Pentanedioic acid, compd. with (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

21 February 1997 and 10 July 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: : recommendations of the Second ICH (ICH 2 1993) for a Fertility and Embryo-foetal development study and the Addendum on Toxicity to Male Fertility at the Third ICH (ICH 3 - 1995).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1592U89 Hemisulfate

IUPAC Name:

1592U89 Hemisulfate

Details on test material:

- Name of test material: 1592U89 Hemisulfate
- Chemical Name: (1S,4R)-4-[2Amino-6-(cyclopropyloamino)-9H-purin-9-yl]-2-cyclopentent-1-methanol hemisulfate
- Molecular formula: C14H18N6O (free base)
- Molecular weight: 286.336 (free base)
- Structural formula attached as image file (if other than submission substance): Please see Attachment 1.
- Lot/batch No.: A96L700/B5/R1/P3 and A96L701/B6/R1/P3
- Storage condition of test material: bulk drug: room temperature; dosing formulations: refrigerated (approx. 5°C), protected from light (amber bottles)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: P animals were approx. 8 weeks old, 8 x wks; (F1) x wks
- Weight at study initiation: (F0) Males: 286.0-339.6 g; Females: 191.1 -237.4 g; Naive females: 215.0 - 267.3 g;
- Fasting period before study: no
- Housing: Males and non-mated females were housed singly, mating pairs were housed as one male and one female, and inseminated F0 females were housed singly, in solid bottom polycarbonate cages with stainless steel wire lids. Untreated females were housed one-three per cage during quarantine, 1:1 with F0 males during mating, and one-three per cage with the same gestational day 0 date, during their gestation through gestational day 15.
- Diet: No. 5002 Purina Certified Rodent Chow was available ad libitum.
- Water: Tap water from the Durham, North Carolina water system was available ad libitum from plastic bottles with stainless steel sipper tubes for all animals during the quarantine and for all animals assigned to study.
- Acclimation period: yes, 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperature was between 18 and 26°C. In animal room 207 (F0 males), temperature was maintained within the range 20.6-24.3°C (69.1-75.8°F). In animal room 206 (F0 females) temperature was between 17.8 and 23.8°C (64.0-75.0°F). In animal room 203 (naive females) temperature remained in range 21-23.8°C (69.8-74.9°F). In animal room 205 (F0 recovery males and naive females) temperature remained in range 20.2-23.9°C (68.4-73.9°F).
- Humidity (%): 30-70%. In animal room 207 (F0 males), humidity was maintained within the range 38.2-70.0%. In animal room 206 (F0 females) humidity was between 35.5-63.8%. In animal room 203 (naive females) humidity remained in range 41.9-59.6%. In animal room 205 (F0 recovery males and naive females) humidity remained within the range apart from single occasions above the range which are believed not to affect the study results.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness

IN-LIFE DATES: From: 1997-02-24 To: 1997-07-10

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Methocel (mehtylcellulose, CAS No. 7732-18-5)

Details on exposure:

The test material, 1592U89 hemisulfate, was formulated in aqueous (deionized/distilled water, CAS No. 7732-18-5) 0.5% Methocel® (methylcellulose; CAS No. 9004-67-5) with the concentration determined by the following formula:

Concentration (mg/ml)= Dose per time (ml/kg) / Dosage volume (10.0 ml/kg)

Dosing formulations were stored in amber bottles under refridgeration prior to use. During use, a bottle of each concentration remained at room temperature and records of dates of use were maintained for each bottle.

Animals were dosed twice daily (morning and afternoon, at least six hours apart). The duration of dosing was as follows: F0 Parental Males: 70 days (10 weeks) prebreed and two 14 days (2 weeks) mating periods, for a total of 98 days (14 weeks); F0 Parental Females: 14 days (2 weeks) prebreed, 14 days (2 weeks) mating and 17 days of gestation (from gd 0 to gd 16) for a total of 45 days (6 weeks). Test and control animals were given a standard dosage volume of 10 mL/kg per time.

Details on mating procedure:

Parental Animals
F0 Mating and Gestation
Animals of the F0 generation were approximately eight (8) weeks of age at the commencement of dosing. Males were dosed for 10 weeks prior to mating, i.e., until they were approximately 18 weeks of age. Females were dosed for two (2) weeks prior to mating, i.e.. until they were approximately 10 weeks old. The animals were then mated on the basis of one male to one female selected randomly within each dose group for a period of 14 days, with no change in mating partners, and dosing continued for both sexes. All females (study and satellite) were examined daily during the cohabitation period by vaginal lavage for the presence of a copulation plug/sperm in the vaginal tract as evidence of successful mating. The day vaginal sperm/plugs were observed was designated gestational day (gd) 0 (Hafez, 1970). Once vaginal sperm or plug were observed, the male and female from that mating pair was individually housed. Dosing continued for each female through gd 16; each study female was terminated on gd 20 (and each satellite female was terminated on gd 16). Dosing continued for each male through both complete mating periods (regardless of whether he had inseminated a female) for a total of at least98 dosing days; 16 F0 males/group were terminated within 24 hours after the last dose (the remaining F0 males/group were retained for recovery). Any study female that did not show evidence of successful mating after 14 days of cohabitation was dosed for no more than 17 days after the last day of cohabitation (designated gd 0) and weighed and feed consumption measured appropriately until termination. If a female without a confirmed gd 0 was, in fact pregnant (based on weight gain and/or palpation), she was euthanized prior to parturition and her gestational information was collected.

