Current Regulatory Requirements in DART Assessments: Segmental

Study Designs Versus The Biologic Continuum

Joseph F. Holson, PhD, DABFEWIL Research Laboratories, LLC

Validity/Predictivity of Animal Models

End Points Comments on Concordance

Carcinogenesis High & multiple studies

Developmental Toxicity

High & multiple studies with only one using power calculations

Developmental Neurotoxicity

•Two, no power considerations, concordance high

Fertility •Highly conserved process, no rigorous studies with systematic process, many anecdotal-type comparisons, considerable experience in dealing with peri-ovulatory endocrine mechanisms•Purely temporal differences between rat and human possible (e.g., squalene synthase inhibition)

Overall Reproductive Toxicity

No rigorous studies considering power, study design and comparability of studies

Safety Pharmacology

No comprehensive studies, power not considered, but concordance considered intermediate to high except for reconciling high dose to low-dose extrapolation

Ontogeny of Physiologic Regulationin Selected Mammals

Implantation

First Heart Beat

Exterioception

Hemoglobin 8% in Blood

Body Weight 1 gm

Thyroid Iodine

Lung Surfactant

Liver Glycogen 0.05%

Birth

Water 85% of Fat-free

Na/K one gm/gm

Anoxia Tolerance 10 min.

Body Fat 5%

Arterial Pr. 50 mm/Hg

Lethal Temp Shift

Resistance to Cooling

Stagemarks

4

Days After Conception

Hamster Rat Rabbit Cat Pig Human

8 10 20 40 80 100 200 400

After Adolph, 1970

Relationship Between Developmentand Phenotypic Diversity

Degree of Phenotypic Variability

Time in Development (Age)

EmbryonicPeriod

FetalPeriod

PostnatalPeriod

Extent of Differentiation

Birth

Holson et al., 2006

Exposure-Based Approach to Study Design

• It is perceived that tests in which animals are treated during defined stages of reproduction better reflect human exposure to medicinal products and allow more specific identification of stages at risk.

• While this approach may be useful for most medicines, long term exposure to low doses does occur and may be represented better by a one- or two-generation study approach.

Adapted from ICH, 1994

Exposures

• Unintended Exposures – Complete (almost) Life-Cycle Study• Industrial and agricultural chemicals

(EPA/OPPTS/OECD)• Food Additives (Redbook 2000)

• Intended Exposures – Segmented Approach• Therapeutics (FDA/ICH)

DART Guideline Studies by Agent/Chemical Use Class (Excluding Medical Devices)

Human Therapeutic Veterinary Therapeutic* Nontherapeutic**

Fertility Assessment Product Use Dependent 1- and 2-Generation

Embryo-Fetal Development in Two Species (One Species for Biologics)

Target Species Prenatal Developmental Toxicity

1- and 2-Generation

and Prenatal Developmental Toxicity in Two Species

Pre- and Postnatal Development

1- and 2-Generation

Pediatric (Juvenile Animals)

Product Use DependentDevelopmental Neurotoxicity (Juvenile Animals)

*Majority of these agents are actually the human formulations modified in dose for the given species**For the sake of brevity, these are condensed but intended to apply to food additives, agrichemicals and industrial chemicals

Reproductive/Developmental Life Stages

A B C D E FPremating to Conception

Conception to Implantation

Implantation to Closure of

Hard Palate

Hard-Palate Closure to End of Pregnancy

Birth to Weaning

Weaning to Sexual Maturity

Reproductive/Developmental Life Stages:Selected Detectable Aberrations and Effects

A B C D E FPremating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Hard-Palate Closure to

End of Pregnancy

Birth to Weaning

Weaning to Sexual

Maturity

Cryptorchidism

Nipple Retention in Males

Hypospadias

Delayed Maturation

(Vaginal Patency/ Balanopreputial

Separation)

Precocious Puberty

(Vaginal Patency/ Balanopreputial

Separation)

