Pre- and postnatal developmental toxicity study design for pharmaceuticals

Review Article

Pre- and Postnatal Developmental Toxicity StudyDesign for Pharmaceuticals

Graham P. Bailey,1� L. David Wise,2 Jochen Buschmann,3 Mark Hurtt,4 and J. Edward Fisher5

1Johnson and Johnson PRD, Janssen Pharmaceutica, Beerse, Belgium2Merck Research Laboratories, West Point, Pennsylvania

3Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany4Pfizer Drug Safety Research & Development, Groton, Connecticut

5FDA/CDER, Silver Spring, Maryland

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the ICHS5(R2) document. The studies that assess the hazard of both pre- and postnatal exposure are predominantly conducted inrodents (rat and mouse). Utilizing the collective experience of the authors, acceptable designs for both the range-finding anddefinitive studies are presented with detailed descriptions for the presentation of data. In addition, the suggested initiationand then total duration of these studies in relation to clinical studies are described. Optional parameters that may beincluded in the studies, as well as possible combination with other study designs are discussed. The details described hereinwill assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agenciesthat ultimately review these studies. Birth Defects Res (Part B) 86:437–445, 2009. r 2009 Wiley-Liss, Inc.

Key words: prenatal; postnatal developmental toxicity; neonatal devel-opment; safety assessment; hazard assessment

INTRODUCTION

The intention of this report is to provide the readerwith a concise outline of adequate designs for pre- andpostnatal developmental toxicity (PPN) studies withsmall-molecule medicinal products (i.e., human pharma-ceuticals), which are primarily governed by guidancefrom the International Conference on Harmonization(ICH). This subject was recently reviewed by Barrow(2009). The present report expands on that work byproviding details of the history, study designs, datapresentation, and timing of the PPN study. It is not ourintention to cover study requirements for environmentalchemicals, including pesticides, which are regulated byother agencies such as the Environmental ProtectionAgency in the United States. Also the design of studies tosupport registration of biopharmaceuticals (i.e., biologicsand vaccines) will not be detailed. Currently, there are noformal guidelines for the design of developmentaltoxicity studies to assess the safety of biopharmaceuti-cals. A valuable points-to-consider report on this topichas recently appeared (Martin et al., 2009), which pointsout that a case-by-case approach is needed.

The history of regulatory guidance as it pertains topreclinical developmental and reproductive toxicitytesting has been previously reviewed in detail (Christianand Hoberman, 1997; Christian et al., 2006). Followingthe first guidance from the U.S. Food and DrugAdministration in 1966, the design of studies to assessthe potential pre- and postnatal developmental toxicity

of medicinal products varied amongst the regulatoryagencies of the United States, Japan, and Europe, andwere generally known as peri- and postnatal studiesas administration to the mothers did not normallycommence until after completion of the period oforganogenesis. These slightly differing approaches wereconsolidated in the early 1990s with the introduction ofthe ICH harmonized guideline ICH S5 (R2) entitled‘‘Detection of Toxicity to Reproduction for MedicinalProducts and Toxicity to Male Fertility,’’ which was firstreleased in June 1993 and last revised in November 2005.This guidance document may be obtained, along with allother guidance documents, at http://www.ich.org. Be-sides outlining the study design to assess pre- andpostnatal developmental toxicity, this guidance docu-ment also addresses fertility and embryo-fetal develop-mental studies, which will be addressed in companionreports. Readers are encouraged to consult the ICHS5(R2) guidance document for additional details andcaveats.

The present report will describe the timing, duration,design, and presentation of PPN studies for conventional

Published online in Wiley InterScience (www.interscience.wiley.com)DOI: 10.1002/bdrb.20217

*Correspondence to: Graham P. Bailey, Johnson & Johnson Pharmaceu-tical Research and Development, A Division of Janssen PharmaceuticaN.V., Turnhoutseweg 30, B-2340 Beerse, Belgium.E-mail: [email protected] 30 October 2009; Accepted 2 November 2009

Birth Defects Research (Part B) 86:437–445 (2009)& 2009 Wiley-Liss, Inc.

small-molecule pharmaceuticals in the most commonlyused species. Study designs for juvenile toxicity and non-human primate studies will be addressed in companionreports. There are similar study design requirements foragricultural chemicals, industrial chemicals, food addi-tives, veterinary drugs, and medical devices, but thisreport will focus on drugs.

TIMING OF PRE- AND POSTNATALDEVELOPMENTAL TOXICITY STUDIES

The time to conduct PPN studies relative to drugdevelopment and clinical trials is described in the ICHM3(R2) document, which reached finalization (i.e., Step 4)of the ICH process as of June 2009. That document isentitled ‘‘Guidance on Nonclinical Safety Studies for theConduct of Human Clinical Trials and MarketingAuthorization for Pharmaceuticals.’’ According to theguidance in that document, the PPN study should becompleted for submission prior to marketing approval.Of course, different companies may require their ownpreferred time-lines for regulatory submissions andsimply by working backward in time from when thestudy reports are needed for regulatory submission onecan determine a fairly definitive start date for each studytype. With the current emphasis on the inclusion ofpediatric prescribing data, pediatric development can nolonger be treated as an add-on, but now as an integral partof the drug-development process. Therefore, PPN studiesmay be performed much earlier than previously seen.This may particularly be the case for dose range-findingstudies (drfPPN), which include pre-weaning adminis-tration to the offspring and the design of these studieswill also be discussed in this report. Thus, the timing ofthe start of drfPPN and PPN studies may also depend onthe anticipated start of clinical studies in the pediatricpopulation.

