study design considerations affecting interpretation of developmental toxicity data

Study Design Considerations Affecting Interpretation of

Developmental Toxicity Data

Joseph F. Holson, Ph.D., D.A.B.F.E.

WIL Research Laboratories, LLC

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Overview

• Concordance and Extrapolability

• Study Design Review

• Endpoint Variability/Sensitivity

• Statistical Considerations

• Rare Events and Historical Control Data

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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

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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

Animal:Human Concordance Studiesfor Prenatal Toxicity

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NCTR Concordance Report:Authors

• Dr. J. Holson

• Dr. C. Kimmel

• Dr. C. Hogue

• Dr. G. Carlo

• Developmental Toxicologist

• Developmental Toxicologist

• Epidemiologist

• Epidemiologist

NCTR Report No. 6015, 1984

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NCTR Concordance Report: Assumptions1) 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.

Holson, et al., 1984

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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

Awareness of Developmental Toxicity of Selected Agents


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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


Effect-Levels for Teratogens in Humans and Test Species

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• Embryo/fetal toxicity in presence of overt maternal toxicity• Aminopterin• Methylmercury• Polychlorinated biphenyls

• Embyro/fetal toxicity in presence of maternal stress (physiological changes)• Steroidal hormones• Ethanol• Cigarette smoking

• Embryo/fetal toxicity without significant maternal effects• Thalidomide• Accutane• Diethylstilbestrol• Ionizing radiation

Relationship between Maternal Toxicity and Fetal Outcome in Humans

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• Why do we know so little?• Early wastage of severely affected embryos• Human exposure information is not well documented• Human exposures may be extremely low• Readily recognizable and consistent lesions are rare• Other types of reproductive problems may be related,

but not thoroughly investigated• Population monitoring is limited• Testing of environmental chemicals and

pharmaceuticals identified potential agents• Pregnant women frequently choose to avoid

exposure to many substances

Human Teratogens

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• 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

Reasons for Apparent Failed Predictions

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Overview






• Interpretational Questions

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Classes of Reproductive and Developmental Toxicity

Reproductive Developmental

Fertility Mortality

Parturition Dysmorphogenesis

Lactation Alterations to Growth

Functional Toxicities

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Possible Interrelationships of Developmental Toxicity Endpoints

Toxic StimulusGrowth

RetardationDeath

Malformation

Functional Impairment

Toxic Stimulus

Malformations

Functional Impairments

Growth Retardation

Death

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 Loss

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

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 Development

Fertility StudyICH 4.1.12W4W

CMAX

AUC

CMAX

AUC

10W

ICH 4.1.3 OECD 414OPPTS 870.3600

870.3700

Standard Study Designs

Endpoints of Developmental Toxicity Studies

Approximate Chronological Order of Collection

Maternal survivalMaternal clinical observationsMaternal body weightMaternal body weight changeMaternal gravid uterine weightMaternal net body weightMaternal net body weight changeMaternal food consumptionMaternal necropsy findingsMaternal clinical pathology*Maternal organ weights*Fetal viabilityCorpora luteaImplantation sitesPreimplantation lossPostimplantation loss- early resorptions- late resorptionsFetal weights- male- female- combinedFetal malformationsFetal developmental variations


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Overview







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Endpoint Variability

• Endpoints may be binary or continuous measures• Fetal body weight is a continuous measure determined

gravimetrically, and as such, displays the least variability of DT endpoints

• Variability may be methodologically or biologically dependent• Endpoints for which both are true may be more variable

• Endpoints are either objectively or subjectively evaluated• Variability may be positively correlated with subjectivity of

evaluation

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Endpoint Variability

• Affects power of statistical evaluation• Should affect group size

• Affects utility of determining causation of rare events

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Ranking by Variability of Developmental Toxicity Study Endpoints

Rat = 1509

Rabbit = 1829

Mouse = 484

Rat = 22074

Rabbit = 10278

Mouse = 5552

Dams Fetuses

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Ranking by Variability of Developmental Toxicity Study Endpoints in the Rat

Studies = 61

Dams = 1509

Fetuses = 22074

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Ranking by Variability of Developmental Toxicity Study Endpoints in the Rabbit

Studies = 81

Dams = 1829

Fetuses = 10278

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Ranking by Variability of Developmental Toxicity Study Endpoints in the Mouse

Studies = 20

Dams = 484

Fetuses = 5552

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Sensitivity & Pattern

• In most cases of developmental toxicity established in humans and laboratory models, fetal weight is the most sensitive and is often associated with arrays of developmental disruption

