Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
28-day repeated dose toxicity study with reproduction/developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2017 - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2017 - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/ developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 261 and 298 g and females weighed between 200 and 245 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA) without cage-enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Results of the analysis were provided by the supplier. Periodic analysis of the water has been performed. It is considered that there were no known contaminants in the feed and in the water that would interfere with the objectives of the study.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 21.6
- Humidity (%): 51 to 77
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jun 2017 To: 25 Aug 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/v) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations (solutions at all dose levels) were prepared daily and dosed within 5 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. For determination of the concentration, samples were collected from all dose groups (one occasion). For determination of dose formulation homogeneity, samples were collected from low and high dose groups (one occasion). The homogeneity results obtained from the top, middle and bottom for the preparations of the low and high dose groups were averaged and utilized as the concentration results.All samples were stored on dry ice immediately after sampling. Upon receipt at the analytical laboratory, the samples were stored in the ultralow freezer ≤ -70 °C until analysis. Analyses were performed by using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity analysis: Duplicate sets of samples were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males.
Main females were treated for 50 - 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 - 15 days after delivery, up to and including the day before scheduled necropsy. The Main female which failed to deliver was treated for 43 days.
Recovery females were treated for 55 days during the same period as Main females. The first day of dosing was designated as Day 1.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Due to an error in the formulation request, Group 4 animals were treated at a dose level of 1400 mg/ kg bw/day, instead of the intended 1000 mg/kg bw/day, during the 14 days of the premating period and on the first day of mating.
No. of animals per sex per dose:
10 (main study); 5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finding study with oral exposure of X-19657 in the rat.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on
the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: 5 Main males and all Recovery males during week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6, 8 and 9), and all Recovery females were tested during the first day a Main female was tested.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pre-test period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. For Recovery females daily vaginal lavage was also performed during the treatment-free recovery period.
In addition, on the day of necropsy a vaginal lavage was taken from all Main and Recovery females to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight.
For the testes of all selected Main males of Groups 1 and 4, and all Main males that failed to sire, additional slides of the testes were stained with PAS/ haematoxylin to examine staging of spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma thyroid hormone levels