DURATION OF PRE- AND POSTNATALDEVELOPMENTAL TOXICITY STUDIES

The durations of some common designs of the drfPPNand PPN studies are shown in Table 1. These areapproximate durations and depend on the number andtype of postnatal assessments conducted and, in thedefinitive study, on the outcome of any mating trial. Thetotal time to a final report is the most variable since itdepends on the interactions of multiple groups (e.g., datacompilation, report writing, review time, quality assur-ance [QA] audit time, and sponsor interface). Only thePPN study is required to be conducted under GoodLaboratory Practice Regulations (OECD: Principles of

Good Laboratory Practice. Number 1, FDA 21 CFR Part58 21).

DESIGN OF PPN STUDIES

The ICH S5 (R2) guideline states that the PPN studyshould be conducted in one species and that thepreferred species is the rat. This does not preclude theuse of an alternative species; however, the alternativewould need to be well characterized in terms of theassessments that are required as part of this study,namely fertility and reproductive and behavioral devel-opment. The mouse makes a suitable alternative speciesbut it would require very special conditions for aninvestigator to embark upon a PPN program using therabbit or the dog. The rabbit is a notoriously difficultspecies in which to do postnatal examinations due tomaternal rejection. Neither the dog nor the rabbit have anestablished and comprehensive behavioral testing bat-tery and assessing the fertility in the dog would best bedescribed as protracted.

The designs of the drfPPN and PPN studies are similarexcept for the number of mated females per group andthe study durations. As mentioned in a companionreport in this issue (Wise et al., pages 418–428), thedrfPPN study design could also be useful for selection ofdose levels for the definitive embryo-fetal developmental(EFD) toxicity study. In most cases, information from theEFD studies is available prior to the start of a PPN study.Although information regarding the maternal and fetalresponse can be appropriate for selecting the dose levels,the continuation of the dosing throughout the parturitionprocess and subsequent consequences on the survivaland viability of the neonates is critical for a successfulPPN study. However, assuming a drfPPN study was notconducted and the rodent EFD study demonstratedadequate maternal toxicity and no significant fetaltoxicity, then appropriate dose levels could be selectedin most cases. For other situations where a drfPPN studyis deemed necessary, the selection of dose levels can bebased on the prior information from developmentaltoxicity studies. Due to the uncertainty in the response inthe early postnatal period, some laboratories may elect toutilize more than 3 drug-treated groups.

There is no ICH guidance for the design of drfPPNstudies; however, a minimum but adequate range-finding study would include at least 6 mated femalesper group and at least 3 drug-treated groups. One suchdesign is presented in Table 2.

An adequate definitive PPN study design is presentedin Table 3.

Table 1Durations of Pre- and Postnatal Developmental Toxicity Studies

In-life

Data compilationand writingF01F1a F1 onlyb QA Total durationa

drfPPN 4 weeks – 40 business days NA 12 weeksPPN 6 weeks C10 weeks 80 business days Varies 32 weeks

aTaken from gestation day 0.bFrom weaning.

438 BAILEY ET AL.

Birth Defects Research (Part B) 86:437–445, 2009

AUTHORS’ NOTES TO TABLES 2 AND 3

A. Note 5 of the ICH S5(R2) guidance lists a number ofpotential species. While the specific guidance for the

PPN study states that the preferred species is the rat, thespecies must still be relevant to humans. The speciesselected most often matches the species utilized forgeneral toxicity studies. In general, outbred stocks of

Table 2Study Design of Dose Range-Finding Pre- and Postnatal Developmental Toxicity (drfPPN) Study

Note

Strain or stock Sprague-Dawley or Wistar AAge on GD 0 10–12 weeksAnimals per group 6Number of groups 4 CDuration of dosing GD 6–PND 6 DF0Physical observations Twice daily EBody weights At least weekly during pregnancy and PND 0 or 1, 4, and 7 FFood consumption Once each week GParturition observations Three times daily from GD 21 until complete HGross necropsy Presumed GD 24 or LD 7 IF1Mortalities and external examination Daily from PND 0 ELitter size, live and dead Day 0 and daily thereafterSex by external exam PND 1 and at changes in the litter sizeBody weights PND 1, 4 and 7Gross examination PND 7 Optional

GD, gestation day; PND, postnatal day; LD, lactation day.

Table 3Study Design of Definitive Pre- and Postnatal Developmental Toxicity Study

Note

Strain or stock Sprague-Dawley or Wistar AAge on GD 0 10–12 weeksAnimals per group 20 BNumber of groups 4 CDuration of dosing GD 6–PND 20 DF0Physical observations Twice daily EBody weights Twice weekly during pregnancy and lactation FFood consumption At least once each week GParturition observations From GD 21 until complete HGross examination Presumed GD 24 or LD 21 IF1 preweaningMortalities and external examination Daily from PND 0 ELitter size, live and dead Daily from PND 0Sex by external exam PND 1 and at changes in the litter sizeBody weights PND 1, 4, 7, 14 and 21Standardisation of litter size Litters culled PND 4 to appropriate even numbers of males and females OptionalReflex ontogeny At least two measures assessed pre-weaning OptionalDevelopmental milestones At least two measures assessed pre-weaning OptionalSelection Litters culled PND 21, at least 1 male 11 female/litter where possible JGross examination PND 21 OptionalF1 from PND 21Animals per group/sex 20 BPhysical observations Daily EBody weights WeeklySexual development Vaginal opening, from Day 30 until complete K

Preputial separation, from Day 40 until completeBehavior and functional tests According to standard procedures of the laboratory LReproductive performance At least 10 weeks old, paired for mating (1M:1F) within the same group (not siblings) MGross examination Females: Caesarean section from GD 13 N

Males: once fertility is confirmed I

GD, gestation day; LD, lactation day; PND, postnatal day.