Naive (untreated females)
F0 study males were approximately eight weeks of age at the commencement of treatment. They were administered test material by gavage at their respective doses for 10 weeks of prebreed exposure and for the two-week mating period to F0 study females. The F0 study males were then cohabited on the basis of one treated male to one naive female for two weeks (14 days) with dosing of the F0 males continuing. Females were examined each morning for the presence of sperm and/or copulation plug in the vaginal tract. The observation of vaginalsperm or a vaginal copulation plug was considered evidence of successful mating. and the date of evidence of copulation was designated gestational day (gd) 0 (Hafez, 1970). Once the male had inseminated the female or the period is completed, the male was individnally housed with dosing continuing until scheduled sacrifice of 16 F0 males/group or with dosing discontinued after the dosing period for the recovery period for eight F0 males/group. The female was housed with up to two other females from the same gestational date, mated to males from the
same dose group. For any female which did not show evidence of successful mating after the two-week cohabitation, the last morning of cohabitation was considered gd 0 for that female and the animal was treated accordingly for subsequent events.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Nominally pure drug substance, 1592U89 hemisulfate in vehicle, was administered to three treatment groups at doses of 60, 160 or 500 mg 1592U89 hemisulfute/kg/day in two doses in the morning and afternoon at least six hours apart. One group received vehicle alone twice daily. The concentration and dosages of the test material are defined as mg 1592U89 hemisulfate/ml, and mg 1592U89 hemisulfate/kg, respectively, with no correction for the hemisulfate salt or other possible contaminants of the drug substance.
Analysis of the dosing formulations for homogeneity, stability and drug concentration are the responsibility of the Sponsor.
Analytical results were to be considered satisfactory if they were within the 10% range of nominal concentrations. The performing laboratory has no official
written knowledge of the methods or results of the analysis and is not responsible for the GLP compliance ofanalyses of the dosing preparations.

Duration of treatment / exposure:

F0 Parental Males: 70 days (10 weeks) prebreed and two 14 days (2 weeks) mating periods, for a total of 98 days (14 weeks);
F0 Parental Females: 14 days (2 weeks) prebreed, 14 days (2 weeks) mating and 17 days of gestation (from gd 0 to gd 16) for a total of 45 days (6 weeks). Test and control animals were given a standard dosage volume of 10 mL/kg per time.

Frequency of treatment:

Animals were dosed twice daily (morning and afternoon, at least 6 hours apart).

Details on study schedule:

Study Groups
The study began with 24 F0 parental animals/sex/group and yielded 23-24 pregnant females/group at scheduled sacrifice and 2-4 pregnant F0 females and 4 F0 males per group as satellite animals. The study was conducted with three (3) treatment groups and one (1) vehicle control group. In addition, there were 28 naive females per group, which were not dosed. Nulliparous animals (F0 parental males and females and naive females) were assigned to the different groups by means of randomization stratified by body weight such that the body weights of all groups were homogeneous by sex at study initiation (F0 parental study and satellite animals) or at the start oflbe second mating of F0 males (naive, untreated females).

Since the start and duration of the prebreed exposure period were different for the F0 males and F0 females, they were assigned study days (sd) based on the start of the exposure period. Once the F0 females were inseminated, they were tracked by gestational days (gd); the F0 males continued to be tracked by study day until scheduled temtination.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
60 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
160 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level for this study was chosen to induce overt parental toxicity, but not to cause a weight loss greater than 20% when compared to concurrent controls, nor to cause greater than 10% parental mortality. This dose was selected by the Sponsor based on data from other studies performed in this laboratory as well as from studies performed elsewhere. The low dose was selected to be a parental/developmental "no observable adverse effect level" (NOAEL). The mid dose was determined as a fraction of the selected high dose.

Examinations

Parental animals: Observations and examinations:

PARENTAL ANIMALS (F0)
CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
Observations for mortality were made twice daily (a.m. and p.m.). The general condition ofall animals was checked daily. Clinical examinations were conducted and recorded at least once daily throughout the course of the study. and at least twice daily, at each dosing, during the time the animals were dosed. This record included the time of onset, degree and duration of symptoms. These cage-side observations included, but were not be limited to, changes in: skin and fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity and behavior pattern.

BODY WEIGHT: Yes
The body weights of the male F0 rats were determined in the morning and recorded initially and then every third day through mating of the F0 and naive females. They were also weighed on the day of terminal sacrifice. The body weights of female F0 rats were recorded in the same manner until the start of the mating period. From the start of the mating period, each female was weighed daily through mating and gestation, and on the day of terminal sacrifice. Body weight gains were computed.

FOOD CONSUMPTION:
Feed consumption measurements were recorded in the morning for all F0 parental males every three (3) days and for all F0 parental females on study day 1, 4, 7, 10 and 13, throughout the prebreed treatment period which encompassed 10 weeks for males and 14 days for females. Feed consumption collection periods corresponded with the collection of the animals' body weight data and were employed to calculate feed intake in g/day and g/kg/day. During pregnancy of F0 females, feed consumption was recorded in the morning on gd 0,.3, 6, 9, 12, 15, 18 and 20. Feed consumption was not measured during the period of cohabitation since two adult animals (one male and one female) were in the same cage, but consumption was visually monitored and the feed was supplemented or changed as necessary.

OTHER:
F0 Male Recovery Animals
A maximum of eight (8) F0 study males per group, randomly selected, were not sacrificed within 24 hours of the last dose after the second mating, but were retained, with no dosing, for a recovery period of four weeks. During this recovery period, the males were singly housed with feed and water ad libitum. They were examined for clinical signs once daily, with body weights and feed consumption recorded weekly. At the time of sacrifice of these animals, at the end of the recovery period, they were subjected to a gross necropsy with retention of selected organs. No effects on seminology (andrologic assessments) were observed in the F0 males sacrificed immediately after the last dose, so these recovery males were not subjected to andrologic assessments.