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Postmortem examinations (parental animals):
Main Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Recovery males: After the recovery period of at least 14 days, which was at least 14 days after the scheduled necropsy of Main males.
Main Females which delivered: PND 14-16.
Main Female which failed to deliver: With evidence of mating: Post-coitum Day 27.
Recovery females: After the recovery period of at least 14 days, which was at least 14 days after the first scheduled necropsy of Main females.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues in accordance with the Guidelines were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-15), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 13-15 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
ducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
-parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
-non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
-incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Salivation seen after dosing among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. shortly after dosing).
All other clinical signs noted occurred incidentally within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of dose response and/or slight change compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of similar findings at other time points (higher mean relative food consumption of 1000 mg/kg bw/day females between post-coitum Days 11-14) or higher dose levels (lower values in 100 mg/kg bw/day females during gestation and lactation).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in hematology parameters (red and white blood cell parameters, number of platelets), all in females treated at 1000 mg/kg bw/day, distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values compared to the control group are indicated between parentheses.
• Higher number of reticulocytes in lactating females (46%) and nulliparous females (26%) at the end of the treatment period. At the end of the recovery period, the numbers of reticulocytes in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Higher red blood cell distribution width (RDW) in lactating females (8%, not statistically significant) and nulliparous females (7%) at the end of the treatment period. At the end of the recovery period, RDW in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Lower haemoglobin concentration in lactating females (8%) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haemoglobin concentration in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• Lower haematocrit in lactating females (5%, not statistically significant) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haematocrit in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• The number of red blood cells tended to be lower in lactating 1000 mg/kg bw/day females (6%) at the end of the treatment period and in nulliparous 1000 mg/kg bw/day females (7%) at the end of the recovery period but statistical significance was not achieved.
The above changes remained within the historical control range except for the higher number of reticulocytes in lactating and nulliparous females at the end of the treatment period, and reductions in haemoglobin concentration, haematocrit and number of red blood cells in nulliparous females at the end of the recovery period.
The reduced level of lymphocytes for high dose females at the end of the recovery phase was not considered toxicologically relevant as this change was not observed at the end of the treatment phase.
There were no treatment-related changes in hematology parameters in males. The statistically significantly lower mean haematocrit value of 300 mg/kg bw/day males at the end of the treatment period was considered unrelated to treatment due to the lack of a dose-related response.
Coagulation parameters (prothrombin time and activated partial prothrombin time) were considered not to be affected by treatment. Isolated statistically significant variations noted in coagulation parameters at the end of the treatment period were considered unrelated to treatment due to the lack of a dose-related response (lower prothrombin time (PT) in 100 mg/kg bw/day males) or a slightly high control value (lower activated partial thromboplastin time (APTT) in nulliparous 1000 mg/kg bw/day females).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lactating females treated at 1000 mg/kg bw/day showed a statistically significantly higher mean plasma level of total bilirubin at the end of the treatment period (relative difference from control mean: 17%). Bilirubin values in 1000 mg/kg bw/day females remained within the historical control range. No differences in bilirubin were noted in nulliparous females at the end of the treatment period or recovery period.
Nulliparous females treated at 1000 mg/kg bw/day showed a statistically significantly higher plasma concentration of cholesterol at the end of the treatment period (relative difference from control mean: 33%). This change was fully reversible and all values remained within the historical control range. The few other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response (cholesterol and inorganic phosphate in males) or lack of a test item-related change in the respective parameter at the end of the treatment period (alkaline phosphatase and inorganic phosphate in males, calcium in females).
Thyroid hormone analyses:
Serum levels of T4 in F0 males were unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A test item-related microscopic change was noted in the thyroid gland of males and females (see 'any other information on results incl tables').
At the end of the treatment period, there was an increased incidence and/or severity of follicular cell hypertrophy in the thyroid of males from 300 mg/kg bw/day onwards and of females from 100mg/kg bw/day onwards. After the 14-day recovery period, follicular cell hypertrophy was present at increased incidence and severity in 1000 mg/kg bw/day males. In 1000 mg/kg bw/day females (nulliparous), no follicular cell hypertrophy was observed at the end of the recovery period.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to be affected by treatment.
At examination of the vaginal smears made during the pre-mating period, an cycle length of 3 or 8 days was noted in 5/15 females treated at 1000 mg/kg bw/day (3 Main group female and 2 Recovery group female). Usually, estrous cycles have a length of 4 or 5 days. Thorough evaluation of the available data indicated that slides used for preparation of vaginal lavages must accidentally have been interchanged on treatment Day 8 and treatment Day 9. This conclusion was based on the following facts:
1) Observed estrous stages had sequences that are biologically impossible. Under ideal conditions all four stages of the estrous cycle can be seen microscopically in the sequence pro-estrus (P), estrus (E), met-estrus (M) and di-estrus (D). However, especially P and M are often missed due to their relatively short duration (P: 12-18 hours; E: 18-24 hours; M: approx. 6 hours; D: 2-3 days). Nevertheless, since the change in cytology from one stage to the other is continuous it is possible to predict the stage of the next smear based on the cytology of the current smear.
2) The interchange occurred when females were group-housed (5 females per cage). Suspicious vaginal smears were from females housed in the same cage.
3) Except for one female which was not pregnant, the remaining three affected Main females had a relatively short mating period (successful mating was confirmed on mating Days 2 or 3) and delivered normal litters (12-14 live pups).
4) During the 14-day treatment-free recovery period, 4 females had regular cycles of four or five days (for one female cycle length in the recovery period could not be determined).
It was therefore concluded that estrous length and regularity were not affected by treatment at 1000 mg/kg bw/day.
All females of the control group and the 100 and 300 mg/kg bw/day groups had regular cycles of four days during the pre-test and pre-mating periods.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in spermatogenic profiling
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time was not affected by treatment. All females showed evidence of mating within five days.
Number of implantation sites was not affected by treatment.
Fertility index was considered not to be affected by treatment. Except for one female at 1000 mg/kg bw/day, all mated females were pregnant. This single case of non-pregnancy was considered unrelated to treatment due to its incidental occurrence and lack of related histopathology changes in reproductive organs. The fertility indices were 90% for the 1000 mg/kg bw/day group and 100% for the other groups.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg instead of at the target dose level of 1000 mg/kg.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg instead of at the target dose level of 1000 mg/kg.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices were 88, 80, 90 and 96% at 0 (control), 100, 300 and 1000 mg/kg bw/day, respectively.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for the 100 and 1000 mg/kg bw/day groups and 99% for the control and 300 mg/kg groups. At first litter check, one pup of the control group and one pup at 300 mg/kg bw/day were found dead. This pup mortality was considered to be unrelated to treatment as it was incidental and showed no dose-related trend.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the control group and 99% for the other groups.
Three pups went missing (presumably cannibalized) on PND 2 or 4 (one of each test group). This post-natal loss was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be adversely affected by treatment.
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
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg instead of at the target dose level of 1000 mg/kg.
Key result
Critical effects observed:
no
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose formulation analyses

accuracy: The concentrations analyzed in the formulations of Groups 2 (low dose), 3 (mid dose) and 4 (high dose) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). Actual data are 100.7%, 101.4% and 100.4% in the low, mid and high dose group formulations, respectively. No test item was detected in the control group formulation.

