Features of safety assessment studies to be considered in assessing thyroid effects of reproduction and their relevance to human health

WIL Research Laboratories

Joseph F. HolsonContributions By: Mark D. Nemec

Chris P. Chengelis

Bennett J. Varsho

Features of safety assessment studies to be considered in assessing thyroid

effects of reproduction and their relevance to human health

National Toxicology Program

Fucus vesiculosus Seaweed Site© 1995-2003 Michael D. Guiry / Seaweed Site/October 09, 1975

Iodide Concentrating Ability Key to Thyroid Function

Fundamental Key isSymporter Molecule

Phylogenetically as early as Brown Algae – Fucus

Iodide Concentrating Occurs also in Salivary Glands, Gastric Mucosa, Choroid Plexus,

Lactating Mammary Tissue

Symporter Origin and Function

Sodium/Iodide Symporter: Transports I-

Na+/K+ ATPase, Quabain-sensitive system in thyroid plasma membrane vesicles

Rat Symporter: 618 amino acids Human Symporter: 643 amino acids

84% Sequence Identity Iodine transport is against an electrochemical gradient Symporter also expected to be in other mammalian

iodide-concentrating tissues: salivary gland, gastric mucosa, choroid plexus & algae such as Fucus sp.

Wolfe, 1998

Phylogeny

http://www.thyroidmanager.org

*

Endostyle

Ammocoete

Importance of iodide sequestration is ubiquitous in our ancestral tree

Prenatal Developmental Defects of Thyroid

Congenital hypothyroidism may be classified as permanent or transient.  The  most common cause of CH is iodine deficiency, which can be eradicated by iodine supplementation.

In iodine sufficient regions thyroid dysgenesis is the most common cause. Thyroid dysgenesis presents as

                 1. Athyrosis (absence of thyroid tissue)                  2. Ectopic thyroid or                  3. Hypoplasia of thyroid

Thyroid dysgenesis account for about 80 - 90% of infants with CH. Familial dyshormonogenesis occur in 10% in infants with CH. Enzyme defects

that affect the synthesis of T4 and T3 lead to hypothyroidism. Hypothalamo-pituitary dysfunction is the cause of CH in a small number of

infants (5 %). In some infants (10 %) hypothyroidism is transient.  Iodine, drugs, maternal

antibodies or transient defect in hormonogenesis can lead to transient hypothyroidism.  These infants also need prompt replacement therapy.

Sperling, 1996

Athyrotic Hypothyroidism Sequence

K. Jones in Smith’s Recognizable Patters of Human Malformation, 1988

K. Jones in Smith’s Recognizable Patters of Human Malformation, 1988

After Cao, et al., 1994

Timing of Vulnerability to Iodine Deficiency in Endemic Cretinism

Time of Treatment(Mo. Before Delivery)

No.Head

CircumferenceDQ Height

No. with Mild Neurologic

Abnormality

6 (included in second trimester)

18 -1.5±1.1 88±8 -2.5 ±0.8 0

>6-7 10 -1.9±1.2 77±9 -3.5 ±0.8 1

>7-8 10 -2.8±1.3 76±11 -2.6 ±0.9 1

>8-9 8 -2.6±1.4 77±16 -3.7 ±2.3 2

P Value (6 vs.>6-9 Mo.)

-- 0.02† <0.001† 0.04† 0.20‡

*Head circumference and height are expressed in standard deviations from the U.S. mean for children of the same age; DQ denotes developmental quotient. For neurologic abnormalities, the number with mild abnormality is shown; none of these children had moderate or severe abnormality.

†By Student’s t-test ‡By the chi-square test

Timing of Vulnerability to Iodine Deficiency in Endemic Cretinism

Cao, et al., 1994

Incidence of Athyrosis in Animal Models and Human Newborns Crl:CD(SD)BR Rats (WIL)

0 Thyroid Findings/39,442 Visceral Exams Crl:CD(SD)BR Rats (MTA/MARTA)

0 Thyroid Findings/24,340 Visceral Exams Crl:CD®(SD)IGS BR Rats (WIL)

0 Thyroid Findings/16,824 Visceral Exams Hra:(NZW)SPF Rabbits (WIL)

0 Thyroid Findings/8,936 Visceral Exams Human Newborns

1 in 4,000 Congenital Hypothyroidism in human newborns worldwide Actual reported ranges 1 in 2,000 in Native Americans

to 1 in 20,000 African Americans Thyroid Dysgenesis accounts for 90%;

of these, 1/3 aplastic and 2/3 rudimentary (Kenneth L. Jones, M.D. – Personal Communication, 2003)

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

From Casarett and Doull, Dr. Capen’s Chapter Species Difference in Thyroid Hormone Economy “The greater sensitivity of the rodent thyroid to derangement by

drugs, chemicals, and physiological perturbations also is related to the shorter plasma half-life of thyroxine T4 in rats than in humans due to the considerable differences in the transport proteins for thyroid hormones between these species (Döhler et al., 1979).”