NAIVE (UNTREATED) FEMALES
CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
Observations for mortality and clinical examinations were conducted and recorded at least once daily throughout the mating and gestational period through gestational day 15. This record included the time of onset, degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity and behavior pattern.

BODY WEIGHT: Yes
Females were weighed prior to pairing (to distribute them into groups), on gd 0 (date of detection of vaginal sperm or copulation plug), and on gd 15 (date of scheduled sacrifice).

FOOD CONSUMPTION:
Feed- consumption was not measured, but it was monitored visually and feed added and/or replaced as necessary.

Oestrous cyclicity (parental animals):

For at least 12 consecutive days prior to the start of mating, study females (excluding satellite females for absorption) were evaluated for estrous cyclicity by taking vaginal smears of sloughed cells once daily. The slides containing the daily smears were stained and subsequently analyzed for the status of the estrous cycle. Recording of cycle stages continued into the mating period but was not be formally analyzed.

Sperm parameters (parental animals):

At the time of sacrifice of F0 parental males, 16 per group within 24 hours of the last dose, one cauda epididymis (the right one) was immediately removed, weighed and seminal fluid from the cauda was assessed for sperm number and motility utilizing an HTM-IVOS computer assisted sperm analysis system (Hamilton-Thorne Research, Beverly, MA) and sperm morphology. Sperm motility was assessed immediately after necropsy; sperm number and sperm morphology (at least 200 sperm per male, if possible) was evaluated at a later date using stained sperm. (The left epididymis per male was retained in fixative for possible subsequent histopathologic examination.) Since no effects were observed in the andrologic assessments of the F0 paternal animals examined immediately after mating, these same assessments were not performed on the F0 recovery animals at sacrifice.

Litter observations:

Not applicable.

Postmortem examinations (parental animals):

PATERNAL AND RECOVERY MALES
SACRIFICE
Within 24 hours of the last dose (after the second mating period), 16 of the surviving paternal animals per group were killed by CO2 asphyxiation, weighed and subjected to a complete gross necropsy.

GROSS NECROPSY
Paternal animals that died during the course of the study were necropsied in an attempt to determine cause of death, with testes, epididymides, prostate, seminal vesicles. pituitary and gross lesions saved. Males that appeared moribund were humanely euthanized by CO2 asphyxiation and necropsied to determine the cause of the morbidity, if possible, with tissues saved as described above. Within 24 hours of the last dose (after the second mating period), 16 of the surviving paternal animals per group were killed by CO2 asphyxiation, weighed and subjected to a complete gross necropsy. The gross necropsy included examination of the external surfaces; all orifices; carcass; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. Testes, seminal vesicles with coagulating glands, epididymides and prostate were weighed (paired organs were weighed individually). Gross lesions and selected organs (testes. epididymides, prostate, seminal vesicles with coagulating glands, pituitary) were retained in appropriate fixative for subsequent histopathologic examination by the Sponsor.

F0 MATERNAL FEMALES
SACRIFICE AND GROSS NECROPSY
No maternal animals died, were moribund, or showed signs of abortion or premature delivery. On gd 20, all maternal animals were killed by CO2 asphyxiation, weighed, thoracic and abdominal cavities and organs were examined, and pregnancy status was confirmed by uterine examination. The liver and uterus of each sperm-positive female were weighed. The ovarian corpora lutea were counted and uterine contents (i.e., number of implantation sites, resorptions, dead conceptuses, live conceptuses) were recorded. Ovaries, uterus, pituitary and any gross lesions were retained in appropriate fixative after gross examination for possible subsequent histologic examination by the Sponsor.

NAIVE (UNTREATED FEMALES)
Each mated female was weighed and sacrificed by carbon dioxide asphyxiation at midpregnancy, on gd 15. The gravid uterus with attached oviducts and one ovary (to ensure correct orientation ofthe uterus during examination) was dissected free and removed. The total number of ovarian corpora lutea were counted. Total implantation sites were also counted and recorded. The status of each implantation site was documented: resorption (early, late and/or total) or live implantation. After the above examination was completed and all data for each female were recorded and verified, each female and all her tissues were discarded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Retained tissues from F0 parental males (taken at sacrifice within 24 hours ofthe last dose for 16 per group and taken at sacrifice after the recovery period for eight per group) and for F0 parental females (taken at sacrifice on gd 20) were preserved as follows: for the males, prostate, seminal vesicles with coagulating glands, pituitary and any gross lesions (except eyes) were fixed in buffued neutral 10% formalin; both testes (recovery males) and the left epididymis
(and any eyes with gross lesions) were fixed in Bouin's solution for 24 hours, rinsed for 20 minutes in tap water and then transferred to 70% ethyl alcohol. In a deviation from the protocol, the testes of F0 study males were inadvertently preserved in 10% formalin instead of Bouin's solution. All retained female tissues were preserved in buffered neutraI 10% formalin (except for any eyes with gross lesions which were retained in Bouin's). After the Bouin's fixed tissues remained for at least 24 hours in 70% ethyl alcohol. most of the alcohol was poured off and the tissues with ethanol and the formaIin-fixed tissues were transferred into heat-sealed bags appropriately labelled and transferred to Ms. Barbara Munch, MSE, Glaxo Wellcome, Inc., RD930l2, Five Moore Drive, Research Triangle Park, NC 27709, with appropriate transfer of custody sheets. These tissues may be subjected to histopathologic examination by the Sponsor; the results of these examinations will be retained by the Sponsor.