homogeneity: The formulations of the low and high dose were homogeneous (i.e. coefficient of variation ≤ 10%). Actual data are 1.1% and 1.3% in the low and high dose formulations, respectively.

Summary Test Item-Related Microscopic Findings - Males

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

2

2

4

3

3

2

     Slight

1

1

1

2

-

3

a = Number of tissues examined from each group.

Summary Test Item-Related Microscopic Findings - Females

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

4

1

3

3

1

-

     Slight

-

3

2

2

1

-

 a = Number of tissues examined from each group.

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental, reproduction and the developmental No-Observed-Adverse-Effect levels (NOAELs) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.
Executive summary:

Repeated dose toxicity X-19657 was studied in a combined 28 -Day repeated dose toxicity study with the reproduction/developmental toxicity screening test followed by a 14 -Day recovery period according to Guidelines and in accordance with GLP principles.

Wistar Han rats were treated with X-19657 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-56 days). The non-pregnant female was treated for 43 days. Additional animals of the control and 1000 mg/kg bw/day groups (5 rats/sex/group) were treated similarly (males for 29 days, females for 55 days), and were terminated after a treatment-free period of 14 days.

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

No parental toxicity was noted up to and including the highest dose level tested (1000 mg/kg bw/day).

Exposure to the test item up to and including 1000 mg/kg bw/day was well tolerated as evidenced by the absence of adverse changes in the parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, body weight, food consumption, clinical pathology, macroscopic examination, organ weights and microscopic examination). A few changes noted in treated rats were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals, in a dose-related manner, was considered a physiological response rather than a sign of systemic toxicity. Microscopic examination revealed a relative slight increase in the incidence and/or severity of follicular cell hypertrophy in the thyroid in males at 300 and 1000 mg/kg bw/day and in (lactating) females at all dose levels at end of treatment. After the 14-day recovery period, no follicular cell hypertrophy was seen in 1000 mg/kg bw/day (nulliparous) females whereas it was still present at increased incidence and severity in 1000 mg/kg bw/day males. Based on its low degree (minimal or slight), the increase in follicular cell hypertrophy in treated animals was regarded as nonadverse.

Females treated at 1000 mg/kg bw/day showed changes in red blood cell parameters, characterized by reductions in haemoglobin concentration, haematocrit and number of red blood cells, and increases in the number of reticulocytes and red blood cell distribution width (RDW). These changes occurred both in lactating females and nulliparous females. Lactating females additionally showed slightly increased plasma levels of bilirubin, suggestive of increased haemolysis of red blood cells. The increases in reticulocytes and RDW were reversible within the 14-day treatment-free period, whereas the magnitude of the reduction in haemoglobin, haematocrit and number of red blood cells was somewhat larger after the recovery period than at the end of the treatment period. As the reductions in haemoglobin concentration, haematocrit and number of red blood cells were less than 10% and values in treated females generally remained within the range of normal biological variation, the effect on red blood cells in 1000 mg/kg bw/day females was regarded as non-adverse.

A higher plasma level of cholesterol was observed in nulliparous 1000 mg/kg bw/day females at the end of the treatment period. Cholesterol levels in treated nulliparous females remained within the historical control range. Moreover, cholesterol in lactating females was not affected and there were no corroborative changes in other endpoints. Therefore, this isolated change, which showed full recovery during the 14-day treatment-free period, was considered nonadverse.

