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# REPRODUCTIV E TOXICIT Y TESTIN G b y CONTINUOU S BREEDIN G 

# f- >

TES T PROTOCO L I N SWIS S (CD-1 ) MIC E

Nationa l Institut e of Environmenta l Healt h Science s

Nationa l Toxicolog yProgra m Developmenta l an d Reproductive Toxicolog yGrou pP. 3



### Jerrol dJ . Heindel 1 

### , J . C . Lamb , IV 1 

### * , Rober t E . Chapin 1 

### , Dushyan t K . Gulati 2 

### , Esthe r Hope 2 

### , Juli e George 3 

### , C . W . Jameson 1 

### , Jane t Teague 4 

### , andB.A . Schwetz 1

1 

### Nationa l Institut e o f Environmenta l Healt hScience s

Nationa l Toxicolog y

Program P.O . Bo x1223 3 Researc hTriangl e Park , N C 2770 9

2 

### Environmenta l Healt hResearc handTesting , inc .

251 4 Regenc y

Roa d Lexington , K Y4050 3

3 

### Researc hTriangl e Institut e P.O . Bo x1219 4 Researc hTriangl e Park , N C 2770 9

4 

### Analytica l Sciences , Inc . 10 0 Capitol a Drive , Suit e 1 Durham , N C 2771 3

4 

### Presen t Address : Jellinek , Schwartz , Connoll y & Freshman , Inc . 135 0 Ne w Yor k Avenue , N.W. , Suit e 40 0 Washington , D C 2000 5 **Th e procedure s describe dher e ar e curren t a s o f Januar y 1989 . Correspondenc e to : Dr . Jerrol dJ . Heinde l

Developmenta l andReproductiv e Toxicolog y Grou p NIEHS/NT P P.O . Bo x1223 3 Researc hTriangl e Park , N C 2770 9TABL E O F CONTENT S P. 4

Topi c Genera l Stud

y Desig n Method s - Genera l Chemica l Characterizatio nan dHandlin g Chemical/Vehicl e Formulation s Animal s Statistica l Method s - Specifi c Tas k 1 - Dos e Rang e Findin g Stud y Tas k 2 - Continuou s Breedin g Tas k 3 - Determinatio no f Affecte dSe x Tas k 3 - Necrops y Sper m Motilit y

Determinatio n Sper m Coun t Determinatio n Sper m Morpholog y

Evaluatio n Vagina l Cytolog y

Evaluatio n Tas k 4 - Offsprin g Assessment Result s - Summar y

Dat a fro

m RAC B Conclusio n Flo w Diagram

o f RAC B Tim e Lin e o f RAC B Mileston e Char t fo r RAC B Reference s Pa^ e 3 5 6 8 1 1 1 3 1 7 1 7 1 9 2 3 2 5 2 6 2 7 2 8 2 8 3 2 3 5 4 1 Figur e 1 Figur e 2 4 2 4 4P. 5

INTRODUCTION

"Reproductiv e Assessmen t b y Continuou s Breedin g (RACB) " i s a ne v standardize d protoco l designe d for routin e reproductiv e toxicit y testing . Th e origina l desig nva s develope d b y th e Nationa l Toxicology Program , Researc h Triangl e Par k (RTP) , NC . The actua l testing an d standardizatio nver e contracte d to tv

o laboratories : Environmenta l Healt hResearc h& Testin g (EHRT) , Lexington , KY , an d Researc hTriangl e Institut e (RTI) , Researc h Triangl e Park , NC . Th e method s fo r statistica l analysi s o f th e result s ver e provide d initiall y b y Progra

m Resources , Inc. , RTP , NC , an d presentl y

b y Analytica l Sciences , Inc. , RTP , NC , unde r th e supervisio no f the Nationa l Toxicolog y

Program . Unti l recently , RAC B va s generall y referre d to a s "Fertilit y Assessmen t b y Continuou s Breedin g (FACB). " Th e titl e va s revise d sinc e th e protoco l provide s informatio no nmor e thanfertility . RAC B share s th e same fundamenta l endpoint s a s reproductiv e toxicit y protocol s recommende d b y th e U.S . Environmenta l Protectio nAgenc y (EPA ) an

d th e Food an

d Dru g Administratio n (FDA ) bu t i s designe d to improve th e reliabilit y

an d sensitivit y

o f reproductiv e testing . Lam b(1985 ) provide d a compariso no f th e RAC B tes t syste

m vs . FD

A segment s I , II , an d II I fo r drugs , FD A multigeneratio nstudies , an d EP A tvo-generatio nstudies . Man y RAC B endpoint s (fertility , pup s pe r litter , pu p veight , sex , an

d survival ) ar e th e sam e a s i nconventiona l EP A an d FD A protocols . I naddition , th e RAC B protoco l test s fertilit y an d reproductiv e outcome unde r constan t challenge . Th e firs t generatio nanimal s (F Q ) ar e house d a s breedin g pair s continuousl y for 1 4 veeks . Thi s approac hno t onl y increase s th

e numbe r o f litter s fro m eac hbreedin g pair , but als o add s th eP. 6

dimensio no f tim

e (progressio no f a nadvers e affect ) t o th e evaluatio no f the reproductiv e performance . Thus , a nearl y effec t ca nb e distinguishe dfro m a later , o r progressive , effect . Standar dmultigeneratio nstudie s fai l t o provid e suc hinformatio nunles s mor e thanon e matin g pe r generatio ni s performed . Furthermore , the statistica l pove r o f the RAC B tes t i s considerabl y highe r tha nthe conventiona l studies . Th e standar dmultigeneratio nstudie s requir e abou t 2 0 successfu l mating s pe r treatmen t grou pand a t mos t tv o litter s pe r matin g pair . Th e RAC B protoco l require s 2 0 male s and2 0 female s pe r treatmen t grou p vit h4 0 contro l breedin g pair s but , becaus e o f the continuou s cohabitatio nfo r 1 4 veeks , a s man y a s 10 0 litter s pe r treatmen t grou pca nb e evaluated . Th e correspondin g numbe r o f litter s fo r th e contro l grou pi s 17 5 t o 200 . Th e purpos e o f thi s articl e i s t o describ e practica l detail s fo r RAC B and thereby : 1 ) serv e a s a referenc e sourc e fo r reproductiv e toxicologist s and regulator y

agencie s intereste di nconductin g o r recommendin g RAC B studies , 2 ) provid e a ninsigh t into , an dknovledg e about , the complexitie s o f the RAC B tes t andassis t i nplannin g an dconductin g thes e studie s successfully . Althoug hRAC B ca nbe , i nfac t ha s been , adapte dfo r us e i nrats , the majorit y

o f th e studie s hav e bee ndon e i nmice . Thi s pape r describe s the standar dmethodolog y fo r conductin g the RAC B stud y i nmice .

GENERA L STUDYDESIGN

Th e RAC B protoco l i s divide dint o fou r task s (Figure s 1 an d2) . Tas k 1 i s a 14-da y

dos e settin g stud

y vhic huse s fiv e dose s anda contro l ( n=P. 7

8/sex/group) . Endpoint s fo r Tas k 1 ar e clinica l signs , bod y veight , andfoo d
o r vate r consumption . Tas k 2 i s the continuou s breedin g phase , consistin g o f
a contro l grou p(4 0 breedin g pairs ) an d3 dos e group s (2 0 pairs/group) . Tas k 2 dos e level s ar e se t s o tha t the highes t dos e vil l b e expecte dt o depres s veigh t gai nb

y approximatel y

10 % andvil l permi t >90 % survival . Th e middl e dos e i s selecte d to produc e littl e o r n o systemi c toxicity , vhil e the lo v dos e i s designe dt o b e a no-effec t level . Afte r 7 day s o f prematin g exposur e to th e tes t agent , th e animal s ar e house da s breedin g pair s fo r 9 8 days . Endpoint s fo r Tas k 2 are : clinica l signs , parenta l bod y veight , fertilit y (numbe r producin g a litter/numbe r o f breedin g pairs) , deliver y interval , litter s pe r pair , liv e pup s pe r litter , proportio no f pup s bor nalive , se xo f liv e pups , the pupbod y veight s vithi n1 8 h r o f birth , and foodandvate r consumption . At the en d o f the 9 8 days , the pair s ar e separate dandhouse d
on e animal/cage . An y litter s bor nafte r th e cohabitatio nphas e ar e reare db y th e da m unti l weanin g (2 1 day s o f age) . Randoml y selecte dF 1 pup s ar e the n expose dt o the tes t agen t a t the same concentratio na s thei r parent s receive d durin g Tas k 2 . Thes e ? l animal s ar e use dfo r assessmen t o f second generatio n fertilit y

(Tas k 4 , belov) . Vhe na positiv e effec t o nfertilit y i s detecte ddurin g Tas k 2 , a 1-vee k
crossove r matin g tria l (Tas k 3 ) i s performe do nth e parenta l animal s t o determin e vhic hse xo r sexe s ma y hav e bee naffecte db y th e chemica l treatment . Tas k 3 i s performe dafte r the las t Tas k 2 litte r i s veaned . Th e crossove r matin g tria l consist s o f 3 group s o f 2 0 pair s each , contro l male s xcontro l females , contro l male s xhigh-dos e females , andcontro l female s xhigh-dos e males . Pair s ar e mate d fo r seve nday s o r unti l a copulator y plu g i s detected , vhicheve r i s first . Animal s ar e no t expose dt o th e tes t chemica l durin g Tas k 3 cohabitation . Dosin g i s resume da t th e appropriat e concentration sP. 8

afte r seve nday s an d continue d unti l necropsy . Endpoint s fo r Tas k 3 ar e th e
sam e a s Task 2 vit hth e additio no f matin g index(numbe r o f female s plu g positive/numbe r pair s cohabited) . A t th e en d o f Tas k 3 , F o male s an d female s

ar e necropsied ; th e endpoint s evaluate d ar e selecte d orga nveight , bod y veight , epididyma l sper m motility , morphology , an d concentration , an d estrou s cyclicit y

a s monitore d b

y vagina l lavag e fo r th e precedin g 1 2 day s (som e studie s hav e use d 7 days) . Selecte d organ s ar e examine d microscopicall y afte r fixatio ni n10 % neutra l buffere d formali no r Bouin s fixativ e an d embeddin g i n paraffi no r plastic ; section s ar e staine d vit hhematoxyli nan d counterstaine d vit heithe r eosi no r PA S accordin g to standar d procedures . Tas k 4 , offsprin g assessment , i s conducte d vhethe r o r no t Tas k 2 shov s reproductiv e toxicity . I nthi s phase , th e las t litte r fro

m Task 2 i s reared , veaned , an

d kep t t o sexua l maturit y (7 4 + 1 0 days ) vhil e house d b y se x2 o r 3
pe r cage , consumin g th e sam e concentratio no f tes t chemica l a s thei r parents . A t thi s time , male s an d female s fro

m differen t litter s bu t vithi nth e same treatmen t group , ar e cohabite d fo r seve nday s unti l a copulator y plu g i s found , an

d the nhouse d singl y unti l delivery . The reproductiv e an d fertilit y endpoint s fo r thi s matin g tria l ar e the sam e a s thos e i nTask 3 . A t th e en d

o f Tas k 4 , the F xmic e ar e necropsied ; th e endpoint s ar e th e same a s fo r th

e necrops y

o f the F o mic e describe d above .