• The critical importance of examining patterns of effect on multiple endpoints and reconciliation of the biological plausibility of the overall effect cannot be overemphasized

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Primary vs. Secondary Effects

• Patterns of effect on multiple endpoints, arising often from depression of fetal body weight, are the key to understanding the arrays of developmental disruption associated with toxic insult to in utero progeny

• Because events such as body cavity development (e.g., gut rotation and retraction) and mid-line closures (neural tube, hard palate, spine, thorax, and abdomen) proceed so systematically and harmoniously, retardation of growth may simply shift the timing of these discrete developmental events by a matter of hours or days in most species

• For example, an insult that manifests as omphalocele or cleft palate may result from developmental delay due to intrauterine growth retardation rather than a direct effect on morphogenesis of those structures

Endpoints of developmental toxicity studies

Approximate chronological order of collection

Ranked by sensitivity(most sensitive to least sensitive)

Maternal survivalMaternal clinical observationsMaternal body weightMaternal body weight changeMaternal gravid uterine weightMaternal net body weightMaternal net body weight changeMaternal food consumptionMaternal necropsy findingsMaternal clinical pathology*Maternal organ weights*Fetal viabilityCorpora luteaImplantation sitesPreimplantation lossPostimplantation loss- early resorptions- late resorptionsFetal weights- male- female- combinedFetal malformationsFetal developmental variations

Fetal weights- male- female- combinedPostimplantation loss- early resorptions- late resorptionsFetal viabilityFetal malformationsFetal developmental variationsMaternal body weightMaternal body weight changeMaternal gravid uterine weightMaternal net body weightMaternal net body weight changeMaternal food consumptionMaternal survivalMaternal clinical observationsMaternal necropsy findingsMaternal clinical pathology*Maternal organ weights*Corpora luteaImplantation sitesPreimplantation loss


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Overview

• Concordance and Extrapolability• Study Design Review• Endpoint Variability/Sensitivity• Statistical Considerations

• Litter Effect• Statistical Power

• Rare Events and Historical Control Data• Interpretational Questions

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Litter as the Experimental Unit

• Dam (litter) is randomized/treated

• Individual fetuses or pups within litters do not respond completely independently

• Fetuses of a given litter tend to exhibit similar responses to toxic insult

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Litter as the Experimental Unit

• Mathematically, using the litter as the experimental unit to account for this influence requires a determination of the percentage of embryos/fetuses within each litter that are affected.

• A grand mean is then calculated from the individual litter means.

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Calculation

Summation per Group (%) =

Σ Preimplantation Loss/Litter (%)

No. Litters/Group

Where:

Preimplantation Loss/Litter (%) =

(No. corpora lutea – No. Implantation sites)/Litter X 100

No. Corpora Lutea/Litter

Example 1: Resorptions (prenatal deaths)

Resorptions

(No.)Implantation sites (No.)

Postimplantation loss (%)

Litter 1 0 15 0%

Litter 2 0 14 0%

Litter 3 5 10 50%

Litter 4 0 13 0%

Litter 5 1 15 7%

Incorrect statistics

Numerator = 6 = Total No. of resorptions

Denominator = 67 = Total No. of implantations

Incidence = 9.0% =Total No. of resorptions/total No. of implantations

Among-litter variability

= -- = Incalculable

In Example 1, the numbers of resorptions are summed and divided by the total number of implantation sites. This calculation [(5+1)/67*100=9.0%] provides a simple incidence, without regard to weighting of effects by litter, as with correct litter-based statistics.

Incorrect Statistics

Example 1: Resorptions (prenatal deaths)

Resorptions

(No.)Implantation sites (No.)

Postimplantation loss (%)

Litter 1 0 15 0%

Litter 2 0 14 0%

Litter 3 5 10 50%

Litter 4 0 13 0%

Litter 5 1 15 7%

Using Example 1, correct litter-based statistics are calculated by summation of the percent per litter postimplantation loss and division by the number of litters, the randomized, true experimental unit [(50%+7%)/5=11.3%].