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated

in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 (PND 13-15) and macroscopic examination).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental, reproduction and developmental No-Observed-Adverse-Effect levels (NOAELs) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2017 - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 261 and 298 g and females weighed between 200 and 245 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp
polycarbonate cage (Ancare corp., USA) without cage-enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Results of the analysis were provided by the supplier. Periodic analysis of the water has been performed. It is considered that there were no known contaminants in the feed and in the water that would interfere with the objectives of the study.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 21.6
- Humidity (%): 51 to 77
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jun 2017 To: 25 Aug 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/v) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations (solutions at all dose levels) were prepared daily and dosed within 5 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. For determination of the concentration, samples were collected from all dose groups (one occasion). For determination of dose formulation homogeneity, samples were collected from low and high dose groups (one occasion). The homogeneity results obtained from the top, middle and bottom for the preparations of the low and high dose groups were averaged and utilized as the concentration results.All samples were stored on dry ice immediately after sampling. Upon receipt at the analytical laboratory, the samples were stored in the ultralow freezer ≤ -70 °C until analysis. Analyses were performed by using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples were sent to the analytical laboratory. Concent ration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity analysis: Duplicate sets of samples were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
Duration of treatment / exposure:
Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males.
Main females were treated for 50 - 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 - 15 days after delivery, up to and including the day before scheduled necropsy. The Main female which failed to deliver was treated for 43 days.
Recovery females were treated for 55 days during the same period as Main females. The first day of dosing was designated as Day 1.
Frequency of treatment:
daily
Duration of test:
males: main groups 29 days; recovery groups 29+14 days
females: main groups 50-56 days; recovery groups 55+14 days
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Due to an error in the formulation request, Group 4 animals were treated at a dose level of 1400 mg/ kg bw/day, instead of the intended 1000 mg/kg bw/day, during the 14 days of the premating period and on the first day of mating.
No. of animals per sex per dose:
10 (main study); 5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finding study with oral exposure of X-19657 in the rat.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: 5 Main males and all Recovery males during week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6, 8 and 9), and all Recovery females were tested during the first day a Main female was tested.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or
AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males
and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma thyroid hormone level

