Current Regulatory Requirements
in DART Assessments: Segmental
Study Designs Versus The
Biologic Continuum
Joseph F. Holso...
Validity/Predictivity of Animal Models
End Points

Comments on Concordance

Carcinogenesis High & multiple studies
Develop...
Ontogeny of Physiologic Regulation
in Selected Mammals
Hamster Rat Rabbit Cat Pig Human

Stagemarks

Implantation
First He...
Relationship Between Development
and Phenotypic Diversity
Extent of Differentiation
Embryonic
Period

Fetal
Period

Postna...
Exposure-Based Approach to Study Design
• It is perceived that tests in which animals are
treated during defined stages of...
Exposures
• Unintended Exposures – Complete (almost)
Life-Cycle Study
• Industrial and agricultural chemicals
(EPA/OPPTS/O...
DART Guideline Studies by Agent/Chemical
Use Class (Excluding Medical Devices)
Human Therapeutic

Veterinary Therapeutic*
...
Reproductive/Developmental Life Stages

A

B

C

D

E

F

Premating to
Conception

Conception to
Implantation

Implantatio...
Reproductive/Developmental Life Stages:
Selected Detectable Aberrations and Effects
A

B

C

D

E

F

G*

Premating to
Con...
Non-Therapeutic Exposures –
“Life-Cycle” Assessment
• The two-generation reproduction study is an apical,
comprehensive te...
Single and Multigenerational Study Designs
A

B

C

D

E

F

Premating to
Conception

Conception to
Implantation

Implanta...
Duration of Phases of 2-Generation Reproductive
Toxicity Study Design (Rats)
PBE
F0

B

10 Weeks

G

L

2W 3W 3W
PBE
F1

I...
Limitations of Current Two-Generation
Guideline
• No measurement of latent effects of endocrine
modulation
• No measuremen...
Tier I Evaluation in ILSI Agricultural Chemical Safety
Assessment (ACSA) Technical Committee Proposal

ClinPath & DNT
Immu...
Case Study
Octamethylcyclotetrasiloxane (D4)
• Two-generation reproduction study of an
ingredient in consumer products including a
va...
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 concu...
Exposure-Based Approach to Study Design
• It is perceived that tests in which animals are
treated during defined stages of...
Intended Exposures – Segmented Approach
• Testing approach uses traditional segmented
reproductive stage approach because
...
Advantages of Three-Segment Design
• Optimize exposures to key events in reproductive
cycle and development, particularly ...
Segmented DART Study Designs
A

B

C

D

E

F

Premating to
Conception

Conception to
Implantation

Implantation to Closur...
Case Study
Iodomethane (MeI)
• “Zero ozone-depletion potential” methyl
bromide soil fumigant replacement
• Example of animal model/de...
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
GD 6-14

GD 15-22

GD 23-24

GD 6-28
Comprehensive

GD 25-26

GD 27-28

GD 6-28
(...
Rabbit Fetal Biomarkers Evaluated with and
without Exposure from GD 21 to 27

TSH, T4, T3
Sloter, 2005
T3
Fetal TSH and Thyroid
Hormones with and
without Iodomethane
Exposure
•Indicates Direct Effect on Fetal Thyroid
Function...
Thyroglobulin (Colloidal) Depletion following
GD 23-26 Exposure to Iodomethane

Sloter, 2005
Rat Two-Generation Pup Survival following
Maternal Iodomethane Exposures
F1 Birth to PND 4
100
90
80

**

% Survival

70
6...
Measurement of T3 in the Perinatal and Adult
Female Rat
180
160
140

ng/dL

120

Fetus/Pup

Dam

100
80
60
40
20
0
GD 20 f...
Case Study
Effects on Prenatal and Postnatal
Development Including Maternal Function
ICH 4.1.2
GD 6

Female (Rat)

PND 20
Gestation

...
ACE Inhibition-Induced Fetopathy
(Human)

