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Human Clinical Relevance of Developmental and Reproductive Toxicology
and Nonclinical Juvenile Testing
Joseph F. Holson, Ph.D.WIL Research Laboratories, Inc.
Presented at Forest Research InstituteMay 13, 2004
Lifetime Costs by Condition, in 1992 Dollars
Comparison of Overall Spontaneous Malformation Rates in Different Species
Species Mean % Range (%) N
Rat 0.33 0-1.6 9643
Mouse 1.2 0-3 5207
Rabbit 3.2 0-10 4708
Dog 5.5 5.3-5.7 167
Human 4.0 3-9 Multiple Surveys
WIL Research Laboratories, Inc. Historical Control Database
Lessons from History
“Those who do not learn from history are doomed to
repeat it”
George Santayana, 1863-1952
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
Standard Designs
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 DevelopmentICH 4.1.3 OECD 414
OPPTS 870.3600 870.3700
Fertility StudyICH 4.1.110W 2W4W
¦ CMAX
AUC
¦ CMAX
AUC
“Chrontogeny” of Reproductive Toxicology
1891
1979
1998
Return of Thalidomide
Effects on Eggs(Dareste)
1855
1997
FDAMA
1996
FQPASWDA
1959
1962
Thalidomide Epidemic
1982
Isotretinoin Approved
1967
First Pregnancy Registry (Atlanta)
1974
First National Pregnancy Registry
1966
1940 First FDA Laboratory Animal Safety Studies
Rubella Epidemic(Gregg)
1941
1981
First ACE-Fetopathy
Case Report
1993
“ACE-Fetopathy”
Coined
Goldenthal Guidelines
1971
DES (Herbst & Scully)
Wilson’s Principles 1963
Conference on Prenatal Drug
Effects
NCTR Collaborative Behavioral
Study
NCTR Collaborative
Study Reported
1985
NCTR Concordance
Study (Teratology vs. Developmental
Toxicology)
Ongoing Methylmercury
and DES Exposures
1973
DBCP
1975
Red Dye No. 2
1906
USDA Bureau of Chemistry
International Concern on Decreasing
Fertility
1992
Agent Orange/2,4,5-T
& TCDD
Karnofsky
1950
Litigation continues - Involves forseeability
Medical community believed DES
promoted progesterone
synthesis by FPU
First randomized control study with
placebo: safe but no efficacy
Present
(Walker - Mouse) Numerous
experimental studies to develop a good
animal model
Litigation
Karnaby Clinical Studies Apparent safety at high doses
Green, et al., Intersexuality
in mice
SYNTHESISfirst orally
active estrogen
mimic
DES-Vaginal CCA link in 8 young women (15-22 years)
Debate about DES use in cattle and residues in meat
1971Herbst & Scully
Not considered a teratogenKalter & Warkany
Prescribed to prevent
spontaneous abortion Based on
Smith
Gabriel-RobezCleft Palates and
heart defects in mice
Wilson’s Principles of Teratology
1. Susceptibility to Teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with adverse environmental factors
2. Susceptibility to Teratogenesis varies with the developmental stage at the time of exposure to an adverse influence
3. Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiated sequences of abnormal developmental events (pathogenesis)
4. The access of adverse influences to developing tissues depends on the nature of the influence (agent)
5. The four manifestations of deviant development are death, malformation, growth retardation, and functional deficit
6. Manifestations of deviant development increase in frequency and degree as dosage increases, from the no-effect to the totally lethal level
Possible Inter-relationships of Developmental Toxicity Endpoints
Toxic Stimulus
Malformations
Functional Impairments
Growth Retardation
Death
Toxic Stimulus GrowthRetardation Death
Malformation
Functional Impairment
Percent Change Percent ChangeFetal Weight Embryolethality
5 10 5 10
Mice A/J 84 22 1176 324 C57BL/6 198 50 992 228 CDI 84 22 805 235
Rats CDb 52 16 858 248 OMc 44 12 723 216
Number of Litters (N)a to Detect Changesin Fetal Weights and Deaths in Mice and Rats
aNumber of litters/groupbCharles River, Wilmington, MAcOsborne-Mendel, Charles River, Wilmington, MA
From Nelson and Holson, 1978
Animal:Human Concordance Studiesfor Prenatal Toxicity
Authors Attributes
Holson, 1980(Proceedings of NATO Conference)Holson, et al., 1981 (Proceedings of Toxicology Forum)NCTR Report No. 6015, 1984
