) and female No. 79 (150 mg/kg bw/day dose group) were euthanized in extremis due to total litter loss. Female No. 90 had a poor clinical condition with rales starting from day 12 of dosing and continuing throughout the study and a treatment-related effect cannot be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significantly lower mean body weight gain compared to the vehicle control group was observed in the 450 mg/kg bw/day male group (p<0.01), associated with a decrease in food consumption.
In the 450 mg/kg bw/day dose post-treatment males, the increase of body weight gain was observed compared to the control group (p<0.05) as a recovery rebound effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain in males was associated with a decrease in food consumption which observed to the fourth week of dosing. A decrease in food consumption was slight (8.8% compared with the control group), and statistically significant only when using the paired t-test (p < 0.05).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male No. 71 (450 mg/kg bw/day) had focal opacity in the right eye.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The test item-related hematological effects were more pronounced in males and were following: decrease in relative and absolute lymphocytes count (males, 150 mg/kg bw/day, p<0.05), increase in relative and absolute number of band (females, 450 mg/kg bw/day dose, not significant) and segmented neutrophils (males, 50, 150 and 450 mg/kg bw/day doses, not significant), increase in RBC count (males, 450 mg/kg bw/day group, p<0.05) and MCHC (females, 450 mg/kg bw/day group, p<0.05), increase of anisocytosis (RDW) in the 450 mg/kg bw/day dose group (female main subgroup, p<0.05, and male recovery subgroup, p<0.05). The observed hematological changes were slight; however, are considered as adverse starting at the dose of 150 mg/kg bw/day.

The hematological findings in blood correlated to the changes in relative cell count on bone marrow. In males, the percentage of total erythrocaryocytes (150 mg/kg bw/day group), band neutrophils (150 and 450 mg/kg bw/day dose groups) and eosinophils (50 and 150 mg/kg bw/day dose groups) was slightly increased (p<0.05), and the fraction of lymphocytes was slightly decreased (50 and 150 mg/kg bw/day doses, p<0.05). The fraction of immature neutrophils and basophils (50 and 150 mg/kg bw/day dose groups), as well as monocytes (150 mg/kg bw/day dose group), was slightly decreased (p<0.05).
Changes in the percentage of cell lines in the bone marrow of females may be associated with a more extended period of test item administration, as well as the specific physiological state of females in this study (pregnancy and lactation).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 450 mg/kg bw/day group males, the glucose level was lower of that in the control group. The decrease was slight (15%, p<0.05), associated with a decrease in body weight gain in males of this group and was not considered as adverse.

Also, males in the test item treated groups had a tendency for a dose-dependent increase in triglyceride levels.

An increase in the mean values of gamma-glutamyl transferase (GGT) in the test item male groups was noted, respectively, in 2, 3 and four males in the 50, 150 and 450 mg/kg bw/day dose groups of relative to the control group with zero value GGT in all males. The increase in the GGT and triglycerides level was associated with microscopic observations in the liver (450 mg/kg bw/day male group) and was considered as adverse.

In 450 mg/kg bw/day dose recovery males, the mean serum chloride ions level was increased (1.7%, p<0.05) when compared to the control group. Although this finding was observed only in a post-treatment period, it can be associated with histological observations in kidneys, so is considered to be treatment-related.

In the test item treated females, the slight decrease in a serum creatinine level was observed in the lower dose group (13.4%). This change was not dose-dependent, can be due of pharmacological action of isoprenol as metabolite intensifying intracellular energy transportation and influence on phosphocreatine-creatine buffer system, and considered to be treatment-related but non-adverse.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Reactivity to handling with vocalization was noted in three males (including dead male No.33) with clinical finding of rales in the 450 mg/kg bw/day dose group, and in one male (No. 66) in the 150 mg/kg bw/day group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males a decrease in thymus weight was associated with hematology changes (decrease in lymphocyte count in the 150 mg/kg bw/day dose group) and is therefore considered to be treatment-related.
In females, organ weight difference considered to be associated with the administration of the test item is the decrease in absolute and relative weight of spleen, revealed in the 150 mg/kg bw/day dose group (p<0.05). Although there were no dose-depended changes in spleen weight, this finding can be associated to macroscopic and microscopic observations in the spleen (incidents of organ deformation in the 150 and 450 mg/kg bw/day dose groups and atrophy of red pulp in the 450 mg/kg bw/day group).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related gross findings during necropsy of schedule euthanized F0 animals were observed in lungs (areas of gray discoloration) and heart (vasodilatation in the ventricles) of two males in the 450 mg/kg bw/day dose group, in the spleen (organ deformation) of two females from the 150 mg/kg bw/day dose group and two female from the 450 mg/kg bw/day dose group. In one male from the 150 mg/kg bw/day dose group, deformation of a kidney was noted. These gross observations correlated to the decreased organ weight (spleen, 150 mg/kg bw/day group), or/and clinical pathology changes and microscopic findings, and assumed as test item related.

