(2S)-3-Methyl-2-[N-({2'-(1H-1,2,3,4-tetrazol-5-yl)-[1,1-biphenyl]-4-yl}methyl)pentanamido]alkanoic acid

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use

Version / remarks:

FDA Introduction to Total Drug; Quality, DHEW Publication No. (FDA) 74-3006, 11/73

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-Pentanoyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-L-valine

EC Number:

604-045-2

Cas Number:

137862-53-4

Molecular formula:

C24 H29 N5 O3

IUPAC Name:

N-Pentanoyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-L-valine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: (Cr1:COB CD[SD]BR

Details on test animals or test system and environmental conditions:

Sixty-three male (with a birthdate of approximately 8/11/92 and weighing 300 to 347 grams shortly after arrival) and 120 female
(with a birthdate of approximately 11/16/92 and weighing 181 to 248 grams shortly after arrival) sexually mature Sprague-Dawley rats (Cr1:COB
CD[SD]BR) were received from Charles River Breeding Laboratories, Kingston, New York on 10/19/92 and 1/18/93, respectively. The rats were acclimated to laboratory conditions for approximately two to four weeks prior to dosing. Certified Purina Rodent Chow #5002, in which the levels of contaminants in the food were specified, and tap water from water bottles and/or automatic watering system were offered ad libitum throughout the study. The food and drinking water were monitored for the levels of contaminants according to the Standard Operating Procedures (SOPs) of the Safety Evaluation Facility (SEF). Upon arrival, 60 male and 120 female rats were housed one per cage (except during mating), assigned random numbers from a computer-generated acclimation cage diagram and identified by cage cards. The three extra male animals were sacrificed via C02 asphyxiation and discarded. Body weights and clinical observations were recorded approximately weekly during the acclimation phase (except males, which were weighed twice during acclimation) of the study and were retained in the raw data file for the study, but are not included in the final report. Only those females found to be cycling normally as demonstrated by vaginal washing cytology and those animals judged healthy based on general observations, physical examinations and body weights performed during the acclimation period were used on this study. The rats were identified by ear tags. The body weight of each Fo parental animal prior to the initiation of the premating phase are included in the final report. All parental females (treated and control) were assigned to one of two groups (N = 12/group) using a program generated by Research Statistics Services (RSS). Sperm-positive females in the first group were dosed through day 19 of gestation and sacrificed on day 20 of gestation. Spermpositive females in the second group were dosed through day 20 of lactation and sacrificed on day 21 of lactation. Females that were not found sperm-positive were maintained until their last possible day 25 of gestation.
At that time they were sacrificed via C02 asphyxiation and necropsied. The disposition of these animals was documented in the raw data file. The 12 males not assigned to the study were sacrificed by C02 asphyxiation and discarded. The 24 females not assigned to the study were transferred to the stock room.
ANIMAL QUARTERS: All rats in this study were housed at the Summit facility in animal rooms (2302A, 2302B) in the SEF. The rooms were maintained
at a daily temperature of 73 ± 5°F and a relative humidity of 50 ± 20%, whenever possible. Room temperature and humidity were monitored throughout the study according to the SOPs of Toxicology. The animal rooms were supplied daily with 14 hours of continual artificial light and were closely monitored so that no unusual activities interfered with the conduct of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0, 1, 5 and 20 mg/ml concentrations of CGP 48933 aqueous suspensions in 0.5% carboxymethylcellulose sodium, type 7HF (CMC) with 0.5% Tween 80.

Details on exposure:

The compound formulations of CGP 48933 were administered as indicated in the dosing schedule for Groups 2, 3 and 4. Group 1 received an equivalent dosing volume of aqueous CMC with Tween 80 and served as a control. Parental males were dosed for 92 days prior to mating, during the three-week mating period and after mating until the Study Director reviewed the data on pregnancy and survival of their derived
litters. All parental females were dosed for 15 days prior to mating and during the three-week mating period. Approximately one-half of
the sperm-positive females in each group were dosed through day 19 of gestation, and the remaining half were dosed throughout gestation and
through day 20 of lactation. Females not found sperm-positive were maintained and dosed until their last possible day 25 of gestation (last dose
day 24 of gestation). All animals were weighed weekly, and the daily dose volume throughout the study was calculated based on the most recent body weight. The formulations of CGP 48933 or aqueous CMC with Tween 80 were administered orally once daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses confirmed the uniformity of the 1 mg/ml and 5 mg/ml suspensions prepared for the initiation of dosing; however, the 20 mg/ml suspension did not meet established guidelines for uniformity during the first three weeks of dosing. Due to the excessive variation in uniformity, the dosing period for all parental: males was extended for three weeks.
Twenty-one samples of CGP 48933 suspensions in aqueous 0.5% sodium carboxymethylcellulose (CMC) with 0.5% polysorbate 80, were tested. Concentrations were 91% to 106% of target concentrations. Seven control samples were tested. No CGP 48933 was detected in the controls.