Interdisciplinary team (epidemiologists & developmental toxicologists)Critical analysis of primary literatureApplied criteria for acceptance of data/conclusions in reports and included power considerationsEstablished and applied concept of multiple developmental toxicity endpoints as representing signals of concordanceQualitative outcomes and external dose comparisons madeNo measures of internal dose
Nisbet & Karch, 1983(Report for the Council onEnvironmental Quality)
Many chemicals/agents addressedLimited review of primary literatureNot a critical analysis of primary literatureRelied on authors’ conclusionsNo power analysesLimited use of internal dose information
Brown & Fabro, 1983(Journal Article)
Not a critical analysis of primary literatureMade use of findings from other reviews Excellent review and presentation of overall concordance issues
Hemminki & Vineis, 1985(Journal Article)
Interspecies inhalatory doses adjustedRelied on authors’ conclusions23 occupational chemicals and mixtures No measures of internal dose
Animal:Human Concordance Studiesfor Prenatal Toxicity
Authors Attributes
Francis, Kimmel & Rees, 1990(Journal Article)
Small number of agents coveredCritical review of the qualitative and quantitative comparability of human and animal developmental neurotoxicityLimited use of internal dose measures
Newman et al., 1993(Journal Article)
Provided detailed informationOnly 4 drugs evaluatedEmphasis on morphologyFocus on NOAELsNo measures of internal dose
Shepard, 1995 (8th Ed.)(Text)
Computer-based annotated bibliographyCatalog of teratogenic agentsNot a critical analysisLimited comments regarding animal-to-human concordance for a limited number of agents No use of internal dose measures
Schardein, 2000 (3rd Ed.)(Textbook)
Extensive compilation of open literatureNot a critical analysisVariably relied on authors’ conclusionsNo measures of internal dose nor criteria for inclusion or exclusion of studiesOnly partially devoted to concordance issues
NCTR Concordance Report:Authors
Dr. J. Holson
Dr. C. Kimmel
Dr. C. Hogue
Dr. G. Carlo
Developmental Toxicologist
Developmental Toxicologist
Epidemiologist
Epidemiologist
NCTR Concordance Report:Assumptions
1) Only agents with an established effect in humans and adequate information for both humans and animals could be evaluated for concordance of effects.
Compounds for which no effect was indicated may actually have been negative or have been a false negative due to the inability to detect effects because of inadequate power of the studies.
2) Statistical power of the study designs had to be considered in order to evaluate adequacy of the data and apparent “species differences” in response.
Situations in which large animal studies may have been matched to a few case reports and a conclusion drawn as to the poor predictability of the animal studies were noted and reevaluated.
3) The multiplicity of endpoints in developmental toxicity comprise a continuum of response (i.e., dysmorphogenesis, prenatal death, intrauterine growth retardation, and functional impairment represent different degrees of a developmental toxicity response).
Although this assumption would be debated by some, the weight of experimental and epidemiological evidence tends to support rather than refute the assumption.The examples of fetal alcohol syndrome, DES, and methylmercury were discussed in support of this assumption.
4) Manifestations of prenatal toxicity were not presumed to be invariable among species (i.e., animal models were not expected to exactly mimic human response).
Also, the human population has exhibited an array of responses that are determined by magnitude of exposure, timing of exposure, inter-individual differences in sensitivities due to genotype, interaction with other types of exposure, and interaction among all of these factors.Just as the human and rat are not the same, all human subjects are not identically responsive to exogenous influences.