METHOD S - GENERAL

Al l Reproductiv e Assessmen t b y Continuou s Breedin g (RACB ) studie s ar e conducte d i naccordanc e vit hGoo d Laborator y Practic e (GLP ) Regulation s establishe d b

y th e FD A fo r Nonclinica l Laborator y Studie s (Federa l Register ,P. 9

Friday , Decembe r 22 , 1978 , Par t II , p p59986-60025 ) an

dvit hth e requirement s
an drecommendation s containe di nthe mos t recen t issu e o f the "Guid e fo r the Car e an dUs e o f Laborator y Animals " b

y Publi c Healt hService , Nationa l Institute s o f Health . CHEMICA L CHARACTERIZATIO N AN D SAFET YAN D HANDLINGPROCEDURE S Th e study

materia l obtaine dfo r th e RAC B studie s ar e representativ e o f th e materia l t o vhic hthe populatio na t ris k ar e exposed . Mos t chemical s obtaine dfo r study

i nthe NT P ar e unformulated , technical-grad e products . Formulate dproduct s ar e avoide dvher e possibl e to minimiz e the numbe r o f component s i nthe stud y materia l vhil e stil l providin g a nevaluatio no f the chemica l (includin g impurities ) t o vhic hpeopl e ar e mos t likel y t o b e exposed . If , hovever , th e chemica l i s bein g teste dprimaril y becaus e o f a ninteres t i n it s molecula r structur e o r becaus e i t i s a nimpurit y i na commercia l product , a pur e for

m o f the chemica l i s obtaine dbecaus e i t i s mor e appropriat e fo r study . Onc e the study

materia l i s procured , a numbe r o f chemica l analyse s and method s developmen t ar e performed . Thes e task s includ e bot hbul k chemica l and dos e formulatio nanalysis . Th e bul k chemica l i s analyze dt o full y characteriz e the study materia l prio r t o us e i nthe RAC B program . Thes e analyse s utiliz e a vid e variet y o f analytica l chemica l technique s includin g chromatography , spectroscopy , elemental analysi s an dphysica l propert y determinations . Thes e analyse s ar e performe d to confirm the identit y

andto determin e th e purit y o f the stud

y material . Impuritie s presen t i nth e study materie l a t level s o f IX o r greate r ar e routinel y identifie dandquantitate d t o assis t i nthe interpretatio no f the RAC B study . A cop y o f the chemica l characterizatio nrepor t i s include di nthe technica l protoco l a s vei l a s the fina l study

report . Procedure s fo r the handlin g o f chemical s ar e establishe dP.1 0

t o ensur e th e safet y o f personne l andthe maintenanc e o f th e purit y and stabilit y

o f the tes t compound . Informatio ncontaine di nth e handlin g protoco l specifie s safet y precaution s t o be observed , includin g protectiv e equipmen t t o be utilize db y personnel . Hazardou s characteristic s o f the chemical , suc ha s flammability , explosiv e properties , an dtoxicit y

ar e listed . Material s o r substance s tha t ar e incompatibl e vit hthe chemica l i nquestio n
ar e liste ds o tha t container s o r equipmen t containin g thes e material s ca nb e avoided . Procedure s fo r th e decontaminatio no f spill s andfo r the disposa l o f vast e chemica l andcontaminate dmaterial s ar e als o recommended , i nadditio nt o th e firs t ai d procedures . Method s fo r mixin g andanalyzin g dos e formulation s ar e develope dprio r to the RAC B studie s t o assur e adequat e dosin g o f the stud y animals . Protocol s
ar e develope dfo r mixin g th e study materia l a t the prope r dos e level s vit hth e appropriat e vehicl e (feed , vater , cor noil) . Homogeneit y o f feedmixture s an d othe r chemica l dos e formulation s tha t ar e no t solution s i s demonstrate dbefor e th e anima l study

begins . Analysi s method s fo r dos e formulation s usuall y involve s isolatio no f th e stud y chemica l b

y extractio nfollowe db y chromatographi c o r spectroscopi c analysi s o f the extract . Stabilit y

o f bot hth e bul k chemica l anddos e formulation s i s als o determine dprio r t o the RAC B studies . Thermall y accelerate d stabilit y studie s
o f th e bul k chemica l ar e performe dt o estimat e the long-term stabilit y

and t o ai di nsettin g storag e condition s fo r th e chemical . Th e bul k chemica l i s the n routinel y

monitore dfo r purit y durin g the study . Th e stabilit y o f dos e formulation s fo r u p t o thre e veek s unde r optimu m storag e condition s an dals o unde r simulate ddosin g condition s i s determined . Th e dat a obtaine d fro m thes e stabilit y

studie s ar e use d t o estimat e th e shel f lif e o f th e dos e formulation s
andth e condition s unde r vhic hi t shoul db e store dt o minimiz e decomposition . Th e dos e formulation s ar e routinel y analyze ddurin g the RAC B studie s to insur e prope r dose s ar e administered .P.1 1

CHEMICAL/VEHICL E FORMULATION Chemical/vehicl e formulation s ar e al l prepare d unde r GL P guidelines . Gavag e Formulatio nan d Administratio n- Th e stud y chemica l i s mixe d i nthe appropriat e vehicl e vit hconcentratio ndetermine d b y the followin g formula :
dos e leve l (mg/kg ) concentratio n(mg/ml ) = dos e volume (10. 0 ml/kg )
Thus , concentratio nvil l var y vit hdose . Eac hconcentratio ni s formulate d independentl y

o na veight/volum e basi s i na quantit y sufficien t t o b e use d
acros s a 14-da y

perio d o f administratio ni f tes t chemical/vehicl e stabilit y permits . Aliquot s o f eac hformulatio no f th e stud y chemica l an

d vehicl e ar e analyze d prio r t o onse t o f exposur e fo r confirmatio no f dos e levels . Distille d vate r i s use d fo r aqueou s formulations . Th e cor noi l use d a s the gavag e vehicle , i s purchase d fres hfro m a loca l groce r ( 1 lot/study) . Eac h lo t o f cor noi l i s monitore d fo r peroxid e conten t ever y mont hvhil e i nuse . I f the peroxid e leve l exceed s 3 meq/k g a fres hlo t o f cor noi l i s obtained . A naliquo t o f eac hformulatio no f tes t chemica l i nvehicl e an d vehicl e ar e archive d a t -20°C . Dosage s ar e adjuste d o na veekl y basis . Animal s ar e veighe d a t the beginnin g o f the vee k an d the ne v

dosag e i s calculate d i nrelatio nt o the bod y veight . Thi s dosag e i s use d unti l the anima l i s veighe d agai ni nseve nday s
o r unti l deliver y

o f a litter . A

dosag e volume o f 1 0 ml/K g bod y veigh t (i.e . IX bod y veight ) i s used . Gavag e i s accomplishe d usin g a lc c Glasspa k syring e fitte d vit ha n18/2 0 guage , 1 1/ 2 inc hcurve d needl e fo r adul t mic e o r a 20/2 2 guage , 1 inc hstraigh t needl e fo r the immatur e mic e i nTas k 4 .P.1 2

Fee dFormulatio nan dDosag e Administratio n- Th e study chemica l i s accuratel y veighe dan

dadde dto a smal l aliquo t o f groun dfee di na beake r andmixe dvit h
a spatul a to mak e a premix . Th e premi xi s the nadde dto a preveighe dgroun d fee dsampl e an

dblende di na Patterson-Kelle y liqui dsolid s tvi nshel l blende r (eigh t quar t model) . Th e blende r i s runfo r fiv e minutes , vit hthe intensifie r ba r on , andfo r 1 0 minute s vithou t the intensifie r (time s ma y be varie dt o obtai nhomogeniety) . Sample s fro m thre e differen t place s i nthe blende r ar e analyze dfro m the firs t mi xo f a ne v study materia l t o assur e homogeneit y

o f mixing . A

differen t concentratio no f tes t chemica l i s require d fo r eac hdos e level . Eac hconcentratio ni s formulate dindependentl y i na quantit y

sufficien t fo r a 14-da y treatmen t perio di f stabilit y o f tes t chemica l i nfee dpermits . Eac hformulate dfee dsampl e i s store dunde r refrigeratio nprio r t o use . Fo r eac hformulation , the quantit y prepare d i s base do nth e assumptio ntha t eac hmous e vil l consume a t mos t 1 0 g pe r day . A n aliquo t o f eac hformulatio no f tes t chemica l i nfee dandthe vehicl e vil l b e kep t fo r analysis . Standar dclea r glass , ambe r glas s o r polycarbonat e feedin g jars , dependin g o ntes t chemica l stability , holdin g approximatel y 3. 5 o z ar e used . A ja r i s fille dvit hthe appropriat e povdere do r granula r die t andth e veigh t recorded . Th e fille dja r i s colo r code daccordin g t o dos e group , andplace d vithi nth e appropriat e cage . Afte r seve ndays , the ja r i s remove dfro m the cag e an

dreveighed . Thi s valu e i s subtracte dfro m the origina l ful l veigh t an dthe differenc e i s the foodconsume ddurin g tha t tim e period . I f additiona l foodi s require dprio r t o the seven-da y

veighing , a veighe dquantity o f fee dvil l b e adde dt o the fee dja r and th e amoun t adde dvil l b e note do nth e appropriat e dat a sheet .P.1 3



