Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.08.2018 - 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:
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 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.
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 a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist.
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 groups: 5 (from the control group and high dose group, not mated)
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
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.

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.

Individual gestation length was calculated using the date when delivery started.
Oestrous cyclicity (parental animals):
Oestrous cycles were monitored before treatment starts to select for the study females with regular cyclicity (4-5 day cycles). monitored before start of the treatment to select for the females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for last week during the pre-mating period with continued monitoring into the mating period until there was evidence of mating. Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.
Litter observations:
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 will be inspected in all males on PND 13 before necropsy.

Before scheduled euthanasia and necropsy on PND 13, the number of nipples / areolae was counted in all males and females
Postmortem examinations (parental animals):
A complete necropsy was 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. Microscopic examination of thyroid glands were done for all adult males and females.
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.

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.
Postmortem examinations (offspring):
All survived pups were subjected to a gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals; thyroid gland were preserved and weighted (after fixation) in 1 male and 1 female from each litter in the control and high-dose groups.

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.
Reproductive indices:
Male Fertility Index, %: (No. of males siring a litter / Total No. of males used for mating) X 100
Female Fertility Index, %: (No. of females with confirmed pregnancy / Total No. of females used for mating) X 100
Female Conception Index, %: (No. of females with confirmed pregnancy / No. of females with evidence of mating) X 100
Gestation index (%, N/N): Number (N) of rats with live offspring / Number of pregnant rats
Viability index (N/N, %): PND 4, 7, 13 (Number of live pups / Number of liveborn pups on PND 0)
Offspring viability indices:
Viability index (N/N, %): PND 4, 7, 13 (Number of live pups / Number of liveborn pups on PND 0)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
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.

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.
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
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.

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects on other parameters of reproductive performance of males were observed at any dosage level.
In the 450 mg/kg bw/day dose group, the mean value of the pre-coital interval was 4.1 ± 1.8 days compared with the value of 2.8 ± 1.5 day in the control group. The increase of pre-coital interval was not statistically significant and is assumed to be associated with a general toxic effect of the test item in the high dose.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related adverse clinical findings in the pups in the 50 and 150 mg/kg bw/day dose groups.
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.

It should be noted that all stillborn were females with one runt among them in the 450 mg/kg bw/day dose. Also, two liveborn pups in 150 and 450 mg/kg bw/day were recognized as runts. All runt pups were without visual malformations. Runts can be born with a low frequency in the population of rats, and the frequency of the runts in test item groups was not significantly different from the control group, which does not allow to make a definite conclusion about the relation with test item administration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The test item in all administrated doses did not affect the survival of the F1 offspring in the postnatal period. 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
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the 450 mg/kg bw/day dose group, the body weight of newborn females was decreased as compared to the control vehicle group (by 8.6%, p < 0.05). The decrease in body weight of newborn males in the high-dose group was small and insignificant (4.6%).
The absolute body weights of male and female pups in the 450 mg/kg bw/day dose group 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.
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:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative anogenital distance in test item-treated groups did not differ significantly from the values in the control group. However, in the 450 mg/kg bw/day dose group, the mean value of normalized AGD was decreased compared to the values in the control group in both males (by 6.5%) and females (by 7.7%).
Nipple retention in male pups:
effects observed, treatment-related
Description (incidence and severity):
In the 450 mg/kg bw/ day dose group, four male pups from three litters had 1-2 areolae. 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. Nevertheless, the relationship of slight increase in nipples/areolae retention in PND 13 male pups with the test item cannot be completely excluded.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In F1 offspring males, the increase in the weight of thyroid glands was observed in the 50 mg/kg bw/day (absolute and relative weights) and 450 mg/kg bw/day (relative weight) dose groups. The change in thyroids weight in males was not associated with any concomitant microscopic changes in thyroid tissue and serum thyroxin level. In this regard, the change in the thyroids weight in low and high dose male groups is considered to be test item unrelated.
Gross pathological findings:
effects observed, non-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. However, the visceral examination of all stillborn and died pups did not indicate any test item-related effects on the morphological development of the offspring.
There were no test item-related gross observations in all pups during scheduled necropsy on PND 13.
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: AGD decreased in male and female at 450 mg/kg bw/day, Visual emaciation of the offspring was found in one litter.

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:
A NOAEL of 150 mg/kg bw/day was set for F0 reproductive 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.

In general, it can be concluded that the test item does not affect the reproductive performance of males and females at any dosage level.

The slight non-significant prolongation of pre-coital interval was observed in the 450 mg/kg bw/day dose group. The increase of pre-coital interval was not statistically significant and is assumed to be associated with a general toxic effect of the test item in the high dose. 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. A visual emaciation of the offspring was found only in one litter from the 450 mg(kg bw/day dose group, was temporary; however, it was considered to be adverse and test item-related.

Therefore the NOAEL of 150 mg/kg bw/day was set for F0 an F1 reproductive toxicity.