5) Sensitivity was based on comparability of the “effect levels” among species.
This was necessary because for most established human developmental toxicants there was still not adequate dose-response information available to compare sensitivities among species.
Summarized from NCTR Report No. 6015 (1984)
Awareness of Developmental Toxicity of Selected Agents
Agent Year First Reported Species*
Alcohol(ism)
Aminopterin
Cigarette Smoking
Diethylstilbestrol
Heroin/Morphine
Ionizing Radiation
Methylmercury
Polychlorinated Biphenyls
Steroidal Hormones
Thalidomide
1957
1950
1941
1940
1969
1950
1953
1969
1943
1961
(gp), ch, hu, mo, rat
(mo & rat), ch, hu
(rab), hu, rat
(rat), hu, mi, mo
(rat), ha, hu, rab
(mo), ha, hu, rat, rab
(rat), ca, hu, mo
(hu), rat
(monk), ha, hu, mo, rat, rab
(hu), mo, monk, rab
*ca - cat, ch - chicken, ha - hamster, gp - guinea pig, hu - human, mi - mink, mo - mouse, monk - monkey, rat - rat, rab - rabbit
Holson, et al.
Effect-Levels for Teratogensin Humans and Test Species
Aminopterin Death/Malformations
Death/Malformations
Agent ResponseHuman
Rat
Species Dose0.1 mg/kg/da
0.1 mg/kg
Diethylstilbestrol Genital Tract Abnormalities/Death
Genital Tract Abnormalities/Death
Human
Mouse
0.8-1.0 mg/kg
1 mg/kg
Ionizing Radiation Malformations
Malformations
Human
Rat/Mouse
20 rads/da
10-20 rads/da
Cigarette Smoking Growth Retardation
Growth Retardation
Human
Rats
>20 cigarettes/da
>20 cigarettes/da
Thalidomide Malformations
Malformations
Malformations
Human
Monkey
Rabbit
0.8-1.7 mg/kg
5.0-45 mg/kg
150 mg/kg
Holson, et al.
WIL Research Laboratories
Joseph F. Holson, Ph.D.WIL Research Laboratories
A probable false positive finding of prenatal toxicity in the rodent model with a high molecular weight protein
oxygen therapeutic: Evidence and Implications
Comparative Early Placentation: Human and Rat Conceptuses
Amniotic Cavity
Extra-Embryonic Coelom
Decidua
Yolk Sac
Uterine Lumen
Uterine Artery
Decidua
Ectoplacenta
Allantois
Visceral Yolk Sac
Vascular Lacuna
Human Conceptus (Pre-Chorioallantoic Placental Stage) Day 10 Rat Conceptus
The inverted yolk sac surrounds rodent embryo but not human
Day 13 Rat Conceptus Treated w/Trypan Blue (GD 11) 6/2003
Mark Hill, UNSW
Comparative Molecular Masses
Agent Molecular Weight
Water 0.018 kDa
Glucose 0.18 kDa
Leupeptin 0.48 kDa
Suramin 1.4 kDa
Inulin 5 kDa
hCG 38 kDa
Purified Bovine Hemoglobin 60 kDa
Trypan Blue + Albumin 1 kDa + 66 kDa = 67 kDa
Alpha-fetoprotein 70 kDa
IgG 150 kDa
Iron Dextran 165 kDa
HBOC-201 250 kDa (each hemoglobin ~ 60 kDa)
Types of Embryonal/Fetal Nutrition
1) Histiotrophic Extracellular material including cellular debris
secreted/deposited in space between maternal/embryonal surfaces in direct contact with trophectoderm Phagocytosis of histiotroph is considered to be a
characteristic of both cellular and syncytial trophoblast.
2) Hemotrophic Materials (O2, electrolytes, amino acids, etc.)
carried in the maternal blood
Wooding and Flint, in Marshall’s Physiology of Reproduction, 1994
Generalized Implications from our Studies and Analysis
There should be no doubt that the InvYSP can be a target for toxicity leading to serious developmental disruption. To the contrary, it has not been demonstrated that the noninverted yolk sac is a similar target.