ACEinh

Fetal
Hypotension

Renal
Compromise
(Anuria)

Calvarial Hypoplasia
Oligo...
ACE Inhibition in Developing Rats
• RAS (renin-angiotensin system) matures around
GD17
• No apparent effect in initial rep...
Comparison of Prenatal and Postnatal
Modes of Exposure
Prenatal

Embryo/Fetus

Placenta

Treatment

Mother

Prenatal

Post...
Life Stages and Toxicity Study Designs
A

B

C

D

E

F

Premating to
Conception

Conception to
Implantation

Implantation...
Deficiencies in Study Designs Leading to Data
Gaps in Risk Assessment
• Early Postnatal Development
• Only potential, gene...
Summation: Factors to Be Considered for Either
Approach (Life Cycle vs. Segmented Approach)

• Many factors must be consid...
Current Regulatory Requirements in Developmental and Reproductive Toxicity Assessments: Segmental Study Designs vs. the Bi...
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Current Regulatory Requirements in Developmental and Reproductive Toxicity Assessments: Segmental Study Designs vs. the Biologic Continuum

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Presented at the Northern California SOT Spring Symposium, SRI International Conference Center, Menlo Park, CA, June 7, 2007.

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  • Current Regulatory Requirements in Developmental and Reproductive Toxicity Assessments: Segmental Study Designs vs. the Biologic Continuum