### 1 0

Determinatio n o f averag e foo d intak e pe r anima l require s divisio n o f tota l foo d consume d i n cag e b ynumbe r o f animal s i n th e cage . Th e tota l foo d intak e pe r anima l divide d b yth e numbe r o f day s vil l estimat e foo d intak e i n gram s pe r anima l pe r day
. Drinkin g Wate r Formulatio n and Administratio n - Th e stud ychemica l i s veighe d an d mixe d vit h deionized/filtere d vate r o n a veigh t t o volum e basis
. A n aliquo t o f eac h formulatio n o f contro l vate r o r stud ychemica l i n vate r i s kep t fo r analysis
. Eac h stoc k solutio n i s hel d i n a polyethylen e o r glas s carbo yan d store d a t 5°C . Fo r eac h formulatio n th e quantit yprepare d vil l b e base d o n th e assumptio n tha t eac h mous e vil l consum e a t mos t 1 5 m l pe r day
. Standar d 50 0 m l clear , glass , vate r bottle s ar e fille d vit h approximatel y 45 0 m l o f contro l vate r o r filtered/deionize d vate r containin g on e o f th e dose s o f th e tes t chemica l an d stoppered
. Th e typ e o f stoppe r an d sippe r tub e
ma yvar yvit h th e toxicit yan d compatabilit yo f th e tes t chemical
. Th e bottl e i s veighe d t o th e neares t 0. 1 g an d place d int o th e cag e top . Afte r a perio d

o f seve n days , th e bottl e i s remove d an d reveighe d vit h th e stoppe r i n place
. Th e amoun t consume d i n gram s pe r animal s i s calculate d subtractin g th e secon d veigh t fro mth e firs t an d dividin g th e numbe r o f animal s i n th e cage . Thi s valu e ca n the n b e divide d b yth e numbe r o f day s th e bottl e va s i n plac e t o estimat e th

e vate r consumptio ni n gram s pe r anima l pe r day
. I t i s assume d tha t 1. 0 g o f dose d vate r i s equivalen t t o a 1. 0 m l volume
. Aclea n vate r bottl e vil l the n b e fille d vit h th e correc t dosin g solutio n an d stoppere d a s befor e an d place d ont o th e cag e top . Not e tha t th e lengt h o f tim e tha t a dose d vate r solutio n ca n b e use d i s dependen t o n stabilit yo f th e tes t chemical
.P.1 4



### 1 1

ANALYSI S O F REPRESENTATIV E DOSIN G FORMULATION S Sample s o f dose d feed , drinkin g vater , o r gavag e solution s ar e sent , unde r predetermine d shippin g conditions , to th e referenc e analysi s laboratory . Transi t tim

e i s usuall y

2 4 hour s an

d neve r exceed s 4 8 hours . Refere e aliquot s

are assaye d a t leas t onc e durin g eac htas k an d approximatel y

onc e a mont h durin g the continuou s breedin g phase . Aliquot s o f the dosin g sampl e sen t ar e retaine d a t th

e RAC B laborator y

unti l th

e analytica l repor t i s received . ANIMAL S Animal s an

d Anima l Husbandr y Caesarean-originate d Barrier-Sustaine d (COB S ) Sviss-derive d CD- I mic e

ar e purchase d a t 6 veek s o f ag e fro m a barrie r facilit y (Charle s Rive r Breedin g Laboratories , Kingston , NY) . A tota l o f 34 0 mic e (17 0 male s an d 17 0 females ) ar e receive d onth e sam e dat e fo r a give nchemica l s o tha t Tas k 1 an d al l subsequen t task s vil l b e conducte d wit hth e same shipmen t o f mice . I f Tas k 1 i s no t t o b e performe d fo r a particula r study , onl y 12 0 male s an

d 12 0 female s are purchased . Immediatel y

onarrival , al l animal s ar e examine d for sickness , dehydration , an

d externa l bod

y characteristic s suc ha s haircoa t condition , physica l abnormalities , sore s and/o r lesion s an d evaluate d fo r parasiti c infection . Mic e ar e initiall y house d 2 pe r cag e i nsoli d botto m polypropylen e o r polycarbonat e cage s (8 " x1 1 1/2 " x6" ) vit hstainles s stee l vire lid s (Lab Products , Inc. , Rochell e Park , NJ) . Ab-Sorb-Dr i cag e litte r (Laborator y Products , Inc. , Garfield , NJ ) i s use d i nal l cages . Cage s ar e place d o n galvanized-stee l rack s (24 " V x60 " L x66 " H ) vit hfive adjustabl e shelves . Deionized/filtere d vate r an d pellete d NIH-0 7 roden t cho v (Ziegle r Brothers ,P.1 5



### 1 2

Inc. , Gardner , PA ) ar e availabl e to bot hmale s an d female s a

d libitum . I f th e tes t chemica l i s to be delivere d vi a th e feed , animal s ar e immediatel y place d

o nground roden t chov o f th e sam e specifications . Anima l holdin g room s ar e equippe d vit hindividua l temperatur e control s an d automaticall y

adjuste d ligh t cycle s (14:10 ) (light s on070 0 to 210 0 hours) . Relativ e humidit y an d temperatur e ar e monitore d i nth e anima l holdin g room s for eac hstudy . Temperatur e i s maintaine d at 2 1 + 2° C an d relativ e humidit y at 3 0 to 70% . Ai r i neac hanima l roo m i s exchange d a minimu m o f 1 2 to 1 4 time s pe r hour . Onarriva l th

e animal s ar e place d i nquarantin e fo r a minimu m o f tv o veek s t o bot hallo w the m t o adap t to ne v

surrounding s (e.g . noise , temperature , humidity , foo d an d caging ) an

d to examin e the

m fo r diseases . Durin g quarantine , tv o male s an

d tv o female s fro

m eac hshipmen t ar e sacrifice d

an d thei r ser a froze nan d sen t to Microbiologica l Associates , Bethesda , MD , fo r determinatio no f antibod y titer s agains t 1 1 mous e viruses . Th e 1 1 viruse s include : Pneumoni a Viru s o f Mic e (PVM) , Reoviru s Typ e 3 , Mous e Encephalomyeliti s (GDVII) , KVirus , Polyom a Virus , Minut e Viru s o f Mic e (MVM) , Ectromelia , Sendai , Mous e Adenovirus , Mous e Hepatiti s Viru s (MHV ) an d Lymphocyti c Choriomeningiti s (LCM) . Onl y a batc ho f animal s vhose representative s ar e negativ e fo r bot hendoparasite s an d th e 1 1 mous e viru s antibodie s ar e use d fo r RAC B studies . Eac hanima l receive s a uniqu e numbe r (earta g o r ea r punch ) vhic h identifie s bot hthe anima l an d the particula r study . Mic e ar e periodicall y examine d fo r retentio no f th e eartag . I f lost , th e earta g i s replace d b y a

ne v tag vit hth

e sam e numbe r an

d attache d to th

e opposit e ea r i norde r to mak e tha t anima l conspiciou s to anima l observer s (Nationa l Ban d an d Ta g Co. , Nevport , KY) .

### 1 3

STATISTICA L PROCEDURE S USE D T O ANALYZ E REPRODUCTIV E AN D FERTILIT YDAT A

Introductio n Mos t hypothese s ar e teste dusin g nonparametri c methods . I ncontras t t o parametri c methods , nonparametri c methods requir e fe v assumption s abou t the distributio no f the populatio nfro m vhic hsample s ar e dravn . I nparticular , nonparametri c method s d o no t assume tha t observation s come fro m a normall y distribute dpopulatio nno r d o they requir e equa l variance s amon g treatmen t groups , a s d o parametri c methods . Sinc e nonparametri c method s ar e nearl y a s efficien t a s parametri c method s vhe nthes e assumption s do hol d and ca nb e substantiall y

mor e efficien t vhe nthey d o not , thes e procedure s provid e superio r contro l ove r erro r rate s associate dvit hhypothesi s test s vhe n distributiona l assumption s ar e ope nt o question . Details o f the nonparametri c method s discusse dbelo v ca nb e foun

di nConove r (1971 ) andHollande r an dUolf e (1973) . Sinc e veigh t i s frequentl y assume dt o b e normall y distributed , parametri c method s ar e use di nsevera l analyse s concerne d vit hbod y andorga nveights . Descriptio no f Statistica l Test s Mos t hypothese s ar e teste dusin g the nonparametri c multipl e comparison s procedure s o f Dun n(1964 ) o r Shirle y (1977) , a s modifie db y William s (1986) . Shirley' s tes t i s designe dt o detec t treatment-relate ddifference s vhe nthe respons e t o treatmen t consistentl y increase s (or decreases ) vit hincreasin g dose . Althoug hthe tes t employ s a smoothin g algorith m t o adjus t fo r dose-respons e inversions , Dunn' s tes t i s mor e appropriat e i f the departur e fror a monotonicit y

i s severe . Jonckheere' s tes t (1954 ) i s use dt o ascertai n whethe r ther e i s sufficien t evidenc e o f a dose-relate drespons e t o appl yP.1 7