439PPN DEVELOPMENTAL TOXICITY STUDY DESIGN


animals are used. While many laboratories obtainnaturally mated females from vendors, which can be amore cost-effective alternative than maintaining a colonyof male breeders to provide in-house mated females, thisdoes have some disadvantages. The most obvious ofthese is the lack of any real acclimatization period priorto the initiation of dosing. Purchased timed-matedanimals would generally be delivered on GD 1 or 2 withdosing then commencing on GD 6.

B. Note 13 of the ICH S5(R2) guidance states thatevaluation of between 16 to 20 litters per group forrodents tends to provide a degree of consistency betweenstudies, and above 20–24 litters per group the consistencyand precision are not greatly enhanced. The group sizesin Table 3 generally assure having these minimums forevaluation of the F1 generation during the growth andmaturation phase and for evaluation at the time of F1caesarean section, assuming appropriate dose selection.

C. Studies with 1 control (usually the vehicle used tosolubilise or suspend the test agent) and 3 drug-treatedgroups are most common. Note 7 of ICH S5(R2) states thatsome minimal toxicity is expected to be induced in thehigh-dose dams. The types of toxicity that may limit thehigh-dose level are stated in Note 7 and include effectssuch as increased or decreased body weight gain, targetorgan toxicity, exaggerated pharmacological response, ormarked pup mortality in a preliminary study. Dose levelsfor use on a PPN study should not induce maternaldeaths or body weight loss that extends for more than 2–3days. Note 7 also establishes 1 g/kg/day as the limit(i.e., highest) dose for developmental and reproductivetoxicity studies under most circumstances. If it is knownor anticipated that 1 g/kg/day will not produce minimaltoxicity in the pregnant female, then it may be possible toinclude only 1 additional lower dose level of the test agent(i.e., 3 total groups). If the toxicity profile is largelyunknown in a given species, then inclusion of 4 or moredrug-treated groups may be warranted.

Note 8 of ICH S5(R2) states that if a ‘‘no observedadverse effect level’’ for ‘‘reproductive aspects’’ is notidentified in a PPN study, then additional studies may beneeded. The designs of those additional studies will becompound specific and will not be discussed here.Ideally, either the low- or mid-dose level will demon-strate no adverse developmental toxicity. While ICHS5(R2) defines the various ‘‘adverse effects to beassessed’’ (e.g., pre- and postnatal death of offspring,altered growth and development), the guidance docu-ment did not attempt a definition for the adjective‘‘adverse.’’ Whether or not a developmental effect isadverse depends on a number of parameter-specificfactors, which can best be addressed by the scientistsconducting the study and/or those persons most knowl-edgeable about other aspects of the compound.

The spacing between dose levels is also an importantdecision. Note 8 states that dose-responses may be steep,and wide intervals between doses would be inadvisable.Narrow intervals may also pose problems when thevariability of a toxicological response overlaps betweenadjacent dose levels. A minimum multiple of 2-fold and amaximum of 4- to 5-fold between dose levels are usuallyadequate.

D. Note 2 of ICH S5(R2) defines GD 0 as the day whenevidence of mating is observed. Vendors that supplymated females must agree to this convention. Note 2 in

the guidance further states ‘‘Unless shown otherwise it isassumed that for rats implantation occurs on day 6–7 ofpregnancy.’’

E. All animals on study must be observed for mortalityand physical signs at least once daily. The best practice isto observe each F0 animal outside the cage during eachday of dosing, and then later a cage-side observation ismade. If significant physical signs are observed, thenmore frequent observations should be implemented todocument approximate onset and duration. Data fromprevious toxicokinetic assessments may be very helpfulin identifying when to perform the post-dose observa-tions (e.g., at Tmax). Moribund animals should beeuthanized as soon as possible. F1 offspring observedwith debilitating abnormalities should be euthanized assoon as possible, and a detailed post-mortem examina-tion is optional.

Observations during the lactation period shouldinclude non-maintenance of the nest and little to nomilk in the pups’ stomach visible through the skin. Theseobservations may aid in assessing if pup mortalities arematernally mediated due to poor maternal care ratherthan a direct effect upon the offspring.

F. ICH S5(R2) suggests that weighing the females twiceweekly is acceptable but more frequent recording of bodyweight provides for accurate adjustment of the dosevolume.

G. ICH S5(R2) indicates that food intake should berecorded at least once weekly. This may be accomplishedby measuring food consumed over 24-hour periods orover multiple days and then calculating grams consumedper day for each pregnant female. Obtaining foodconsumption data is fairly labor intensive and is onlysemi-quantitative due to wastage that is difficult, if notimpossible, to measure. It is not necessary to record foodintake during late lactation as the offspring also startconsuming the food.