Duration of treatment / exposure:

The study was initiated 10/20/92. Dosing of males and females started 11/16/92 and 2/1/93 (first dose administration on day 0 of premating), respectively, and ended 4/18/93 (last dose on day 20 of lactation). Dosing of the males terminated 3/21/93. The date of completion of the in-life phase of this study was 7/14/93.

Frequency of treatment:

Parental males were dosed for 92 days prior to mating, during the three-week mating period and after mating until the Study Director reviewed the data on pregnancy and survival of their derived litters. All parental females were dosed for 15 days prior to mating and during the three-week mating period. Approximately one-half of the sperm-positive females in each group were dosed through day 19 of gestation, and the remaining half were dosed throughout gestation and
through day 20 of lactation. Females not found sperm-positive were maintained and dosed until their last possible day 25 of gestation (last dose day 24 of gestation).

Details on study schedule:

The compound formulations of CGP 48933 were administered as indicated in the dosing schedule for Groups 2, 3 and 4. Group 1 received an equivalent dosing volume of aqueous CMC with Tween 80 and served as a control. Parental males were dosed for 92 days prior to mating, during the three-week mating period and after mating until the
Study Director reviewed the data on pregnancy and survival of their derived litters. All parental females were dosed for 15 days prior to mating and during the three-week mating period. Approximately one-half of the sperm-positive females in each group were dosed through day 19 of gestation, and the remaining half were dosed throughout gestation and through day 20 of lactation. Females not found sperm-positive were maintained and dosed until their last possible day 25 of gestation (last dose day 24 of gestation). All animals were weighed weekly, and the daily dose volume throughout the study was calculated based on the most recent body weight. The formulations of CGP 48933 or aqueous CMC with Tween 80 were administered orally once daily according to the following schedule:

Number of Rats Daily Dosing Compound
Group Male Female Dose Volume Conc .
(mg/kg) (ml/kg) (mg/ml)
1 12 24 0 10 0
2 12 24 10 10 1
3 12 24 50 10 5
4 12 24 200 10 20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

20 mg/kg diet

No. of animals per sex per dose:

12 males / 24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to evaluate CGP 48933, an angiotensin II antagonist, for effects on fertility and/or reproductive performance in one generation of rats and their offspring. The compound was suspended in aqueous 0.5% carboxymethylcellulose sodium (CMC) with 0.5% Tween 80 and administered orally by gavage at doses of 10, 50 or 200 mg/kg/day to three groups of sexually mature male (N = 12/group) and female (N - 24/ group) rats. A fourth group (control) received an equivalent dosing volume (10
ml/kg) of aqueous 0.5% CMC with 0.5% Tween 80 and served as a control. The males were treated for at least 90 days prior to mating, during the three-week mating period and approximately three weeks thereafter. The females were treated for at least 14 days prior to mating, during mating and until day 19 of gestation or day 20 of lactation. Approximately half of the parental females were sacrificed on gestational day 20 and the remaining half were sacrificed on lactational day 21. Day 20 fetuses were examined grossly for anomalies. Approximately one-third of the kidneys from gestational day 20 control and 200 mg/kg fetuses were examined grossly and microscopically for potential effects on fetal kidney development. Behavioral testing (grip strength, open field motor activity and passive avoidance) was conducted on randomly selected F1 animals from all groups. Following sexual maturation of the F1 generation, selected males .and females within each group were mated to evaluate fertility and reproductive performance.

Examinations

Parental animals: Observations and examinations:

Physical Condition, Body Weight, Food Consumption, Or~an Weights, Vaginal Washinas,

Oestrous cyclicity (parental animals):

Only those females found to be cycling normally as demonstrated by vaginal washing cytology and those animals judged healthy based on general observations, physical examinations and body weights performed during the acclimation period were used on this study. Sperm-positive females in the first group were dosed
through day 19 of gestation and sacrificed on day 20 of gestation. Spermpositive females in the second group were dosed through day 20 of lactation and sacrificed on day 21 of lactation. Females that were not found sperm-positive were maintained until their last possible day 25 of gestation. At that time they were sacrificed via C02 asphyxiation and necropsied.