GROSS EXAMINATION OF DEAD PUPS: yes.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
-parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
-non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
-incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Salivation seen after dosing among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight
severity and the time of occurrence (i.e. shortly after dosing).
All other clinical signs noted occurred incidentally within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and
showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of dose response and/or slight change compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of similar findings at other time points (higher mean relative food consumption of 1000 mg/kg bw/day females between post-coitum Days 11-14) or higher dose levels (lower values in 100 mg/kg bw/day females during gestation and lactation).
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in hematology parameters (red and white blood cell parameters, number of platelets), all in females treated at 1000 mg/kg bw/day, distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values compared to the control group are indicated between parentheses.
• Higher number of reticulocytes in lactating females (46%) and nulliparous females (26%) at the end of the treatment period. At the end of the recovery period, the numbers of reticulocytes in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Higher red blood cell distribution width (RDW) in lactating females (8%, not statistically significant) and nulliparous females (7%) at the end of the treatment period. At the end of the recovery period, RDW in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Lower haemoglobin concentration in lactating females (8%) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haemoglobin concentration in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• Lower haematocrit in lactating females (5%, not statistically significant) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haematocrit in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• The number of red blood cells tended to be lower in lactating 1000 mg/kg bw/day females (6%) at the end of the treatment period and in nulliparous 1000 mg/kg bw/day females (7%) at the end of the recovery period but statistical significance was not achieved.
The above changes remained within the historical control range except for the higher number of reticulocytes in lactating and nulliparous females at the end of the treatment period, and reductions in haemoglobin concentration, haematocrit and number of red blood cells in nulliparous females at the end of the recovery period.
The reduced level of lymphocytes for high dose females at the end of the recovery phase was not considered toxicologically relevant as this change was not observed at the end of the treatment phase.
There were no treatment-related changes in hematology parameters in males. The statistically significantly lower mean haematocrit value of 300 mg/kg bw/day males at the end of the treatment period was considered unrelated to treatment due to the lack of a dose-related response.
Coagulation parameters (prothrombin time and activated partial prothrombin time) were considered not to be affected by treatment. Isolated statistically significant variations noted in coagulation parameters at the end of the treatment period were considered unrelated to treatment due to the lack of a dose-related response (lower prothrombin time (PT) in 100 mg/kg bw/day males) or a slightly high control value (lower activated partial thromboplastin time (APTT) in nulliparous 1000 mg/kg bw/ day females).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lactating females treated at 1000 mg/kg bw/day showed a statistically significantly higher mean plasma level of total bilirubin at the end of the treatment period (relative difference from control mean:
17%). Bilirubin values in 1000 mg/kg bw/day females remained within the historical control range. No differences in bilirubin were noted in nulliparous females at the end of the treatment period or recovery period.
Nulliparous females treated at 1000 mg/kg bw/day showed a statistically significantly higher plasma concentration of cholesterol at the end of the treatment period (relative difference from control mean: 33%). This change was fully reversible and all values remained within the historical control range. The few other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response (cholesterol and inorganic phosphate in males) or lack of a test item-related change in the respective parameter at the end of the treatment period (alkaline phosphatase and inorganic phosphate in males, calcium in females).
Thyroid hormone analyses:
Serum levels of T4 in F0 males were unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Any statistically significant differences noted in absolute or relative organ weights were considered unrelated to treatment due to the lack of a dose-related response or lack of a test item-related change in the respective organ weight at the end of the treatment period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A test item-related microscopic change was noted in the thyroid gland of males and females (see 'any other information on results incl tables').
At the end of the treatment period, there was an increased incidence and/or severity of follicular cell hypertrophy in the thyroid of males from 300 mg/kg bw/day onwards and of females from 100 mg/kg bw/day onwards. After the 14-day recovery period, follicular cell hypertrophy was present at increased incidence and severity in 1000 mg/kg bw/day males. In 1000 mg/kg bw/day females (nulliparous), no follicular cell hypertrophy was observed at the end of the recovery period.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices were 88, 80, 90 and 96% at 0 (control), 100, 300 and 1000 mg/kg bw/day, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Compared to controls, mean litter size at 100 mg/kg bw/day was slightly lower (9.0 versus 11.1, not statistically significant). This could be explained by the small size of two litters (which had three and four pups, respectively). Such small litter sizes occasionally occur in untreated controls. As mean litter sizes at the higher dose levels were close to the mean control value or even above (10.5 at 300 mg/kg bw/day and 12.0 at 1000 mg/kg bw/day), the apparently lower value at 100 mg/kg bw/day was considered unrelated to treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for the 100 and 1000 mg/kg bw/day groups and 99% for the control and 300 mg/kg bw/day groups. At first litter check, one pup of the control group and one pup at 300 mg/kg bw/day were found dead. This pup mortality was considered to be unrelated to
treatment as it was incidental and showed no dose-related trend.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment. Except for one female at 1000 mg/kg bw/day, all mated females were pregnant. This single case of non-pregnancy was considered unrelated to treatment due to its incidental occurrence and lack of related histopathology changes in reproductive organs. The fertility indices were 90% for the 1000 mg/kg bw/day group and 100% for the other groups.
Other effects:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time was not affected by treatment. All females showed evidence of mating within five days.
Number of implantation sites was not affected by treatment.
No deficiencies in maternal care were observed, except for one low dose female. At first litter check one (male) of her 11 pups was found with the placenta still attached, many bite marks and no milk. One day later, this pup was missing. As this was an isolated observation in the lowest dose group only, it was considered to be unrelated to treatment.
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure pe riod, male and female parental rats were inadvertently treated at 1400 mg/kg instead of at the target dose level of 1000 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be adversely affected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the control group and 99% for the other groups.
Three pups went missing (presumably cannibalized) on PND 2 or 4 (one of each test group). This post-natal loss was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Compared to controls, mean litter size at 100 mg/kg bw/day was slightly lower (9.0 versus 11.1, not statistically significant). This could be explained by the small size of two litters (which had three and four pups, respectively). Such small litter sizes occasionally occur in untreated controls. As mean litter sizes at the higher dose levels were close to the mean control value or even above (10.5 at 300 mg/kg bw/day and 12.0 at 1000 mg/kg bw/day), the apparently lower value at 100 mg/kg bw/day was considered unrelated to treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the control group and 99% for the other groups.
Three pups went missing (presumably cannibalized) on PND 2 or 4 (one of each test group). This post-natal loss was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose formulation analyses

accuracy: The concentrations analyzed in the formulations of Groups 2 (low dose), 3 (mid dose) and 4 (high dose) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). Actual data are 100.7%, 101.4% and 100.4% in the low, mid and high dose group formulations, respectively. No test item was detected in the control group formulation.

homogeneity: The formulations of the low and high dose were homogeneous (i.e. coefficient of variation ≤ 10%). Actual data are 1.1% and 1.3% in the low and high dose formulations, respectively.

Summary Test Item-Related Microscopic Findings - Males

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

2

2

4

3

3

2

     Slight

1

1

1

2

-

3

a = Number of tissues examined from each group.

