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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Study period:

November 26 1996 and July 22 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: Testing strategy proposed by the International Conference on Harmonisation (ICH; Commission ofthe European Communities, 1993; ICH, 1996) and the U.S. FDA (ICH, 1994).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Abacavir Hemisulphate

IUPAC Name:

Abacavir Hemisulphate

Constituent 2

Reference substance name:

188062-50-2

Cas Number:

188062-50-2

IUPAC Name:

188062-50-2

Details on test material:

- Name of test material (as cited in study report): 1592U89 Hemisulfate
- Chemical Name: (1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]2- cyclopentene-1-methanol hemisulfate
- Molecular formula (if other than submission substance): Please see Attachment 1.
- Molecular weight (if other than submission substance): Please see Attachment 1.
- Structural formula attached as image file (if other than submission substance): Please see Attachment 1.
- Manufacturer's Batch No.: B41R11P3
- Lot/batch No.: A96L630
- Storage condition of test material: Bulk Drug: Room temperature; Dosing formulations: Refrigerated ( approximately 5°C); protected from light (amber bottles)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

The proposed test animal was Caesarean-originated. Virus Antibody Free (VAF) outbred albino rats (Crl:CD® (SD)BR) supplied by Charles River Laboratories. Inc., Raleigh, NC.
One hundred seventy (170) nulliparous female rats, nine weeks of age (date of birth, September 30, 1996; 63 days old upon arrival) and approximately 201-225 grams upon arrival, were purchased for this study, and 170 arrived at RTI on December 2, 1996. They were eartagged with unique numbers during quarantine. One hundred (100) male rats from the RTI breeding colony, originally from the same supplier (which arrived at RTI on October 21, 1996; date of birth, August 12, 1996; 70 days old upon arrival), were used to generate timed-mated females. All animals were housed in the Animal Research Facility as follows:

F0 females (and their F1 litters) were housed in room 305 from December 2, 1996 (date of arrival at RTI) to December 9, 1996 (end of quarantine) and from December 10, 1996 (first gd 0) through January 25, 1997 (last pnd 21 for F1 offspring);
F1 males were housed in room 205 from January 22, 1997 (first pnd 21) through May 19, 1997 (last sacrifice date);
F1 females (and their F2 litters) were housed in room 204 from January 22 (first pnd 21) through May 19, 1997 (last sacrifice date).

Female rats were approximately 9-11 weeks of age and approximately 221.7-280.3 grams in weight on gestational day (gd) 0. Ninety-six (96) sperm-positive female rats, designated the F0 generation, were used in this study (i.e., four groups of 24 sperm-positive dams each).

During an approximately seven-day quarantine period animals were randomly assigned to cages. Males were housed singly in solid bottom polycarbonate cages (8" x 19" x 10 1/2"). Non-mated females were group housed (maximum 3 per cage) and mated females were singly housed in solid bottom polycarbonate cages (S" x 19" x 10 1/2") with stainless steel wire lids (Laboratory Products. Rochelle Park, NI). Ab-Sorb-Dri® cage litter (Laboratory Products. Garfield. NI) was used in all cages. Feed (#5002 Purina Certified Rodent Chow®) and tap water from the Durham, North Carolina water system in plastic bottles with stainless steel sipper tubes, was available ad libitum for the initial study females during quarantine and the F0 females and the F1 males and females to the end of the study.
Breeding males used to generate timed-pregnant F0 females were on ad libitum feed and water; the water for the males was provided by an automatic watering system (Edstrom Industries, Inc., Waterford, WI); F0 females during the initial mating period only (to be designated F0) were also on the automatic watering system.

Environmental conditions in the Animal Research Facility animal room(s) were continuously monitored, recorded. and controlled (Barber-Colman Network 8000 System, Loves Park, IL) during the course of the study. Animal rooms used for this study (305, 206 and 207) were maintained on a 12:12 hour light:dark cycle. Target conditions for temperature and relative humidity in the animal room(s) were between 68-75°F and 40-70%, respectively, with 10-15 air changes per hour. Temperature and/or relative humidity excursions more than 5°F or 10% above or below the target ranges and excursions that lasted more than two hours were documented in the Study Records and discussed below.