H. At least 3 times per day should be scheduled forobservations of delivery. The onset and completion ofdelivery and any signs of difficulty in parturition shouldbe noted if possible. Based on unpublished data from ourlaboratories, approximately 80% of Sprague-Dawleyfemales will initiate parturition during the lights-onperiod (7 AM to 7 PM), and mean duration of parturitionis approximately 2 hours. For practical purposes, it isacceptable to assign a whole day value (e.g., 21.0 or 22.0)to females that have completed delivery at the firstobservation of the day. Females that complete deliveryduring the work-day are assigned a half-day value (e.g.,21.5 or 22.5).

I. A gross (macroscopic) examination of the adultthoracic and abdominal viscera is performed by trainedpersonnel. Organs with macroscopic findings andsamples from concurrent control females are preservedin an appropriate fixative for possible histologicalevaluation. Apparently non-pregnant uteri on presumedGD 24 may be briefly immersed in B10% ammoniumsulphide solution in order to visualize early implantationsites. In females that have littered, the number ofimplantation sites (as represented by metrial glands)should be recorded to enable a comparison with thenumber of pups born and allow an assessment of the inutero and neonatal loss (when females deliver at night,dead pups are often cannibalized by the dam before thefirst inspection of the litter).

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J. Selection of the F1 generation for maturation toadulthood should be made on the same basis as forstandardization of the litter in that it should be on a totalrandomization basis without body weight or other bias.

K. The study must assess the onset of puberty in bothsexes and Note 21 of ICH S5(R2) strongly suggestsmonitoring vaginal opening of females (Adams et al.,1985) and cleavage of the balanopreputial gland (i.e.,preputial separation) of males (Korenbrot et al., 1977) forthis assessment. Testis descent is not a recommendedmethod of assessment as this can be variable and verysubjective in comparison to balanopreputial separation.The latter is associated with increasing testosterone levelswhereas testis descent is not. As with other physicalmilestones, these parameters are affected by generalgrowth and therefore it is recommended that the bodyweight of the animal be recorded at the time ofattainment to differentiate between specific compoundeffects and those related to growth. Some laboratoriesexamine animals every other day for attainment of theselandmarks. There is one reference that states that theprocess of preputial separation can be accelerated byperforming the examination, so, at least, it is best tominimize the days of examination before separation isexpected (Clark, 1999).

L. Note 21 of ICH S5(R2) avoids recommending anyparticular tests for the assessment of functional effectsbut points out that in the past, tests have been directedalmost exclusively to behavior. Lastly, this note states‘‘Investigators are encouraged to find methods that willassess sensory functions, motor activity, learning andmemory.’’ While a good source of information on thevarious appropriate tests available is contained withinthe EPA Developmental Neurotoxicity Guideline (OPPTS870.6300) and the OECD Developmental NeurotoxicityGuideline (OECD 426), the investigator should beconfident of the validation of the test within thelaboratory and the tests should be capable of detectingboth increases and decreases in ability (Crofton et al.,2008; Tyl et al., 2008). An adequate historical controldatabase for each test is a requirement.

There is a tremendous diversity of ‘‘behavioral’’ testsemployed throughout laboratories in industry, govern-ment, and academia. Table 4 shows how a number oflaboratories, both from the authors and others, conductpostnatal behavioral tests. This list should providereaders with a starting point for what is consideredadequate and acceptable.

While traditionally a number of simple water mazes(‘‘T,’’ ‘‘E,’’ ‘‘M,’’ and ‘‘Y’’) have been used for learningand memory, these are now generally considered not

sophisticated or insufficiently complex for a comprehen-sive assessment. The tendency is now for more complexlearning and memory mazes to be requested such as theCincinnati, or Biel multiple ‘‘T’’ mazes or the Morriswater maze (Morris, 1984; Vorhees, 1987, 1990, 1997);however, these are logistically more complicated, parti-cularly when testing larger numbers of animals. Othersuitable behavioral tests are auditory startle-responseassessment and pre-pulse inhibition, investigation of(spontaneous) locomotor activity, or functional observa-tional battery (e.g., Gad, 1982; Moser et al., 1991)including determination of grip strength (Meyer et al.,1979).

M. ICH S5(R2) does not specify details of howreproductive performance should be assessed, but itwould seem appropriate to utilize the basic design of theFertility and Early Embryonic Development Study (seeLerman et al., pages 429–436, this issue). Males andfemales of the same dose group should be paired on a 1:1basis (avoiding the pairing of siblings) at approximatelyPND 70 and pairing should continue until evidence ofcopulation is observed whereupon the pair should beseparated as soon as possible. If, however, a female has notmated after 2 weeks or has exhibited 3 estrous periods, thepair should be separated. The option then exists to re-pairthe female with a male from the same group which haspreviously mated and to pair the male with an untreatedfemale. This would clarify if any effect on fertility or libidoexists and in which sex any effect is manifest.

N. Termination of mated F1 females is generallybetween GD 13–17. For each litter, the numbers ofcorpora lutea, resorptions, live and dead implantationsare recorded.

COMBINED STUDY DESIGNS

The ICH S5(R2) guidance allows for conducting thePPN study in combination with the fertility study and/orthe EFD study. The companion reports published in thisissue on fertility and developmental toxicity studydesigns should be consulted. Although the guidelinesallow for these combinations, there are few advantages ofcombining the PPN with either of the other studies.