Caution: To demonstrate directly true species differences in sensitivity, the following should be shown:

Internal DoseAUC/Cmax

Response[T3, T4] Δ

Rat is most “successful” of mammals in terms of number and environment

Guideline Developmental (Prenatal) Reproduction Study*

OECDMaternal & Fetal:

None Specified (N.S.)1-G (1983) Adult/Weanling: N.S.

2-G (2001) Weight Only

ICH (1994) Maternal & Fetal: N.S.Fertility: Adult: N.S.

Pre-/Postnatal: Adult/Weanling: N.S.

JMAFF (2001) Maternal & Fetal: N.S.Adult: Weight Only

Weanling: N.S.

EC (1989) Maternal & Fetal: N.S. Seg I/Seg III: Adult/Weanling: N.S.

OPPTS (1998) Maternal & Fetal: N.S. 2-G: Adult/Weanling: N.S.

MHLW (1994) Maternal & Fetal: N.S. Seg I/Seg III: Adult/Weanling: N.S.

Redbook I (1984) Maternal & Fetal: N.S. Adult/Weanling: N.S.

Redbook 2000 Maternal & Fetal: N.S. Adult/Weanling: N.S.

Guideline-Required Thyroid Evaluations for Developmental and Reproductive Toxicity Studies

*Target organs are identified by further study in generation suspected to be affected, but not required transgenerationally

Guideline-Required Thyroid Evaluations for Subchronic and Chronic Toxicity Studies

Agency/ Guideline

Thyroid Weights

Histopathology Hormones

OECD Non-rodents Control and High Dose Animals

If effect suspected

EPA Non-rodents Control and High Dose Animals

If effect suspected

FDA Redbook II

Rodents and Non-rodents

All Animals If effect suspected

MOHW Often on Rodents and Non-rodents

Control and High Dose Animals

If effect suspected

JMAFF Non-rodents Control and High Dose Rodents and All Non-rodents

If effect suspected

1) Class Effects2) SAR3) Physiologic/Pharmacologic Data4) Results from Previous Studies

In our experience, most often from 28- or 90-day studies

Snapshot of Major Toxicologic Studies: 10 Years at CRO - WIL

Study Type Number

SubchronicGeneral Toxicology

777

Chronic/OncoGeneral Toxicology

67

Reproduction 145

Developmental 531

Total 1520 (912 Drugs/608 Chem)

Experience by Agent or Type

Expected and for Validation of EPA DNT Protocol PTU Methimazole

Anticipated Resorcinol Underway, Historical Human-Use Drug – Tetracycline Class Effects Pesticide Replacement Chemical - Iodinated

Unanticipated Drug – Miscrosomal Enzyme Inducer Drug – GABAnergic Agonist Herbicide – Unknown and ceased development 3 Drugs suspected of microsomal enzyme induction

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 Repro/Development 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

Basic (EPA) Developmental Neurotoxicity Study Design

Conception to

Implantation

Implantation to Closure of Hard

Palate

Hard-Palate Closure to End of

PregnancyBirth to Weaning

Weaning to Sexual Maturity

Indirect Exposure (Maternal)

Gestational and LactationalGD 6 PND 11 PND 72

10/sex/group for each assessment: PND 4, 11, 21, 35, 45, 62 - FOB PND 20, 60 - Auditory Startle PND 13, 17, 21, 61 - Locomotor Activity PND 22, 62 - Learning and Memory

On PND 11, 10 pups/sex/litter Brain weights From these, 6/sex in control and high dose: Neuropathology, Morphometry

On PND 72, 10 pups/sex/litter Brain weights From these, 6/sex in control and high dose: Neuropathology, Morphometry

Schematic Depiction of Study Design/Exposure Regime of Report by R. Lavado-Autric, et al.,

