Segment I and Segment III Female Reproductive Toxicity Studies

Background: The Segment Framework

• Segment I (FEED) covers Stages A-B
• Segment II (EFD) covers Stages C-D
• Segment III (PPND) covers Stages C-F

SEGMENT I - Fertility and Early Embryonic Development (FEED) Study

Purpose

Species Used

• Primarily rodents (rats preferred)
• Non-rodent species may be added if data warrants

Female-Specific Dosing Timeline

• Treatment begins 2 weeks before mating (approximately 2 estrous cycles) to capture the window of folliculogenesis and oocyte maturation
• Dosing continues through mating and up to gestational day 6-7 (implantation)
• Some designs extend dosing to GD 13-15 to differentiate live implantations from resorption sites

Female-Specific Endpoints Assessed

• Vaginal cytology is examined daily during pre-mating and mating phases
• Evaluates regularity, phase duration, and any disruption of the estrous cycle
• Disruption indicates hypothalamic-pituitary-gonadal (HPG) axis interference

• Females are co-housed with untreated males (or treated males in full fertility studies)
• Pre-coital interval (days from pairing to mating) is recorded
• Copulation index = % females that mated / % females paired

• % of mated females that became pregnant
• Indicates whether successful fertilization occurred

• Corpora lutea count (per ovary) - indicates number of released eggs
• Pre-implantation loss = (corpora lutea - implantation sites) / corpora lutea × 100
• Ovarian weight and histopathology

• Total implantation sites
• Live implantations
• Early resorptions (dead implants before GD 13)
• Post-implantation loss rate

• Ovaries, uterus, vagina, oviducts - examined microscopically
• Follicle counts (primordial, primary, secondary, Graafian follicles) - identifies follicular depletion or atresia
• Corpus luteum quality

• Evaluates oviductal function (movement of fertilized egg toward uterus)

Key Calculations in Segment I (Female)

Dose Selection

• Minimum 3 dose groups + vehicle control
• Highest dose: maximally tolerated dose (MTD) or limit dose (1000 mg/kg)
• NOAEL and LOAEL are derived from these dose groups

Regulatory Context

SEGMENT III - Pre- and Postnatal Development (PPND) Study

Purpose

1. The pregnant/lactating female
2. Parturition (the birth process)
3. Offspring development from birth through sexual maturity and reproductive capability

Species Used

• Primarily rats (rodents)
• Non-human primates (NHPs) may be used for biologics (ePPND study)
• Rabbits are typically used for Segment II, not Segment III

Female Dosing Timeline

• Dosing begins on Gestational Day 6-7 (post-implantation, to avoid confounding implantation effects which are Segment I territory)
• Continues through parturition and lactation until postnatal day (PND) 21 (weaning)
• This covers: late organogenesis, fetal growth, birth, and nursing

Female-Specific Endpoints - Maternal Assessment

• Body weight and food consumption throughout gestation and lactation
• Clinical observations for signs of enhanced toxicity in the pregnant/lactating state (pregnant animals may be more susceptible due to altered PK/PD)

• Number of days from confirmed mating to delivery
• Prolonged or shortened gestation indicates interference with hormonal parturition signals (prostaglandins, oxytocin, progesterone withdrawal)

• Duration and ease of delivery
• Dystocia (difficult or obstructed labor)
• Number of stillbirths at birth
• Cannibalism behavior by dam (reflects maternal stress/neglect)

• Nursing behavior and litter care
• Milk production (assessed indirectly by pup weight gain)
• Milk yield via pup weight on PND 1 vs. PND 4 (pre-weaning weight gain index)

• Uterine involution
• Return to estrous cyclicity after weaning

Offspring (F1 Generation) Endpoints

• Viability index: pups alive on PND 4 / total born alive × 100
• Weaning index: pups alive at PND 21 / pups alive on PND 4 × 100
• Pup body weights at PND 1, 4, 7, 14, 21 - tracks growth trajectory

• Vaginal opening (females) - indicator of sexual maturation timing
• Balanopreputial separation (males)
• Eye opening, pinna unfolding, incisor eruption - general developmental markers

• Neurobehavioral: motor activity, learning and memory (Morris water maze or passive avoidance), sensory function (acoustic startle response, visual placing)
• Physical development: body weight, organ weights at necropsy

• At least 1 male and 1 female per litter are selected and reared to adulthood
• These F1 adults are then mated to assess their own fertility (this is what extends the study to sexual maturity)
• Endpoints mirror Segment I: fertility index, litter size, implantation data

• Brain, reproductive organs, liver, kidneys of selected pups

Key Indices in Segment III

Regulatory Context

• Segment III (PPND) is generally required before Phase 3 trials and before marketing authorization
• Under ICH M3(R2), PPND is not essential for Phase 2 trials but must be complete prior to marketing
• For anticancer drugs targeting advanced cancer (ICH S9), PPND is generally not required to support marketing