Caution should be exercised In generalizing too broadly the findings of studies of this product, which by design, was given at high doses (mass) of hemoglobin protein, 6 g/kg.
Large and/or proteinaceous agents 1) with no pharmacologic action on the biochemical modalities of the InvYSP or 2) which do not contain a moiety with toxic properties would not be expected to exert similar effects.
The former types of agents would appear to represent a small number of the universe of xenobiotics and no broad sense lessens the value of current models.
Synopsis of Regulatory Assessment Process
Guideline Study
Hazard Identification Data
Animal-Human ConcordanceDevelopmental - High
Reproductive - Less Certain
Regulatory Analysis & Decision
Toxicologist/Regulatory
Regulator
Com
mun
icat
ion
Selected Reproductive Endpoints Exhibiting Strong Signals from Rare Events/Low Incidence
EndpointExamples from WIL Research Historical Control in Crl:CD(SD)IGS BR
Mean Viable Litter Size
13.9 1.02 decrease of 1
Mortality PND 4Mean = 96.2% Min/Max 91-95%
91%
DystociaMean = 0.36%
(4/1100)≥1 is significant signal
Total Litter Loss Mean = 0.94% (10/1061) 1 is equivocal 2 is more significant signal
Newborn Pup Weights
Mean = 7.0g 0.23 range 6.5-7.4g n = 1100 litters
6.5g strong signal
Holson, et al.
Reasons for Apparent Failed Predictions
Appropriate studies not conducted Incidence of effect too low for experimental
detectionUnknown/unstudied type(s) of effectHypersensitive individuals in human population Interaction of multiple agentsUnfounded/nonexistent claims or effectsHuman exposure is overestimated by
experimental design
Relevancy & Risk Analysis
BiologicDynamics &Dimensions
Integrity of Data Base
Regulators’ Questions
1) was an established, validated model used? 2) was a NOAEL (NOEL) demonstrated? 3) does increasing dose increase severity/incidence?
4) when was the when effect exerted? 5) is the effect reversible? 6) are there indications of sensitization (generational effects or imprinting)? 7) is the effect gender specific?
8) appropriate TK (AUC/CMAX) comparison between experimental study and
estimated human PK or exposure scenarios? 9) are there significant differences in the pattern, timing or magnitude of
exposure between guideline studies and human scenarios?10) is there concordance of effects among species?11) is the mode of action known or deducible?12) is the mechanism of action known?
Extent to Which Guideline Studies Answer Key Regulatory Questions
Fert. P/P DT DT 2-G DNT DT 1-G 2-G Screen DNT4.1.1 4.1.2 4.1.3 3700 3800 6300 414 415 416 421 426
1Validated
Model
2NOAEL
Determined
3Rare Event
w/Dose
4Insult Timing
Elucidated
5 Reversibility
6Imprinting
Phenomenon
7 Gender Basis
8 TK Profiled
9Exposure Mimicked
? ? ? ? ? ? ? ?
10Interspecies
Concordance
11Mode of Action
12Mechanism of
Action
What do regulators want to
know?
OECDEPAICH
Postnatal Models and Ontogeny
Attributes of Successful Modelsfor Safety Assessment
ValiditySensitivityReproducibilityPracticability
Ontogeny Recapitulates Phylogeny
Haeckel
Maturational Data for Various Species
Human toAnimal
Life Span
267 20 22 32 63 16716
Human Mouse Rat RabbitGuinea
PigRhesusMonkey
SyrianHamster
MinimalBreeding
Age(weeks)
Gestation(days)
728 7 10.52832
410
2185
6.5
1.0 44 33 12 17 4.466
Background
Adolph (1949) showed that metabolic rates scale across species according to (body weight)0.73.
Boxenbaum (1982) demonstrated that the disposition kinetics of xenobiotics in species is scaled by the same relationship.