In animal room 305, the temperature remained in range throughout the animal residence time, at 70.6-74.9°F; relative humidity also remained in range. at 43.4- 62.7%. In animal room 205, the temperatures remained in range throughout the animal residence time, at 70.7-73.0°F; relative humidity remained in range, at 39.4-69.8%, except for two (2) brief excursions: on February 14, 1997, for one hour up to 77.2%; and on March 18, 1997, for one hour at 71.5%. In animal room 204, the temperature remained in range throughout the animal residence time, at 69.5-73.9°F; relative humidity also remained in range, at 43.6-63.6%. These few, brief and minor excursions in relative humidity in one of the animal rooms did not affect the design, conduct or conclusions of this study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous (distilled/deionized water) 0.5% methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Nominally pure drug substance, 1592U89 hemisulfate in vehicle, was administered to three treatment groups at doses of 60, 160 or 500 mg I592U89 hemisulfate/kg/day in two doses in the morning and afternoon at least six hours apart. One group received vehicle alone.
The concentration and dosages of the test material are defined as mg 1592U89 hemisulfate/mL, and mg 1592U89 hemisulfate/kg, with no correction for the
hemisulfate salt or other possible contaminants of the drug substance.

VEHICLE
The test material, I592U89 hemisulfate, was formulated in aqueous (deionized/distilled water, CAS No. 773218 ·5) 0.5% Methocel® (methylcellulose; CAS No. 9004-67-5) with the concentration determined by the following formula:

Concentration (mg/mL) = Dose per time (mg/kg) / Dosage volnme (10.0 mL/kg)

Aliquots of each dose level and vehicle from both fonnulation dates were transferred to the Sponsor for analysis on cold packs. Dosing formulations were stored in amber bottles in the refrigerator 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing preparations were formulated at Research Triangle Institute and analyzed by the Sponsor. Analytical results were to be considered satisfactory if they were within the 10% range of nominal concentrations. The performing laboratory has no knowledge of the methods or results of the analysis and is not responsible for the GLP compliance of analyses of the dosing preparations.

Details on mating procedure:

For breeding, individual females were placed in the home cage of singly-housed males (i.e., one male and one female). On the following morning and each morning thereafter, the females were examined for the presence of vaginal sperm or a vaginal copulation plug (Hafez, 1970). The day on which vaginal sperm or plugs were found was designated as gestational day (gd) 0. These females were presumed pregnant. The initial sperm-positive females (dams) designated the F0 generation, were housed individually or with their litters until scheduled sacrifice. Sperm-negative females were retained in the same male's cage and checked for sperm or vaginal plug on successive mornings until insemination occurs or the treatment groups were filled, whichever came first. When all treatment groups were filled, surplus sperm-negative females were sacrificed by asphyxiation with CO2. Selected male and female offspring, designated the F1 generation, were mated 1:1 within dose groups and housed as described above. The fate of all animals was fully documented.

Duration of treatment / exposure:

Dosing period: 6 - 20 (gd - pnd)

Frequency of treatment:

Dams were dosed twice daily (morning and afternoon, at least six hours apart) from gestational day (gd) 6 (day of natural insemination is designated gd 0) through postnatal day (pod) 20 inclusive (day of littering is designated pnd 0). Test and control animals were given a standard dosage volume of 10 mL/kg per time. The most recent body weights (taken in the morning prior to the first of the two daily doses) were used to calculate the dose volume administered.

Duration of test:

6 months

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:

The study was conducted with three (3) treatment groups and one (1) vehicle control group, each comprised initially of 24 presumed pregnant (sperm-positive) rats.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected by the Sponsor based on a previous developmental toxicity study on 1592US9 succinate (GW TOX775) where doses of 100, 300 and 1000 mg/kg/day (salt form) for 12 days (gestation days 6 through 17) caused dose-related effects on body weight gain and feed consumption in pregnant rats. Fetal body weights were reduced and the incidences of fetal malformations were increased at 1000 mg/kg/day.

- Rationale for animal assignment (if not random): All sperm-positive F0 female rats (dams) were assigned to treatment groups by a stratified randomization method designed to provide uniform mean body weights across dosage groups at the initiation of gestation, gd 0.

Examinations

Maternal examinations:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations of F0 maternal animals were documented at least once daily on gd 0-5 (prior to dosing period) and on pnd 21 (after the dosing period), and at least four times daily, at and after each dosing, throughout the dosing period (gd 6 through pnd 20). The examining technicians were unaware of dosage levels. Observations were made for (but not limited to):

a. Any response with respect to body position, activity, coordination, or gait.
b. Any unusual behavior such as head flicking, compulsive biting or licking, circling, etc.
c. The presence of:
1. Convulsions, tremors or fasciculations
2. Increased salivation
3. Increased lacrimation or red colored tears
4. Increased or decreased urination or defecation (including diarrhea)
5. Piloerection
6. Mydriasis or miosis (enlarged or constricted pupils)
7. Unusual respiration (fast, slow, labored. audible, gasping, or retching)
8. Vocalization

BODY WEIGHT: Yes
All F0 dams were weighed on gd 0, and daily during the dosing period, gd 6 through pnd 20 for calculation of dosing volume. F0 maternal weight gains were calculated for gd 0-6 (pre-treatment), 6-9, 9-12, 12-15, 15-18, 18-20 pnd 0-4, 4-7, 7-14, 14-20, gd 0-20 (gestation period), gd 6-20 and pnd 0-20 (treatment period).