Postmortem examinations (offspring):

SACRIFICE, GROSS NECROPSY AND HISTOPATHOLOGY
Live F1 fetuses (from F0 male and F0 female mating) were dissected from the uterus and immediately placed on a moist paper towel over a tray of ice, a procedure which induces anesthesia by lowering the core body temperature below 25°C (Lomb and Jones, 1973; Blair, 1979). All live fetuses were weighed individually and were examined for external morphological abnormalities, including cleft palate. Each placenta was also weighed individually. All live fetuses per litter were examined for visceral malformations and variations and sex determined using a fresh tissue dissection method (Staples, 1974; Stuckhardt and Poppe, 1984). Approximately half of the fetuses per litter were decapitated prior to dissection, and the heads were fixed in Bouin's solution for free hand sectioning and examination (Wilson, 1965). All fetal carcasses were eviscerated, macerated and stained with alcian blue/alizarin red S and approximately half of the fetuses per litter (intact, not decapitated) were examined for skeletal malformations and variations (Marr e/ aI., 1988). Malformations were documented with photographs and/or sketches if an abnormality was unusual and/or a written description alone was inadequate; there were no unusual malformations. All maternal and non-retained fetal organs and maternal carcasses were destroyed by incineration. All stained fetal carcasses (those examined and those not examined) were stored in glycerin:70% ethanol (1:1); fetal head sections were stored in 70% ethanol after examination.

Statistics:

The unit of comparison was the male, the female, the pregnant female or the litter, as appropriate. Mating and gestational data for the mated naive females were related to the appropriate study male. Quantitative continuous data (e.g., parental body weights, feed consumption, fetal body weights, placental weights, naive female gestational data, etc.) were compared among the three treatment groups and the one vehicle control group by the use of Bartlett's test for homogeneity of variances.

Please see "Any other information on materials and methods" for further information.

Reproductive indices:

Twenty-five (25) to twenty-eight F0 males per group were paired with F0 dosed females in the same group. Since seven F0 males died or were sacrificed moribund during the premating period, two at 0 mg/kg/day, three at 160 mg/kg/day and two at 500 mg/kg/day, and no F0 females died during this period, there were not 28 males available in each group to pair with the 28 females available in each group. Therefore, initially, all available males, 26, 28, 25 and 26, were paired with females, at 0, 60, 160 and 500 mg/kg/day, respectively. Once a female was found sperm-positive at 0, 160 and 500 mg/kg/day, the successful male was paired with an initially unpaired female within the same dose group, until all females had been paired with males. This action resulted in pregnancy indices (defined as number of pregnant females/number of males that mated) greater than 100.0%.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Please see details below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please see details below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Please see details below.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please see details below.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Please see details below.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Please see details below.

Reproductive performance:

no effects observed

Description (incidence and severity):

Please see details below.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

F0 MALES AND SATELLITE MALES:
Twenty-eight (28) F0 males per group were placed on study. Twenty-four (24) males per group were designated as F0 males. and four (4) males per group were designated as satellite males. Eight males died or were euthanized moribund during the study with seven dying or sacrificed moribund prior to the mating (on sd 70) with F0 females, as follows: at 0 mg/kg/day, one male was euthanized moribund on sd 45 (male no. 217) from a suspected misdirected dose (technician intubation error), and one male was found dead on sd 68 (male no. 61), cause of death unknown; at 60 mg/kg/day, there were no F0 male deaths; at 160 mg/kg/day, three males were euthanized moribund, one each on ad 38 (no. 117), on ad 39 (no. 59), and on sd 61 (no. 165), all from suspected misdirected doses (technician intubation error); and at 500 mg/kg/day, two males were euthanized moribund, one each on sd 37 (no. 11) and on sd 66 (no. 195), both from suspected misdirected dose (technician intubation error). In addition, one male (no. 161) was found dead on sd 87 after the initial mating to F0 females and after he had inseminated "his" naive female during the second mating period, cause of death unknown. Rooting in bedding post-dosing was observed in all drug-exposed groups on study days (sd) 2 through 14, and in the low dose group on sd 15. On sd 2 through 11, the incidence of this finding was dose related; for example, 4, 17 and 23 F0 males exhibited rooting in bedding post-dosing on ad 2 at 60, 160 and 500 mg/kg/day, respectively. This clinical sign is NOT interpreted as toxicity per se, but as an indication of taste aversion. It is a common finding in studies with oral dosing as the route of administration. Other clinical observations appeared to be unrelated to treatment.
F0 FEMALES AND SATELLITE FEMALES:
Twenty-eight (28) F0 females per group were placed on study. Twenty-four (24) females per group were designated as F0 dosed females scheduled for sacrifice on gd 20. Four (4) females per group were designated as satellite females scheduled for sacrifice on gd 16. No females died or were removed from study during this period. Rooting in bedding postdosing was observed in all drug-exposed groups on sd 1, 2, 5, and 6, and in the 160 and 500 mg/kg/day dose groups on sd 3 and 4. The incidence of this finding was dose related on sd 1 (1,3, and 9 F0 females at 60, 160 and 500 mg/kg/day, respectively), and on sd 3 (5 and 8 F0 females at 160 and 500 mg/kg/day, respectively). Other clinical observations appeared to be unrelated to treatment.
NAIVE FEMALES:
Twenty-five (25) to twenty eight (28) naive females per group were mated to F0 dosed males after their initial mating to F0 dosed females, and were scheduled for sacrifice at mid pregnancy, on gd 15. No naive females died or were removed from study during this period. Clinical observations were notable for only one naive female. That female, mated to a 60 mg/kg/day F0 male, exhibited scabs between the eye and ear. This clinical observation was unrelated to treatment.