1. Information from the fertility and developmentaltoxicity studies is required much earlier during thedrug development timetable to enable clinical trials tobe performed in women of childbearing potentialwhereas the PPN is not required until Phase III trialsare ongoing.

Table 4Examples of Postnatal Behavioral Tests Employed in the PPN Study

Sensory functions tests Motor activity tests Learning and memory tests

Auditory startle habituation/pre-pulseinhibition

Open fieldFigure ‘‘8’’ activity maze

Passive avoidance‘‘M’’ water maze

Automated open field ‘‘E’’ water mazeAccelerating rotarod Automated activity cage ‘‘Y’’ mazePreyer response Automated motor activity Biel water mazePupillary reflex Accelerating rotarod Cincinnati ‘‘T’’ maze

Other multiple ‘‘T’’ mazesMorris maze



2. Combining with the developmental toxicity studydoes not result in any savings in animals, in theamount of test substance, or in time. Double thenumber of females would be required at commence-ment and while there may be some economies of scale,these would be small in comparison. And, aspreviously discussed, dose selection may differ con-siderably between the EFD and PPN components

3. Combination with the fertility would reduce thenumber of animals required but would not reducethe test article requirement.

4. If an effect on the fertility phase was observed suchthat insufficient females became pregnant, then therewould be no point continuing with the PPN phase ofthe study.

5. This combined study would have a total duration ofapproximately 45 weeks, which would seriously delaythe reporting of the findings of the fertility assess-ment. The report by Wise et al. (pages 418–428, thisissue) discusses the pharmacokinetic considerationsfor such extended dosing periods.

For these reasons, PPN combination studies are lesscommon than other combination studies.

OPTIONAL PARAMETERS FOR PPN STUDIES

1. Maternal Toxicokinetics: Neither the ICH S5(R2) or ICHS3A (entitled ‘‘Note for Guidance on Toxicokinetics:The Assessment of Systemic Exposure in ToxicityStudies’’) guidance documents require assessment ofmaternal systemic exposure. Usually toxicokineticassessments will have been conducted as part of theEFD study so if the PPN dose levels are similar, thenthose data would cover the pregnant phase of the PPNstudy. Despite the changes in the maternal conditionrelating to the milk production and hormone changesduring the lactation period, it is uncommon to collectfurther samples during this period.

2. In compounds that may alter androgen status, post-natal measurement of anogenital distance can bemade and this is usually done on PND 0 or 1(McIntyre et al., 2001).

3. Adjustment of litter size (culling): Note 20 of ICHS5(R2) states that ‘‘The value of culling or not cullingfor detection of effects on reproduction is still underdiscussion. Whether or not culling is performed, itshould be explained by the investigator.’’ The prosand cons of culling were discussed in back-to-backpapers (Agnish and Keller, 1997; Palmer and Ulbrich,1997), yet the issue has not yet been resolved.Standardization or culling of the litter size by randomselection to yield, as nearly as possible, 8 pups(4 females, 4 males), is commonly performed althoughthe guidance suggests that this is optional. The usualargument in favor of standardization is that since pupweight is related to litter size, a more uniform pupweight at weaning should be obtained. If litters areculled, then this should be on a total randomizationbasis without body weight or other bias. If the optionto standardize the litters is taken, then this should bejustified and the process selected should be describedin detail. If standardization is not performed, then the

litter size should be included in any statisticalanalyses of the data as a covariant.

It is suggested in Note 19 of ICH S5(R2) that at the timeof weaning (i.e., PND 21), 1 animal per sex is selectedfrom each litter. While it is feasible to conduct allsubsequent evaluations using only these animals, theoption exists to randomly select more than 1 animal/sex/litter. Doing so allows the option of having naı̈veanimals for at least one behavioral test and they couldalso provide animals for a second mating trial forreproductive performance should issues be encounteredduring the first mating.

1. It is accepted that the best indicator of physicaldevelopment is body weight and Note 21 of ICHS5(R2) states that the acquisition of pre-weaningdevelopmental milestones is highly correlated withthe body weight and that this weight should berelated to post-coital time rather than postnatal time,especially where significant differences in gestationlength occur. This does not preclude the monitoring ofphysical milestones and reflex acquisition but due tothis close correlation many laboratories utilize bodyweight alone as the indicator of postnatal growth anddevelopment. Physical parameters suggested formonitoring include pinna unfolding, hair growth,incisor eruption, and eye opening (Adams et al.,1985). When reflex ontogeny is included, it is usual toinclude at least two measures of reflex ontogeny andthese may include surface righting, auditory startle,negative geotaxis, air righting, Preyer response, orpupillary reflex. The option also exists to monitormore complex forms of behavior and a good exampleof this is the ontogeny of swimming behavior, forwhich seven different stages can be clearly distin-guished and easily evaluated in rats and mice (Klausand Hacker, 1978).

2. While there is no requirement of individual identifica-tion of F1 offspring prior to weaning, it is feasible toidentify individual pups within each litter. This isperformed either at or shortly after birth or afterculling on PND 4 by foot or toe tattoo. The advantageof marking the animals on PND 0 or 1 is that it enablesindividual assignment of data collected before PND 4,e.g., surface righting, forelimb grip strength, pinnadetachment, and can be conducted without adverseeffects on pup development. This practice, whilebeing time-consuming, does allow one to track theprogress of the pup through all of the preweaning andpostweaning developmental milestones and reflexacquisition and allows comparison with the indivi-dual body weight during lactation.

