2-[(2S,3S)-3-[[(1,1dimethylethoxy)carbonyl]amino]-2-hydroxy-4-phenylbutyl]-2-[[4-(2pyridinyl)phenyl]methyl]-,1,1-dimethylethyl ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2017 - 29 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl 2-{(2S,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-4-phenylbutyl}-2-[4-(pyridin-2-yl)benzyl]hydrazinecarboxylate

EC Number:

606-396-7

Cas Number:

198904-86-8

Molecular formula:

C32H42N4O5

IUPAC Name:

tert-butyl 2-{(2S,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-4-phenylbutyl}-2-[4-(pyridin-2-yl)benzyl]hydrazinecarboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage condition of test material: At room temperature protected from light

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study, as demonstrated in a separate study

OTHER SPECIFICS:
- Test item handling: use amber glassware or wrap container in aluminum-foil

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and eproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: not applicable, reproduction/developmental toxicity screening study
- Age at study initiation: (P) males 10 weeks, females 13 weeks at the start of dosing
- Weight at study initiation: (P) Males: 273-306 g; Females: 204-236 g;
- Fasting period before study: none
- Housing:
Pre-test (females only) and pre-mating: group-housed (up to 5 animals/sex/group) in polycarbonate Macrolon cages
During mating: males and females on 1:1 basis in Macrlon cages
Post-mating: males in their home cages (up to 5 animals/sex/group), females individually in Macrolon palstic cages
During lactations: female dams with pups in Macrolon plastic cages, equipped with water bottles and appropriate bedding.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum, except during designated procedures
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 48-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 June 2017 To: 17 August 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial preparations performed at the test facility
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Specific gravity: 1.036

Details on mating procedure:

- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as day 0 post-coitum
- Further mating if unsuccessful after 14 days: no
- After successful mating each pregnant female was caged individually in Macrolon palstic cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by LC using a validated analytical procedure. Accuracy and homogeneity were determined for formulations on the first day of preparation and towards the end of the study. Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory at the test facility. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Duration of treatment / exposure:

A minimum of 28 days (males for 29 days, up to and including the day before the scheduled necropsy; females that delivered, for 50-63 days; females that failed to deliver, for 41-43 days; one female that was found dead on day 2 of mating, was treated for 15 days).

Frequency of treatment:

Once daily, 7 days/week. Pups were not dosed, but could have been exposed to the test substance via lactation.

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks males, 15 weeks females.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle controls (Group 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on information provided by the Sponsor (28-day repeated dose toxicity study with oral administration of BMS-233101-01 in rats), and in an attempt to produce graded responses to the test item. In this 28-day study, dose levels of 0, 15, 150 and 1000 mg/kg bw/day were tested and a NOEL of 1000 mg/kg bw/day was established. This is the highest dose to be tested according to the applicable OECD 421 test guideline.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

Not applicable.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

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

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

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

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

OTHER:
Thyroid hormone examination: measurement of T4 was conducted for F0-males. Blood was collected on a day of scheduled necropsy. F0-animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except forone female in low dose group that died spontaneously.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight. For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup), but no T4 assessment was performed.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
Measurement of T4 was conducted for PND 13-15 pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; pups were also sexed. Possible cause of death was determined for pups born or found dead. The stomach was examined for the presence of milk, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after a minimum of 28 days of exposure.
- Maternal animals: All surviving animals at PND 14-16
- Females that failed to deliver: post-coitum day 25-26

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: the following organ weights were determined for all scheduled euthanasia animals: epididymis, coagulation gland, thyroid, including parathyroid, prostate, seminal vesicles, testes.

HISTOLOGY: the following tissues were examined by histopathologist:
All animals: thyroid gland and gross lesions/masses
Males and females of controls and high-dose group: epididymides, ovaries and testes
Unscheduled deaths (1 low-dose female found dead): cervix, mammary gland, ovaries, uterus, vagina, gross lesions/masses.
Males that failed to sire and females that failed to deliver healthy pups: cervix, coagulation gland, prostate gland, seminal vesicle gland, uterus and vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on PND 13-15.
- Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.


HISTOPATHOLOGY / ORGAN WEIGTHS: not performed.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

The following indices were calculated:
Mating index = 100% x (number of females mated/number of females paired)
Precoital time = number of days between initiation of cohabitation and confirmation of mating
Fertility index = 100% x (number of pregnant females/number of females mated)
Gestation index = 100% x (number of females with living pups on PND1/number of pregnant females)
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