Comparison: Segment I vs. Segment III

Why Both Studies Are Needed

• ICH S5(R3) - Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals
• ICH M3(R2) - Nonclinical Safety Studies for Clinical Trials
• ICH S9 - Anticancer Pharmaceuticals

Segment I and Segment III Female Reproductive Toxicity Studies - Per OECD Guidelines

OECD Guideline Framework Overview

SEGMENT I - Female Reproductive Toxicity

Applicable OECD Test Guidelines

1. OECD TG 415 - One-Generation Reproduction Toxicity Study

• Begins at least 2 weeks before mating (to cover at least 2 complete estrous cycles)
• Continues through mating, gestation, and lactation until weaning

• Estrous cyclicity - vaginal smears taken daily to characterize cycle length, phase duration, and regularity before and during treatment
• Body weight and clinical signs monitored weekly

• Pre-coital interval - days from pairing to confirmed mating (vaginal plug or sperm-positive smear)
• Copulation index = (females mated / females paired) × 100
• Fertility index = (pregnant females / females mated) × 100

• Females weighed on GD 0, 7, 14, and 20/21
• Food consumption measured weekly
• Duration of gestation recorded

• Ease and duration of delivery
• Dystocia observations
• Cannibalism rate

• Maternal behavior and nursing
• Litter care assessment

• Ovarian weight and histopathology
• Corpora lutea count per ovary (number of eggs released)
• Follicle counts (primordial, primary, secondary, antral) to detect follicular depletion
• Uterine weight and gross appearance
• Histopathology of: ovaries, uterus, cervix, vagina, oviducts, mammary glands

• Total number of implantation sites
• Number of live implants (live fetuses/resorptions)
• Pre-implantation loss = (corpora lutea - implants) / corpora lutea × 100
• Post-implantation loss = (implants - live fetuses) / implants × 100

2. OECD TG 416 - Two-Generation Reproduction Toxicity Study

• Dosing begins 10 weeks before mating (to encompass full folliculogenesis cycle)
• Continues throughout mating, gestation, and lactation of the F1 litter
• Estrous cycles monitored via daily vaginal cytology during pre-mating, mating, and into gestation
• Gestation weights: GD 0, 7, 14, 20/21
• Lactation weights: same schedule as litter weighings

• F1 pups are selected at weaning to become the F1 parents
• F1 females are dosed from weaning (PND 21) through mating, gestation, and lactation of F2 litter
• Vaginal opening (sexual maturation marker) is monitored daily from PND 25-30
• First estrous cycle post-vaginal opening is recorded
• Estrous cycle monitored for at least 2 weeks before F1 mating
• F1 females are assessed for all the same endpoints as P females

• Anogenital distance (AGD) measured at birth and PND 4 - indicator of androgenic/estrogenic disruption
• Number of nipples/areolae in male pups - indicator of anti-androgenic effects
• Vaginal cytology of F1 females during adulthood

3. OECD TG 421 - Reproduction/Developmental Toxicity Screening Test

• 2 weeks before mating, through mating, gestation, and at least 13 days post-partum

• Estrous cycles: monitored from pre-dosing period to select females with regular cyclicity; vaginal smears daily from start of treatment to evidence of mating
• Mating and fertility indices
• Gestation weights (GD 0, 7, 14, 20; within 24 h of parturition; PND 0/1, 4, 13)
• Food consumption weekly
• Parturition observation: timing, duration, stillbirths
• Pup viability on PND 4 and 13
• Uterine examination at necropsy: number of implantation sites, corpora lutea

4. OECD TG 422 - Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test

5. OECD TG 443 - Extended One-Generation Reproductive Toxicity Study (EOGRT)

• Optional Cohort 2 (developmental neurotoxicity) and Cohort 3 (developmental immunotoxicity)
• Ovarian primordial follicle quantification is mandatory
• Thyroid hormones (T3, T4, TSH) in dams and F1 pups
• Expanded developmental landmarks and functional endpoints

SEGMENT III - Female Reproductive Toxicity (Pre- and Postnatal Development)

Primary OECD Test Guideline: TG 426 - Developmental Neurotoxicity Study

OECD TG 426 - Developmental Neurotoxicity (DNT) Study

• General maternal toxicity during gestation and lactation
• Offspring developmental and neurofunctional endpoints
• Offspring reproductive competence (when F1 animals are evaluated through adulthood)

Female (Dam) Endpoints in OECD TG 426

• Body weight: recorded at GD 0, 6, 9, 12, 15, 17, 20 and PND 1, 4, 7, 14, 21
• Food consumption: weekly throughout gestation and lactation
• Clinical signs: daily observation; onset, degree, duration of any toxic signs recorded individually
• Comparison of toxicity in pregnant vs. non-pregnant state is mandatory - to detect whether the gravid/lactating female is more susceptible (enhanced toxicity)