3. Offspring Toxicokinetics: While the ICH S5(R2)guidance as well as the ICH S3A guidance do notrequire any neonatal exposure assessment, the latterguidance document states ‘‘Toxicokinetics may in-volve exposure assessment of dams, embryos, fetusesor newborn at specified days. Secretion in milk maybe assessed to define its role in the exposure ofnewborns. In some situations, additional studies maybe necessary or appropriate in order to study embryo/fetal transfer and secretion in milk.’’

The usefulness of these data is considerable whenunexpected findings are observed such as poor

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survival or decreased body weight gain in theoffspring. Rather than conducting an additional studyas suggested, it is possible to obtain blood samplesfrom the offspring culled at PND 4 to give anassessment of the transfer via the milk. The pups canbe removed from the litter at time intervals post-dosing to enable an approximation of the plasma drugprofile in the offspring. While these data are extre-mely variable, they do give a good indication of theexposure via the milk, which can be investigated in aseparate study if more information is required. Thereis little point collecting blood samples from the pupsat terminal necropsy on PND 21 as both milkproduction and suckling are much reduced fromPND 15/16 onwards.For the above assessment to be meaningful, bloodsamples are also required from the dam. Dependingon the complexity of the desired analyses, it may beappropriate to include satellite females with theirlitters specifically for this purpose. In addition tosampling maternal blood, it is not uncommon to alsosample the milk to provide the following ratios forcomparison: maternal plasma/milk, milk/ pup plas-ma, and maternal plasma/pup plasma. To enablesampling of the milk, females are pre-treated withoxytocin to promote excess milk production prior tosampling (Buschmann et al., 2001). It is sometimespreferable to sample the mother the day followingremoval of the pups. There are a number of excellentdiscussions of this general subject (Wier, 2006).

4. Although not required or suggested by ICHS5 (R2),the monitoring of estrous cycles in the F1 generationfemales prior to pairing may be considered if the testarticle is suspected of influencing the reproductivesystem or if triggered by precocious or delayed onsetof puberty. This would normally be conducted byassessment of vaginal cytology by means of vaginalsmears for a period of between 10 and 14 daysimmediately before pairing commenced. For moredetail on the collection and interpretation of thesmears, the companion paper (see Lerman et al.,pages 429–436, this issue) on the design of fertilitystudies should be consulted.

5. Should poor fertility be observed, which by re-matingis considered a male-mediated effect, then histo-pathology and/or sperm evaluations may be per-formed at termination of the F1 males. Methods havebeen described by Seed et al. (1996).

6. While ICH S5(R2) guidance does not specify or detailhow the fertility of the F1 generation should beassessed, it is generally understood that an assessmentbased on the guidance for the fertility study shouldsuffice. In some cases, however, it may be moreappropriate to allow the F1 generation to litter andassess the pregnancy and lactation performance of theF1 together with the survival growth and develop-ment of the F2 generation up to LD 7. This may beparticularly appropriate where significant problemshave been observed in the pregnancy performance ofthe F0 generation or the sexual development andfertility of the F1 generation females.

7. When unanticipated effects are encountered in a PPNstudy, such as pup deaths or marked decreased pupbody weight gain in the high-dose group, a fostering/cross-fostering study may be necessary to clarify if the

effects are due to exposure during gestation and/orlactation (i.e., for better assessment of human rele-vance). In this study design, there are 20–22 matedfemales per group, but only 2 groups (vehicle controland high-dose). Dams are treated for the sameduration as in the PPN study. On the day of birth,all pups are transferred to different mothers in thefollowing combinations: control pups to both controland high-dose dams, and high-dose pups to bothcontrol and high-dose dams. The combination ofcontrol pups cross-fostered to the high-dose damsexamines the effects of lactation-only exposure, whilethe combination of high-dose pups to control damsexamines the effects of gestation-only exposure.

PRESENTATION OF DATA FROM PPNSTUDIES

There are no specific requirements detailed in the ICHS5(R2) guidance document regarding the presentation ofdata; however, section 6 calls for ‘‘tabulation of indivi-dual values in a clear concise manner to account forevery animal that was entered into the study.’’ It shouldbe possible to follow individual animals initially from thematernal data and through birth, lactation, and subse-quent maturity such that a detailed history of eachindividual can be constructed and their contribution tothe study assessed. Logically, therefore, all the tables ofindividual findings should identify the F0 female/litteror the selected F1 males and females.

Graphical presentation of continuous data (i.e., valuesfor data collected on multiple days or within a specifictime frame such as mean maternal body weights, F1generation body weights, activity scores across time, orstartle response across multiple trials) is often moreclearly visualized in a line graph. Summary tables of allmajor categories of data are essential but the individualdata that contribute to these means must be in a form thatmakes the review of the data easy. It is now common thatthese data are captured by online systems designed toenable rapid and accurate data capture. Unfortunately,the formatting of the reporting tables and appendicesoften do not receive the same attention and data appearto be presented in a particular way that suits thecomputer system rather than focusing on the ease ofthe subsequent use of the data presentations. Meanabsolute body weights, body weight gains, and meanfood consumptions are generally included. Summaryand individual tables of physical observations may alsobe included. Summary tables for data collected atlittering and during lactation would include thoseparameters shown in Table 5. We consider it mandatorythat when collected, preweaning developmental mile-stones and reflex acquisition data be calculated with thelitter, and not the individual pup, as the unit of measure.