These concepts led to the mathematical relationships that are used to standardize experimental dose regimens and to scale across species in PBPK models.
Physiologic Time
A method for scaling the lifespan of different species so that comparable stages of maturation are congruent, regardless of chronological age
An example of the concept of physiologic time that is intrinsic to PBPK models:
T1/2 = Body Weight rat
Body Weight human
0.25
Time to Develop Adult Characteristics
%Adult Status
Age (years)
0
Rat
Human
100
0 2015105
Comparative Age Categories Based on Overall CNS and Reproductive
Development
Days
Months
Years
Weeks
Weeks
Pre-Term Neonate
Term Neonate Infant/Toddler Child Adolescent
161220.8B
483660.5B
2820630.5B
261442B
90452110< 9B
B Birth
Ontogeny
Minipig
Rat
Dog
Nonhuman Primate
Human
J. Buelke-Sam
Review of Adolph’s Seminal Work
Implantation
First Heart Beat
Exterioception
Hemoglobin 8% in Blood
Body Weight 1gm
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
Ontogeny of Physiologic Regulationin Selected Mammals
Stagemarks
4
Days After Conception
Hamster Rat Rabbit Cat Pig Human
8 10 20 40 80 100 200 400
After Adolph 1970
Comparative Perinatal Water Content
After Adolph and Heggeness, 1971
Hamster
Rat
Rabbit
GP
PigHuman
= Birth
% WaterIn a
Fat-FreeBody
95
90
85
75
Days After Conception
80
10 30 100 300
Water fraction decreases with age in all species
*
*
*
**
*
*
Comparative Ontogeny of Fat Content
Fetal Guinea Pig and human deposit fat prior to birth
% FatIn
Body
30
25
20
15
10
5
010 30 100 300
Days After Conception
Hamster
Rat
Rabbit
Guinea Pig
Cat
Pig
Human
= Birth
After Adolph and Heggeness, 1971
*
*
*****
*
Difficulties in a priori Selection of Models for Nonclinical Juvenile Toxicity
Relationship Between Developmentand Phenotypic Diversity
Degree of Phenotypic Variability
Time in Development (Age)
EmbryonicPeriod
FetalPeriod
PostnatalPeriod
Extent of Differentiation
Birth
Critical Periods for Structuraland Functional Effects
Sensitivity
Time
Organogenesis
Structural Development
Functional Development
Why are we interestedin organ system maturation?
It is essential for comparing postnatal toxicity among species
Effects on Prenatal and Postnatal Development Including Maternal Function
ICH 4.1.2 (Segment III)
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
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) effects from neonatal sensitivity
Prenatal Treatment Postnatal
Embryo/Fetus Placenta Mother Mammae Neonate
Comparison of Prenataland Postnatal Toxicity Profiles
Toxicity
Log of Dose
Maternal
Developmental
Prenatal – valid and insightful – Embryonic exposure – Mode of action
Postnatal – valid only – when xenobiotic level is measured in both mother and
offspring
Presence of Enzymes During Embryonic (E), Fetal (F), and Neonatal (N) Periods
Data extracted from Juchau et al., Kulkarni, 1997; Miller et al., 1996; Oesterheld, 1998; Raucy and Carpenter, 1993. CYP=cytochrome P450
Human
E F N
G. Pig
E F N
Rabbit
E F N
Hamster
E F N
Mouse
E F N
Rat
E F NCYP1A1
CYP1A2
CYP1B1
CYP2E1
CYP3A4
CYP3A5
CYP3A7
CYP2C8
CYP2C9
CYP2D6
Flavin-containing monooxygenase
Prostaglandin synthetase
Lipoxygenase
Perosidase
Epoxide hydrase
GSH-S-transferase
UDP-glucuronyltranferase Sulfotransferases
+–+
–––
++
+–++–––
–+
++
+–+
–––
+
+
++–––
+
–
++
+
+
++
++
+–++–++
–
+–++–+++–++
++++++
–+
+++–+++
Messages from Case Studies
ACE Inhibitors Low-incidence effects and temporal exposure issues in
experimental models Quinilones
Finding proper model and early clinical alert apparently preventing human damage
Fluoxetine For risk assessment, need for updated guideline studies on
older, but widely used products Complexity of risk/benefit issues in multiple therapeutic
populations Isotretinoin/Neurobehavioral
Animal model/human confirmation of experimental model in developmental neurotoxicity arena
Examples of Perinatal/Juvenile Toxicants
The following examples are not the result of an exhaustive literature search.