FOOD CONSUMPTION: Yes
FO maternal feed consumption was evaluated for gd 0-6 (pre-treatment), gd 6-9, 9-12, 12-15, 15-18, 18-20, pnd 0-4, 4-7, 7-14, 14-20, gd 0-20 (gestation period), gd 6-20 and pnd 0-20 (treatment period).

POST-MORTEM EXAMINATIONS: Yes
Dams that had not produced a litter by calculated gestation day 26 were necropsied. Any dams whose whole litters were born dead or died prior to pnd day 21 were sacrificed.
Necropsy of F0 females:
F0 females which were moribund, aborted, delivered early (with offspring not viable outside the uterus) were sacrificed by CO2 asphyxiation, necropsied and discarded. Intact fetuses (in utero, aborted or delivered early) were examined externally and discarded. Any F0 dams whose whole litters were born dead or died prior to pnd 21 were sacrificed and examined as described below for F2 progeny.
On pnd day 21, all F0 dams were sacrificed with CO2; the thoracic and abdominal organs were examined for grossly evident morphological changes, and implantation scars were counted and recorded. Organs or tissues showing macroscopic abnonnalities were retained in neutral buffered 10% fonnalin. Microscopic examination of the tissues may be undertaken only if considered necessary to interpret the findings of the study. Uteri from any F0 females who appeared nonpregnant were stained with 10% ammonium sulfide (Salewski, 1964) for confirmation of pregnancy status and count of implantation sites, if any. F0 maternal carcasses and nonretained tissues were discarded. A sample of mammary gland tissue (one abdominal mammary gland with nipple, surrounding skin and underlying mammary tissue) was retained in buffered neutral 10% fonnalin from each F0 female on study.

OTHER: PARTURITION AND LACTATION
Beginning on gd 20, each female was observed twice daily (a.m. and p.m.) for evidence of littering. Dosing continued through parturition through pnd 20. If the dam was in the process of littering at the usual time of dosing, she was not dosed at that time but was dosed at the next scheduled morning or afternoon dosing time. Signs of dystocia or other signs of difficulty at parturition were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Dams that had not produced a litter by calculated gestation day 26 were necropsied. Apparently nonpregnant uteri were stained in 10% ammonium sulfide (Salewski. 1964) to determine pregnancy status. Any dams whose whole litters were born dead or died prior to pnd day 21 were sacrificed, the number of implantation scars were recorded and a sample of mammary tissue (one abdominal mammary gland with nipple, surrounding skin and underlying mammary tissue) was retained in buffered neutral 10% formalin for possible future examination.

Fetal examinations:

All pups were counted, weighed, sexed and examined externally as soon as possible on the day of birth (designated postnatal day [pnd] 0) to determine the number of total, viable and stillborn members of each litter. Grossly malformed pups were sacrificed and examined externally and viscerally.

Pups that were stillborn or died before pnd 4 were examined externally and viscerally by Staples' technique (Staples, 1974), and any abnormal tissues or specimens were retained in buffered neutral 10% formalin until the study report was finalized. Pups were examined daily and were counted, weighed, sexed, and examined externally on pnd 4, 7, 14, and 21. Litters were not culled. Pups that died or were sacrificed moribund on pnd 5-21 were necropsied; any abnormal tissues or specimens were retained in buffered neutral 10% formalin until the study report was finalized. Survival indices were calculated on pnd 0, 4, 7 and 14 and at weaning, pnd 21. The body weights and sexes of the F1 pups were recorded on an individual basis, but the pups were not uniquely identified at this stage. All pups were examined for physical abnormalities at birth and throughout the lactation and post-wean period.

