Pathologic conditions affecting developmental disturbances and anomalies
Study Design Considerations Affecting Interpretation of Developmental Toxicity Data
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Transcript of 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
2
Overview
• Concordance and Extrapolability
• Study Design Review
• Endpoint Variability/Sensitivity
• Statistical Considerations
• Rare Events and Historical Control Data
3
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
Holson, et al., 1984
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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
Holson, et al., 1984
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
12
Overview
• Concordance and Extrapolability
• Study Design Review
• Endpoint Variability/Sensitivity
• Statistical Considerations
• Rare Events and Historical Control Data
• Interpretational Questions
13
Classes of Reproductive and Developmental Toxicity
Reproductive Developmental
Fertility Mortality
Parturition Dysmorphogenesis
Lactation Alterations to Growth
Functional Toxicities
14
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
Holson, et al., 2005
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Overview
• Concordance and Extrapolability
• Study Design Review
• Endpoint Variability/Sensitivity
• Statistical Considerations
• Rare Events and Historical Control Data
• Interpretational Questions
18
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
25
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
Holson, et al., 2005
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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
33
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
36
Unbalanced Design?
37
Overview
• Concordance and Extrapolability
• Study Design Review
• Endpoint Variability/Sensitivity
• Statistical Considerations
• Rare Events and Historical Control Data• Case Studies
• Interpretational Questions
38
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
39
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
40
• 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
41
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
43
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
45
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
46
Rare Events: Control vs. Treated Groups
3
1
3:1 Probability that spontaneous event will occur in treated group
Low Mid
HighControl
47
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
48
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
49
• 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
Holson, et al., 2002
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
52
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.
53
Overview
• Concordance and Extrapolability
• Study Design Review
• Endpoint Variability/Sensitivity
• Statistical Considerations
• Rare Events and Historical Control Data• Case Studies
• Interpretational Questions
54
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.
55
Interpretational Questions
Question Implications/Comments
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.
56
Interpretational Questions
Question Implications/Comments
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.
57
Interpretational Questions
Question Implications/Comments
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.
58
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.
59
Interpretational Questions
Question Implications/Comments
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.
60
Interpretational Questions
Question Implications/Comments
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.
61
Interpretational Questions
Question Implications/Comments
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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Interpretational Questions
Question Implications/Comments
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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Interpretational Questions
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Interpretational Questions
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Interpretational Questions
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Interpretational Questions