Summary Test Item-Related Microscopic Findings - Females

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

4

1

3

3

1

-

     Slight

-

3

2

2

1

-

 a = Number of tissues examined from each group.

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental, reproduction and the developmental No-Observed-Adverse-Effect levels (NOAELs) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.
Executive summary:

Repeated dose toxicity X-19657 was studied in a combined 28 -Day repeated dose toxicity study with the reproduction/developmental toxicity screening test followed by a 14 -Day recovery period according to Guidelines and in accordance with GLP principles.

Wistar Han rats were treated with X-19657 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-56 days). The non-pregnant female was treated for 43 days. Additional animals of the control and 1000 mg/kg bw/day groups (5 rats/sex/group) were treated similarly (males for 29 days, females for 55 days), and were terminated after a treatment-free period of 14 days.

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

No parental toxicity was noted up to and including the highest dose level tested (1000 mg/kg bw/day).

Exposure to the test item up to and including 1000 mg/kg bw/day was well tolerated as evidenced by the absence of adverse changes in the parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, body weight, food consumption, clinical pathology, macroscopic examination, organ weights and microscopic examination). A few changes noted in treated rats were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals, in a dose-related manner, was considered a physiological response rather than a sign of systemic toxicity. Microscopic examination revealed a relative slight increase in the incidence and/or severity of follicular cell hypertrophy in the thyroid in males at 300 and 1000 mg/kg bw/day and in (lactating) females at all dose levels at end of treatment. After the 14-day recovery period, no follicular cell hypertrophy was seen in 1000 mg/kg bw/day (nulliparous) females whereas it was still present at increased incidence and severity in 1000 mg/kg bw/day males. Based on its low degree (minimal or slight), the increase in follicular cell hypertrophy in treated animals was regarded as nonadverse.

Females treated at 1000 mg/kg bw/day showed changes in red blood cell parameters, characterized by reductions in haemoglobin concentration, haematocrit and number of red blood cells, and increases in the number of reticulocytes and red blood cell distribution width (RDW). These changes occurred both in lactating females and nulliparous females. Lactating females additionally showed slightly increased plasma levels of bilirubin, suggestive of increased haemolysis of red blood cells. The increases in reticulocytes and RDW were reversible within the 14-day treatment-free period, whereas the magnitude of the reduction in haemoglobin, haematocrit and number of red blood cells was somewhat larger after the recovery period than at the end of the treatment period. As the reductions in haemoglobin concentration, haematocrit and number of red blood cells were less than 10% and values in treated females generally remained within the range of normal biological variation, the effect on red blood cells in 1000 mg/kg bw/day females was regarded as non-adverse.

A higher plasma level of cholesterol was observed in nulliparous 1000 mg/kg bw/day females at the end of the treatment period. Cholesterol levels in treated nulliparous females remained within the historical control range. Moreover, cholesterol in lactating females was not affected and there were no corroborative changes in other endpoints. Therefore, this isolated change, which showed full recovery during the 14-day treatment-free period, was considered nonadverse.

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated

in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 (PND 13-15) and macroscopic examination).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental, reproduction and developmental No-Observed-Adverse-Effect levels (NOAELs) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Batch No.of test material: X-019657-00-00
Pale yellow liquid, UVCB

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 261 and 298 g and females weighed between 200 and 245 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp
polycarbonate cage (Ancare corp., USA) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier. Periodic analysis of the water has been performed. It is considered that there were no known contaminants in the feed and in the water that would interfere with the objectives of the study.

For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 21.6
- Humidity (%): 51 to 77
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jun 2017 To: 25 Aug 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/v) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations (solutions at all dose levels) were prepared daily and dosed within 5 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. For determination of the concentration, samples were collected from all dose groups (one occasion). For determination of dose formulation homogeneity, samples were collected from low and high dose groups (one occasion). The homogeneity results obtained from the top, middle and bottom for the preparations of the low and high dose groups were averaged and utilized as the concentration results.All samples were stored on dry ice immediately after sampling. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70 °C until analysis. Analyses were performed by using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity analysis: Duplicate sets of samples were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males.
Main females were treated for 50 - 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 - 15 days after delivery, up to and including the day before scheduled necropsy. The Main female which failed to deliver was treated for 43 days.
Recovery females were treated for 55 days during the same period as Main females. The first day of dosing was designated as Day 1.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Due to an error in the formulation request, Group 4 animals were treated at a dose level of 1400 mg/kg bw/day, instead of the intended 1000 mg/kg bw/day, during the 14 days of the premating period and on the first day of mating.
No. of animals per sex per dose:
10 (main study); 5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finding study with oral exposure of X-19657 in the rat.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled necropsy (main and recovery groups)
- Animals fasted: Yes (o/n, with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: 5/sex/group (main groups) and all recovery males and females
- Parameters according to Guidelines were determined