Weaning
At weaning, pnd 21, at least one female and one male (whenever possible) from each F1 litter, were selected on a random basis to become parents of the next generation (F1 parents to produce F2 litters), for a total of 20/sex/dosage level. All pups were available for selection. The parentage of each weanling was ascertained to avoid brother-sister matings, if possible. Following this selection, the remaining offspring were examined for gross external abnormalities, euthanized by CO2 asphyxiation and discarded after concurrence of the Sponsor's Representative. The selected F1 animals were held for a minimum of 70 days until all selected F1 pups were at least 91 days old. They were not dosed.
F1 prewean developmental landmarks
Pinna Detachment - Each pup was observed on pnd 1 for detachment of the pinnae. Pups which did not have both pinnae detached on the initial day of observation were observed daily until detachment was present in all pups of the same sex and litter.
b. Eye Opening - Each pup was observed beginning on pnd 11 for eye opening. The characteristic was scored as present when both eyes were open. The number of pups having both eyes greater than one-half open were recorded daily until all pups of the same sex and litter had this response.
F1 postwean observations and procedures
All F1 pups were weaned on pnd 21. Selected F1 pups, 20/sex/group, were retained for a minimum of 70 days with no dosing, prior to mating. Selected F1 pups were eartagged with a unique number as well as assigned a unique study number. The pups were examined daily for adverse effects and were weighed weekly during this period.
a. Eye Examination - The eyes of control and high dose pups were examined by a certified veterinary ophthalmologist after the pups were at least 21 days old (23-26 days old). The eyes of low and mid dose pups were to be examined only if abnormalities were found in the high dose group. The pups' eyes were dilated by topical application of 0.5% tropicamide. "Mydriacyl" (Alcon Pharmaceuticals), approximately one drop per eye, prior to examination. (The protocol specified 1.0% tropicamide. but the RTI attending veterinarian decided to order and use the 0.5% solution to minimize stress. This is therefore a protocol deviation that had no effect on results of eye examination or study outcome.) Biomicroscopy and indirect ophthalmoscopy were used for the examination.
b. Auditory Function - The auditory function was assessed by observation of the startle reflex in pups that were at least 21 days old (21-23 days old). Startle
reflex was measured by placing animals individually in an isolation cabinet with a force detector attached to the bottom (San Diego Instruments, Inc., San Diego, CA). The startle reflex was automatically monitored by an IBM-AT computer (startle response measurement system, San Diego Instruments. Inc., Version 2.1. San Diego, CA 92126). After a 5-minute acclimation period, an approximately 120 dB tone was presented to the animal for 50 milliseconds. The computer system monitored response for an additional 50 milliseconds after cessation of the tone and then paused for an 8-second intertrial interval. The procedure was repeated 50 times per session, for one session per animal.
c. Vaginal Opening (patency) - Each female pup was observed beginning at 22 days of age for vaginal opening. The number of female pups with the vagina
open was recorded daily until all female pups had this response.
d. Cleavage of the Balanopreputial Gland (preputial separation) . Each male pup was observed beginning at 35 days of age. The characteristic was present when the prepuce could be completely retracted to expose the glans penis. The number of male pups with this separation was recorded daily until all male pups had this response.
e. Motor Activity - All retained male and female pups were observed in a motor activity maze for one hour on day 34 ± 1 of age. Figure 8 mazes were used to
determine animal activity. The apparatus detected the movement of the animal through a maze that was designed to simulate the natural burrows of a rodent. Movement was detected when the animal broke photocell beams that were spaced such that not more than one beam could be broken at a time. For each
pup, a continuous one hour test period was used, broken into twelve 5-minute data collection segments; activity was collapsed into six 10-minute segments for reporting (Photobeam Activity System. Version 1.0. San Diego Instruments. Inc., San Diego, CA 92126).
f. Learning and Memory - All retained male and female pups were tested in a water filled M (Morris)-maze (Morris, 1981; Whishaw. 1985; Nanry et al., 1989;
Tilson et aI., 1990). when at least 40 days of age (pnd 48-53). Each rat was given 8 runs/day in the maze for 2 consecutive days. The inter-trial interval was
a maximum of 15 minutes.

Necropsy Of F1 parental males and females:
F1 males or females which died or were sacrificed moribund prior to scheduled sacrifice were sacrificed by CO2 asphyxiation (if moribund), and necropsied. Any gross lesions were retained in fixative for possible subsequent histopathologic evaluation. Animal carcasses and nonretained tissues were discarded.
On pnd 4 of each F2 litter, when the pups were euthanized and examined, each F1 dam was sacrificed by CO2 asphyxiation and weighed. Nonpregnant F1 females or females whose whole litters were born dead or died prior to pnd 4 were sacrificed at or after their calculated pnd 4 date. Thoracic and abdominal cavities and organs were examined with any organs or tissues with gross lesions retained in buffered neutral 10% formalin for possible subsequent histopathologic examination. Special attention was paid to reproductive organs. A sample of mammary gland tissue (one abdominal mammary gland with nipple, surrounding skin, and underlying mammary tissue) was retained in buffered neutral 10% formalin from any F1 female whose litter was born dead or died prior to pnd 4. Uteri of any F1 females who appeared nonpregnant were stained with 10% ammonium sulfide (Salewski, 1964) to confirm pregnancy status and to count implantation sites, if any.
At or after the pnd 4 date of their F2 litter. F1 males were euthanized by CO2 asphyxiation, weighed. and necropsied. Thoracic and abdominal cavities and organs were examined with any organs or tissues with gross lesions retained in buffered neutral 10% formalin for possible subsequent histopathologic examination. Special attention was paid to reproductive organs. Carcasses and nonretained tissues from F1 males and females were discarded.