Correct litter-based statistics

Numerator = 57% =Sum of postimplantation loss (%) per litter

Denominator = 5 = Total No. of litters

True %PL = 11.3% =Sum of postimplantation loss (%) per litter/total No. of litters

Among-litter variability

= 21.8% =Standard deviation of postimplantation loss

Litter-Based Statistics

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Statistical Power

• Power is the probability that a true effect will be detected if it occurs

• Defined as 1-ß, where ß is the probability of committing a Type II error (false negative)

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Statistical Power

• Dependent on:• Sample size• Background incidence• Variability of endpoint• Significance (α) level of hypothesis test

• Example: • The sample size (number of litters) needed to detect a 5%

or 10% change in an endpoint is dramatically lower for a continuous measure with low variability than for a binary response with high variability

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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

aNumber of litters/groupbCharles River, Wilmington, MAcOsborne-Mendel, Charles River, Wilmington, MA

From Nelson and Holson, 1978

Number of Litters (N)a to Detect Changesin Fetal Weights and Deaths in Mice and Rats

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Unbalanced Design?

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Overview





• Rare Events and Historical Control Data• Case Studies


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Multiple Surveys3-94.0Human

1675.3-5.75.5Dog

47080-103.2Rabbit

52070-31.2Mouse

96430-1.60.33Rat

NRange (%)Mean %Species

WIL Research Laboratories, LLC Historical Control Database

Comparison of Overall Spontaneous Malformation Rates in Different Species

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FDA Definition

Rare Event – “an endpoint that occurs in less than 1 percent of the control animals in a study and in historical control animals”

Reviewer Guidance(Draft)

Integration of Study Results to AssessConcerns About Human Reproductive

And Developmental Toxicities

CDER, 10/2001Pharmacology/Toxicity

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• Disbelief, rely on statistical insignificance• Comparison to concurrent control• Comparison to historical control (HC)• Comparison to other HC databases• Ask experience/opinions of others• Construct explanation to negate• Agency rejects• Re-do study or label appropriately

Rare Events (Low-Incidence Findings): Typical Reaction to, and Subsequent Scenario

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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 91.3-99.3%

91%

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

Selected Reproductive Endpoints Exhibiting Strong Signals from Rare Events/Low Incidence

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• 2-generation with second mating phase of F1, vapor inhalation, used industrially, OTC pharmaceutically

PPM 0 70 300 500 700

F0 0 0 0 2/24 3/26

F1-1st 0 0 0 0 1/17

F1-2nd 0 0 1/21 1/18 0/12

• HC then: 2/333 = 0.60%• HC now: 4/1100 = 0.36%

Case Study: Dystocia, Extended Parturition and/or Pregnancy

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Case Study: Functional Alteration Example 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

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• RAS (renin-angiotensin system) matures around GD17

• No ‘apparent’ effect in initial reproductive studies

• Nonstatistically significant increase in postnatal mortality (~8%)

• Subsequent postnatal studies with direct administration to pups

• Growth retardation

• Renal alterations (anatomic and functional)

• Mortality increased to more than 30%

Case Study: Functional Alteration Example ACE Inhibition in Developing Rats

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Historical Control Data

Malformation TotalMean% PL

Min Max

Retroesophageal Aortic Arch

2/16,824 0.01% 0.0% PL 0.3%PL

Rat Study Data

Malformation 1 2 3 4

Retroesophageal Aortic Arch

0 01

(0.3%PL)1

(0.3%PL)

Case Study: Malformation Example Topical Antibiotic for Oral Mucosa

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Rare Events: Control vs. Treated Groups

3

1

3:1 Probability that spontaneous event will occur in treated group

Low Mid

HighControl

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MalformationIncidence (%PL)

Rat Rabbit

Ventricular Septal Defect 0 0.02

Cleft Lip/Palate 0.02 0.04

Abdominal Wall Defect Including Gastroschisis

0.04 0.06

Hydrocephaly 0.03 0.20

Spina Bifida 0 0.17

Renal Agenesis 0.01 0.02

Diaphragmatic Hernia 0 (2/39442) 0.04

Malformations can involve any tissue or structure and may constitute a rare event issue

Malformations which Have Occurred as Rare Events in Numerous Scenarios

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Control Low Mid High

0 0 0 1

0 1 0 0

0 0 1 0

0 0 1 1

1 0 0 1

Rare Event Manifestation Matrix

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• Comparison to concurrent control• Evaluate dose-responsiveness including TK, AUC/Cmax• Compare to HC range and mean, consider other statistical tests,

including Monte-Carlo Analysis• Evaluate signals of developmental toxicity among dose groups• Compare to second species• Compare to findings in the combined pre-/postnatal study• Perform confirmatory study:

• Increasing N• Increasing number of concurrent controls• Increasing dose (based on TK: AUC/Cmax)• Consider unbalanced study design• Delimited exposure regime• Evaluate pharmacologic action relative to ontogeny of receptors, etc. and

reconcile with modified dosing regime• Label and follow-up in birth defects registry