• Gestation length (number of days from confirmed mating to delivery)
• Parturition duration and ease - prolonged or difficult delivery noted
• Number of pups born alive vs. stillborn
• Post-partum observations for cannibalism or neglect

• On or before PND 4, litter size is adjusted (culled) to the average litter size for the strain (typically 8-12 pups per litter), with equal sex ratio
• This step normalizes nutritional availability through maternal milk - critical to prevent confounding

• Nursing posture, pup retrieval, nest building
• Maternal milk sufficiency assessed indirectly through pup weight gain

Offspring (F1) Endpoints in OECD TG 426 - Sex-Specific Developmental Landmarks

• Motor activity: measured using automated activity monitors; assessed at approximately PND 21, 40-45, and 60-70; should show habituation (decrease in activity within session)
• Acoustic startle response: assessed at approximately PND 22-24 and post-weaning; includes prepulse inhibition
• Cognitive function: learning and memory tests (passive avoidance or Morris water maze) at approximately PND 60-75
• Sensory function: visual and auditory assessments

• Perfusion-fixed brains at PND 11 (neonatal) and PND 70+ (adult)
• Brain weight and morphometric analysis
• Brain regions examined histologically: cerebral cortex, hippocampus, cerebellum, striatum, brainstem, olfactory bulb
• Morphometric measurements: cortical thickness, hippocampal areas, cerebellar volume

Segment III Endpoints Embedded within OECD TG 416 (Two-Generation Study)

• Live birth index, viability index, lactation index (formulae listed above)
• Pup weights on PND 1, 4, 7, 14, 21
• Anogenital distance (AGD) at PND 1 and 4 (sex-specific index of endocrine disruption)
• Number of nipple/areolae anlagen in male pups
• Sex ratio at birth and on PND 4
• Developmental landmarks: vaginal opening (females), testis descent and balanopreputial separation (males)

• Vaginal opening monitored daily from PND 25-30
• First estrous cycle after vaginal opening recorded
• F1 females mated to produce F2 litter - all parturition/litter data collected again (confirms the next generation's fertility)

Summary Comparison Table: OECD-Based Segment I vs. Segment III

Regulatory Notes (OECD Context)

• TG 415 is the older (1983) one-generation design and is being phased out in favor of TG 443
• TG 416 (two-generation) is still widely used but TG 443 is now the preferred choice under OECD because it reduces animal use while providing richer data (neurotoxicity, immunotoxicity cohorts)
• TG 421/422 are screening tests only - they cannot substitute for TG 414, 415, 416, or 443 for regulatory submissions
• OECD guidelines align closely with ICH S5(R3) for pharmaceuticals, but OECD TGs also apply to industrial chemicals, pesticides, and food additives under REACH, CLP, and biocidal product regulations

• The oldest and simplest design; females dosed 2 weeks pre-mating through lactation
• Only one generation assessed; no F1 adult mating
• Being phased out - limited current regulatory acceptance

• Adds a full F1 generation of adult females dosed from weaning, mated to produce an F2 litter
• Captures vaginal opening, first estrous cycle, and F1 female fertility
• Still widely used under REACH for industrial chemicals and pesticides

• The current preferred OECD standard - replaces TG 416 for most purposes
• Mandatory primordial follicle quantification and thyroid hormones in dams/F1
• Modular cohort system: Cohort 2 (neurotoxicity) and Cohort 3 (immunotoxicity) can be triggered
• Reduces total animal use vs TG 416 while providing deeper mechanistic data

TG 415 (Teal - top section)

• P female row: Orange pre-mating bar (14 days, 2 estrous cycles) → mating → green gestation (GD 0-21) → blue lactation (PND 0-21) → red necropsy marker
• F1 litter row: Shows pup monitoring PND 0-21; annotated with live birth index, viability index, pup weights, sex ratio
• Status badge: ⚠ OUTDATED - limited current regulatory use

TG 416 (Blue - middle section)

• P female row: 70-day pre-mating bar (10 weeks for full folliculogenesis) → mating → gestation → lactation → necropsy
• F1 daughters row: Dosed from weaning (Day 117) → pre-mating with vaginal opening monitoring → mating → gestation → lactation → necropsy
• F2 litter row: PND 0-21 with AGD, organ weights annotated
• Status badge: ✔ Still accepted - REACH / pesticides / industrial chemicals

TG 443 EOGRT (Purple - bottom section)

• P female row: Pre-mating with mandatory primordial follicle count + T3/T4/TSH annotation → gestation → lactation → necropsy
• Cohort 1A (F1 fertility): Post-wean growth → adult pre-mating → mating → gestation → F2 litter → necropsy
• Cohort 2 (DNT - triggered): Extends from PND 21 to ~Day 140 with motor activity, startle, learning/memory, brain morphometry
• Cohort 3 (DIT - triggered): Extends to ~Day 126 with lymphocyte subsets, NK cell activity, T-cell antibody response
• Status badge: ★ PREFERRED CURRENT STANDARD