### 1 4

Shirley' s test . I f the p-valu e fro m Jonckheere' s tes t i s les s tha n0.10 , Shirley' s tes t i s used ; othervise , Dunn' s tes t i s applied . I nthe crossove r matin g tria l (Tas k 3) , the Kruskal-Walli s tes t (Kruskal-Wallis , 1952 ) i s use dt o tes t equalit y o f respons e amon g dos e groups , vhil e multipl e comparison s test s us e th e metho d o f Dunn . I f Tas k 4 i s performe dvit honl y on e dos e grou pan

dth e contro l group , Wilcoxon' s tes t (Conover , 1971 ) i s used . Fo r dat a expresse da s a proportion , suc ha s numbe r fertile/numbe r cohabited , the Cochran-Armitag e tes t (Armitage , 1971 ) i s use dt o tes t fo r a dose-relate dtrend , andpairvis e comparison s ar e performe dusin g a chi-squar e tes t (Conover , 1971) . I nTas k 3 , vher e dos e group s d o no t represen t increasin g dos e levels , a chi-squar e tes t i s use d t o tes t simultaneou s equalit y

o f respons e acros s group s an dfo r pairvis e comparisons . t Sinc e the numbe r o f pup s i na litte r may influenc e th

e averag e pupweigh t i na litter , a parametri c analysi s o f covarianc e (Nete r andWasserman , 1974 ) i s use dt o tes t overal l equalit y i naverag e pupveight , afte r adjustmen t fo r averag e litte r size . Pairvis e comparison s ar e performe dusin g Dunnett' s test . Vagina l cytolog y

dat a ar e analyze dusin g a multivariat e analysi s o f varianc e (Morrison , 1976 ) t o tes t fo r the simultaneou s equalit y o f measurement s acros s dos e levels . Befor e applyin g the test , a narcsin e transformatio ni s performe d t o brin g the dat a int o close r conformanc e vit h normalit y

assumptions .
Applicatio no f Statistica l Test s

Fertilit y

an d Matin g Indice s

Th e Cochran-Armitag e tren dtes t i s use dto tes t fo r a dose-relate d decreas e i nfertilit y an dmatin g indices . Pairvis e comparison s betvee nP.1 8



### 1 5

contro l anddos e group s ar e mad e usin g a nunadjuste dchi-squar e test . I nTas k 3 , vher e dos e group s d o no t represen t increasin g dos e levels , a chi-squar e tes t i s use d t o tes t fo r a noveral l equalit y amon g group s an

dfo r pairvis e comparisons . Siz e andNumbe r o f Litter s Litte r size s an

d numbe r o f litter s i ndos e group s ar e compare dt o control s usin g Dunn' s o r Shirley' s test . T o examin e potentia l difference s i n treatmen t effect s o nmale s an dfemales , numbe r o f mal e pups , numbe r o f femal e pup s andtota l numbe r o f pup s i nlitter s i ntreate dgroup s ar e compare dto controls . Proportio no f Liv e Pup s andSe xRati o Th e rati o o f the numbe r o f pup s bor naliv e to the tota l numbe r o f pup s carrie dt o ful l ter m i s compute dfo r al l fertil e pairs . Th e se xratio , expresse da s th e proportio no f males , i s compute dfo r al l fertil e pair s vit h
a t leas t on e liv e pup . Shirley' s o r Dunn' s tes t i s use dto compar e dose d group s t o control s base do nJonckheere' s test , a s describe dabove . I nTas k 3 , Dunn' s tes t i s used , an di nTas k 4 vit htv o groups , Wilcoxon' s tes t i s applied . Pu pBody

Weigh t T o adjus t fo r the potentia l effec t o f the numbe r o f pup s pe r litte r o n averag e pu p veight , a nanalysi s o f covarianc e i s performed . Th e covariat e
use di s averag e litte r size , includin g liv e an ddea dpups . Leas t square s estimate s o f dos e grou pmean s adjuste dfo r litte r siz e ar e compute dandteste d fo r overal l equality usin g a nF-tes t an dpairvis e equalit y usin g Dunnett' s test . Unadjuste dveight s ar e analyze dvit hShirley' s o r Dunn' s test . T o examin e potentia l differentia l effect s onmale s an dfemales , analyse s o n males , females , andbot hsexe s ar e performed .P.1 9



### 1 6

Fina l Tas k 2 Litte r Dat a Averag e pu pveigh t fo r the las t Tas k 2 litte r o f eac hbreedin g pai r i s
measure da t variou s ages , includin g birth , veanin g andmating . Also , th e numbe r o f pup s survivin g a t eac htim e point , divide db

y the numbe r o f pup s bor nalive , i s compute dfo r eac hlitte r andthe nth e averag e fo r eac hdos e grou pi s determined . A t eac htim e point , dose d group s ar e compare d t o th e control s b

y Shirley' s o r Dunn' s test . Dam Weight s Dam s ar e veighe dimmediatel y followin g deliver y o f eac hlitter . Shirley' s o r Dunn' s tes t i s applie dbase do nth e resul t o f Jonckheere' s test , a s describe dabove . I nTas k 3 , onl y Dunn' s tes t i s used . Cumulativ e Day s t o Litte r Tas k 2 dat a ar e analyze dfirs t vit hJonckheere' s tes t and the nvit h eithe r Shirley' s o r Dunn' s test . Tas k 3 da m veight s ar e analyze dvit hDunn' s test . Wilcoxon' s tes t i s use di nTas k 4 vhe nonl y tv o group s ar e t o b e compared . Necrops y

Weight s Absolut e orga nan

dbod y veights , an

dorga nveight s relativ e t o bod y veigh t ar e analyze db y Shirley' s o r Dunn' s test . Sper m andVagina l Cytolog y Dat a Sper m concentration , sper m motilit y

an dpercen t abnorma l sperm ar e analyze db

y Shirley' s o r Dunn' s test . A narcsin e transformatio ni s applie d t o vagina l cytolog y

data , representin g the proportio no f th e observatio nperio d tha t a nanima l va s i na give nestrou s stage , t o brin g th e dat a int o close r conformanc e vit hnormalit y assumptions . Treatmen t effect s ar e investigate db y applyin g a multivariat e analysi s o r varianc e t o tes t fo r the simultaneou s

### 1 7

equalit y

o f measurement s acros s dos e levels . Cycl e lengt hi s analyze dusin g Shirley' s o r Dunn' s test . Foo d an dWate r Consumptio n Usin g eithe r Shirley' s o r Dunn' s test , foo dan dwate r consumptio ndat a
ar e analyze dpe r anima l pe r day an dpe r gram

o f bod y veigh t pe r day . Cumulativ e Day s t o Litte r Th e dat a ar e firs t analyze dvit ha Jonckheere' s tes t andthe nvit heithe r
a Shirley' s o r Dunn' s tes t dependin g o nthe result s o f th e Jonckheere' s test . Treatmen t effect s ar e investigate db y applyin g a multivariat e analysi s o f varianc e t o tes t fo r the simultaneou s equalit y o f measurement s acros s dos e levels . Cycl e lengt hi s analyze dusin g Williams' s test .

METHOD S - SPECIFI C

TAS K1 - DOS E RANG E FINDINGSTUD Y(Se e Figur e 2 andthe mileston e char t fo r a composit e time line ) Selectio no f Animals fo r Tas k 1 A tota l o f 10 0 animals ar e use di nTas k 1 ; 9 6 fo r treatmen t an dfou r fo r qualit y control . On e vee k afte r arrival , eac ho f the 34 0 animal s purchase d (17 0 male s and17 0 females ) i s eartagge d andassigne da rando m numbe r fro

m a tabl e o f random

number s (Snedeco r andCochran , 1967) . Th e 5 0 male s vit hthe highes t rando

m number s vil l b e assigne dt o Tas k 1 . O f thes e 50 , the tv o vit h th e highes t rando

m number s vil l b e use dfo r qualit y control . Th e same proces s vil l b e follove dfo r the assignmen t o f Tas k 1 females . Randomizatio nint o Treatmen t Group s i nTas k 1 Th e animals use d i nTas k 1 ar e veighe da t th e conclusio no f veek s 1 and 2
o f quarantine . I f n o healt hproblem s ar e observe ddurin g quarantine , Tas k 1P.2 1



### 18

animal s vil l b e ranke d b y the vee k 2 bod

y veight s an

dassigne dt o treatmen t group s b

y a stratifie drandomizatio nproces s s o tha t the mea nbod y veight s o f
eac hgrou p ar e statisticall y similar . Pairin g o f Animal s Tv o animal s o f the same se xar e house dpe r cag e durin g Tas k 1 . T o selec t cagemate s vithi na treatmen t group , previousl y assigne drando

m number s ar e arrange di nascendin g order , male s an dfemale s separately . Th e male/femal e vit hthe loves t rando m numbe r i s paire dvit hthe male/femal e vit hthe nex t highe r rando

m number . Al l animal s ar e paire di nthi s manner . Study Result s Th e follovin g dat a ar e collecte dandevaluate ddurin g Tas k 1 : 1 . Body

veigh t o ndays 0 , 7 , an d14 . 2 . Averag e dail y

intak e o f feedan

ddrinkin g vate r fo r eac hvee k i n orde r t o asses s tes t agen t intake . 3 . Mortalit y

durin g the 1 4 day s o f treatment . 4 . Clinica l symptom s o f toxicit y (tvic e daily) . Th e percen t veigh t chang e ove r the tvo-vee k perio di s analyze db y a tvo-va y analysi s o f varianc e (dos e vs . sex ) andpercen t mortalit y i s analyze d b y th e Chi-Squar e Test . L D i s determine dbase do nth e mortalit y data . Th e probi t analysi s i s don e t o determin e LD 1 Q , i f deat hoccur s i ntv o o r mor e group s (Snedeco r andCochran , 1967) . I nadditio nt o the probi t analysis , the chang e i nbody

veigh t i s considered . Fro m thes e data , the MT D (maximu m tolerate ddose) , define da s th e highes t dos e tha t ca nb e administere dfo r tv o veek s vithou t depressin g veigh t b y mor e than10 % o r resultin g i nsignifican t mortalit y