BODY WEIGHT (PARENTAL ANIMALS)

F0 MALES AND SATELLITE MALES:
F0 male and satellite male body weights were equivalent across all groups for the entire study. Male body weight changes were equivalent across all groups for all intervals up to sd 21. F0 male body weight change exhibited a significant increase at 160 mg/kg/day on sd 21-24, and sd 24-27, and at 500 mg/kg/day on sd 30-33 and 33-36. Significant decreases in F0 male body weight change were observed at 500 mg/kg/day on sd 27-30, sd 36-39. and sd 78-81. F0 male body weight change exhibited significant decreases in all drug-exposed groups on sd 75-78. For the entire premating period, sd 0-69, there were no differences among groups for body weight change.
F0 MALES AND NAIVE FEMALES:
F0 male body weights were equivalent across all groups during the mating period to naive females (sd 87-99). Male body weight changes were equivalent across all groups for all intervals from sd 87 to sd 99.
The sacrifice body weights of the F0 males, 16/group sacrificed within 24 hours of the last dose after the second mating (to naive females), were statistically equivalent across all groups, although there was a significant (p<0.05) trend; the mean body weight at 500 mg/kg/day was 96.20% of the mean control value (NOT statistically significantly different).

BODY WEIGHT (PARENTAL ANIMALS): PREMATING PERIOD
F0 FEMALES AND SATELLITE FEMALES:
F0 dosed female and satellite female body weights were equivalent across all groups for the premating period. Female body weight changes were also equivalent across all groups for all intervals during the premating period.

BODY WEIGHT (PARENTAL ANIMALS): GESTATION
F0 FEMALES AND SATELLITE FEMALES:
F0 dosed female and satellite female body weights during gestation were equivalent across all groups. F0 maternal body weight changes during gestation were equivalent across all groups on gestational days (gd) 0-3, 3-6, 9-12, and 18-20. In addition, maternal body weight change was equivalent across all groups for the whole gestational period, gd 0-20, and for corrected maternal body weight change (maternal gestational body weight change minus gravid uterine weight). On gd 6-9, F0 maternal body weight change exhibited significant decreases at 60 and 160 mg/kg/day but not at 500 mg/kg/day. On gd 12-15, significant decreases in maternal body weight gain were observed in the 160 and 500 mg/kg/day dose groups. Maternal body weight gain was also significantly decreased in the 500 mg/kg/day dose group on gd 15-18. At scheduled sacrifice on gd 20, mean gravid uterine weight was significantly reduced at 500 mg/kg/day and mean maternal absolute and relative liver weights were both significantly increased at 500 mg/kg/day.
NAIVE FEMALES:
Naive female body weights were equivalent across all groups for measurements taken on gd 0 and on gd 15. Body weight changes for naive females during gestation to gd 15 were equivalent across all groups.

FOOD CONSUMPTION (PARENTAL ANIMALS)

F0 MALES AND SATELLITE MALES:
F0 male and satellite male feed consumption during the premating period. expressed as g/day, was unaffected at 60 and 160 mg/kg/day for all intervals. F0 male feed consumption as g/day at 500 mg/kg/day was significantly decreased for sd 0-3, but was significantly increased for sd 33-36, 48-51, 51-54, and was unaffected for all other intervals during the premating period. When the data were expressed as g/kg/day, F0 male feed consumption was significantly decreased at 500 mg/kg/day on sd 0-3. It was significantly increased at 500 mg/kg/day for sd 6-9, 9-12, 12-15, 15-18, 24-27, 27-30, 33-36, 45-48, 48-51, 51-54,54-57,60-63,63-66,66-69, and sd 0-69 (premating period); it was significantly increased at 160 mg/kg/day for sd 12-15, 15-18, 24-27, and 33-36; and significantly increased at 60 mg/kg/day for sd 15-18,24-27,33-36,45-48,60-63, and 0-69 (premating period).

FOOD CONSUMPTION (PARENTAL ANIMALS): PREMATING PERIOD
F0 FEMALES AND SATELLITE FEMALES:
F0 female (F0 dosed females and satellite females) feed consumption dnring the premating period (sd 0 to 12), expressed as g/day, was equivalent across all groups for all intervals. When the data were expressed as g/kg/day, feed consumption was increased at 60 mg/kg/day for sd 0-3, but was equivalent across all groups for all other intervals during the premating period.