Statistics:

Please see "any other information on materials and methods" for details.

Indices:

F0 and F1 Maternal Generations: Mating index (%) = (No. females sperm - positive / No. females paired) x 100 (F1 only)
Fertility index (%) = (No. females pregnant / No. females spenn - positive) x 100
Prenatal (postimplantation) loss (%) = (No. implantation scars - No. pups born live / No. implantation scars) x 100

F1 Parental Generation: Mating index (%) = (No. males impregnating females / No. males paired) x 100
Fertility index (%) = (No. males siring litters / No. males impregnating females) x 100
Pregnancy index (%) = (No. pregnant females / No. males impregnating females) x 100

F1 and F2 Offspring Generations: Gestational index (%) = (Number of females with live litters / Number of females pregnant) x 100
Stillbirth index (%) = (Number of dead pups at birth / Total number of pups at birth) x 100
Live birth index (%)= (Number of live pups at birth / Total number of pups born) x 100
4 - day survival index (%) = (Number of pups surviving 4 days / Total number of live pups at birth) x 100

F1 Generation Only: 7-day survival index (%) = (Number of pups surviving 7 days / Total number of live pups at 4 days) x 100
14-day survival index (%) = (Number of pups surviving 14 days / Total number of live pups at 7 days) x 100
21-day survival index (%) = (Number of pups surviving 21 days / Total number of live pups at 14 days) x 100
Lactation index, pnd 4-21 (%) = (Number of pups surviving 21 days / Total number of live pups at 4 days) x 100
Lactation index, pnd 0 - 21 (%) = (Number of pups surviving 21 days / Total number of live pups at birth) x 100

Historical control data:

Large historical data bases for reproductive performance and prevalence of spontaneous malformations in control rats were available from studies conducted at RTI (currently based on over 300 control litters) as well as from the supplier (Charles River, 1988).
Functional testing by the experimental laboratory has been completed using, when possible, comparisons of historical data analyses using SAS against analyses using the SUDAAN(R) functions.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
F0 Females
Gestation - Clinical Observations, Body Weights, Feed Consumption
Twenty-four (24) sperm-positive females, presumed pregnant, per group were placed on study. No females died or were removed from study during this period. Pregnancy rate was high and equivalent across all groups (91.7-100.0%) with 22, 23, 22 and 24 pregnant and 2, 1.2. and 0 nonpregnant at scheduled sacrifice at 0, 60, 160 and 500 mg/kg/day, respectively. Rooting in bedding post-dosing was observed in all drug-exposed groups in a dose related time to initial observation, i.e., in the top dose group starting on gd 6, in the mid dose group starting on gd 7, and in the low dose group by gd 9. Clinical weight loss (equal to or > 5 g during any weigh period) was observed predominantly at 160 and 500 mg/kg/day, with very occasional occurrences at 0 and 60 mg/kg/day. The other clinical observations appeared unrelated to treatment.
Maternal body weights were equivalent across all groups for gd 0 and 6 prior to the initiation of dosing. There were no also significant differences in periodic body weights among groups at any time point duting gestation.
Maternal weight changes during gestation were equivalent across all groups for gd 0-6 prior to the initiation of dosing. Maternal weight change exhibited a significant reduction at 500 mg/kg/day for gd 6-9 (first dosing interval). Maternal weight changes during all other intervals at 500 mg/kg/day, for all intervals at 60 and 160 mg/kg/day and during the gestational dosing period, gd 6-20 and for gestation, gd 0-20, were equivalent across all groups.
Maternal feed consumption during gestation, expressed as g/day, was equivalent across all groups for gd 0-6 prior to the initiation of dosing, and was significantly reduced at 500 mg/kg/day for gd 6-9, the first dosing interval. It was significantly increased at 160 mg/kg/day for gd 12-15, 15-18, 18-20, gd 6-20 (gestational dosing period) and gd 0-20 (gestational period). Feed consumption in g/day was equivalent across all other intervals for 160 and 500 mg/kg/day, and was unaffected at 60 mg/kg/day. When the data were expressed as g/kg/day, maternal feed consumption was unaffected for gd 0-6 across all groups and was significantly reduced for gd 6-9 at 500 mg/kg/day. It was significantly increased at 500 mg/kg/day for gd 18-20, at 160 mg/kg/day for gd 12-15, 15-18,
18-20,6-20 and 0-20, and at 60 mg/kg/day for gd 9-12. The increases in feed consumption were not considered to be of biological significance. Feed consumption in g/kg/day was equivalent across groups for all other intervals.