In order to detect treatment-related effects, all para-meters should be compared initially against the con-current control group. Further comparisons againstrecent historical control values (i.e., approximately 3–5years) can be useful in terms of single parameters, whereno clear trend in other similar parameters is observed,and knowledge of trends and variations within thespecies and strain of animals in the testing laboratory.A table detailing the range of values from historical



control values for all offspring development parameterscan be helpful.

A description or discussion of appropriate statisticalanalyses of data regenerated from pre- and postnataldevelopmental toxicity studies is beyond the scope ofthis report. It should be stated that statistical analyses arenot required by the ICH guidelines.

THE FUTURE OF PRE- AND POSTNATALDEVELOPMENTAL TOXICITY STUDIES

The design and usefulness of PPN studies is notenvisioned to change markedly in the foreseeable future.The basic PPN study has been and will continue to be thebasis for evaluating adverse effects on the pregnant/lactating female and on development of the conceptusand the offspring following exposure during pregnancyand during the neonatal period via the milk.

The importance of the PPN study in the currentclimate of pediatric drug development may diminishparticularly in terms of postnatal development. With

regulatory authorities now generally requesting theconduct of juvenile studies with dosing commencingduring the early neonatal period, the role of the PPNstudy in the assessment of exposure via the milk duringthis period may be superseded. However, the PPN studyremains the stalwart of assessing postnatal effectsfollowing in utero and lactational exposure.

In contrast, the role of the drfPPN study may assumegreater importance as it presents an opportunity toinvestigate the consequence of exposure during the earlyneonatal period as well as the garnering of data for thedefinitive PPN study. This particularly applies when theprovision for direct dosing of the offspring is included(De Schaepdrijver and Bailey, 2005) and this modifiedstudy design is currently being used in a few labora-tories. The approach involves modifying the existing pre-and postnatal developmental toxicity range–findingstudy and including a simple ‘‘add-on’’ to the end ofthe study. This combined range-finding study not onlyprovides justification for the selection of dose levels forthe definitive PPN study but also generates early

Table 5Routine Tabulation of Pre- and Postnatal Developmental Toxicity Data Collecting During Littering and Lactation

F0 parameter Details

Numbers of females in each group Mated females assigned (may exclude TK females)Unscheduled deaths (i.e., found dead or euthanized early)Non-pregnant femalesPregnant females successfully litteringFemales with no live young

Parameters from pregnant femalesGestation length In half days, see Note HImplantations (metrial glands) Mean (7SD)Gestation index Percentage live born litters

F0, F1litter observations and pre-weaning assessmentsPost-implantation survival indexa Mean percent (7SD)Live pups delivered Mean percent (7SD)Live pups per litter on PND 0, 3/4 (pre- and postcull), 7, 14, 21 Mean (7SD)Live birth index Mean percent (7SD)Dead pups PND X to Yb Total and mean percent (7SD)Milestone acquisition Mean day (7SD), optionalReflex ontogeny Mean day (7SD), optionalF1 postweaning parametersFunctional, behavioral, and sensory assessments Mean (7SD) of appropriate values for each testSexual development Mean (7SD) day of occurrenceCopulation (mating) indexc PercentagePrecoital interval (number of days needed for mating) Mean (7SD)Pregnancy (fecundity) indexd PercentageFertility indexe PercentageF1 generation pregnancy dataCorpora lutea Mean (7SD) of number per pregnant femaleImplantations Mean (7SD) of number per pregnant female% Pre-implantation lossf Mean percent (7SD) of all pregnant females% Early resorptionsg Mean percent (7SD) of implantations per litter%Late resorptions Mean percent (7SD) of implantations per litter% Postimplantation lossh Mean percent (7SD) of all pregnant femalesLive embryos Mean (7SD) of number per pregnant female

a(live pups delivered/the higher of [metrial glands or total pups delivered])� 100.bFor example, PND 1–3, 4–7, 8–14, 15–21, and 4–21.c[number mated/total number cohabited]� 100.d[number pregnant/total number mated]� 100.e[number pregnant/total cohabited]� 100.f[(no. corpora lutea � no. implantations)/no. corpora lutea]� 100.gImplant with no visible embryo morphology.h[(no. implantations � no. live embryos)/no. implantations]� 100.

444 BAILEY ET AL.


scientific data on exposure and potential toxicity injuvenile animals. In summary, the study is composed oftwo control groups and a number of treatment groups,and during the lactation study phase dosing ceases forthe maternal females at LD 6. After completion ofmaternal treatment, pups are selected for a subsequentjuvenile study phase. After an appropriate wash-outperiod, groups of pups, including those from one of thetwo control groups (providing initially naive animals),are dosed directly with the test material until a suitablepoint post-weaning. Toxicokinetic data can be collectedfrom unselected pups (exposure via the maternal milk)and from directly dosed animals for single dose and‘‘steady state’’ assessments. This study design candifferentiate between maternally mediated dosing effectsand those related to direct pup dosing, and allows anearly assessment of potential safety concerns. In addition,this combination approach represents a decrease in drugdevelopment time and a clear reduction in animal usageand cost.