FOOD CONSUMPTION (PARENTAL ANIMALS): GESTATION
F0 FEMALES AND SATELLITE FEMALES:
F0 female feed consumption during gestation (gd 0-20 for study females and gd 0-16 for satellite females), expressed as g/day, was increased at 500 mg/kg/day for gd 9-12, and 18-20, but was equivalent across groups for all other intervals during gestation. When the data were expressed as g/kg/day, feed consumption was significantly increased at 500 mg/kg/day for gd 9-12,18-20, and gd 0-20 (gestational period), and at 160 mg/kg/day for gd 18-20. F0 maternal feed consumption (g/kg/day) was unaffected at 60 mg/kg/day.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 FEMALES AND SATELLITE FEMALES:
Both the stages and the duration of the cycle were examined. Twenty-four (24) F0 dosed females per group were examined for vaginal cytology to evaluate estrous cyclicity. All of the females in each group were cycling. and all had normal cycles. Mean cycle length was 4.09. 4.09. 3.95, and 4.10 days for the 0, 60, 160, and 500 mg/kg/day groups, respectively. There were no effects of treatment on any estrous cycle parameter.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no effect of treatment on the percent motile sperm, percent progressively motile sperm, epididymal sperm concentration, or percent abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
REPRODUCTIVE INDICES
F0 MALES AND SATELLITE MALES:
The mating index was high and equivalent across all groups (92.2-1000/0). In addition, the fertility index was high (96.2-100%), with 26, 25, 25, and 26 males siring litters at 0, 60, 160, and 500 mg/kg/day, respectively. The pregnancy index was 107.7, 96.2, 112.0, and 107.7% at 0, 60, 160, and 500 mg/kg/day, respectively. The male fecundity index was also high and equivalent across groups, 100.0, 89.3, 100.0 and 100.0% at 0, 60, 160 and 500 mg/kg/day respectively.
F0 MALES AND NAIVE FEMALES:
Twenty-five (25) to twenty-eight F0 males were paired and mated with naive females. One male (no. 161) at 500 mg/kg/day was found dead on gd 87 after he had inseminated "his" naive female during this second mating, so he is included in the reproductive indices. The mating index was high and equivalent across
all groups (96.4-100%). In addition, the fertility index was high (96.3-100%), with 26, 26, 25, and 26 males siring litters at 0, 60, 160, and 500 mg/kg/day, respectively. The fecundity index was also high and equivalent across groups, 100.0, 92.9, 100.0 and 100.0% at 0, 60, 160 and 500 mg/kg/day, respectively. The pregnancy index was 100.0, 96.3, 100.0, and 100.0 at 0, 60, 160, and 500 mg/kg/day, respectively.
F0 FEMALES AND SATELLITE FEMALES:
Twenty-eight (28) F0 dosed females per group (including satellite females) were placed on study. The mating index was high and equivalent across all groups (92.9-100%). In addition, the fertility index was high and equivalent across all groups (96.2-100.0%), resulting in 28, 25, 28, and 28 pregnant females at 0, 60, 160, and 500 mg/kg/day, respectively. The fecundity index was also high and equivalent across groups, 100.0, 89.3, 100.0 and 100.0% at 0, 60, 160 and 500 mg/kg/day, respectively. There were no effects of treatment on F0 female reproductive indices.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 MALES:
There were no significant treatment-related changes in paired testes weight, left epididymis weight, prostate weight, or the weight of seminal vesicles with coagulating gland. When F0 male reproductive organ weights were expressed relative to terminal body weights, there were also no differences among dose groups. There was a significant (p<0.01) dose-related upward trend for relative left epididymis weight, in the absence of any significant pairwise comparisons to the control group value.

GROSS PATHOLOGY (PARENTAL ANIMALS)

F0 MALES:
F0 males that were found dead or sacrificed moribund (i.e., died on study; unscheduled deaths) exhibited no significant treatment-related necropsy findings. All findings were consistent with misdirected dose (technician gavage error). In addition, no treatment-related findings were .observed for the F0 study males, I6/group, at scheduled sacrifice.
F0 FEMALES AND SATELLITE FEMALES:
All F0 dosed females (24/group) and satellite females (4/group) survived to scheduled sacrifice (gd 20 for F0 dosed females; gd 16 for satellite females) and there were no treatment-related findings at necropsy. One satellite female in the 60 mg/kg/day group had a uterus that was slightly distended with fluid. One F0 dosed female in the control group had hydronephrosis of the right kidney. At 500 mg/kg/day, maternal absolute liver weight (in grams) and liver weight relative to terminal body weight were significantly increased, and gravid uterine weight was significantly decreased, most likely due to reduced fetal body weights at this dose.
NAIVE FEMALES:
All naive females (25-28/group) survived to scheduled sacrifice on gd 15 and there were no treatment-related findings at necropsy. At scheduled sacrifice, one naive female mated to a male in the 160 mg/kg/day group had an enlarged spleen, and one female mated to a male in the control group had thick, brown mucus in the vagina.

HISTOPATHOLOGY (PARENTAL ANIMALS)

F0 FEMALES AND SATELLITE FEMALES:
UTERINE PARAMETERS
The number of ovarian corpora lutea (a measure of the number of eggs ovulated), and of uterine implantation sites and therefore preimplantation loss were unaffected by treatment. The incidence of resorptions per litter and the number of litters with one or more resorptions were significantly increased at 500 mg/kg/day. Resorptions were exclusively limited to early resorptions (evidence of metriaI glands or with maternal and/or fetal placental remnants with no embryo/fetal tissue) in all groups. In addition, one female at 500 mg/kg/day (dam no. 136) also carried a dead fetus on gd 20 (defined as a conceptus greater than 0.800 grams in weight for rats with no vital signs and discernible digits on limbs). Because of the increase in resorptions, the incidences of nonlive implants per litter (defined as resorptions plus dead fetuses) and of adversly affected implants per litter (defined as nonlive plus malformed implants) were also significantly increased at 500 mg/kg/day. Also because of the increase in resorptions, percent postimplantation loss was significantly increased at 500 mg/kg/day. The number of live fetuses per litter was significantly reduced at 160 mg/kg/day (but not at 500 mg/kg/day), due to the significant decrease in the number of implantation sites per litter at 160 mg/kg/day and to the slightly (not statistically significantly) reduced number of corpora lutea per dam at 160 mg/kg/day. As these parameters were not significantly decreased at 500 mg/kg/day, the findings of decreased numbers of implantations, and therefore of live fetuses per litter at 160 mg/kg/day, are not considered to be related to treatment with 1592U89 hemisulfate. Sex ratio (% male fetuses) was unaffected by treatment. Mean fetal body weight per litter, for all fetuses and separately by sex, was significantly reduced at 500 mg/kg/day; mean placental weight per litter, for all fetuses, or separately by sex, was equivalent across all groups.
UTERINE PARAMETERS: NUMBER OF CORPORA LUTEA
The number of corpora lutea per F0 dosed dam was statistically equivalent across all groups although slightly reduced at 160 but not at 500 mg/kg/day. These data did not include the satellite females.
UTERINE PARAMETERS: NUMBER OF IMPLANTATIONS
The number of implantation sites per litter for F0 females was significantly decreased at 160 mg/kg/day, but was unaffected at 60 and 500 mg/kg/day. These data included the satellite females.
UTERINE PARAMETERS: PREIMPLANTATION LOSS
The percent preimplantation loss per litter was not significantly affected by drug treatment. These data did not include the satellite females.
UTERINE PARAMETERS: POSTIMPLANTATION LOSS
The percentage post-implantation loss per litter was significantly increased at 500 mg/kg/day, but was not affected at 60 or 160 mg/kg/day. Data from the satellite females were included in this parameter.