In most instances, the cause of postnatal morbidity/ mortality has not been investigated or is not known.
The absence of standard blood biochemistry/hematology assays and target organ pathology hinders the identification of sites and modes of action.
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.
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.
Critical Aspects of Renal Development to Juvenile Model Use
In the conventional rodent model (rat), awareness of rapid and major renal maturational dynamics occur between PNDs 14 and 21 Primary organ for xenobiotic clearance GFR relatively greater than in adult Often a target organ itself All these being interrelated
Particular example for ACE inhibitors Experimental impact of influence on AUC and Cmax Refer to upcoming Teratology Society Meeting paper by
Beck, et al.
Selective Juvenile Toxicity of Quinilones
Drug
Ofloxacin (and other quinilones)
Modified from Stahlmann, et al., 1997
Species &Treatment
Multiple Species,postnatal exposure.20mg/kg (dog, 3 mo.)600mg/kg (rat, 5 wk)
Effects
Chondrotoxic effects. Cartilage erosion in weight-bearing joints.
Gait alterations in juvenile dogs only.
Remarks
Human relevance unknown; drugs contraindicated in juvenile patients.
Mechanism: Probable deficiency of bioavailable Mg2+ in cartilage (quinilones chelate divalent cations).
No effect in routine segment III studies.
Sufficient evidence exists for the Panel to conclude that fluoxetine exhibits developmental toxicity as characterized by an increased rate of poor neonatal adaptation (e.g., jitteriness, tachypnea, hypoglycemia, hypothermia, poor tone, respiratory distress, weak or absent cry, diminished pain reactivity, or desaturation with feeding) at typical maternal therapeutic doses (20–80 mg/day orally). These effects appear to result more readily from in utero exposure late in gestation. The observed toxicity may be reversible, although long-term follow-up studies have not been conducted to look for residual effects. The evidence suggests that developmental toxicity can also occur in the form of shortened gestational duration and reduced birth weight at term.
CERHR Fluoxetine Report:Overall Conclusions – Developmental Toxicity
Isotretinoin
Animal research on retinoic acids (RA) has helped to establish a principle of neurobehavioral teratology: embryonic exposure to a CNS teratogen produces a continuum of outcomes, ranging from death, to malformation, to subtle functional alterations. Demonstrating this principle in human studies was problematic prior to the introduction in 1982 of 13-cis RA (Accutane, Hoffman-LaRoche) for the treatment of severe cystic acne. 13-cis RA is a potent human teratogen that causes a 40% rate of spontaneous abortion, and, among liveborn infants, a 35% rate of major malformation (Lammer et al, 1985; 2001). The characteristic pattern includes hindbrain, craniofacial, cardiac, and thymic abnormalities.
Longitudinal follow-up of this original cohort and the matched controls has focused on neuropsychological characteristics at 5 and 10 years of age, and the relationships between malformation and cognitive status. Of 35 children exposed to RA between postconception days 14 and 60 and tested at age 5, 46% scored in the below average range of general mental ability, as compared to 10% of the controls. Boys were more frequently affected than girls with respect to major malformations and reduced intelligence. Presence of a major malformation was associated with reduced intellectual performance, however, 6 of 16 children scoring in the below average range had no detectable malformations. Thus, as in the animal studies, features at birth, such as major malformations, do not fully characterize the adverse consequences of embryonic RA exposure. Likewise, the effects cannot be fully characterized by examining only reduced intelligence. As compared to control children, RA exposed children with average mental ability were three times more likely to have a specific, non-verbal learning disability and boys were again over-represented.