Paradigm to Evaluate Rare Findings

Approach to Determining Biological Relevance of Rare-Event Findings

Stepwise Approach Consideration/Caution

Dose Response(Including TK)

Possibility of any rare event occurring is 3:1 in favor of test article-treated groups

Historical Control Comparison

Mean and range values (lab’s own data, then others); Values near or outside upper or lower limits of historical control range provide a strong signal that rare event is “real”

Additional Statistical Tests

Monte-Carlo Analysis to examine sampling error and predict population estimates relative to sampling values

Second Species Comparison (Including TK Data)

Establishment of similar internal dose (Cmax and/or AUC) critical

Approach to Determining Biological Relevance of Rare-Event FindingsStepwise Approach Consideration/Caution

Other Studies Comparison

Concordant outcome from embryo/fetal and pre/postnatal studies (e.g., slight decrease in fetal BW in developmental study, along with slight decrease in PND 1 BW in reproductive study) is considered to be “real” event

Confirmatory Study Increased N/group; increased N in control group; increased dose; limited exposure regime (if developmental timing of organ/system affected is known, only administer doses to achieve same AUC on limited regime, which could illuminate whether or not the underlying embryology and the outcome make sense)

Mechanistic Studies Evaluate pharmacological action relative to ontogeny of receptors, signaling pathways, and other possible modes of action

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The Bottom Line

• With rare events, the best practice for resolution of the relationship to treatment is through a large historical control database developed at the same laboratory, using consistent methodology and conditions in conjunction with appropriately designed confirmatory study.

• Human risk assessment and management may require study in human registries.

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Overview





• Rare Events and Historical Control Data• Case Studies


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Interpretational Questions

Question Implications/Comments

Are minor test article-treated group changes inappropriately weighted by uncommonly low/high control group values?

Although the data from test article-treated groups initially should be compared to the concurrent control group, use of the laboratory’s historical control data will enable identification of atypical concurrent control values.

Is the pregnancy rate significantly reduced (either relative to the concurrent control group or in all groups)?

Reduced pregnancy rates caused by the test article are rare, considering the onset of treatment in most developmental toxicity studies (typically after implantation). The power of the study (ability to detect dose-response relationship) may be impaired.

Are clinical signs of toxicity present?

Severe clinical signs of toxicity that disrupt maternal homeostasis and nutritional status may result in subsequent insults to the products of conception. Conversely, frank increases in terata in the absence of significant maternal toxicity probably signal an exquisite effect on morphogenesis and suggest that the embryo/fetus is more sensitive.

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Is there an increased incidence of abortion (in

rabbits)?

Related maternal data must be examined carefully (females may have stopped eating), including the historical control rate of abortion and temporal distribution of events (abortions during GD 18 to 23 are rare, compared to those occurring later in gestation).

Does decreased maternal body weight gain correlate with similar changes in food consumption?

Concomitant reductions in body weight and food consumption usually indicate systemic toxicity, and in some cases signal a maternal central nervous system (CNS) effect.

Do body weight deficits occur in a dose-related manner?

Dose-related effects on body weight generally indicate a compound-related effect. If maternal mortality is present at the high-dose level, effects on body weight gain may not manifest because of elimination of sensitive members of the group. In this case, body weight effects occurring only at lower doses may represent an adverse effect.

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Does maternal body weight gain “rebound” after cessation of treatment?

A rebound in body weight gain following treatment may indicate recovery from the effects of the compound.

Do maternal body weight gain and food consumption decrease following cessation of treatment?

This may indicate a withdrawal effect during the fetal growth phase because of maternal CNS dependency (e.g., opioid compounds). However, it is difficult to correlate such an effect with fetal outcome.

Is maternal net body weight affected?

A decrease in maternal net body weight is most likely an indicator of systemic toxicity. However, reduced maternal body weight in the absence of an effect on maternal net body weight indicates an effect on intrauterine growth and/or survival, rather than maternal systemic toxicity.

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Is food consumption reduced?

Changes in food consumption generally signal either palatability issues or systemic toxicity caused by the test substance. Reduced food consumption without a concomitant effect on body weight gain is likely a transient effect and probably not adverse. If food utilization is unaffected when food intake is reduced, the test article is probably affecting caloric intake (i.e., a test article or vehicle with high caloric content may cause an animal to consume less food with minimal or no net effect on body weight gain).