NAIVE FEMALES:
UTERINE PARAMETERS
There were no effects of treatment to the males on any gestational parameters in naive females. There were no effects on pre- or postimplantation loss, and the calculated dominant lethal factors, FL% (Ehling et al., 1978; EPA, 1982; Green et aI., 1985) were obviously unaffected by the male exposures, equaling 1.43, -2.79 and -0.83 at 60, 160 and 500 mg/kg/day, respectively.
UTERINE PARAMETERS: NUMBER OF CORPORA LUTEA
The numbers of ovarian corpora lutea per naive female were equivalent across all groups.
UTERINE PARAMETERS: NUMBER OF IMPLANTATIONS
The number of implantation sites per litter for naive females was equivalent across all groups.
UTERINE PARAMETERS: PREIMPLANTATION LOSS
The percent preimplantation loss per litter was not significantly affected by drug treattnent.
UTERINE PARAMETERS: POSTIMPLANTATION LOSS
The percent postimplantation loss per litter was equivalent across all groups.
UTERINE PARAMETERS: NUMBER OF LIVE IMPLANTS
The number of live implants per litter was equivalent across all groups.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects body weight; food consumption; number of implantation sites; litter weight;

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Please see details below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please see details below.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Please see details below.

Histopathological findings:

not examined

Details on results (F1)

F0 FEMALES AND SATELLITE FEMALES
VIABILITY (OFFSPRING)
NUMBER OF LIVE FETUSES
The number of live fetuses per litter was significantly reduced at 160 mg/kg/day, but was not significantly affected at 60 or 500 mg/kg/day. Data from the satellite females were included in this parameter.
PERCENT OF MALE LIVE FETUSES
The percentage of male live fetuses per litter was equivalent across groups. In the 0, 60, 160, and 500 mg/kg/day groups, 45.36, 50.75, 50.26, and 49.21% male fetuses were observed, respectively. Data from the satellite females were not included in this parameter, since the satellite females were necropsied on gd 16, prior to term on gd 20 with no fetal evaluations performed.

BODY WEIGHT (OFFSPRING)
FETAL BODY WEIGHT
Average fetal body weight per litter (sexes combined or separately) was significantly decreased at 500 mg/kg/day, but was not affected at 60 or 160 mg/kg/day. Data from the satellite females were not included in this parameter, since the satellite females were necropsied on gd 16, prior to term on gd 20.
PLACENTAL WEIGHT
Mean placental weight was equivalent across all groups and was not affected by treatment. Data from the satellite females were not included in this parameter, since the satellite females were necropsied on gd 16, prior to term.on gd 20.

GROSS PATHOLOGY (OFFSPRING)
FETAL EXAMINATION
A total of 311, 318, 302, and 281 fetuses were examined for external and visceral malformations and variations in the 0, 60, 160, and 500 mg/kg/day groups, respectively. A total of 157, 160, 152 and 137 fetuses (approximately 50% per litter) were examined for skeletal malformations and variations in the 0, 60, 160 and 500 mg/kg/day groups, respectively. These fetuses comprised the 22, 23, 24, and 22 litters examined from the 0, 60, 160, and 500 mg/kg/day groups, respectively.
MAJOR ABNORMALITIES
The incidence ofexternal malformations was 0/311, 0/318, 0/302, and 1/281 fetuses in the 0, 60, 160, and 500 mg/kg/day groups. The one fetus in the 500 mg/kg/day group with an external malformation had an umbilical hernia. Visceral malformations were observed in 7/311, 10/318,8/302, and 13/281 fetuses in the 0, 60, 160, and 500 mg/kg/day groups. They included situs inversus, hydronephrosis (left, right, and bilateral), and hydroureter (left, right, and bilateral). Hydronephrosis and hydroureter are common findings in control CD® rat fetuses at term. No skeletal malformations were observed in this study. The percent malformed fetuses per litter (external, visceral, and skeletal malformations) was equivalent across groups and was not affected by treatment. There was no difference in the incidence of malformations between male and female fetuses.
MINOR DEFECTS (VARIATIONS)
The incidence ofexternal variations was 1/311, 0/318, 0/302, and 1/281 fetuses in the 0, 60, 160, and 500mg/kg/day groups. The fetus in the control group and the fetus in the 500 mg/kg/day group both had hematomas. Visceral variations were observed in 37/311,37/318,37/302, and 49/281 fetuses in the 0, 60, 160, and 500 mg/kg/day groups. They included enlarged lateral ventricle of the cerebrum, cysts in the lower jaw, agenesis of the innominate artery, extra tissue on median liver lobe, and distended ureter. Skeletal variations were observed in this study in 15/157, 8/160, 6/152, and 9/137 fetuses in the 0, 60, 160, and 500 mg/kg/day groups. The skeletal variations observed included: misaligned sternebrae, extra rib on the lumbar I vertebra, short rib XIII, and variations in the ossification of thoracic or lumbar centra, all common fmdings in control CD® rat fetuses at term. The percent fetuses per litter with variations (external, visceral, and skeletal variations) was equivalent across groups and was not affected by treatment. There was no difference between the incidence of variations between male and female fetuses.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects number of implantation sites; litter weight;

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Frequency of dominant lethal factors

The frequency of dominant lethal factors was equivalent across all groups. Exposure of the males did not cause a dominant lethal effect.