ACKNOWLEDGMENTS

The authors appreciate the review and comments fromthe other contributors to this series of papers. Theopinions and processes described herein are those ofthe authors and do not necessarily represent an officialposition of their place of employment.

REFERENCES

Agnish ND, Keller KA. 1997. The rationale for culling of rodent litters.Fundam Appl Toxicol 38:2–6.

Adams J, Buelke-Sam J, Kimmel CA, Nelson CJ, Reiter LW, Sobotka TJ,Tilson HA, Nelson BK. 1985. Collaborative behavioral teratologystudy: protocol design and testing procedure. Neurobehav ToxicolTeratol 7:579–586.

Barrow PC. 2009. Reproductive toxicity testing for pharmaceuticals underICH. Reprod Toxicol 28:172–179.

Buschmann J, Bartsch W, Dasenbrock C, Fuhst R, Pohlmann G, Preiss A,Berger Preiss E. 2001. Cross-fostering inhalation toxicity study withhcfc-123 in lactating Sprague Dawley rats. Inhal Toxicol 13:671–687.

Christian MS, Hoberman AM. 1997. Perspectives on the U.S., EEC, andJapanese developmental toxicity guidelines. In: Hood RD, editor.Handbook of developmental toxicology. Boca Raton, FL: CRC Press,p 551–595.

Christian MS, Hoberman AM, Lewis EM. 2006. Perspectives on thedevelopmental and reproductive toxicity guidelines. In: Hood RD,editor. Developmental and reproductive toxicology, a practicalapproach. Boca Raton, FL: CRC Press, p 733–798.

Clark RL. 1999. Endpoints of reproductive system development. In:Daston G, Kimmel C, editors. An evaluation and interpretation ofreproductive endpoints for human health risk assessment.Washington, DC: International Life Sciences Institute Press. p 10–21.

Crofton KM, Foss JA, Hass U, Jensen KF, Levin ED, Parker SP.2008. Undertaking positive control studies as part of develop-mental neurotoxicity testing: a report from the ILSI ResearchFoundation/Risk Science Institute expert working group on neuro-developmental endpoints. Neurotoxicol Teratol 30:266–287.

De Schaepdrijver L, Bailey GP. 2005. An elegant study design to generatejuvenile animal data early in drug development: current experiences.Reprod Tox 20:481.

Gad SC. 1982. A neuromuscular screen for use in industrial toxicology.J Toxicol Environ Health 9:691–704.

Klaus S, Hacker E. 1978. The development of swimming behavior in mice asa behavior test in toxicology. Z Versuchtriek 20:81–87.

Korenbrot CC, Huhtaniemi IT, Weiner RW. 1977. Preputial separation asan external sign of pubertal development in the male rat. Biol Reprod17:298–303.

Martin PL, Breslin W, Rocca M, Wright D, Cavagnaro J. 2009.Considerations in assessing the developmental and reproductivetoxicity potential of biopharmaceuticals. Birth Defects Res (Part B)86:176–203.

McIntyre BS, Barlow NJ, Foster PMD. 2001. Androgen-mediateddevelopment in male rat offspring exposed to flutamide in utero:permanence and correlation of early postnatal changes in anogenitaldistance and nipple retention with malformations in androgen-dependent tissues. Toxicol Sci 62:236–249.

Meyer OA, Tilson HA, Byrd WC, Riley MT. 1979. A method for theroutine assessment of fore- and hindlimb grip strength of rats andmice. Neurobehav Toxicol 1:233–236.

Morris R. 1984. Developments of a water-maze procedure for studyingspatial learning in the rat. Neurol Sci Methods 11:47–60.

Moser VC, McDaniel KM, Philips PM. 1991. Rat strain and stockcomparisons using a functional observational battery: baselinevalues and effects of amitraz. Toxicol Appl Pharmacol 108:267–283.

Palmer AK, Ulbrich B. 1997. The cult of culling. Fundam Appl Toxicol38:7–22.

Seed J, Chapin RE, Clegg ED, Dostal LA, Foote RH, Hurtt ME, Klinefelter GR,Makris SL, Perreault SD, Schrader S, Seyler D, Sprando R, Treinen KA,Veeramachaneni DNR, Wise LD. 1996. Methods for assessing spermmotility, morphology, and counts in the rat, rabbit and dog: a consensusreport. Reprod Toxicol 10:237–244.

Tyl RW, Crofton K, Moretto A, Moser V, Sheets JP, Sobotka TJ. 2008.Identification and interpretation of developmental neurotoxicity: areport from the ILSI Research Foundation/Risk Science Instituteexpert working group on neurodevelopmental endpoints. Neuro-toxicol Teratol 30:349–381.

Vorhees CV. 1987. Maze learning in rats: A comparison of performance intwo water mazes in progeny pre-natally exposed to different dosesof phenytoin. Neurotoxicol Teratol 9:235–241.

Vorhees CV. 1990. Validity of the Biel water maze for detectingdevelopmentally induced learning impairments. NeurotoxicolTeratol 12:567.

Vorhees CV. 1997. Methods for detecting long-term CNS dysfunction afterprenatal exposure to neurotoxins. Drug Chem Toxicol 20:387–399.

Wier P. 2006. Use of toxicokinetics in developmental and reproductivetoxicology. In: Hood RD, editor. Handbook of developmentaltoxicology. New York: CRL Press, Inc. p 571–597.



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Pre- and postnatal developmental toxicity study design for pharmaceuticals