(>10%) , i s computed . Thes e dat a ar e thenutilize dt o calculat e the dose s fo r Tas k 2 s o tha t the highes t dos e vil l no t depres s weigh t b y mor e than 10 % ove r the 98-da y continuou s breedin g study .P.2 2



### 19

I t shoul d be note d tha t i f sufficien t informatio ni s availabl e i nth e literatur e o r fro

m previou s studie s to estimat e th e MTD , Tas k 1 i s no t conducted . TAS K2 - CONTINUOU S BREEDIN G

Quarantin e The animal s fo r Task 1 an d 2 ar e usuall y

purchase d a t th

e same time ; therefore , th

e animal s ar e hel d i nisolatio nfor five veek s prio r t o commencin g Task 2 , (i.e . tv o veek s precedin g Tas k 1 , th e tvo-vee k perio d durin g vhic hTask 1 i s conducte d an d on e vee k durin g vhic hth e Task 1 dat a ar e bein g analyzed , an d the dos e level s fo r Task 2 selected) . Mic e ma y als o b e purchase d separatel y fo r Task 2 i nvhic hcas e the y receiv e th

e usua l 2 vee k quarantin e period . Randomizatio nInt o Treatmen t Group s The 24 0 animals , 12 0 male s an d 12 0 females , maintaine d i nquarantin e throughthe conclusio no f Tas k 1 ar e reveighe d an d assigne d to treatmen t group s b

y a stratifie d randomizatio nprocedur e base d onveigh t a s exemplifie d b y the follovin g example . Th e animal s o f eac hse xar e ranke d accordin g to veight . Surplu s animal s (onl y 10 0 male s an

d 10 0 female s vil l be utilized )

ar e remove d fro

m th e to p an d botto

m o f th e rando

m numbe r list . Beginnin g a t the top o f the lis t o f randomize d animal s th e male s ar e divide d int o subgroup s

o f five each . O f thes e five males , th e tv o animal s vit hth e highes t rando

m number s ar e assigne d to th e contro l group ; th e anima l vit hth

e third highes t goe s int o th

e lo v dos e group , th

e fourt hhighes t to th e mi d dos e an

d th e loves t t o th

e highdos e group . Thi s proces s i s repeate d vit hth e nex t five animal s an

d s o onunti l al l male s hav e bee nplace d int o a treatmen t grou p (4 0P.2 3



### 2 0

i ncontro l and 2 0 i neac ho f the group s receivin g th e tes t chemical) . Th e
same procedur e i s use dto assig nfemale s to th e fou r treatmen t group s (on e contro l grou pvit h4 0 female s an dthre e dos e level s vit h2 0 female s pe r dose) . Initiatio no f Treatmen t vit hTes t Chemical : Wee k 1 Animal s ar e 1 1 veek s o f ag e a t th e initiatio no f treatment . Durin g the firs t seve nday s o f treatment , the sexe s ar e house dseparatel y i ngroup s o f tv o i n7 1/2 " x1 1 1/2 " x5 " polypropylen e o r polycarbonat e cages . Th e tes t chemica l i s administere dvi a gavage , feedo r drinkin g vater . Continuou s Breedin g (Cohabitation ) fo r 9 8 Day s Follovin g treatmen t vit hthe tes t chemica l fo r seve ndays , on e mal e an d
on e femal e ar e selecte drandoml y (se e belov ) fro

m the same dos e grou p and cohabite dfo r 9 8 day s (1 4 veeks) . Ther e ar e 4 0 breedin g pair s i nthe contro l grou pand2 0 pair s i neac hgrou preceivin g the tes t chemica l (thre e dos e levels ) fo r a tota l o f 20 0 mic e (10 0 male s an d10 0 females) . Th e animal s vil l b e 1 2 veek s o f ag e a t the commencemen t o f cohabitation . Random Selectio no f Breedin g Pair s T o selec t breedin g pairs , the mal e vit hthe loves t rando m numbe r vithi na treatmen t grou pi s paire dvit hthe femal e vit hth e loves t rando

m number . Th e mal e vit hthe secon d loves t rando m numbe r vit hth

e correspondin g anima l fro m the femal e list , ands o on . Housin g and Identificationo f Breedin g Pair s Eac hbreedin g pai r i s assigne da numbe r vhic hi s place donthe cag e car d displayin g the numbe r o f the mal e an dfemal e an

da colo r code ddos e leve l indicator . Th e pair s vil l b e house di n7 1/2 " x1 1 1/2 " x5 " polycarbonat e cages . Othervise , housin g condition s ar e the same a s thos e describe d previously .P.2 4



### 2 1

Dos e Selectio nfo r Tas k 2 Th e dat a fro

m Tas k 1 ar e utilize dto determin e the appropriat e dose s fo r Tas k 2 . Dose s ar e selecte da s follovs : Th e hig hdos e i s selecte d s o tha t i t vil l produc e some genera l toxicit y (i.e . maxima l tolerate ddose ) a s define db y specifi c orga ntoxicit y (dependin g o nthe chemical ) o r bod y veigh t depressio n
o f abou t 10 % ove r the cours e o f the study . Th e mid-dos e i s expecte dto produc e n o systemi c toxicit y an dthe lov

dos e i s a nestimate dno-effec t level . Dat a Collectio nDurin g the 98-Da y Cohabitatio nPerio d Th e follovin g criteri a ar e evaluate ddurin g Tas k 2 : 1 . Body

Weigh t - Al l experimenta l animal s ar e veighe d o nday - 0 , 7 , 14 , 21 , 35 , 63 , 9 1 and12 6 (beginnin g o f veek s 1 , 2 , 3 , 5 , 9 , 13 , and18 , respectively) . Refe r t o mileston e char t i nappendi xfo r generi c veig hdates . 2 . Pup s Delivere dDurin g Days 0 t o 9 7 - Al l cage s ar e inspecte da t leas t tvic e a day

t o chec k fo r the presenc e o f pups . Afte r the da m ha s complete d delivery , the pup s ar e counted , sexed , and veighe d a s describe dlate r i nthi s section . Th e pup s ar e the nsacrifice d b y asphyxiatio nvit hcarbo ndioxide . I f a femal e ha s no t complete d deliver y

b y evenin g healthsurveillance , the pup s ar e checke d the
nex t da y

andthi s day

vil l b e considere dpostnata l da y 0 . I nth e
even t a pregnan t femal e mous e die s prio r t o o r durin g parturition , a n immediat e necrops y

i s performe dand , i f possible , the numbe r o f liv e
o r dea d pup s i nthe uteru s i s counted . 3 . Evaluatio no f Pup s - Th e day o f deliver y

i s designate da s postnata l day 0 . Afte r the da

m ha s delivere dan

dha s cleane d he r pups , sh e i s veighed . Th e veigh t an dtim e i s recorded . Th e tota l numbe r o f pups , numbe r o f liv e pups , andminimu m numbe r o f dead/cannibalize dpup s i sP.2 5



### 2 2

noted
. Al l pup s ar e examine d fo r externa l abnormalities
. Liv e pup s
ar e sexed
. Th e se x o f a pu p i s determine d externall yb yth e distanc e betvee n th

e urogenita l an

d ana l opening s (mal e distanc e i s tvic e th e femal e distance , usuall y2 m mvs . 1 mm ) an

d mal e perineu

mappear s slighl ysvolle n (sit e o f futur e scrota l sacs) . Withi n a litter , al l male s ar e place d o n th e balanc e an

d th e veigh t i s recorde d i n gram s t o tv o decima l places
. Th e sam e procedur e i s follove d fo r femal e pups . Dead/cannibalize d pup s ar e no t sexe d o r veighed
. Atissu e i s place d on th

e balanc e t o preven t chilling o f pups . 4 . Th e numbe r o f day s t o litte r i s calculate d fo r eac h breeding pair
. 5 . Da mveight s ar e measure d afte r eac h litter
. I f th e rout e o f tes t chemica l i s b

ygavage , th

e da mveigh t i s used fo r determining dosag e volume unti l th

e nex t veighing sessio n i s scheduled
. 6 . A n aliquo t o f eac h o f th e representativ e dosing solution s i s submitte d fo r referenc e chemica l analysis
. Th e specifi c veek s vhe n sample s ar e submitte d ar e 1 , 6 , 10 , an d 14 . 7 . Dose d feed/vate r consume d i s monitore d fo r a vee k onc e a mont h throughou t cohabitatio n (veek s 1 , 2 , 5 , 9 , 13 , an d optionall y18) . Separatio n o f Breeding Pair s Immediatel yfollovin g the 98-da

ycohabitatio n period , eac h pai r i s separate d an

d the mal e an

d femal e ar e house d separatel yi n cage s o f the same dimension s an

d othervis e identica l condition s a s th e 98-da ycohabitatio n period
. Al l animal s ar e continue d o n thei r respectiv e treatment s fo r a perio d

o f 2 1 days
. Pup s delivere d during the holding perio d ar e allove d t o remai n vit h thei r dams
. Thes e offspring ar e counted , sexed , an d veighe d on day s 0 , 4 , 7 , 14 , an d 2 1 an d the percen t surviva l i s calculated
. Pos t partu mda mveigh t i s als o recorded
.