J. Adams & E.J. LammerJ Neurochem 2002 Jun;81(Suppl 1):113
Primary Reasons that Experimental ModelsAppear to be Invalid
Findings at, or extrapolated to, exaggerated doses
Exposure to and internal dose of noxious agent not measured
Timing of exposure does not coincide with the appearance of the developmental target
Duration of exposure not scaled to physiologic time
Incorrect / unvalidated endpoints assessed
Too little knowledge / data concerning mode of action
Challenges of “Mining” the Literature
Limited attention given to the issue of postnatal models for safety assessment
There is a paucity of reviews / data compilations
Isolated key information is embedded in papers addressing other concerns
Analysis requires interdisciplinary expertise and commitment of resources
Many and substantial data gaps (species and organ systems) exist
Conclusions
Parallelism exists among species regardless of lifespan.
Additional measurements and changes to current guidelines could increase our ability to predict postnatal toxicity.
Molecular biology and genomics have influenced pharmaceutical development toward agents with increasing specificity.
For novel, selective pharmaceutical agents, nonclinical testing must be preceded by literature mining and analysis.
Examples of Perinatal/Juvenile (?) Developmental Toxicants
Exposure Time ofToxicant Period Species Endpoint Manifestation Reference
Estrogen PND1-5 mouse cervical/vaginal adult Dunn & Green, 1963;cancer Takasagi & Bern, 1964
DES prenatal human vaginal cancer/ pubescence Herbst & Skully, 1970
reprod. tract effects
DES PND1-5 mouse vaginal adenosis adult Forsberg, 1976
Sex hormone PND1-5 mouse vaginal adenosis/ adult Bern et al., 1976
(DES) cancer
DES GD15, 16, 17 mouse vaginal adenosis, adult Walker, 1980
transverse ridges (14 mo.)
Reasons for Increased Attention toJuvenile Toxicity
New Trends in Drug Discovery
Chiral molecules
Rational, structure-based molecular design
Targeted pharmacology
Attention to Sensitive Subpopulations in Human Risk Assessment
Food Quality Protection Act
FDA Modernization Act
Challenges
Identifying and managing risks Modulation of growth Alteration of functional maturation
Examples: EGF, TGF, Leptin, KGF, CRF
Knowledge of whether condition, agent, procedure, chemical/drug exerts adverse effects on reproduction or development?
What is relative risk to human beings?
Sufficient degree of comfort to enable sound decision-making Guideline studies Additional evaluations Burden of proof is on industry
Reproductive ToxicologyWhat Do the Regulators Want?
Validity of Animal Models, Concordance with Human Outcomes and Factors Affecting Study Effectiveness
Event
Germ cells in genital ridges
Gonads begin sexual differentiation
Leydig cells differentiate
Sertoli cells proliferate
Oocytes initiate meiosis
Arrest of meiosis in females
Testes descend into scrotum
Pubertal period: females
Pubertal period: males
Rat
gd 13
gd 13-14
gd 17
gd 15 - pnd 16
gd 17
pnd 5
pnd 21
pnd 30-38
pnd 35-60
Human
gd 35-37
gd 40-42
gd 60-70
fetal - to puberty?