Death

Dystocia

Interference with Lactation

Growth Retardation

Failure to Thrive

MorphologicalCNS Disruption

Abnormal Maternal Behavior

Death

Interference with Histogenesis

Spontaneous Abortion

Growth Retardation

Premature Delivery

Functional Deficit

Toxemia

Postimplantation Loss

Dysmorphogenesis

Growth Retardation

Dominant Lethality

Spontaneous Abortion

Mutagenesis

Male-Mediated Developmental

Toxicity

Accelerated Tubal Transport

Failed Uterine Deciduation

Failure to Implant

Ectopic Pregnancy

Chromosomal Aberrations

Death

Effects on Libido/Intromission

Impaired Sperm Motility

Decreased Sperm Count

Female Reproductive Cycle

Disruption

Ovulatory Blockade

Ovarian Dysfunction

Pseudopregnancy

G*Maturity to

Reproductive Senescence

Appearance of Latent and

Nonreproductive Effects

Premature Menopause

Accelerated Aging

Neoplasia (e.g., Mammary

Glands)

*These type of effects not

addressed by current standard

study designs

Non-Therapeutic Exposures – “Life-Cycle” Assessment

• The two-generation reproduction study is an apical, comprehensive test, evaluating long-term, low-level exposures

• Human exposures are involuntary, largely uncontrolled and often unavoidable (e.g., residues in food)

• Evaluates fertility, gestation, lactation, offspring maturation through two generations (in essence, covering all life stages from conception through early adulthood)

• Clarification and enhancement of effects that were marginal or not obvious in the first generation

Adapted from Cooper et al., 2006

Denotes Dosing Period

A B C D E F

Premating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Hard-Palate Closure to End of Pregnancy

Birth to Weaning Weaning to Sexual Maturity

ParturitionGestation Length Pup Viability Litter SizeLandmarks of Sexual Development Pup WeightNeurobehavioral Assessment Organ Weights Acoustic Startle Response F1 Mating and Fertility Motor Activity Hormonal Analyses Learning & Memory Ovarian QuantificationHistopathology Premature Senescence

Optional

Postimplantation LossViable FetusesMalformationsVariationsFetal Weight

Estrous CyclicityMatingFertilityCorpora LuteaImplantation SitesPre-Implantation LossSpermatogenesis

Single- and Multigenerational

Satellite Phase

OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB

F1

F2 ????????????????

????????????????

Modified from: Holson et al., 2006

Single and Multigenerational Study Designs

Duration of Phases of 2-Generation Reproductive Toxicity Study Design (Rats)

10 Weeks 2W 3W 3WF0

F1 10 Weeks 2W 3W 3WIU/L

IU/LF2

PBE B G L

PBE B G L

PBE = Prebreeding ExposureM = Breeding PeriodG = Gestational PeriodL = Lactational PeriodIU/L = Potential In Utero/Lactational Exposure

Approximately Nine Months

Limitations of Current Two-Generation Guideline

• No measurement of latent effects of endocrine modulation

• No measurement of functional deficits and organ system maturation

• Limited developmental neurotoxicity evaluation

• Limited developmental immunotoxicity evaluation

Cooper et al., 2005

Tier I Evaluation in ILSI Agricultural Chemical Safety Assessment (ACSA) Technical Committee Proposal

Dellarco et al., 2005

Extended One-Generation Study

ClinPath & DNT

Immunotox

SMVCE/Breeding

Case Study

Octamethylcyclotetrasiloxane (D4)

• Two-generation reproduction study of an ingredient in consumer products including a variety of personal care products and pharmaceuticals

• Standard guideline approach

• Low incidence of dystocia observed in F0 and F1 generations

D4

Holson et al., 2006

D4

• Laboratory’s historical control rate of dystocia was 0.6%.• Because of the lack of evidence of dystocia in the concurrent control

groups for either generation and the very low incidence of dystocia in the historical control data, the staff at the conducting laboratory viewed dystocia as a treatment-related effect.

• This conclusion was challenged because no statistical significance of dystocia occurred in any generation, there was no consistent pattern across generations and/or matings, and there was no apparent dose-response pattern. There were, however, plausible explanations for dystocia being a treatment-related effect in this study. Statistical significance was not (and would never have been) detected because the effect occurred at such a low incidence. In addition, the offspring of those animals exhibiting dystocia in the F0 generation were not represented in the F1 generation because of death. Therefore, an apparently decreased response in the first F1 mating was thought to be the result of loss of the more sensitive animals from the second generation. When two more instances of dystocia were observed in the F1 generation second mating (including one at a lower exposure level than previously observed), the laboratory’s conclusion was strengthened, thereby confirming the generational effect.