### 23

Dispositio no f Animal s I f n o effec t onreproductiv e performanc e i s observed , the n
a ) Randoml y

selecte d offsprin g o f the contro l and high-dos e animal s ar e utilize d fo r Tas k 4 . Th e othe r litter s ar e humanel y sacrifice d vhe n they ar e 2 1 day s o f age . b ) Th e contro l lov , mi d an d high-dos e animal s ar e humanel y sacrificed . I f a neffec t o f reproductiv e performanc e i s observed , then :
a ) Tas k 3 i s conducte d vit hthe contro l and hig hdos e animal s afte r vhic hthe

y ar e necropsied , b ) Th e lov

an d mid-dos e animal s ar e humanel y sacrificed . c ) Randoml y

selecte d offsprin g o f the control , lov , mi d a s vei l a s hig h dos e animal s ar e utilize d fo r Tas k 4 . Statistica l Evaluatio no f Tas k 2 Dat a vil l b e totale d an d summarize d veekl y an d a t th e conclusio no f the 98-da y cohabitatio nperiod . Th e breedin g pair s ar e the independen t samplin g uni t an d reproductio n parameter s fo r eac hpai r ar e summe d acros s litter s i norde r t o carr y ou t statistica l analysi s o f the dat a (Se e Statistica l Procedures , p . 13) .

TAS K3 (DETERMINATION O F TH E AFFECTE D SEX ) Whe nth e las t litte r o f Tas k 2 i s 2 1 day s old , the pup s ar e veaned . I f
a neffec t o nreproductio ni s observe d i nTas k 2 , Tas k 3 i s the nconducte d usin g the controls an d high-dos e animals . Mid-dos e animal s may b e use d i f th

e effec t o f the toxican t va s sever e i nthe high-dos e grou p (i.e . n o litter s a t al l o r onl y

a firs t o r second litte r o r hig hmortalit y o r sever e generalize d toxicity ) an

d the mid-dos e animal s shove d a significan t reductio ni n fertility .

### 24

Random Assignmen t o f Breedin g Pair s O f the 4 0 contro l male s an d4 0 contro l females , 2 0 o f eac hse xar e use d fo r a contro l xcontro l cros s an d2 0 o f eac hse xar e bre dwit hhig hdos e animals . Th e 2 0 contro l female s vit hthe highes t rando m number s (eithe r assigne dearlie r fo r Tas k 2 o r a ne v rando m number ) an

d2 0 contro l male s vit h the loves t rando

m number s ar e use dfo r th e contro l xcontro l mating . Th e 2 0 loves t female s an

d2 0 highes t male s ar e use dfo r breedin g vit hthe hig hdos e animals . Th e breedin g pair s ar e determine db y a randomizatio nprocedur e simila r t o tha t o f Tas k 2 (p . 18) . Breedin g fo r Tas k 3 Breedin g pair s ar e checke dtvic e dail y fo r th e presenc e o f a copulator y plug . Cohabitatio no f breedin g pair s continue s fo r seve nday s o r detectio no f
a copulator y

plug , vhicheve r i s first . Animal s ar e no t expose dt o the tes t chemica l fo r thi s perio d o f cohabitation . Exposur e i s reinstate dafte r seve n day s and continue s unti l the conclusio no f the Task . Follovin g separatio nthe mal e andfemal e ar e house dseparatel y (a s fo r Tas k 2 las t litter ) unti l a t leas t day

2 8 o f Tas k 3 . Durin g thi s perio dfertilit y dat a ar e collecte d o n
any litters produce da s describe dfo r Tas k 2 andthe litte r ar e sacrificed . Postpartu

m da m veight s ar e determine dafte r deliver y o f the litter . I n addition , al l animal s ar e veighe do nthe firs t day o f cohabitatio nandonc e a
vee k thereafte r unti l sacrific e andthe amoun t o f feed/vate r consume di s monitore dveekly . Th e follovin g informatio ni s als o calculated :

Fertilit y

inde x= (No . fertile/No. cohabited ) x10 0 (No . fertil e i f define da s no . producin g a t leas t on e litter ) Matin g inde x= (No . vit hcopulator y plugs/No . cohabited ) x10 0 Randomizatio no f Animal s Prio r to Schedule dNecrops y a t the En

do f Tas k 3 A rando m numbe r i s assigne dt o eac hanima l schedule dfo r necropsy . Random number s ar e sorte di nascendin g order . Thi s lis t represent s the orde r
o f necropsy .P.2 8



### 2 5

Tas k 3 (Necropsy ) Afte r Tas k 3 litter s hav e bee nsacrifice d the mal e an d femal e mic e ar e necropsie d i nth

e presenc e o f a pathologist . Th e pathologis t examines the entir e anima l fo r abnormalities . Th e complet e procedur e shoul d tak e n o longe r tha ntvent y

minute s fo r eac hanimal . Durin g the schedule d mal e necropsy , th e follovin g parameter s ar e evaluated : 1 . Sper

m motility , sper

m densit y

(caud a epididyma l sper m number/mg tissue) , an

d sper m morphology . 2 . Body

veight . 3 . Live r veight . 4 . Righ t testi s veight . 5 . Ventra l prostat e veigh t (pair) . 6 . Semina l vesicl e veigh t (pai r includin g th e coagulatin g glands) . 7 . Epididyma l veight . 8 . Combine d kidne y (lef t an

d right ) veigh t vit hadrena l glan d attached . 9 . Othe r selecte d orga nveights , base d o nthe specificit y o f the tes t chemical , ma y

als o b e measured . Afte r the anima l i s veighed , the prostate , semina l vesicles epididymi s
an d testi s vit hattache d epididymi s i s excised , veighe d an d fixe d i nBouin s fo r embeddin g i nparaffi no r i n10 % neutra l buffere d formali ni f they ar e t o b e embedde d i nglyco l methacrylate . Th e live r an d kidney s ar e fixe d i n10 % neutra l buffere d formalin . Fo r th e females , estrou s cyclicit y i s measure d b

y vagina l smear s fo r 1 2 day s prio r t o necropsy . Th e follovin g parameter s ar e thenevaluate d durin g necropsy :P.2 9



### 2 6

1 . Bod y veight . 2 . Live r veight . 3 . Combine d kidne y (lef t an

d right ) veigh t vit hadrena l gland s attached . 4 . Righ t ovary . 5 . Th e reproductiv e trac t i s excise d an d fixed i n10 % neutra l buffere d formali nbut no t veighed . Th e righ t ovary fro m eac hfemal e i s fixe d i nBouin' s solution . Histopatholog y o f selecte d tissu e i s conducte d as/whe nneeded . Sper m Motilit y

Determinatio n Sper m motilit y

i s extremel y

sensitiv e to p

H an d temperature . Therefore , th e follovin g material s ar e assemble d an d maintaine d a t 37°C : phosphat e buffe r microscop e slide s an d coverslip s Pasteu r pipet s petr i plate s microscop e stag e varme r modifie d Tyrode' s eg g yolk solutio nadjuste d to 7.2 . pH (Environmenta l Healt han

d Testing , Lexington , KY) . Afte r th

e righ t caud a epididymi s i s excise d an d veighed . The tissu e i s place d i na dis hvit hsalin e a t 37° C an d picke d vit ha forcep s to releas e some sperm . Sper

m motilit y

i s assesse d a s quickl y a s possible . A

smal l dro p o f semina l flui d i s presse d int o a dro p (80ul ) o f Tyrode s solutio nona prevarme d microscop e slide . Tv o independen t slide s ar e prepare d to estimat e sper m motility . A var m coversli p (2 4 x4 0 mm ) i s place d onth e slid e an

d th e slid e i s place d o na varme d microscop e stag e (Bailey Instruments , Inc. , Saddl e Brook , NJ ) to maintai nth e sper m at 37 ° durin g th e motilit y

evaluation .P.3 0



### 27

T o estimat e sper

m motility , th

e actua l numbe r o f motil e andnonmotil e sperm fo r eac hfiel do f visio ni s determined . A han dcounte r i s use dt o coun t th e numbe r o f motil e andnonmotil e sperm . Th e fina l motilit y i s base do nten independen t field s an d observation s mad e b y tv o independen t viever s usin g a 40 Xobjective . A

minimu m o f 20 0 tota l sper

m mus t b e observe d i nmale s vit h lo v sperm counts . Sper m ar e considere dmotil e i f the y sho v an y movemen t a t all . Sinc e percen t motilit y

decrease s vit htime , evaluatio ntim e i s critica l an

dmus t b e accomplishe d a s quickl y a s possibl e (i.e. , 3- 5 min.) . Similarl y temperatur e
andp H mus t b e carefull y controlled . Sper m Coun t Afte r the slide s fo r sper m motilit y

evaluatio nhav e bee nprepared , the entir e caud a i s immediatel y place dint o 2. 0 m l o f phosphat e buffere d salin e (PBS ) (p

H 7.4 ) i na 3 5 x1 0 m m plasti c petr i dish . Th e caud a i s gentl y mince d vit ha scalpel , thereb y releasin g the cauda l spermatozoa . Th e petr i dis h content s ar e svirle d severa l time s an dplace di na nincubato r (37°C ) fo r 1 5 minute s i norde r t o allo v tim e fo r aggregate s o f sper m to disperse . A t the conclusio no f incubation , the content s ar e svirle dagai nt o evenl y distribut e the sperm suspensio nan

d0. 5 m l o f the sper m suspensio ni s transferre dt o a tes t tub e an ddilute dvit hPBS . Th e volum e o f Tyrode' s solutio nadde dma y

b e varie daccordin g t o the densit y o f th e sperm suspension . Th e sperm

ar e the nkille db

y placin g the tes t tub e i nboilin g vate r fo r on e minut e andon e dro p o f the suspensio ni s place di neac ho f tv o hemocytomete r chambers . Tv o count s ar e performe dusin g separat e sample s o f dilute dsper m suspension . Consistenc y i nthe preparatio no f sper m sample s fo r densit y determination s i s essentia l to obtai naccurat e results .P.3 1