Is maternal body weight gain reduced during a specific period of gestation?

Decreased maternal body weight gain during the late gestational period may be associated with reduced fetal skeletal mineralization. In all cases, risk to the developing embryo/fetus is greater the longer the reduction in maternal body weight gain is sustained.

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Interpretational QuestionsQuestion Implications/Comments

Is gravid uterine weight affected?

Reduced gravid uterine weights are usually due to reduced viable litter size and/or reduced fetal weights. In rare cases, effects on placentae or the uterus may be initially recognized by non-fetus-related changes in gravid uterine weight.

Is the percentage of affected litters increased relative to controls?

This may indicate a maternal dimension if the proportion of fetuses in those litters is not similarly affected.

Is fetal weight subtly decreased in the high-dose group (e.g., mean rat fetal weights of 3.6, 3.6, 3.6, and 3.4 g in control, low-, mid-, and highdose groups, respectively)?

If other signals of developmental toxicity are present, the decrease in fetal weight is probably adverse. If no other signals of developmental toxicity exist, and the low fetal weight is within the laboratory’s historical control range, it is probably not an adverse effect. A robust, highly consistent historical control database in this instance would indicate that fetuses weighing 3.4 g are able to survive and thrive, and therefore the weight reduction is likely to be temporary. Such a conclusion may be corroborated with data from postnatal assessment studies.

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Is viable litter size decreased?

Decreased viable litter size generally represents either increased resorption rate (usually attributable to the test agent) or decreased numbers of corpora lutea/implantation sites (not likely to be a test substance-related effect unless treatment began at or prior to fertilization).

Is the resorption rate increased?

Complete litter resorptions are rare (3 in 1452 Crl:CD®(SD)IGS BR rat litters). Therefore, even one completely resorbed rat litter in the high-dose group merits attention. Furthermore, an increased incidence of control female rats with more than 3 resorptions usually constitutes a signal of developmental toxicity.

Is the percentage of affected fetuses per litter increased relative to controls?

This probably indicates a proximate fetal effect.

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Does a correlation exist between low-weight-for-age fetuses and dysmorphogenesis?

If malformations are limited to low-weight-for-age fetuses, the malformations may be due to generalized growth retardation (e.g., omphalocele in rabbits or cleft palate in rats).

Is a syndrome or spectrum of effects present?

A syndrome of effects indicates multiple insults on a specific organ system during development (e.g., tetralogy of Fallot, or a cascade of events during heart development, beginning with ventricular septal defects and including pulmonary stenosis and valvular defects). A spectrum of dysmorphogenic effects implies a less targeted, more generalized response (e.g., vertebral agenesis occurring with ocular field defects).

Is the total malformation rate per group affected?

Several organ systems may have slight malformation rate increases that may not be outside the historical control range when evaluated individually. However, in summation they may signal an increased generalized dysmorphogenic effect.

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Are there qualitative differences in the relative functional utility of affected structures/systems?

Bent long bones (e.g., femur) are of greater concern than bent ribs. Unossified centra or sternebrae are not of great import if the underlying cartilaginous structures are present and properly articulated. Malformed caudal vertebrae or facial papillae in rats would not merit the concern that similar alterations to analogous human organs/structures would elicit.

Is fetal ossification generally retarded (evidenced by Alizarin Red and/or Alcian Blue uptake)?

Delayed ossification may be due to developmental delay resulting from intrauterine growth retardation or may stem from properties of the test agent expected to cause delayed mineralization of skeletal structures (e.g., calcium chelation, altering of blood urea nitrogen, alkaline phosphatase inhibition, parathyroid hormones, vitamin D, etc.).

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Is a dose-related response observed when the endpoint of “affected implants” is evaluated?

For this endpoint, each implantation is counted once (tabulated as “affected”), whether the outcome is an early/late resorption, malformed fetus, or dead fetus. As the rate of malformation increases, the rate of late resorption declines. Likewise, as the number of early resorptions rises, the numbers of late resorptions and malformed fetuses decline.

Are there similar effects in other studies?

Comparison to effects in a second species developmental toxicity study, malformations manifested as anatomic/functional changes in the pre- and postnatal development study, and maternal toxicity relative to systemic toxicity observed in subchronic toxicity studies may indicate that the pregnant animal is more susceptible to the test agent than the nonpregnant animal.

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study design considerations affecting interpretation of developmental toxicity data

Health & Medicine

human concordance studies

rat rat

rat mo rat

human response

nctr concordance report

concordance of effects

rab rat

overview concordance