    1. 1. Current Regulatory Requirements in DART Assessments: Segmental Study Designs Versus The Biologic Continuum Joseph F. Holson, PhD, DABFE WIL Research Laboratories, LLC
    2. 2. 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
    3. 3. Ontogeny of Physiologic Regulation in Selected Mammals Hamster Rat Rabbit Cat Pig Human Stagemarks 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 4 8 10 20 40 80 100 200 400 Days After Conception After Adolph, 1970
    4. 4. Relationship Between Development and Phenotypic Diversity Extent of Differentiation Embryonic Period Fetal Period Postnatal Period Degree of Phenotypic Variability Birth Time in Development (Age) Holson et al., 2006
    5. 5. 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
    6. 6. 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)
    7. 7. 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 1- and 2-Generation Pre- and Postnatal Development Pediatric (Juvenile Animals) Product Use Dependent Developmental 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
    8. 8. Reproductive/Developmental Life Stages 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
    9. 9. Reproductive/Developmental Life Stages: Selected Detectable Aberrations and Effects A B C D E F G* 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 Maturity to Reproductive Senescence Effects on Libido/Intromission Male-Mediated Developmental Toxicity Postimplantation Loss Death Death Cryptorchidism Interference with Histogenesis Dystocia Nipple Retention in Males Appearance of Latent and Nonreproductive Effects Impaired Sperm Motility Decreased Sperm Count Female Reproductive Cycle Disruption Ovulatory Blockade Ovarian Dysfunction Accelerated Tubal Transport Failed Uterine Deciduation Failure to Implant Ectopic Pregnancy Dysmorphogene sis Growth Retardation Dominant Lethality Spontaneous Abortion Mutagenesis Chromosomal Aberrations Pseudopregnancy Death Spontaneous Abortion Growth Retardation Interference with Lactation Growth Retardation Failure to Thrive Premature Delivery Functional Deficit Toxemia Morphological CNS Disruption Abnormal Maternal Behavior Hypospadias Delayed Maturation (Vaginal Patency/ Balanopreputial Separation) Precocious Puberty (Vaginal Patency/ Balanopreputial Separation) Premature Menopause Accelerated Aging Neoplasia (e.g., Mammary Glands) *These type of effects not addressed by current standard study designs
    10. 10. 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
    11. 11. Single and Multigenerational Study Designs 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 Single- and Multigenerational OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB Estrous Cyclicity Mating Fertility Corpora Lutea Implantation Sites Pre-Implantation Loss Spermatogenesis Postimplantation Loss Viable Fetuses Malformations Variations Fetal Weight F1 ???????????????? F2 Satellite Phase ???????????????? Parturition Gestation Length Pup Viability Landmarks of Sexual Development Neurobehavioral Assessment Acoustic Startle Response Motor Activity Learning & Memory Histopathology Optional Litter Size Pup Weight Organ Weights F1 Mating and Fertility Hormonal Analyses Ovarian Quantification Premature Senescence Denotes Dosing Period Modified from: Holson et al., 2006
    12. 12. Duration of Phases of 2-Generation Reproductive Toxicity Study Design (Rats) PBE F0 B 10 Weeks G L 2W 3W 3W PBE F1 IU/L B 10 Weeks F2 Approximately Nine Months PBE = Prebreeding Exposure M = Breeding Period G = Gestational Period L = Lactational Period IU/L = Potential In Utero/Lactational Exposure G L 2W 3W 3W IU/L
    13. 13. 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
    14. 14. Tier I Evaluation in ILSI Agricultural Chemical Safety Assessment (ACSA) Technical Committee Proposal ClinPath & DNT Immunotox SMVCE/Breeding Extended One-Generation Study Dellarco et al., 2005
    15. 15. Case Study
    16. 16. 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
    17. 17. D4 Holson et al., 2006
    18. 18. 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 doseresponse 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
    19. 19. 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
    20. 20. 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
    21. 21. 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)
    22. 22. Segmented DART Study Designs 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 Fertility Study 10W 4W 2W Estrous Cyclicity Mating Fertility Implantation Sites Pre-Implantation Loss Spermatogenesis ICH 4.1.1 Corpora Lutea Prenatal Development ƒ CMAX AUC ICH 4.1.3 Rats: Therapeutic: GD 6-17 Nontherapeutic: GD 6-19 (or 20) OECD 414 OPPTS 870.3600, 870.3700 Rabbits: Therapeutic: GD 7-20 Nontherapeutic: GD 7-28 Postimplantation Loss Viable Fetuses Malformations & Variations Fetal Weight F0 ƒ CMAX AUC Pre- and Postnatal Development ICH 4.1.2 F1 Parturition Gestation Length F1 Mating and Fertility ???????????????? Litter Size Pup Viability Pup Weight Organ Weights Landmarks of Sexual Development Neurobehavioral Assessment Acoustic Startle Response Motor Activity Learning & Memory Denotes Dosing Period Modified from: Holson et al., 2006