### 28

Th e tota l numbe r o f sper m pe r caud a epididymi s an d pe r m g o f caud a epididymi s i s the ncalculated . Sper m Morpholog y Th e procedure s an

d criteri a o f Wyrobe k an d Bruc e (1975 ) an

d Soare s e t al . (1979 ) ar e use d t o evaluat e sper m morphology . Th e suspensio no f sper m i s transferre d t o a tes t tub e an d on e dro p o f 1 % Eosi nYstai ni nwate r i s added . Th e content s o f the tube ar e gentl y agitate d an

d allowe d to stai nfo r approximatel y

4 5 minutes . Th e sper m ar e gentl y

resuspende d wit ha Pasteu r pipet , place d o na microscop e slide . Fou r slide s ar e prepare d fro m eac hsperm suspension . Afte r smear s hav e bee nmade , the slide s ar e place d a t a 3 0 t o 4 0 degre e angl e o na tra y s o tha t exces s suspensio nca ndrai noff . Th e slide s
ar e air-drie d overnigh t i na dust-fre e atmosphere an d coverslippe d using Permoun t o r suitabl e mountin g mediu m an d xylene . Fiv e hundre d sper m ar e evaluate d pe r sper

m suspensio n(12 5 sper m pe r slid e i s a minimu m requirement )
a t 400 Xmagnification . Ther e ar e seve ncategorie s fo r sper m morphologica l evaluation : normal ; blun t hook ; banana ; amorphous ; pin-head ; tv o heads/tv o tails ; an

d tailles s sperm . Th e dat a ar e reporte d a s percen t abnorma l sperm pe r sample . Vagina l Cytolog y

Evaluatio n Vagina l smear s obtaine d fro m mic e hav e bee nfoun d t o correlat e vei l vit h change s occurrin g i nthe reproductiv e trac t an d vit hthe secretio no f the ovaria nhormones . Th e mos t commo ntype s o f cell s presen t i na vagina l smea r
are :
a . Polymorphonuclea r leucocyte s b . Nucleate d epithelia l cell s
c . Larg e squamou s epithelia l cells , als o calle d cornifie d cell s (nucleate d o r non-nucleated )

### 29

Th e estrou s cycl e o f the mous e i s complete di nfou r t o fiv e day s althoug h the timin g o f the cycl e may b e influence db

y exteroceptiv e factor s (light , temperature , etc.) . Th e cycl e i s roughl y divisibl e int o fou r mai nphases . Estru s - Thi s i s th e perio do f "heat" , andcopulatio ni s possibl e onl y
a t thi s time . Thi s phas e last s fro m 9 t o 1 5 hour s an

d i s characterize d b y a hig hrat e o f runnin g activity . Many mitose s occu r i nthe vagina l
mucosa , anda s ne v

cell s accumulate , the superficia l layer s becom e squamou s and cornified . Th e latte r cell s ar e exfoliate dint o the vagina l lume nandthei r presenc e i nvagina l smear s i s indicativ e o f estrus . Durin g lat e estru s ther e ar e "chees y masses " o f cornifie dcell s vit h degenerat e nucle i presen t i nth e vagina l lumen , bu t fev , i f any , leucocyte s ar e foun ddurin g estrus . Muc ho f th e lumina l flui di nth e uteru s i s los t befor e ovulation . Metestrou s - Thi s occur s shortl y afte r ovulatio nand i s intermediat e betvee nestru s anddiestrus . Thi s phas e last s fo r 1 0 to 2 4 hour s an d matin g i s usuall y

no t permitted . Many leucocyte s appea r i nthe vagina l lume nalon g vit ha fev cornifie dcells . Diestru s - Thi s phas e last s fo r 6 0 t o 7 0 hours , durin g vhic hfunctiona l regressio no f th e corpor a lute a occurs . Th e vagina l mucos a i s thi nand leucocyte s migrat e throughit , givin g a vagina l smea r consistin g almos t entirel y

o f leucocytes . Proestru s - Thi s herald s the nex t "heat " an di s characterize d b y functiona l involutio no f th e corpor a lute a an dpreovulator y

svellin g o f th e follicles . Flui d collect s i nth e uter i an

dthey become highl y contractile . Th e vagina l smea r i s dominate db y nucleate depithelia l cell s vhic hoccu r singl y o r i nsheets . Th e duratio ni s approximatel y 1 2 hours .P.3 3



### 3 0

1 . Obtainin g a Vagina l Smea r fro m Mic e A vagina l smea r fro m eac hanima l i s prepare d betvee n8 an d 1 0 a.m . fo r 1 2 consecutiv e day s befor e termina l sacrifice . Full y froste d slide s

ar e use d t

o mak e vagina l smear s (Dakin-Erie , Cat . #2958) . Al l smear s ar e mad e onth

e froste d fron t o f th e slide . Si xday s o f smear s ar e incorporate d onon e slid e vhic hcanbe staine d a t on e time . On e dro p o f 0.9 % salin e o r PB S solutio ni s place d o nth e microscop e slid e i nth e appropriat e square . Next , a smal l amoun t (-5 0 pl ) o f 0.9 % salin e o r PB S solutio ni s aspirate d int o a 3-4 " medicin e dropper . The medicin e droppe r i s place d jus t insid e th e vagina l openin g o f th e mous e an

d th e vagina l fluid s lavage d severa l times . Th e content s o f th e medicin e droppe r ar e transferre d ont o the slid e an d allove d to air-dry . I f a non-disposabl e eye droppe r i s used , i t i s thoroughl y vashe d betvee nanimal s i n95 % alcoho l follove d b y saline . A

t th e en d o f eac h day , th

e smear s ar e fixed b y lightl y

sprayin g fixativ e (Spray Cyt e I I b

y Cla y Adams , #7180) . I norde r to fi xonl y th e specifi c squar e onth e slide , a 2 " x 4 " fil e card vit ha vindo v i s used . Afte r al l smear s hav e bee nmade , th e slide s ar e allove d to dry completel y

an d staine d vit hToluidin e Blu e th e same da y o r vithi nth

e nex t fe

v days . Fo r staining , slide s ar e loade d i nglas s rack s an d dippe d i nth

e follovin g solutions :

- 95 % Ethano l - 3 0 minute s

- 80 % Ethano l - 1 minut e vit hgentl e shakin g

- 70 % Ethano l - 1 minut e vit hgentl e shakin g

- 50 % Ethano l - 1 minut e vit hgentl e shakin g

- 20 % Ethano l - 1 minut e vit hgentl e shakin gP.3 4



### 3 1

- 0.5 % Toluidin e Blu e i n20 % Ethano l - 30-4 5 second s Persona l judgemen t shoul db e use dbecaus e Toluidin e Blu e improve s wit h age .

- Runnin g tapwate r rins e - unti l tapwate r run s clea r (a t thi s point , i f slide s ar e no t dar k enough , they ar e dippe d i n20 % Ethano l andpu t bac k i nthe stain . This proces s i s repeate dunti l desire dcolo r i s obtained) . Adequac y o f stainin g shoul dbe judge dbase do nthe metestrou s o r diestru s smears . Th e staine dslide s ar e gentl y blotte dvit hbibulou s paper . Paper s ar e
no t reuse d becaus e materia l o na slid e ca nb e tranferre d t o othe r slides . Slide s ar e allove dt o dr y completel y

andthe ncoverslippe dusin g Permoun t (o r anothe r suitabl e mountin g medium ) an dxylene . Evaluatin g Vagina l Smear s Th e slide s ar e code dandevaluate dusin g a lov pove r objectiv e fo r scannin g anda 20 Xobjectiv e to mak e the fina l judgement . Eac hsmea r i s assigne dt o on e o f the estrou s stage s (proestrus , estrus , metestrus , diestrus )
a s previousl y

defined . Whe ni t i s difficul t t o mak e an y judgemen t fro

m the
smear , i t i s designate d'no t clear. ' Th e estrou s cycl e lengt hi s estimate da s follovs : the numbe r o f days betvee ntv

o consecutiv e proestru s (P ) stage s beginnin g vit hth e firs t P ar e counted . Th e secon d P i s no t counte da s a da y i nth e cycl e length ; i t i s th e beginnin g o f th e nex t cycle .

- Whe ntv

o P s ar e no t present , tv o nonconsecutiv e Estru s Stage s ar e use d follove db

y tv o Metestru s Stages , the ntv o Diestru s Stages .

- Th e estrou s cycl e lengt hi s considere dno t clea r vhe na nanima l i s i n the same estrou s phas e fo r seve nconsecutiv e days .P.3 5



### 32

Pathologi c Observation s A t necropsy , i nadditio nt o th e detaile dexaminatio no f th e reproductiv e organs , gros s lesion s o f al l tissue s ar e noted . A complet e histopathologica l examinatio no f the liver , kidney , reproductiv e tissue s andothe r selecte d tissue s may

b e performed .

TAS K4 (OFFSPRINGASSESSMENT ) I f chemica l exposur e cause dn o significan t effect s o nfertilit y and reproductiv e performanc e i nTas k 2 , the nrandoml y selecte dmember s o f eac hse x fro m the fina l litte r (i.e . the litte r bor ndurin g the holdin g perio dafte r the 98-da y

cohabitatio nperio di nTas k 2 ) fro m th e contro l an

dhig hdos e grou p
ar e raise dunti l 7 4 + 1 0 day s o f age . I f th e chemica l va s shovnt o be a reproductiv e toxicant , the nrandoml y selecte dmember s o f the fina l litte r fro m th e contro l an

dal l treatmen t group s
ar e save d an

draise dunti l 7 4 + 1 0 day s o f age . Animal s ar e dose dvit hth e same tes t articl e a s thei r parent s fro m veanin g throughou t Tas k 4 . Random Selectio no f Weanling s fo r Offsprin g Assessmen t Al l animal s i nthe las t litter s o f eac htreatmen t grou par e no t neede d fo r th e Tas k 4 assessment ; therefore , a t 2 1 day s o f ag e al l pup s ar e assigne d
a rando m number . I ngeneral , vithi neac hlitter , rando m number s ar e the n arrange di ndescendin g order, male s an dfemale s separately . Th e tv o mal e pup s vit hthe highes t rando m number s ar e selecte dto conduc t Tas k 4 . Th e tv o femal e pup s vit hthe highes t rando m number s ar e selecte dsimilarly .P.3 6



