Human Clinical Relevance of Developmental and Reproductive Toxicology and Nonclinical Juvenile Testing

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