Recovery males

After the 16 F0 males/group were selected for necropsy within 24 hours after the last dose (after the second mating), remaining F0 males, five to eight per group, were designated recovery males and were retained for four weeks with no dosing.

Clinical observations

No F0 recovery males died prior to scheduled necropsy. One animal in the 160 mg/kg/day group exhibited alopecia. There were no other clinical observations.

Body Weight

Recovery male body weights were equivalent across all dose groups for sd 101-129. Body weight change was also unaffected.

Feed Consumption

Recovery male feed consumption (g/day) during intervals from sd 101 to sd 129 was unaffected. When feed consumption was expressed as g/kg/day, feed consumption was increased in the 60 and 500 mg/kg/day groups, but not the 160 mg/kg/day group for sd 108 -115, and was unaffected during other intervals.

Terminal Examination of Recovery Males

Gross Necropsy of Recovery Males

Gross necropsy findings were limited to kidney stones, with enlarged kidney and nephrosis in one animal in the control group and urinary bladder stones in one animal in the 60 mg/kg/day group.

Body weight

The sacrifice body weights of the recovery males were equivalent across all groups.

Reproductive organ weights

There were no significant treatment related changes in paired testes weight, left epididymis weight, prostate weight, or the weight of seminal vesicles with coagulating gland. When F0 male reproductive organ weights were expressed relative to terminal body weights, there were also no differences among dose groups.

Seminology

Since there were no effects of treatment on seminology parameters for the F0 males sacrificed within 24 hours after the last dose, no seminology (sperm assessment) was conducted on the recovery males.

Applicant's summary and conclusion

Conclusions:

At the 500 mg/kg/day 1592U89 hemisulfate/kg/day dose level, there was some evidence of systemic toxicity (transient reductions in body weight change and feed consumption in F0 males during premating, and transient reductions in body weight change in F0 females during gestation) and developmental toxicity expressed as increased resorptions (in utero early deaths of conceptuses) and reduced fetal body weights per litter, associated with reduced gravid uterine weight at this dose. There was no evidence of teratogenicity at any dose evaluated. There were no effects on male or female reproductive functions including male seminology. There was also no evidence of a dominant lethal effect from long term oral exposure of males to 1592U89 hernisulfate at any dose tested.

The no toxicologically significant effect level for F0 animals was therefore considered to be at or above 160 mg/kg/day.

The no toxicologically significant effect level in the F1 fetuses was considered to be at or above 160 mg/kg/day. No effects on fetal malformations or variations were observed in this study.

No significant findings were reported in this study to contraindicate the therapeutic use of 1592U89 hemisulfate in humans.

Executive summary:

The antiviral agent 1592U89 Hemisulfate in vehicle (0.5 % aqueous methyl cellulose) was administered orally by gavage twice daily to CD® (Sprague-Dawley) rats at doses of 0, 60, 160 or 500 mg/kg/day. The possible effects of treatment on the parental animals and on maturation of gametes, mating behavior, fertility, preimplantation, and implantation through organogenesis were assessed. Dominant lethal effects were also evaluated, F0 parental males were exposed for 10 weeks prior to mating, during the two-week mating period to exposed F0 parental females and for the subsequent two-week mating period to naive (unexposed) females for a total of approximately 14 weeks of exposure. F0 parental females were exposed for two weeks prior to mating, through the two-week mating period and through organogenesis (through gestational day 16) for a total of approximately six weeks of exposure. Parental F0 females and their litters were evaluated on gestational day 20. The naive females, mated to F0 males to assess dominant lethal effects, were necropsied on gestational day 15 for evaluation of pregnancy status and products of conception. F0 males, 16 of 24 per group, were sacrificed for histologic examination immediately after completion of the second mating within 24 hours of the last dose. The remaining F0 males per group (five to eight) were retained for a four-week recovery period without dosing. Satellite F0 males and F0 females, treated the same way as the parental F0 animals, were used to evaluate absorption and therefore systemic exposure of the test material.

At the 500 mg/kg/day 1592U89 hemisulfate/kg/day dose level, there was some evidence of systemic toxicity (transient reductions in body weight change and feed consumption in F0 males during premating, transient reductions in body weight change in F0 females during gestation. and increased liverweight in pregnant females) and developmental toxicity expressed as increased resorptions (in utero early deaths of conceptuses) and reduced fetal body weights per litter. There was no evidence of teratogenicity at any dose evaluated. There were no effects on male or female reproductive functions including male seminology. There was also no evidence of a dominant lethal effect from long term oral exposure of males to 1592U89 hemisulfate at any dose tested.

The no toxicologically significant effect level for F0 animals was therefore considered to be at or ahove 160 mg/kg/day. The no toxicologically significant effect level in F1 fetuses was considered to be at or above 160 mg/kg/day. No effects on fetal malformations or variations were observed in this study.

No significant findings were reported in this study to contraindicate the therapeutic use of 1592U89 hemisulfate in humans.