Litter observations:

Sex and Number of Live Pups, Pur, Bodv Weights, Mortalitv and Clinical Observations, Postvartum Develo~mental Parameters, Behavioral, Post~artum Fertility Assessment

Postmortem examinations (parental animals):

evaluation of the animals' visceral organs, particularly the reproductive organs

Postmortem examinations (offspring):

Renal Gross Examination and Histopathology

Statistics:

Parental Body Weight and Food Consumption Parameters and Organ Weights and Reproductive and F1 Generation Parameters:
All analyses were conducted in accordance with the SOPS of RSS. Statistical analysis of pup and fetal weights were performed using a TEROS program on the MCS that employed Healy's weighted analysis and was transferred to RSS for interpretation and reporting. The Study Director, after consultation with RSS,
decided what further statistical evaluations were to be performed, and the statistical tests applied are referenced in the statistics section of the final report.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: A treatment-related clinical sign of salivation (after dosing) was observed in males at doses 2 50 mg/kg. Animal #317 in the 10 mg/kg group had salivation on day 30 (12/16/92). Since this sign was noted prior to dosing and at the same frequency as control males, it was considered incidental to treatment. All other clinical signs (i.e., alopecia, chromodacryorrhea, diarrhea, staining, stool changes and broken or missing teeth) were considered incidental based on a low incidence of
occurrence, the absence of a dose-response relationship or because they were associated with the non-compound-related death of #341.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males: No compound-related deaths occurred during the study; however, two incidental mortalities were noted in males at 200 mg/kg. No treatment-related deaths occurred in a previously conducted 13-week oral toxicity study with CGP 48933 in rats at doses up to 600 mg/kg which further indicates the above mortalities were incidental to treatment.
Females: A treatmentrelated clinical sign of salivation was observed in females at 200 mg/kg. Salivation noted in one female (#3168) in the 50 mg/kg was considered incidental as it was noted on one day only. All other clinical signs (alopecia, distended stomach and decreased stool) were considered incidental based on absence of a dose-response relationship. In addition, umbilical hernia was noted in two control females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: A slight, non-statistically significant reduction in body weight (approximately 5% to 9%) occurred in males at 200 mg/kg on days 42 to 126. There were no treatment-related changes in male body weight at 10 or 50 mg/kg. Treatment-related, statistically significant and nonsignificant, decreases in body weight gain (approximately 9% to 78% of concurrent control) were noted in males at doses 2 50 mg/kg from day 0 through day 77 with a tendency toward reduction overall days 0 to 126. The mean body weight loss noted at 200 mg/kg on days 56 to 63 was due to the 104g loss of animal #341 that died on day 69. Since this animal ' s demise was not considered a result of treatment, mean body weight loss was also not considered a compound-related effect. Exclusion of animal #341 resulted in a mean body weight gain of 3.6 + 7.2 grams for days 56 to 63. Therefore, a reduced body weight gain was still apparent for this group during this interval. All other changes in body weight gain from days 77 to 126 (significant loss at 200 mg/kg on days 91 to 98 and significant increases at 10 and 50 mg/kg on days 105 to 112) were considered incidental due to sporadic occurrences, absence of a dose-response relationship or possibly due to a low control value for this parameter.
Females: Treatment-related, statistically significant decreases in body weight (approximately 7% to 11% of the concurrent control value) occurred at 200 mg/kg on premating days 7 and 14, gestation days.0, 6, 13 and 20, corrected gestation day 20, and lactation day 0. A significantly lower body weight (less than approximately 5%) was noted on premating day 0 prior to the dosing period. However, since concomitant reductions in body weight gain were also noted (see below), the effects on body weight during the dosing period noted above could be supported as compoundrelated. Treatment-related, statistically significant reductions in body weight gain (approximately 22% to 62% of the concurrent control value) were noted at 200 mg/kg on premating days 0 to 7, gestation days 0 to 6, and corrected gestation body weight gain days 0 to 20 (non-statistically significant). A statistically significant increase in body weight gain at 200 mg/kg on lactation days 0 to 7, 7 to 14 and overall days 0 to 21 was not considered a compound-related effect. This difference was attributed to biological variation resulting in a low control value for these intervals. There were no treatment-related changes in body weight parameters in females at 10 or 50 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: No compound-related changes in food consumption were apparent in males at any dose level.
Females: Compound-related, statistically significant decreases in food consumption were noted in females at 200 mg/kg during premating days 0 to 14 and gestation days 0 to 6. Non-statistically significant reductions occurred during gestation days 6 to 13 and 13 to 20. All decreases ranged from approximately 7% to 14% of the concurrent controlvalues. No compound-related changes in food consumption were noted in females during lactation or for any period at 10 or 50 mg/kg.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no significant compound-related effects on any examined reproductive parameters, on fetal sex ratios, or on fetal weights in animals C-sectioned on gestational day 20. A higher absolute and percent preimplantation loss at 10 mg/kg was attributed to two dams . that had 67% and 89% loss and was incidental based on the lack of a doserelationship. The numbers of corpora lutea, number of implantations and live fetuses were slightly lower than control at 10 and 200 mg/kg; however, these observations were not dose-related and were considered incidental. Furthermore, there were no effects on pre- and postimplantation losses. A higher absolute and percent postimplantation loss at 50 mg/kg was not dose-related and incidental to treatment. Gross examination of fetuses did not reveal any compound-related findings. A single, nondose-related finding of domed head in the 50 mg/kg group was considered incidental. Two fetuses in the control group were observed with a shared placenta.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No compound-related effects on reproductive parameters for full-term females were evident at any dose level. Higher absolute and percent stillbirths at 50 mg/kg were considered incidental due to the lack of a dose-response relationship.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no compound-related changes in any Fo parental fertility parameter evaluated. Although a lower percentage of females was inseminated (87.5%) at 200 mg/kg, this was considered within normal range for this species.