    23. 23. Case Study
    24. 24. 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
    25. 25. Effect of Iodomethane on Postimplantation Loss in the Rabbit * * * Sloter, 2005
    26. 26. Phased-Exposure Study to Determine Window of Sensitivity Sloter, 2005
    27. 27. Fetal Death during Phased-Exposure Study GD 6-14 GD 15-22 GD 23-24 GD 6-28 Comprehensive GD 25-26 GD 27-28 GD 6-28 (Summed) Standard ICH Guideline would not detect this effect Sloter, 2005
    28. 28. Rabbit Fetal Biomarkers Evaluated with and without Exposure from GD 21 to 27 TSH, T4, T3 Sloter, 2005
    29. 29. T3 Fetal TSH and Thyroid Hormones with and without Iodomethane Exposure •Indicates Direct Effect on Fetal Thyroid Function Reveals Onset of Thyroid Function in Fetal Rabbt These Effects Would be Missed in ICH Regimen due to Eight Fewer Days of Exposure T4 TSH Sloter, 2005
    30. 30. Thyroglobulin (Colloidal) Depletion following GD 23-26 Exposure to Iodomethane Sloter, 2005
    31. 31. Rat Two-Generation Pup Survival following Maternal Iodomethane Exposures F1 Birth to PND 4 100 90 80 ** % Survival 70 60 50 40 30 20 10 0 0 ppm 5 ppm 0 ppm 5 ppm 20 ppm 20 ppm 50 ppm 50 ppm •Remember, these exposures ceased at GD 20 because of birth
    32. 32. Measurement of T3 in the Perinatal and Adult Female Rat 180 160 140 ng/dL 120 Fetus/Pup Dam 100 80 60 40 20 0 GD 20 fetus PND 4 PND 21 GD 20 dam LD 21 Age at Evaluation Ontogeny of thyroid function in the rat begins between GD 20 and PND 4 compared to GD 22-23 in the rabbit
    33. 33. Case Study
    34. 34. Effects on Prenatal and Postnatal Development Including Maternal Function ICH 4.1.2 GD 6 Female (Rat) PND 20 Gestation Lactation (Macroscopic Pathology) F1 Denotes Treatment Period Denotes Possible Transfer Via Milk Weaning Growth PN day 21 9 wks PN day 17 Mating 2 wks PN day 80 Gestation 3 wks F2 Behavioral/Anatomic Measures Motor Activity Auditory Startle Water Maze Developmental Landmark Vaginal Patency Preputial Separation Holson et al., 2006
    35. 35. ACE Inhibition-Induced Fetopathy (Human) ACEinh Fetal Hypotension Renal Compromise (Anuria) Calvarial Hypoplasia Oligohydramnios Neonatal Anuria IUGR Death • Organogenesis (classically defined) is unaffected • Effects are severe • Risk is low • Caused by ACEinh that cross placenta Holson et al., 2006
    36. 36. 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
    37. 37. Comparison of Prenatal and Postnatal Modes of Exposure Prenatal Embryo/Fetus Placenta Treatment Mother Prenatal Postnatal Mammae Neonate Postnatal Drug Transfer to Offspring Nearly all transferred Apparent selectivity (“barrier”) Drug Levels in Offspring Cmax and AUC measured Not routinely measured Maternal Blood vs. Offspring Levels Maternal often a surrogate Maternal levels probably NOT a good predictor Exposure Route to Offspring Modulated IV exposure, via placenta Oral, via immature GI tract Commentary Timing of exposure is critical Extent of transfer to milk and neonatal bioavailability is key to differentiating indirect (maternal) effects from neonatal sensitivity Holson et al., 2006
    38. 38. Life Stages and Toxicity Study Designs 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 Fertility Study 10W 4W 2W ICH 4.1.1 Estrous Cyclicity Mating Fertility Implantation Sites Pre-Implantation Loss Spermatogenesis Corpora Lutea Prenatal Development ƒ Deficiency: CMAX AUC ICH 4.1.3 OECD 414 OPPTS 870.3600, 870.3700 Postimplantation Loss Viable Fetuses Malformations & Variations Fetal Weight Unknown Extent (ifC Any) Pre- and Postnatal Development F ICH 4.1.2 ofƒExposure AUC F ???????????????? to the Test Article in Single- and Multigenerational Preweaning 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB OECD Animals Satellite Phase F ???????????????? MAX 0 1 Parturition Gestation Length F1 Mating and Fertility Estrous Cyclicity Mating Fertility Corpora Lutea Implantation Sites Pre-Implantation Loss Spermatogenesis Litter Size Pup Viability Pup Weight Organ Weights Landmarks of Sexual Development Neurobehavioral Assessment Acoustic Startle Response Motor Activity Learning & Memory 1 Postimplantation Loss Viable Fetuses Malformations Variations Fetal Weight Parturition F2 ???????????????? Gestation Length Pup Viability Litter Size Landmarks of Sexual Development Pup Weight Neurobehavioral Assessment Organ Weights Acoustic Startle Response F1 Mating and Fertility Motor Activity Hormonal Analyses Learning & Memory Ovarian Quantification Histopathology Premature Senescence Denotes Dosing Period Modified from: Holson, et al., 2006
    39. 39. 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)
    40. 40. 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?

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