R. Lavado-Autric, et al., JCI, 2003

M C F1

1M C F1

M C F1

2M C F1

M C F1

3M C F1

M C F1

4

M C F1

= 10 Days of LID + 1% KClO4

M = Mated with Untreated Males

C = Cull on PND 3-4 to 8 Pups/Dam

Euthanize 4 Dams/Subgroup - T4 & T3

Stratal Analysis of Cortical Cell Migration

Group Gestation LactationPre-Breeding Post-weaning

KEY

GD 21

Euthanasia

Immunohistochemistry

PND 21

Weaning

PND 40

Pup Weight

BrdU on GD 14, 15 or 16

BrdU on GD 17, 18 or 19

BrdU on GD 14, 15 or 16

BrdU on GD 17, 18 or 19

BrdU on GD 14, 15 or 16

BrdU on GD 17, 18 or 19

BrdU on GD 14, 15 or 16

BrdU on GD 17, 18 or 193 Months of LID + KI (~10ug I/Day)

3 Months of LID

3 Months of LID + 0.005% KClO4

Untreated Control Group

10 D

10 D

10 D

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

Proposed 15-day Intact Male Screening Assay (Currently undergoing multi-lab evaluation)

15 Intact Males/Group

BW & Dose 15 Days

Necropsy & OW following

Last Dosing

Collect Blood for Hormone Analyses

Spectrum of Effects

(10 weeks old)

O’Connor, et al., 1999

Hormonal Fingerprint

Thyroid Agent TDHT

E2

PRL

LH

FSH

TSH

T3

T4

Excretion Enhancer

Synthesis Inhibitor

O’Connor, et al., 1999

Relationship Between Developmentand Phenotypic Diversity

Phenotypic Expression

and Diversity

Time in Development (Age)

EmbryonicPeriod

EmbryonicPeriod

FetalPeriodFetal

PeriodPostnatal

PeriodPostnatal

Period

Extent of Differentiation

BirthBirth

Commentary

Thyroid essential immediately postnatal for thermiogenic shivering providing thermiogenesis

But rarely is cause of death in progeny of reproduction studies established or even investigated

Essential role of thyroid postnatally logically makes a probable contributor to postnatal wastage

The former vulnerability may not be analogous to the human situation Important questions need to be answered relative to the role of

brown adipose fat in different species Interestingly Leptin affected white but not brown adipose tissue

in extensive reproduction studies at WIL Research

Needs for Thyroid Toxicity Assessment

Our ability to detect, demonstrate, validly identify and apply appropriate risk analyses of adverse reproductive effects of anti-thyroidal agents and conditions is dependent on the following: Strong scientific knowledge base of thyroid function Conduct of the appropriate studies, exposure regime

important Utilization of protocols guiding valid measures Appropriate use of experimental models Good risk assessment processes

To do the former for the thyroid in reproductive toxicology, the inherent problems differ little from other arenas of debate in toxicology and risk assessment

Commonalities include: Use of validated models Inter-study (species) comparisons and generalization must be

cognizant of internal dose (exposure) variables Care to make comparisons from studies with equivalent powers In particular with rodents and the thyroid, sensitivity to metabolic

induction (T3 & T4) concomitant with high applied dose (pk issues) studies

Need to ensure study integrity and quality In particular for this instance the validity and sensitivity of several of the

reproductive measures to human reproduction Neonatal adaptation Postnatal exposure via milk vs. human in-utero exposures

Needs for Thyroid Toxicity Assessment (continued)

Summary and Inferences

Review of one CRO’s database indicates that primary guideline-driven detectors of anti-thyroid agents are subchronic toxicity studies

Subclinical hypothyroxinemia-inducing agents may be detected by the former type studies

The recent work by Lavado-Autric, if confirmed in other laboratories and current DNT protocols applied to those findings and are shown to be sensitive to detect to detect such consequences of subclinical hypothyroxinemia, these developmental CNS derangements would represent the most sensitive of hypothyroidal conditions

A somewhat modified two-generation reproductive toxicity study would be the best extant guideline-driven protocol to detect such effects of xenobiotics because of the early and protracted premating exposure regime in conjunction with the extended generational exposure and the addition of the DNT component to the second generation progeny

The thyroid, unlike much of the other endocrine glands functions in a more tonic fashion and is less evaluable from an acute exposure and pathogenesis perspective

Gull WW: On a cretinoid state supervening in adult life in women. Trans Clin Soc London 1874; 7: 180.

“My theory maintains that if the thyroid is impaired, or as in this

instance, removed entirely, then iodine will accumulate in

the female body…causing impairment of the brain with

consequential loss of faculties.

Does not the concept spur you to HUMILITY?

Think of it Acland…less than a thimble full of iodine divides the intellectual from the imbecile.”