Details on results (P0)

There was no effect of treatment on gestation duration for full-term females.

Effect levels (P0)

Key result

Dose descriptor:

NOEC

Effect level:

< 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg diet

System:

gastrointestinal tract

Organ:

salivary glands

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related postpartum clinical signs in the F1 generation animals.
Abnormal incisors at 200 mg/kg and blood in the urine at 50 mg/kg were considered incidental to
treatment due to the low frequency of occurrence. One control pup was observed with eye agenesis.
There were no compound-related effects on evaluated developmental parameters in either sex at any
dose level. Statistically significant increases in time to ear canal opening at 2 50 mg/kg were noted in
females only and was not considered to be biologically significant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in mean litter size, pup sex ratio or survival at any dose level during this study. At 200 mg/kg, the 94.3% female survival on days 0 to 4 (preculling), althou gh lower than control, was considered to be incidental to treatment due to lack of a dose-response relationship. Furthermore, this value represents the loss of only one female pup in each of four litters.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related effects on pup weight. In all treated groups, pup weight tended to be slightly lower compared with controls. However, these changes were generally not dose-related. Furthermore, review of day 0 lactation litter size revealed non-dose-related and incidentally larger litter sizes in all treated groups. Since the Healy analysis generates pup weight means that are weighted by litter size, additional statistical analyses to generate unweighted means pup weights were performed. Unweighted pup means from treated groups still tended to be lower than controls, indicating that pup weight decreases could in part, be attributed to litter size effects that occurred during early lactation. In addition, significantly lower female pup weights on day 35 were attributed to biological variation resulting in a higher control value.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The kidneys of fetuses from females receiving 0 and 200 mg/kg CGP 48933 harvested on gestation day 20 were evaluated for potential effects of the compound on fetal kidney development. There were no compound-related gross or microscopic abnormalities. Grossly, the kidneys appeared normal.
Microscopic examination revealed a similar incidence, in the control and treated groups of minimal to mild dilatation of the renal pelvis. The change in the kidneys was considered a normal developmental variation and has been reported to be due to a disparity, during late gestation, in growth rates between the renal papilla and renal parenchyma. The result is a relative, transient enlargement of the renal pelvis

Gross pathological findings:

not specified

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes considered treatmentrelated, noted in either sex at any dose level, during open field motor activity, passive avoidance or grip strength testing. During open field motor activity testing, a significant increase in peripheral and total beam breaks occurred with females at 200 mg/kg. This finding did not occur with tested males, was not biologically dose-related in the females and
was therefore considered incidental. Passive avoidance testing of females at 200 mg/kg resulted in an increase in trials to success (criterion) during the retention (memory) phase. However, this finding was not considered compound-related for the following. First, there were no significant differences between the retention score (average latency per trial) in this group compared with control. Second,
the difference between the learning and retention trials, which is.an indication of memory relative to learning, was not significantly different in the 200 mg/kg group female versus controls. Finally, the change in retention was not noted in tested males.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

There was no effect of treatment on F1 generation fertility at any dose level

Effect levels (F1)

Key result

Dose descriptor:

NOEC

Generation:

F1

Effect level:

< 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (F1)

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

50 mg/kg bw (total dose)

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Administration of CGP 48933 produced parental effects at doses 2 50 mg/kg. There were no effects on fertility or reproductive performance in any generation and no effects on F1 generation development.