Lactation· Clinical Observations, Body Weights, Feed Consumption
Dams with live litters on pnd 0 included 22, 21, 22 and 24 at 0, 60, 160 and 500 mg/kg/day, respectively. (Two additional dams at 60 mg/kg/day were, in fact, pregnant, but carried implant sites only [dam no. 2] or one retained dead fetus [dam no. 15] at scheduled sacrifice.) No dams died, were removed from study. or were sacrificed moribund to the end of in-life, until scheduled sacrifice. Rooting in bedding post-dosing was observed in all drug-exposed groups in a dose-related incidence on pnd 0 through 20. No other clinical observations appeared treatment related.
Maternal body weights were equivalent across all groups at timepoints examined during lactation, pnd 0, 4, 7, 14 and 20.
Maternal weight change during lactation was equivalent across all groups for all lactational intervals examined, pnd 0-4, 4-7, 7-14, 14-20 and for the lactational period, pnd 0-20.
Maternal lactational feed consumption expressed as g/day was equivalent across all groups for all lactational intervals. When the data were expressed as g/kg/day, maternal feed consumption was significantly reduced at 500 mg/kg/day for pnd 7-14 and for pnd 0-20.

At F0 necropsy
There were no treatment-related necropsy findings in F0 females at scheduled necropsy on pnd 21 of their F1 litters. One dam at 0 mg/kg/day exhibited bilateral mottled kidneys and one dam at 60 mg/kg/day exhibited severe bilateral hydronephrosis of the kidneys. There were no necropsy findings at 160 or 500 mg/kg/day.

F0 Reproductive and Lactational Indices
There were no significant differences among groups for mating or gestational indices, or for gestational length, number of implantation sites per litter, percent post-implantation loss or for number of live pups per litter on pnd 0. At 500 mg/kg/day, the stillbirth index was significantly increased and the live birth index was significantly reduced (both due to the increase in stillborn pups at this dose). There were no effects on these parameters at 60 or 160 mg/kg/day. Survival indices. including day 4, 7, 14, 21 and lactational indices (for pnd 0-21 and for pnd 4-21), were equivalent across all groups.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 160 mg/kg bw/day

Based on:

other: indirect exposure

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Postnatal Observations (F1 offspring)
F1 litter size, weight and sex ratio
Average numbers of pups and sex ratio (% males) per litter were unchanged across groups throughout lactation. Average pup body weights per litter by sex (males and females separately) or with sexes combined, were significantly reduced at 500 mg/kg/day for all timepoints measured during lactation, pnd 0, 4, 7, 14 and 21. At 160 mg/kg/day, only total pup body weight per litter (not for males and females separately) was significantly reduced only on pnd 4. There were significant reductions in combined sex body weights per litter at 500 mg/kg/day for all timepoints and at 160 mg/kg/day on pnd 4. There were no effects of treatment at 60 mg/kg/day.

F1 clinical observations during lactation
Pups found dead on pnd 0 (including stilIborn pups) included 2, 4, 1 and 12 at 0, 60, 160 and 500 mg/kg/day, respectively; an additional pup at 0 mg/kg/day was euthanized moribund. During the rest of lactation through pnd 16 (no pups died after pnd 16), the numbers of pups found dead or missing and presumed dead included 6, 6, 3 and 12 at 0, 60, 160 and 500 mg/kg/day, respectively. The only other possible treatment-related finding was pale appearance predominantly at 500 mg/kg/day on pnd 0 and 1. Thin fur was observed at 60 mg/kg/day on pnd 14. In addition, one female pup at 0 mg/kg/day exhibited short string-like tail and anal atresia; it was euthanized moribund on pnd 0, and one male pup at 160 mg/kg/day exhibited a small growth on the lower lip, left side, on pnd 13, 14 and 16.
Necropsy of pups that died or were sacrificed moribund during lactation: There were no treatment-related findings; all observations were typical of dead perinatal pups, were observed predominantly early in the lactation period, pnd 0-4, and included no air in lungs (atelectasis), patent (open) ductus arteriosus, and no milk in stomach. In addition, one female pup at 0 mg/kg/day exhibited anal atresia and short string-like tail and was euthanized moribund on pnd 0 (see above), one male pup at 0 mg/kg/day exhibited double outlet right ventricle and interventricular septal defect (pnd 9) and one female pup at 0 mg/kg/day exhibited a 2x2 mm cream colored area on the median liver lobe. None of these findings were considered treatment related.

