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

1. Current Regulatory Requirements
in DART Assessments: Segmental
Study Designs Versus The
Biologic Continuum
Joseph F. Holson, PhD, DABFE
WIL Research Laboratories, LLC

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Case Study

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. D4
Holson et al., 2006

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. 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. 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. 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. 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. Case Study

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. Effect of Iodomethane on
Postimplantation Loss in the Rabbit
*
*
*
Sloter, 2005

26. Phased-Exposure Study to Determine
Window of Sensitivity
Sloter, 2005

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. Rabbit Fetal Biomarkers Evaluated with and
without Exposure from GD 21 to 27
TSH, T4, T3
Sloter, 2005

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. Thyroglobulin (Colloidal) Depletion following
GD 23-26 Exposure to Iodomethane
Sloter, 2005

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. 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. Case Study

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. 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. 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. 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. 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. 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. 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?