Changes in implicit relationship to test item administration were observed in the thyroids and ovaries.
Thyroids were visually enlarged in three animal in the 450 mg/kg bw/day group and one female in the 50 mg/kg bw/day group. These incidental gross findings were not correlated with the mean organ weight, serum thyroxin level, and microscopic observations. In two females from the 450 mg/kg bw/day dose group extra nodes were founded in one of the ovaries caused by single or multiple cysts. These gross observations were not correlated with any changes in reproductive performance.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related histologic findings were revealed in the liver, spleen, kidneys, lungs, and heart. Histologic changes with an unclear relationship to test item administration were observed in the ovaries.

Microscopic findings in the heart and lungs in the 450 mg/kg bw/day administrated males (bronchiolar epithelial hyperplasia with a plethora of vessels, subepicardial necrosis with mononuclear and neutrophils infiltration in the right ventricle) correlated to the in-life clinical observations and gross observation in dead males, considered test item-related and adverse.

Atrophy of red and white pulp of spleen with fibrosis in some males and females in the 150 and 450 mg/kg bw/day were correlated with gross finding during necropsy, changes in organ weight and hematology data, were considered adverse and test item-related.

Subcapsular necrosis in kidneys (one male in the 150 mg/kg bw/day dose group) and necrosis of tubules (one female in the 50 mg/kg bw/day dose group) correlated in males with gross observation, slight non-significant decrease in organ weight and clinical serum chemistry, were considered adverse and test item-related.

Hepatocellular vacuolation (males) and angiectasis (females) findings in the 450 mg/kg bw/day dose group correlated with a non-significant increase in serum triglyceride and GGT in high dose male group; were slight, not observed after the recovery period, considered non-adverse. The subcapsular hepatocellular necrosis was noted in one female in the 50 mg/kg bw/day and female from the control recovery subgroup, which allows considering this finding as of uncertain relationship to test item administration in females.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
other: Effects are not limited to one organ but various systems are affected.
Organ:
blood
bone marrow
kidney
liver
spleen
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, 50 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for males systemic toxicity and the lowest-observed-adverse-effect-level (LOAEL) for F0 female systemic toxicity (pregnant and lactating with more extended dosing period).
Executive summary:

This study was designed to evaluate the potential toxic effects of the test item Isoprenol (IPN) when administered to rats for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

Adverse respiratory and cardiovascular-related clinical findings with mortality in males, lower mean body weight gain, and/or reduced food consumption were noted in the 450 mg/kg bw/day group males and females during the treatment periods.

Microscopic findings in the heart and lungs in the 450 mg/kg bw/day administrated males (bronchiolar epithelial hyperplasia with a plethora of vessels, subepicardial necrosis with mononuclear and neutrophils infiltration in the right ventricle) correlated to the in-life clinical observations and gross observation in dead males, considered test item-related and adverse. Hepatocellular vacuolation (males) and angiectasis (females) findings in the 450 mg/kg bw/day dose group correlated with a non-significant increase in serum triglyceride and GGT in high dose male group; were slight, not observed after the recovery period, considered non-adverse. The subcapsular hepatocellular necrosis was noted in one female in the 50 mg/kg bw/day and female from the control recovery subgroup, which allows considering this finding as of uncertain relationship to test item administration in females. Atrophy of red and white pulp of spleen with fibrosis in some males and females in the 150 and 450 mg/kg bw/day were correlated with gross finding during necropsy, changes in organ weight and hematology data, were considered adverse and test item-related. Subcapsular necrosis in kidneys (one male in the 150 mg/kg bw/day dose group) and necrosis of tubules (one female in the 50 mg/kg bw/day dose group) correlated in males with gross observation, slight non-significant decrease in organ weight and clinical serum chemistry, were considered adverse and test item-related.

The observed hematological changes were slight; however, are considered as adverse starting at the dose of 150 mg/kg bw/day. The hematological findings in blood correlated to the changes in relative cell count on bone marrow. ). The direction of relative changes in the bone marrow cell lines was most pronounced in the dose of 150 mg/kg bw/day.

No effects on FOB parameters and no effects on locomotor activity were noted at any dose level.

The slight non-significant prolongation of pre-coital interval was observed in the 450 mg/kg bw/day dose group. There were no test item-related effects on other parameters of F0 reproductive performance, gestation length, parturition, or reproductive organs at any dosage level. Also, there were no changes in a mean number of pups born, live litter size, and postnatal survival. However, the body weight of newborn females (and less in males) was reduced in the 450 mg/kg bw/day dose group; and normalized anogenital distance was slightly non-significantly decreased in females as well as males newborns. The slight increase in the percentage of newborn males was noted in litters in the 150 and 450 mg/kg bw/day dose groups. The number of F1 males with retention of areolae on PND 13 was slightly non-significantly increased compared to the control group and historical control data.

Therefore, under the conditions of this screening study, 50 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for males systemic toxicity and the lowest-observed-adverse-effect-level (LOAEL) for F0 pregnant and lactating female with more extended dosing period.