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: 5 Main males and all Recovery males during week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6, 8 and 9), and all Recovery females were tested during the first day a Main female was tested.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: reproductive and developmental parameters (see sections 7.8.1. and 7.8.2.)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
-parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
-non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
-incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Salivation seen after dosing among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. shortly after dosing).
All other clinical signs noted occurred incidentally within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of dose response and/or slight change compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
Incidental statistically significant differences from the control group were considered unrelated to treatment due to the lack of similar findings at other time points (higher mean relative food consumption of 1000 mg/kg bw/day females between post-coitum Days 11-14) or higher dose levels (lower values in 100 mg/kg bw/day females during gestation and lactation).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in hematology parameters (red and white blood cell parameters, number of platelets), all in females treated at 1000 mg/kg bw/day, distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values compared to the control group are indicated between parentheses.
• Higher number of reticulocytes in lactating females (46%) and nulliparous females (26%) at the end of the treatment period. At the end of the recovery period, the numbers of reticulocytes in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Higher red blood cell distribution width (RDW) in lactating females (8%, not statistically significant) and nulliparous females (7%) at the end of the treatment period. At the end of the recovery period, RDW in nulliparous 1000 mg/kg bw/day females and controls did not differ significantly.
• Lower haemoglobin concentration in lactating females (8%) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haemoglobin concentration in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• Lower haematocrit in lactating females (5%, not statistically significant) and nulliparous females (4%) at the end of the treatment period. At the end of the recovery period, haematocrit in nulliparous 1000 mg/kg bw/day females was still lower than that of controls (9%) but statistical significance was not achieved.
• The number of red blood cells tended to be lower in lactating 1000 mg/kg bw/day females (6%) at the end of the treatment period and in nulliparous 1000 mg/kg bw/day females (7%) at the end of the recovery period but statistical significance was not achieved.
The above changes remained within the historical control range except for the higher number of reticulocytes in lactating and nulliparous females at the end of the treatment period, and reductions in haemoglobin concentration, haematocrit and number of red blood cells in nulliparous females at the end of the recovery period.
The reduced level of lymphocytes for high dose females at the end of the recovery phase was not considered toxicologically relevant as this change was not observed at the end of the treatment phase.
There were no treatment-related changes in hematology parameters in males. The statistically significantly lower mean haematocrit value of 300 mg/kg bw/day males at the end of the treatment period was considered unrelated to treatment due to the lack of a dose-related response.
Coagulation parameters (prothrombin time and activated partial prothrombin time) were considered not to be affected by treatment. Isolated statistically significant variations noted in coagulation parameters at the end of the treatment period were considered unrelated to treatment due to the lack of a dose-related response (lower prothrombin time (PT) in 100 mg/kg bw/day males) or a slightly high control value (lower activated partial thromboplastin time (APTT) in nulliparous 1000 mg/kg bw/day females).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lactating females treated at 1000 mg/kg bw/day showed a statistically significantly higher mean plasma level of total bilirubin at the end of the treatment period (relative difference from control mean: 17%). Bilirubin values in 1000 mg/kg bw/day females remained within the historical control range. No differences in bilirubin were noted in nulliparous females at the end of the treatment period or recovery period.
Nulliparous females treated at 1000 mg/kg bw/day showed a statistically significantly higher plasma concentration of cholesterol at the end of the treatment period (relative difference from control mean: 33%). This change was fully reversible and all values remained within the historical control range. The few other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response (cholesterol and inorganic phosphate in males) or lack of a test item-related change in the respective parameter at the end of the treatment period (alkaline phosphatase and inorganic phosphate in males, calcium in females).
Thyroid hormone analyses:
Serum levels of T4 in F0 males were unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Any statistically significant differences noted in absolute or relative organ weights were considered unrelated to treatment due to the lack of a dose-related response or lack of a test item-related change in the respective organ weight at the end of the treatment period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A test item-related microscopic change was noted in the thyroid gland of males and females (see 'any other information on results incl tables').
At the end of the treatment period, there was an increased incidence and/or severity of follicular cell hypertrophy in the thyroid of males from 300 mg/kg bw/day onwards and of females from 100 mg/kg bw/day onwards. After the 14-day recovery period, follicular cell hypertrophy was present at increased incidence and severity in 1000 mg/kg bw/day males. In 1000 mg/kg bw/day females (nulliparous), no follicular cell hypertrophy was observed at the end of the recovery period.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg instead of at the target dose level of 1000 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Dose formulation analyses

accuracy: The concentrations analyzed in the formulations of Groups 2 (low dose), 3 (mid dose) and 4 (high dose) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). Actual data are 100.7%, 101.4% and 100.4% in the low, mid and high dose group formulations, respectively. No test item was detected in the control group formulation.

