Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422, Combined 28-Day Repeated Dose Oral (Gavage) 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
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 will be used: 58
Number of females will be 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
Actual 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.

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 were9 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 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.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the F0 males and females avoiding sibling mating (animals were received from Breeding Center with indication of litters). Each female will be housed in the home cage of the male. In Group 4, three males (No. 9, 14 and 23) were cohabited with two females, because as three males in this group died before mating. Positive evidence of mating was confirmed by the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0 (G0).
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 Group: 5 (from control and high dose group, not mated)
Control animals:
yes, concurrent vehicle
Details on study design:
For dose range finding study see 7.8.1 Toxicity to reproduction, DRF OECD 422.

Examinations

Maternal examinations:
All rats were observed twice daily for morbidity and mortality. Each F0 female was 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. Detailed physical examinations were recorded for all female (F0) animals before first dosing and regularly on a weekly basis throughout the study. 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.
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.

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 females, randomly selected from each group on the last day before euthanasia.

Blood samples for clinical pathology evaluations (hematology, coagulation and serum chemistry, T4) were collected.

Individual gestation length was calculated using the date when delivery started.
Ovaries and uterine content:
Organ weights, tissue collection macroscopic and microscopic examination.
For F0 females, the number of former implantation sites was recorded.
Fetal examinations:
When parturition was judged to be complete (day 0 or 1 post-partum), the sex of each pup, anogenital distance (AGD) and body weight was determined, pups were examined for gross malformations and the number of stillbirths and live pups was recorded. Any changes or abnormalities in nesting and nursing behavior were recorded. Each litter was examined daily for survival and abnormalities, and all deaths were recorded.

Blood samples were collected from two of the surplus pups per litter euthanized during litter adjusting on PND 4, and from two of pups per litter at termination on PND 13.

The external genitalia were inspected in all males on PND 13 before necropsy.

All survived pups will be subjected to a gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals; thyroid gland will be preserved and weighted (after fixation) in 1 male and 1 female from each litter.

Pups found dead on PND 0-1 had lungs removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup was reported as stillborn.
Stillborn pups, pups found dead and any pups that are euthanized in extremis will be necropsied using a fresh dissection technique.
If a skeletal anomaly was suspected, the pups were eviscerated, cleared, and stained with Alizarin Red (Dawson’s technique). Organs/tissues were saved for possible histological examination in 10% neutral-buffered formalin only as deemed necessary by the gross findings. Cannibalized pups were discarded without necropsy.
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. The litter, rather than the pup, will be considered as the experimental unit.
Indices:
Pregnancy (<= 21 days, = 22 days, >= 23 days)
Assigned to natural delivery (N)
Pregnant (N, %)
Delivered a litter (N, %)
Duration of gestation, days
The time elapsed between confirmed mating and the day of first pup was delivered
Pre-delivery findings
Significant findings during parturition
Implantation sites (a) per delivered litter
Dams with live pups (N, %) (at 4 and 13 postpartum day)
Dams with stillborn pups (N, %)
Dams with no liveborn pups (N, %)
Gestation index (%, N/N)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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 females starting from the 4-6 weeks of dosing.

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 females starting from the third week of dosing, and in the 450 mg/kg bw/day 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 female groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
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:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In the lactation period, a slight decrease in food consumption compared with the control group was observed in the 450 mg/kg bw/day group on days 4-5 (9.5%) and days 12-13 (9.1%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The test item-related hematological effects were following: Increase in relative and absolute number of band neutrophiles (450 mg/kg bw/day dose, not significant), increase in MCHC (450 mg/kg bw/day group, p<0.05), increase of anisocytosis (RDW) in the 450 mg/kg bw/day dose group (main subgroup, p<0.05). The observed hematological changes were slight; however, are considered as adverse.

The hematological findings in blood correlated to the changes in relative cell count on bone marrow. 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 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:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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):
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.

Test item-related gross findings during necropsy of schedule euthanized F0 animals were observed 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. 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.
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.

Changes of unclear relationship to test item administration were found in the ovaries and uterus. Two females in the 450 mg/kg bw/day dose group had ovarian cysts. These findings were not seen in other females, were not correlated with any changes in reproductive performance and assumed as incidental. Also, one female from the high dose satellite subgroup had the cyst of the corpus uterus which considered not treatment-related.

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.

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.

Angiectasis findings in the 450 mg/kg bw/day dose group were slight, not observed after the recovery period and considered non-adverse.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A single female (No. 85) in 450 mg/kg bw/day dose group did not deliver, being gravid and having one implantation site. There were no associated gross observations, or microscopic findings were observed in this female; however, a treatment-related effect cannot be excluded.
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. Each of these females had one implantation site and delivered one unviable pup. There were no adverse microscopic observations in female No. 79, associated with the total litter loss or that considered to be treatment-related. Female No. 90 had a poor clinical condition with rales starting from day 12 of dosing and continuing throughout the study.
However, the number of females with pre-natal, as well as postnatal loss of offspring and the number of females with stillborn pups were approximately the same between groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean values of all dead pups and litters survival on postnatal day 0 in all dose treated groups did not differ from the values in the control group.
The mean number of liveborn pups, live litter size and postnatal survival in the all dose-treated groups did not significantly differ from the values in the control group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 450 mg/kg bw/day dose group, the absolute body weights of male and female pups were slightly lower compared to the control values through the postnatal day 4 (by 6.2%, not significant). However, at the postnatal day 13, the absolute body weights and final body weight gains of male and female pups in all dose treated groups did not differ from the values in the control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
The mean number of females was decreased compared to the control group in the 150 mg/kg bw/day dose group (p < 0.05) and 450 mg/kg bw/day dose group (not significantly). As a result, in these groups, the percentage of born males per litter was slightly increased (without statistical significance when comparing ANOVA with the Dunette test). It should be noted, that in the control group of this study, the absolute and percentage number of males per litter are slightly reduced relative to the historical control data (respectively, MEAN + S.D. (N=44), 6.4 + 2.3 and 50.2 + 13.9). Nevertheless, the mean values of the percentage of males born in the 150 and 450 mg/kg bw/day groups were still slightly higher relative to the mean value of the historical control (approximately by 8%).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During necropsy of pup No. 90-1m (50 mg/kg bw/day dose group), which was the only one in the litter, was liveborn and found dead soon after birth on PND 0, a widespread subcutaneous hematoma of the pelvic region was found without any visual malformations. This incident potentially cannot be excluded as attributed to F0 female test item administration.
Two alive pups were recorded as runts without visual malformations. One of them from 450 mg/kg bw/day dose group was cannibalized on PND 2, and the other one from the 150 mg/kg bw/day dose group survived until scheduled euthanasia on PND 13.
In the 450 mg/kg bw/day dose group, the emaciation, and dehydration of pups in one litter were observed starting from PND 3 through PND 7. Visual emaciation of the offspring was found only in one litter, was temporary; however, it was considered to be adverse and test item-related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was 50 mg/kg bw/day for F1 developmental toxicity.
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.

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, the no-observed-adverse-effect-level (NOAEL) was 50 mg/kg bw/day for F1 developmental toxicity.