F1 developmental landmarks (pnd 1-49)
All available animals were evaluated as indicated below until all animals had acquired the specific landmark. There were no statistically significant or biologically relevant differences among groups in acquisition of pinna detachment (evaluated on pnd 1 through 4), eye opening (evaluated on pnd 8-21 pre-wean), vaginal patency in females (evaluated on pnd 22 through pnd 37 post-wean) and preputial separation in males (evaluated on pnd 35 through 49 post-wean) during the prebreed period.

F1 eye examination (pnd 23-26)
Examination of F1 eyes in the control group (0 mg/kg/day, Rx Code 66523) and the high dose group (500 mg/kg/day, Rx Code 00697) was performed during the prebreed period when the F1 offspring were 23-26 days old (on January 27, 1997). The conclusion was that there were no treatment-related effects; i.e., "no significant observations".

F1 behavioral analyses
Auditory startle (pnd 21-23)
There were no statistically significant or biologically relevant differences among groups for any parameters associated with auditory startle: force of jump, and duration of jump during five (5) time blocks.
Motor activity (pnd 33-35)
Motor activity was evaluated for a total of one hour in 12 five-minute intervals and collapsed into six 10-minute intervals for reporting. All values for all parameters were statistically and biologically equivalent across all groups. Animals in all groups exhibited comparable patterns of habituation to a novel environment over the one-hour period.
Learning and memory (pnd 48-53)
Learning and memory were assessed by the Morris Water Maze on animals which were at least at pnd 40. Animals were evaluated on pnd 48-53 over February 25-28 and March 3-6, 1997. Each animal received eight trials on the first day (to assess learning) and eight trials on the second day (to assess memory).
Test performance in all groups was comparable and showed no dose relationship.

F1 Prebreed Period
All F1 litters reached pnd 21 and were weaned within one calendar week (over four days, January 22-25,1997, Wednesday - Saturday). Therefore, all selected F1 pups, 20/sex/group, were weighed on the same Wednesday following their weaning, January 29, 1997, designated study day (sd) 7, and then weekly through the F1 prebreed period through sd 70 (until each pup was at least 91 days old; 21 + 70 = 91 days). Clinical observations were recorded daily during this prebreed period. These selected F1 animals were not dosed with the test material.

F1 males
Body weights
F1 male weekly body weights were significantly reduced at 500 mg/kg/day for all weekly timepoints evaluated, sd 7 through 70 (prebreed), 77-84 (mating) and 84-112 (holding prior to scheduled termination). F1 male weekly body weight changes were also significantly reduced at 500 mg/kg/day for sd 7 through 49 and for sd 7-70 (entire prebreed). There were no effects on weight change at 500 mg/kg/day after sd 70, and no effects of treatment on body weights or weight changes at 60 or 160 mg/kg/day.
Clinical observations
Clinical observations were documented daily throughout these periods until scheduled demise on sd 91-93.

F1 females
Body weights
F1 female body weights at 500 mg/kg/day were significantly reduced for all weeks of the prebreed period, sd 7-70. There were no differences among groups during the two-week mating period, sd 77 and 84. For all subsequent weeks, through sd 112, the group sizes were very small, n=1-5, so the data were not analyzed statistically. F1 female weekly body weight changes were unaffected for all analyzed weeks except for a significantly reduced value at 160 mg/kg/day (but not at 500 mg/kg/day) for sd 21-28. There were no other effects at 160 mg/kg/day and no effects at 60 mg/kg/day.
Clinical observations
Clinical observations were recorded daily during the prebreed period, through mating and gestation, for females not inseminated, and through the four-day lactational period for females without litters. One F1 female (no. 142) at 60 mg/kg/day had her leg caught in the cage top; the veterinarian diagnosed the
leg as broken and she was euthanized on sd 5. There were no treatment- or dose-related incidences in clinical observations for the F1 females.
Estrous cyclicity
During the last 12 days of the prebreed period, F1 female offspring were assessed daily for estrous cyclicity by vaginal lavage. There were no statistically significant differences among groups for any estrous cycle parameters.
There were no differences among groups in the percentage of females cycling (all groups at 100.0%), in the percentage of cycling females with abnormal cycles (5.0, 0.0, 15.0 and 5.0% at 0, 60, 160 and 500 mg/kg/day, respectively) or in the average cycle length in days (4.04, 4.04,4.45 and 4.39 days at 0,60,160 and 500 mg/kg/day, respectively). Consistent with the estrous cyclicity data, there was no evidence of impaired reproductive performance in F1 animals, based on mating index (and number of females who did not mate) and fertility index (and number of females with live litters).