Holson et al., 2006

Exposure-Based Approach to Study Design

• It is perceived that tests in which animals are treated during defined stages of reproduction better reflect human exposure to medicinal products and allow more specific identification of stages at risk.

• While this approach may be useful for most medicines, long term exposure to low doses does occur and may be represented better by a one- or two-generation study approach.

Adapted from ICH, 1994

Intended Exposures – Segmented Approach

• Testing approach uses traditional segmented reproductive stage approach because exposures can generally be controlled temporally relative to reproductive stage

• Most commonly used models (rodent and rabbit) are amenable temporally, statistically and economically to segmented design

Advantages of Three-Segment Design

• Optimize exposures to key events in reproductive cycle and development, particularly if rapid enzyme induction is anticipated

• Therapeutic entities in general are designed to have relatively short half-lives and low potential for bioaccumulation

• Logistically more manageable than full life-cycle studies

• Allows mimicking of certain therapeutic regimens • Allows differentiation and examination of populations

at risk (biological model drives this)

A B C D E F

Premating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Hard-Palate Closure to End of Pregnancy

Birth to Weaning Weaning to Sexual Maturity

Parturition Litter Size Landmarks of Sexual DevelopmentGestation Length Pup Viability Neurobehavioral Assessment F1 Mating and Fertility Pup Weight Acoustic Startle Response

Organ Weights Motor Activity Learning & Memory

Postimplantation LossViable FetusesMalformations & VariationsFetal Weight

Estrous Cyclicity Mating Corpora Lutea Fertility Implantation SitesPre-Implantation Loss Spermatogenesis

Pre- and Postnatal Development

F1

ICH 4.1.2F0

????????????????

Prenatal Development

ICH 4.1.3

Fertility StudyICH 4.1.12W4W

CMAX

AUC

CMAX

AUC

10W

OECD 414 OPPTS 870.3600, 870.3700

Modified from: Holson et al., 2006

Segmented DART Study Designs

Rats:Therapeutic: GD 6-17Nontherapeutic: GD 6-19 (or 20)

Rabbits:Therapeutic: GD 7-20Nontherapeutic: GD 7-28

Denotes Dosing Period

Case Study

Iodomethane (MeI)

• “Zero ozone-depletion potential” methyl bromide soil fumigant replacement

• Example of animal model/developmental schedule apparent difference

• Initial reviews and results of general toxicity studies indicated low toxicity and absence of overt thyroid effects

• Rat two-generation reproduction and rabbit developmental toxicity studies conducted in parallel

Sloter, 2005

*

Effect of Iodomethane on Postimplantation Loss in the Rabbit

Sloter, 2005

*

*

Phased-Exposure Study to Determine Window of Sensitivity

Sloter, 2005

Fetal Death during Phased-Exposure Study

Standard ICH Guideline would not detect this effectSloter, 2005

GD 23-24 GD 25-26

GD 6-28Comprehensive

GD 6-28(Summed)

GD 6-14 GD 15-22 GD 27-28

Rabbit Fetal Biomarkers Evaluated with and without Exposure from GD 21 to 27

Sloter, 2005

TSH, T4, T3

Sloter, 2005

Fetal TSH and Thyroid Hormones with and without Iodomethane Exposure

T3

T4

TSH

•Indicates Direct Effect on Fetal Thyroid FunctionReveals Onset of Thyroid Function in Fetal RabbtThese Effects Would be Missed in ICH Regimen due to Eight Fewer Days of Exposure

Sloter, 2005

Thyroglobulin (Colloidal) Depletion following GD 23-26 Exposure to Iodomethane

Rat Two-Generation Pup Survival following Maternal Iodomethane Exposures

F1 Birth to PND 4

0

10

20

30

40

50

60

70

80

90

100

0 ppm 5 ppm 20 ppm 50 ppm

% S

urv

ival

0 ppm 5 ppm 20 ppm 50 ppm

**

•Remember, these exposures ceased at GD 20 because of birth

Measurement of T3 in the Perinatal and Adult Female Rat

Ontogeny of thyroid function in the rat begins between GD 20 and PND 4 compared to GD 22-23 in the rabbit

0

20

40

60

80

100

120

140

160

180

GD 20 fetus PND 4 PND 21 GD 20 dam LD 21

Age at Evaluation

ng

/dL

Fetus/Pup Dam

Case Study

Effects on Prenatal and Postnatal Development Including Maternal Function

ICH 4.1.2

Denotes Treatment Period

GD 6 PND 20

Gestation Lactation

Weaning Growth Mating GestationPN day 21 9 wks 2 wks 3 wks

F1

F2

Female (Rat)