homogeneity: The formulations of the low and high dose were homogeneous (i.e. coefficient of variation ≤ 10%). Actual data are 1.1% and 1.3% in the low and high dose formulations, respectively.

Summary Test Item-Related Microscopic Findings - Males

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

2

2

4

3

3

2

     Slight

1

1

1

2

-

3

a = Number of tissues examined from each group.

Summary Test Item-Related Microscopic Findings - Females

 

Main

Recovery

Dose level (mg/kg):

0

100

300

1000

0

1000

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

  Follicular cell hypertrophy

 

 

 

 

 

 

     Minimal

4

1

3

3

1

-

     Slight

-

3

2

2

1

-

 a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental No-Observed-Adverse-Effect level (NOAEL) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.
Executive summary:

Repeated dose toxicity X-19657 was studied in a combined 28 -Day repeated dose toxicity study with the reproduction/developmental toxicity screening test followed by a 14 -Day recovery period according to Guidelines and in accordance with GLP principles.

Wistar Han rats were treated with X-19657 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (for 50-56 days). The non-pregnant female was treated for 43 days. Additional animals of the control and 1000 mg/kg bw/day groups (5 rats/sex/group) were treated similarly (males for 29 days, females for 55 days), and were terminated after a treatment-free period of 14 days.

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

No parental toxicity was noted up to and including the highest dose level tested (1000 mg/kg bw/day).

Exposure to the test item up to and including 1000 mg/kg bw/day was well tolerated as evidenced by the absence of adverse changes in the parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, body weight, food consumption, clinical pathology, macroscopic examination, organ weights and microscopic examination). A few changes noted in treated rats were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals, in a dose-related manner, was considered a physiological response rather than a sign of systemic toxicity. Microscopic examination revealed a relative slight increase in the incidence and/or severity of follicular cell hypertrophy in the thyroid in males at 300 and 1000 mg/kg bw/day and in (lactating) females at all dose levels at end of treatment. After the 14-day recovery period, no follicular cell hypertrophy was seen in 1000 mg/kg bw/day (nulliparous) females whereas it was still present at increased incidence and severity in 1000 mg/kg bw/day males. Based on its low degree (minimal or slight), the increase in follicular cell hypertrophy in treated animals was regarded as nonadverse.

Females treated at 1000 mg/kg bw/day showed changes in red blood cell parameters, characterized by reductions in haemoglobin concentration, haematocrit and number of red blood cells, and increases in the number of reticulocytes and red blood cell distribution width (RDW). These changes occurred both in lactating females and nulliparous females. Lactating females additionally showed slightly increased plasma levels of bilirubin, suggestive of increased haemolysis of red blood cells. The increases in reticulocytes and RDW were reversible within the 14-day treatment-free period, whereas the magnitude of the reduction in haemoglobin, haematocrit and number of red blood cells was somewhat larger after the recovery period than at the end of the treatment period. As the reductions in haemoglobin concentration, haematocrit and number of red blood cells were less than 10% and values in treated females generally remained within the range of normal biological variation, the effect on red blood cells in 1000 mg/kg bw/day females was regarded as non-adverse.

A higher plasma level of cholesterol was observed in nulliparous 1000 mg/kg bw/day females at the end of the treatment period. Cholesterol levels in treated nulliparous females remained within the historical control range. Moreover, cholesterol in lactating females was not affected and there were no corroborative changes in other endpoints. Therefore, this isolated change, which showed full recovery during the 14-day treatment-free period, was considered nonadverse.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, including a 14-day recovery period, the parental No-Observed-Adverse-Effect level (NOAEL) of X-19657 was established to be at least 1000 mg/kg bw/day.

note: During the first 15 days of the exposure period, male and female parental rats were inadvertently treated at 1400 mg/kg bw/day instead of at the target dose level of 1000 mg/kg bw/day.