F1 Females During Gestation
Body weights
F1 maternal body weights at 500 mg/kg/day were significantly reduced for all timepoints during gestation, gd 0, 6, 9, 12, 15, 18 and 20, and at 60 mg/kg/day for gd 0 only. Maternal gestational weight change was equivalent across all groups for all intervals, including gd 0-6, 6-9, 9-12, 12-15, 15-18, 19-20 and gd 0-20 (gestational period).
Clinical observations
Clinical observations were recorded daily during gestation, gd 0-21. Alopecia at various locations was observed throughout gestation in only four females, as follows: female no. 174 at 60 mg/kg/day on limb(s); female nos. 182 and 192 at 160 mg/kg/day on neck, limbs and multiple areas; and female no. 250 at 500 mg/kg/day on multiple areas. There were clearly no treatment- or dose-related incidences of clinical observations during gestation.

F1 Females During Lactation
Body weights
Maternal body weights on pnd 0 and 4 at 500 mg/kg/day were significantly reduced. Maternal body weight on pnd 0 was also significantly reduced at 60 mg/kg/day (most likely due to two resorbed litters at this dose). There were no effects at 60 mg/kg/day on pnd 4 and no effects at 160 mg/kg/day. There were no differences among groups for maternal weight change, pnd 0-4.
Clinical observations
There were no treatment- or dose-related incidences (or severities) ofclinical observations during this period.

F1 Reproductive and Lactational Indices
There were no effects of treatment or dose on F1 male or female mating index or fertility index, on female gestational index, or on gestational length in days. There were no significant effects of treatment or dose on live birth or stillbirth indices on pnd 0 or on the 4-day survival index (pnd 0-4).

F2 Offspring
Litter size and body weights
There were no effects of treatment or dose on any of these parameters.
Clinical observations
There were no treatment- or dose-related differences in observations. A "milk band", the visualization through the skin of a milk-filled stomach in pups, is a good indication of a lactating mother and surviving pups nursing early in the postnatal period. The finding of "no milk band" was made in pups at 0 and 500 mg/kg/day. The numbers of pups found dead, or missing and presumed dead, were 23, 3, 11 and 15 at 0, 60, 160 and 500 mg/kg/day. In addition, one pup at 0 mg/kg/day on pnd 1, and two pups at 500 mg/kg/day on pnd 2 were moribund and euthanized.

Gross necropsy findings
Unscheduled deaths
As expected, pups died in all groups. F2 pups which were stillborn, died or were sacrificed moribund, were examined externally and retained in fixative, but were not examined internally. Therefore, these pups were not necropsied.
Scheduled deaths on pnd 4
There were no gross findings at scheduled necropsy on pnd 4 in either male or female F2 pups.

Necropsy of F1 Parental Animals
F1 males
All selected F1 males, 20/group, survived to scheduled sacrifice and there were no treatment-related findings at necropsy.
F1 females
All F1 females except one (no. 142) at 60 mg/kg/day, who was euthanized due to a broken leg and therefore not necropsied, survived to scheduled sacrifice and there were no treatment-related findings at necropsy. At scheduled sacrifice, one female (no. 226) at 500 mg/kg/day exhibited uterus filled with fluid.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

At the 500 mg 1592U89 hemisulfate/kg/day dose level, toxicity to the F0 parental females was evidenced by transient decreases in body weight change during the dosing period. This maternally toxic dose was associated with an increase in stillbirths and decreased body weights of the F1 offspring during the postnatal period.
In the treated F0 dams, the "no toxicologically significant effect level" is considered to be at or below 160 mg/kg/day.
The "no toxicologically significant effect level" for the F1 generation animals. exposed only indirectly to test material, was considered to be at or above a maternal treatment level of 160 mg/kg/day.
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 timed-pregnant CD® (Sprague-Dawley) rats during gestation and lactation periods (from gd 6 through pnd 20) at doses of 0, 60, 160 or 500 mg/kg/day. The possible effects of treatment on the parental females and the growth and development of their offspring exposed to test material either in utero or via maternal milk were assessed.

At the 500 mg 1592U89 hemisulfate/kg/day dose level, toxicity to the F0 parental females was evidenced by transient decreases in body weight change during the dosing period. This maternally toxic dose was associated with an increase in stillbirths and decreased body weights of the F1 offspring during the postnatal period.

In the treated F0 dams, the ''no toxicologically significant effect level" is considered to be at or below 160 mg/kg/day.

The "no toxicologically significant effect leve1" for the Fl generation animals, exposed only indirectly to test material, was considered to be at or above a maternal treatment level of 160 mg/kg/day.

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