(Macroscopic Pathology)

PN day 17 PN day 80

Behavioral/Anatomic Measures

Motor ActivityAuditory StartleWater MazeDevelopmental Landmark

Vaginal PatencyPreputial Separation

Denotes Possible Transfer Via Milk

Holson et al., 2006

ACE Inhibition-Induced Fetopathy (Human)

• Organogenesis (classically defined) is unaffected

• Effects are severe

• Risk is low

• Caused by ACEinh that cross placenta

ACEinhFetal

Hypotension

RenalCompromise

(Anuria)Oligohydramnios

Calvarial Hypoplasia

Neonatal Anuria

IUGR

Death

Holson et al., 2006

ACE Inhibition in Developing Rats

• RAS (renin-angiotensin system) matures around GD17

• No apparent effect in initial reproductive studies

• Subsequent postnatal studies with direct administration to pups

• Growth retardation

• Renal alterations (anatomic and functional)

• Death

Holson et al., 2006

Comparison of Prenatal and Postnatal Modes of Exposure

Drug Transfer to Offspring

Drug Levels in Offspring

Maternal Blood vs.Offspring Levels

Exposure Route toOffspring

Commentary

Prenatal

Nearly all transferred

Cmax and AUC measured

Maternal often a surrogate

Modulated IV exposure, via placenta

Timing of exposure is critical

Postnatal

Apparent selectivity (“barrier”)

Not routinely measured

Maternal levels probably NOT a good predictor

Oral, via immature GI tract

Extent of transfer to milk and neonatal bioavailability is key to differentiating indirect (maternal) effectsfrom neonatal sensitivity

Prenatal Treatment Postnatal

Embryo/Fetus Placenta Mother Mammae Neonate

Holson et al., 2006

A B C D E F

Premating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Hard-Palate Closure to End of Pregnancy

Birth to Weaning Weaning to Sexual Maturity

Parturition Litter Size Landmarks of Sexual DevelopmentGestation Length Pup Viability Neurobehavioral Assessment F1 Mating and Fertility Pup Weight Acoustic Startle Response

Organ Weights Motor Activity Learning & Memory

ParturitionGestation Length Pup Viability Litter SizeLandmarks of Sexual Development Pup WeightNeurobehavioral Assessment Organ Weights Acoustic Startle Response F1 Mating and Fertility Motor Activity Hormonal Analyses Learning & Memory Ovarian QuantificationHistopathology Premature Senescence

Postimplantation LossViable FetusesMalformations & VariationsFetal Weight

Postimplantation LossViable FetusesMalformationsVariationsFetal Weight

Estrous Cyclicity Mating Corpora Lutea Fertility Implantation SitesPre-Implantation Loss Spermatogenesis

Estrous CyclicityMatingFertilityCorpora LuteaImplantation SitesPre-Implantation LossSpermatogenesis

Denotes Dosing Period

Single- and Multigenerational

Satellite Phase

OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB

F1

F2 ????????????????

????????????????

Pre- and Postnatal Development

F1

ICH 4.1.2F0

????????????????

Prenatal Development

ICH 4.1.3

Fertility StudyICH 4.1.12W4W

CMAX

AUC

CMAX

AUC

10W

OECD 414 OPPTS 870.3600, 870.3700

Modified from: Holson, et al., 2006

Life Stages and Toxicity Study Designs

Deficiency:

Unknown Extent (if Any)

of Exposure to the

Test Article in

Preweaning Animals

Deficiencies in Study Designs Leading to Data Gaps in Risk Assessment

• Early Postnatal Development• Only potential, generally unquantified lactational

exposure• No direct exposure

• Need arose for studies of direct exposure during early postnatal development• DNT (following FQPA)• Juvenile Toxicity (Following Pediatric Rule)

Summation: Factors to Be Considered for Either Approach (Life Cycle vs. Segmented Approach)

• Many factors must be considered in the final experimental design• What are the triggers to deviate from standard

designs?• Duration and timing of human therapy/exposure• Knowledge of related agents• Key findings during study progress

• What is the therapeutic indication?• How restrictive is it?• Are you looking for Mode of Action?