gd 84
by pnd 56
gd 220-225
12-13 years
13-15 years
Selected Milestones of Reproductive Development in Rats and Humans
Comparison of Timesin Male Sexual Development
3 Days 50 Days19 Days
Human
Rat
14 Days 14 Years8 Months
Genital TubercleFormation
Conception
Genital Development StaticSecondary Sexual
Characteristics
BirthAdult Status
CERHR Fluoxetine Report:Expert Panel
Ronald N. Hines, Ph.D. (Chair) Medical College of Wisconsin, Milwaukee, WIJane Adams, Ph.D. University of Massachusetts, Boston, MAGermaine M. Buck, Ph.D. National Institute of Child Health & Human
Development, Rockville, MDWillem Faber, Ph.D. WFT Consulting, LLC, Victor, NYJoseph F. Holson, Ph.D. WIL Research Laboratories, Inc., Ashland, OHSandra W. Jacobson, Ph.D. Wayne State University School of Medicine, Detroit, MIMartin Keszler, M.D. Georgetown University Hospital, Washington, DCKenneth McMartin, Ph.D. LSU Health Sciences Center, Shreveport, LARobert Taylor Segraves, M.D., Ph.D. MetroHealth Medical Center, Cleveland, OHLynn T. Singer, Ph.D. Case Western Reserve University, Cleveland, OHI. Glenn Sipes, Ph.D. University of Arizona, Tucson, AZPaige L. Williams, Ph.D. Harvard School of Public Health, Boston, MA
CERHR Fluoxetine Report:Recent Fluoxetine Prescriptions
According to the FDA (11), 1.2 billion tablets (or teaspoons) of fluoxetine were sold to U.S. pharmacies in 2002.
Fluoxetine was the most commonly prescribed SRI in 1998 and dropped to the third most commonly prescribed SRI during the past 3 years.
In 2002, about 26.7 million prescriptions were dispensed for fluoxetine, with 1.2 million dispensed to pediatric and adolescent patients (1–18 years old) and 8.4 million dispensed to women of child bearing age (19–44 years old).
CERHR Fluoxetine Report:Overall Conclusions – Reproductive Toxicity
The Expert Panel concluded that there is sufficient evidence in humans that fluoxetine can produce reproductive toxicity in men and women as manifested by reversible, impaired sexual function, specifically orgasm.
The mechanism(s) by which fluoxetine can cause reproductive and developmental toxicity is unknown. However, the Panel suspects both the adverse and desired pharmacological actions of this and other SRIs are mediated by their serotonergic activity.
The Panel concluded there are insufficient data to draw conclusions regarding concern for drug-induced toxicity in infants exposed to fluoxetine through breast milk or children on fluoxetine therapy. There also are insufficient data on possible drug associations with maternal and/or embryonic/fetal toxicity leading to pregnancy loss.
CERHR Fluoxetine Report:Overall Conclusions – Reproductive Toxicity
Data from prospective cohort studies of women planning pregnancies to capture all hCG-detected pregnancies and determine effects of fluoxetine on critical windows of human development including at or shortly after conception
Additional data on the possible effects of fluoxetine on gestational length, prematurity, fetal growth, and neonatal adaptation
Data from longitudinal prospective studies on whether prenatal fluoxetine exposure affects postnatal growth, neuroanatomy, and neurobehavioral development
Data from studies on neonatal growth and neurobehavioral function in neonates exposed to fluoxetine through breast milk
Data from longitudinal prospective studies on neuropsychological functioning using standardized and sensitive measurements in children taking the medication
CERHR Fluoxetine Report:Critical Data Needs – Human DT
Data from rodent studies that comply with current testing guidelines
Data from developmental neurobehavioral studies, including brain histology
Data examining prenatal exposure effects on hippocampal development
CERHR Fluoxetine Report:Critical Data Needs – Animal DT
Data on the effects of fluoxetine on male and female fertility
Data on spontaneous abortion that can address separation of the effects of medication from effects of the underlying disorder
Additional data from sexual function studies based on underlying disease (indication for therapy)
CERHR Fluoxetine Report:Critical Data Needs – Human RT
Data on the effects on semen quality, ovulation, conception, and pregnancy loss
CERHR Fluoxetine Report:Critical Data Needs – Animal RT
Case Studies
ACE InhibitorsQuinilonesFluoxetineIsotretinoin
Acknowledgements
John M. DeSesso Catherine F. Jacobson Amy L. Lavin
Bennett J. Varsho
Patrick J. Wier
Judy Buelke-SamToxicologyServices