“From Hell” Moore & Campbell, 1999

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

Reasons for Apparent Failed Predictions

Appropriate studies not conducted Incidence of effect too low for experimental

detection Unknown/unstudied type(s) of effect Hypersensitive individuals in human population Interaction of multiple agents Unfounded/nonexistent claims or effects Human exposure is overestimated by

experimental design

Animal:Human Concordance Studies for Prenatal Toxicity

Nisbet & Karch, 1983 Many chemicalsRelied on authors’ conclusionsEmphasis on fertilityNo measures of internal dose

Attributes

Interdisciplinary team Criteria for acceptance of data/conclusionsConcept of multiple developmental toxicology endpoints No measures of internal dose

Authors

Holson et al., 1981 (Tox Forum)Kimmel et al., 1984 (NCTR Report)

Attributes

Interspecies inhalatory doses adjustedRelied on authors’ conclusions23 occupational chemicals and mixtures No measures of internal dose

Provided detailed informationOnly 4 drugsEmphasis on morphologyFocus on NOAELsNo measures of internal dose

Many chemicals and agentsVariably relied on authors’ conclusionsNo measures of internal dose nor criteria for inclusion or exclusion of studies

Authors

Hemminki & Vineis, 1985

Newman et al., 1993

Schardein, 1995 & 2000*

Animal:Human ConcordanceStudies for Prenatal Toxicity

*Misnomer to refer to thousands of experimental developmental toxicants

WIL Recommendations

Dose GD 6 through LD 20 Pre-weaning motor activity: PND 13, 17, 21 No reflex ontogeny Assign 1 pup/sex/litter/test for behavior

(20/sex/group) Behavioral tests performed on same animals for

all but one session of learning and memory Neuropathology after last dose (PND 21) and at

young adulthood (72 days of age) using 10 rats/sex/group, with morphometry at both ages

Figure 14-10. A schematic and integrated representation of the three sets of clinical conditions that may affect thyroid function in mother alone, fetus alone, or both (i. e. the feto-maternal unit), to show how the relative contributions of an impaired maternal and/or fetal thyroid function may eventually lead to alterations in the fetal thyroxinemia during its development. (reproduced from Glinoer & Delange, Ref N° 63, with permission of the authors and the Editor).

www.thyroidmanager.org

Comparison of Prenataland Postnatal Toxicity Profiles

Toxicity

Log of Dose

Maternal

DevelopmentalDevelopmental

Prenatal – valid and insightful – Embryonic exposure – Mode of action

Postnatal – valid only – when xenobiotic level is measured in both mother and

offspring

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

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

Selective Juvenile Toxicity of Quinilones

Drug

Ofloxacin (and other quinilones)

Modified from Stahlmann et al., 1997.

Species &Treatment

Multiple species,postnatal exposure.20 mg/kg (dog, 3 mo.)600 mg/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.

EPA vs. OECD Harmonization Issues Dose GD6 through LD 10 Pre-weaning motor activity: PND

13, 17, 21 No reflex ontogeny

10/sex/group

Neuropathology after last dose using 6 rats/sex/group, with morphometry

Assign 1 pup/litter/test for behavior Behavioral tests performed on

different animals

Dose GD 6 through LD 20 Pre-weaning motor activity: once

before PND 21 Two measures of reflex ontogeny -

i.e., righting reflex, air righting 20/sex/group for all behaviors

except learning and memory - at least 10/sex/group

Neuropathology on PND 11 and optional after last dose using 10 rats/sex/group, tiered approach to morphometry

Assign 1 pup/sex/litter/test for behavior

Behavioral tests performed on same animals for all but one session of learning and memory

Sledgehammer Thyroid Toxicity

Short Ventral Limb of Dentate Gyrus

HypercellularControlPND 11

100 ppm MethimazolePND 11

Figure 15-7. Brain neurologic development relative to thyroid function in the rat and human. TH, thyroid hormones; dpc, days postconception; dpn, days postnatal. (From Porterfield and Hendrich, with permission.)

Figure 15-6. Maturation of thyroid hormone effects in the human fetus and neonate. The left edge of the bars indicate the approximate time the effects of thyroid hormone become manifest (Used with permission from Fisher DA, Polk DH, Wu SY: Fetal thyroid metabolism: a pluralistic system. Thyroid 4:367,1994 ).

www.thyroidmanager.org

www.thyroidmanager.org

www.thyroidmanager.org

The Symporter

www.colostate.edu

Rat; GD20; Bouin’s Fixed; Wilson’s Sectioning Technique

Rat; GD20; Fresh Dissection Technique

Size: 0.5 to 1 mm

Rat; PND 4; Fresh Dissection Technique

Rat; PND 21; Fresh Dissection Technique