The following indices were calculated:
Post-implantation survival index = 100% x (total number of offspring born/total number of uterine implantation sites)
Live birth index = 100% x (total number of live offspring on PND 1/total number of offspring born)
Percentage of live males at first litter check = 100% x (number of live male pups at first litter check/number of live pups at first litter check)
Percentage of live females at first litter check = 100% x (number of live female pups at first litter check/number of live pups at first litter check)
Viability index = 100% x (number of live offspring on PND 4 before culling/number of live offspring on PND 1 after littering)
Lactation index = 100% x (nueber of live offspring on PND 13/number of live offspring on PND 4 after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations.
Salivation seen after dosing among animals at dose levels starting at 100 mg/kg bw/day on several occasions during the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect, its time of occurrence (i.e. after dosing) and in the absence of a dose-related response. This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other incidental findings that were noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.
During dosing on the first day of pre-mating, one mid-dose female bit on her tongue, followed by a short period of bleeding (approximately 1 minute).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 100 mg/kg bw/day was found dead on Day 2 of the mating period. The day preceding the spontaneous death, slight rales were noted for this female, and on the next morning slightly laboured respiration and piloerection were observed. At necropsy, dark-red discoloration of the lungs and many reddish foci on the thymus were noted. The discoloration of the lungs together with the respiratory problems may be indicative for an oral gavage accident, although no cause of death could be determined at microscopic examination. Based on the single occasion and as a dose-relationship was lacking, this mortality was considered not to be related to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were slightly lower for females at 1000 mg/kg bw/day during the lactation phase (not always reaching statistical significance).
Body weights and body weight gain of males up to and including 1000 mg/kg bw/day and females up to and including 300 mg/kg bw/day remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was slightly lower for females at 300 and 1000 mg/kg bw/day during the lactation phase, however generally without being statistically significant and lacking a dose-relationship.
Food consumption before or after correction for body weight was similar to the control level for males up to and including 1000 mg/kg bw/day and females at 100 mg/kg bw/day over the treatment period.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone (T4) was considered not to be affected in males up to and including 1000 mg/kg bw/day. The statistically significantly lower T4 level for males at 100 mg/kg bw/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with BMS-233101-01 were noted in the thyroid gland of the 1000 mg/kg bw/day group males and females.
An increased incidence and severity of follicular cell hypertrophy was present in males and females at 1000 mg/kg bw/day (minimal-slight in males, 7/10, in total, vs. 5/10 in controls; minimal to moderate in females, 8/10 vs. 9/10 (minimal/slight) in controls). In addition, colloid alteration was present in the thyroid gland in males at 1000 mg/kg bw/day (minimal to slight, 3/100, vs. 0 in controls). There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected bytreatment.
Most females had regular cycles of 4 to 5 days. Extended di-estrus occurred in two females at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day with a regular cycle, and in one control female with an irregular cycle. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not considered to be affected by treatment. All females, except one, showed evidence of mating. One female at 100 mg/kg bw/day was found dead on Day 2 of mating.
Precoital time was not considered to be affected by treatment. Most females showed evidence of mating within 4 days. Two females at 100 mg/kg bw/day showed evidence of mating after 13 days only, after extended di-estrus. In the absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Fertility index was not considered to be affected by treatment. One female at 300 mg/kg bw/day was not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.
Number of implantation sites was not considered to be affected by treatment. One female at 100 mg/kg bw/day had a single implantation site only. For this female, a mummified fetus was found in the uterus. And one female at 1000 mg/kg bw/day had three implantation sites only and delivered two live pups. At these single occurrences in absence of a dose response relationship, this was considered to be unrelated to treatment.
Gestation index and duration of gestation were not considered to be affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects observed at the highest dose level (slightly lower body weights in females, minimal increased incidence and severity of follicular cell hypertrophy in thyroids in both sexes and colloid alteration in males) were considered to be non-adverse

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose level

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

System:

other: no adverse effects at the highest dose level

Organ:

other: no adverse effects at the highest dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of all clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
Two pups at 300 mg/kg bw/day from two litters were found dead on PND 2 or 4. One pup of the control group and one pup at 300 mg/kg bw/day were found missing on PND 4 and 2, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. One pup at 100 mg/kg bw/day was found missing (i.e. most likely cannibalised) on PND 9. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not considered to be affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose level

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

System:

other: no adverse effects at the highest dose level

Organ:

other: no adverse effects at the highest dose level

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Any other information on results incl. tables

Analytical determination:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (at the first week of preparation, mean accuracies were 90, 88 and 91%, respectively; at the last week of preparation, mean accuracies were 100, 99 and 99%, respectively; n = 6 in low and high dose, 2 in mid-dose).

The formulations were considered to be homogeneous (at the first week of preparation, coefficients of variations were 3.7 and 3.2% in low- and high-dose solutions; at the last week of preparations, they were 3.8 and 1.9%, n = 6).

Applicant's summary and conclusion

Conclusions:

In the OECD guideline 421 study with rats, performed under GLP, oral gavage administration of the test item in propylene glycol at dose levels of 0 (vehicle controls), 100, 300 and 1000 mg/kg bw/day caused no adverse effects on reproduction and development at the highest dose tested. The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day. Slightly lower body weights and lower food intake in parental animals and minimal changes in thyroid (sligthly increased incidence and severity of follicular cell hypertrophy in both sexes and minimal to slight colloid alteration in 3/10 males) were considered to be non-adverse. Therefore the NOAEL for general toxicity was also set at 1000 mg/kg bw/day.