### 3 3

Pu pmortalit y

i s closel y

monitore dprio r to andafte r veaning . I f the pupmortalit y

i nthe treate dgrou pappear s to be significantl y highe r tha nthe contro l group , mor e tha ntv o pup s o f eac hse xmay b e save dfro

m eac hlitter . Also , mor e animal s ma y b e selecte dfro

m a litte r i f the chemica l i s administere d vi a gavage , rathe r tha nthe fee do r vater . Frequently , dosin g
error s occu r b

y gavage , resultin g i nthe deat ho f the weanling . Th e animal s
no t selecte d fro

m the litter s ar e kille db y asphyxiatio nwit hC0 2 . Housin g Randoml y

Selecte dWeanling s Weanling s ar e house d tv o o r thre e pe r cag e b y se xan dtreatmen t i n 7 1/2 " x1 1 1/2 " x5 " polycarbonat e cage s unde r the condition s describe d previousl y

i nTas k 1 . Whe nonl y on e puppe r se xpe r litte r i s selected , non-littermate s o f th e same se xandtreatmen t ar e house dtogether . I f tv o o r fou r animal s pe r se xpe r litte r ar e selecte dthey ar e house di npair s b y se x
andtreatmen t a s littermates . I f thre e animal s pe r se xpe r litte r ar e randoml y

selected , tv

o o f the

m ar e house dtogether . Th e thirdveanlin g wil l b e house dsingly , i f n o othe r litter s fro m tha t treatmen t grou par e t o b e
veane dtha t day , o r paire dvit ha nanima l fro m a differen t litte r o f th e same
se xandtreatment . Al l Tas k 4 animal s ar e hel di nthi s manner , andcontinu e t o receiv e treatmen t unti l 7 4 + 1 0 day s o f age . Tas k 4 . Random

Selectio no f F t Matin g Pair s Rando m number s previousl y assigne dt o selec t pup s fo r th e Tas k 4 study
ar e als o use dt o selec t matin g pair s vhe nthe animal s ar e 7 4 + 1 0 day s old . Th e mal e and femal e pup s vit hthe tv o highes t rando

m number s ar e chose nt o represen t eac hlitter . I t i s preferre dtha t tvent y (20 ) mal e and tvent y (20 ) femal e pup s vithi na grou prepresen t tvent y (20 ) differen t litters . Sinc e thi s ma y

no t alvay s b e possible , a secondmal e (or female ) pupma y b e selecte d fro m a litter . On e va y t o for m pair s i s a s follovs . Animal s ar e paire dP.3 7



### 34

vithi na treatmen t grou pb y matchin g the mal e vit hthe highes t rando m numbe r to the femal e vit hthe highes t rando m numbe r an

ds o on . Siblin g mating s ar e avoided . Fo r example , i f the mal e wit hthe thirdhighes t rando m numbe r i s siblin g t o the femal e wit hthe thirdhighes t rando m number , i t i s paire dwit h the femal e vit hthe fourt hhighes t rando m number . Similarly , the femal e mous e vit hthe thir dhighes t numbe r i s mate dvit hthe mal e vit hthe fourt hhighes t rando m number . Tas k 4 . Breedin g Assessmen t Al l breedin g pair s ar e house da s describe dfo r Tas k 3 . The y ar e separate d afte r seve nday s o r upo nobservatio no f a vagina l copulator y plug , vhicheve r i s first , and house dseparatel y fo r 2 1 day s follovin g conclusio no f the cohabitatio nperiod . Durin g thi s period , fertilit y dat a ar e collecte do n
an y litter s produce da s describe dfo r Tas k 2 an dthe litte r i s sacrificed . Postpartu

m da m veight s ar e measure dafte r deliver y o f the litter . I n addition , al l animal s ar e veighe donthe firs t day o f cohabitatio nandonc e a
vee k thereafte r unti l necrops y an dthe amoun t o f dose dfeed/drinkin g vate r
consume di s monitore do na veekl y basi s unti l necropsy . Fertilit y inde xand matin g inde xi s als o calculated . Randomizatio nan

dNecrops y Th e ? 1 parent s ar e randomize dfo r necrops y an d necropsie da s detaile dfo r Tas k 3 . T o asses s estrou s cyclicity , a vagina l smea r i s obtaine ddail y fo r th e las t 1 2 day s prio r t o sacrific e o f the females . Statistica l Analysi s (Se e page s 12-16. )P.3 8



### 3 5

8 . RESULT S Summary Dat a fro

m RAC B Studie s Th e follovin g sectio ndetail s i novervie v fashio nal l the result s obtaine d fro

m a RAC B study . I norde r to bette r understan d hov thi s dat a i s organize d an

d utilize d i na nactua l study , i t may b e helpfu l t o examin e a n NTI S technica l repor t fro m a RAC B study . 1 . Analysi s o f dosin g sample s (Qualit y Control ) Tas k 1 - Wee k 1 Tas k 2 - Wee k 1 Tas k 2 - Wee k 4 Tas k 2 - Wee k 8 Tas k 2 - Wee k 1 2 Tas k 2 - Wee k 1 6 Tas k 3 - Wee k 2 3 Tas k 4 - Wee k 2 0 Tas k 4 - Wee k 2 4 2 . Summar y

o f bod y veigh t an

d dail y feed/vate r consumptio nfo r Tas k 1 b y dos e an d se x Percen t mortalit y Percen t chang e i nbod y veigh t Estimate d dail y

dos e leve l o f the tes t articl e Body veigh t onday s 0 , 7 an d 1 4 Numbe r o f liv e animal s onday s 0 , 7 an d 1 4 Averag e dail y

consumptio no f dose d feed/vate r fo r vee k 1 an d
vee k 2 3 . Fertilit y

o f pair s durin g Continuou s Breedin g (Tas k 2 ) Treatmen t grou p vs . Fertilit y inde xP.3 9



## 3 6

4 . Reproductiv e performanc e o f fertil e pair s durin g Continuou s Breedin g (Tas k 2 ) b

y treatmen t grou p Litters/pai r Liv e pups/litte r Proportio no f pup s bor naliv e Sexo f pup s bo m

aliv e Liv e pup veigh t - male , femal e combine d Liv e pu

p veigh t adjuste d fo r litte r siz e 5 . Postpartu

m da m veigh t (Tas k 2 ) 6 . Cumulativ e day s t o litte r Tas k 2/pai r 7 . Matin g an

d fertilit y

o f pair s afte r a matin g tria l to determin e affecte d se x

Contro l xContro l Matin g Inde xan

d Fertilit y

Inde xContro l M xTreate d F Contro l F xTreate d M

8 . Reproductiv e performanc e o f fertil e pair s afte r a matin g tria l to determin e affecte d sex . Litters/pai r Liv e pups/litte r Proportio no f pup s bor naliv e Se xo f pup s bor naliv e Liv e pu p veigh t - male , female , combine d Liv e pup veigh t adjuste d fo r litte r siz eP.4 0



### 37

9 . Femal e bod

y an d orga nveight s at necrops y (Tas k 3 ) Actua l an d adjuste d fo r bod y veigh t 

### Body Live r Kidne y 10 . Mal e bod

y an d orga nveight s at necrops y (Tas k 3 ) Actua l an d adjuste d fo r bod y veigh t Bod y (g ) Live r (mg ) Kidne y (mg ) Semina l vesicl e (mg ) Testi s (mg ) Epididymi s (mg ) Caud a epidydimi s (mg ) Prostat e (mg ) 11 . Summary

o f dat a fro

m sper m studie s (Tas k 3 ) Sper m motilit y

(% ) Sper m concentration/m

g cauda l epididymi s Abnorma l sper

m (% ) 12 . Summary

o f dat a fro

m vagina l cytology

studie s (Tas k 3 ) Relativ e frequenc y

o f estrou s cycles/stages/grou p 13 . Matin g and fertilit y o f second generatio nbreedin g pair s (Tas k 4 ) Matin g Inde xan

d Fertilit y

Inde x 14 . Reproductiv e Performanc e o f Secon d Generatio nBreedin g Pair s (Tas k 4 ) Litters/pai r Liv e pups/litte r Proportio no f pup s bor naliv e Sexo f pup s bor naliv e Liv e pup veigh t - male , femal e combine d Liv e pu p veigh t adjuste d fo r litte r siz eP.4 1



### 38

15 . Secon

d generatio

nfemal e bod

y an d orga nveight s a

t necrops

y (Task 4 ) Actua l an

d adjuste

d fo r bod y veigh t Bod y (g ) Liver (mg ) Kidne y (mg ) Righ t ovar y 16 . Secon

d generatio

nmal e bod

y an d orga nveight s a

t necrops

y (Task 4 ) Actua l an

d adjuste

d fo r bod y veigh t Bod y (g ) Liver (mg ) Kidne y (mg ) Semina l vesicl e (mg ) Testi s (mg ) Epididymi s (mg ) Caud a epidydimi s (mg ) Prostat e (mg ) 17 . Summar

y o f dat a o

nsper m studie s (Task 4 ) Sper m motilit

y (% ) Sper m concentration/m

g cauda l epididymi s Abnorma l sper

m (% ) 18 . Summar

y of dat a o

nvagina l cytolog

y studie s (Task 4 ) Relativ e frequenc

y o f estrou s cycles/stages/grou pP.4 2



### 39

19 . Bod

y veigh t dat a (pair ) (F Q animals ) Wee k 0 Wee k 1 Wee k 2 Wee k 3 Wee k 5 Wee k 9 Wee k 1 3 Wee k 1 8 Wee k 2 2 Wee k 2 3 Wee k 2 4 Wee k 2 5 20 . Dail y

feed/vate r consumption/pai r (F Q animals ) Wee k 1 Wee k 2 Wee k 5 Wee k 9 Wee k 1 3 Wee k 1 8 Wee k 2 2 Wee k 2 3 Wee k 2 4 Wee k 2 5P.4 3



### 4 0

21 . Secon

d generatio n bod

yveigh t an

d surviva l dat a (Fina l Tas k 2 litter ) Bod yveigh t o n day s 1 , 4 , 7 , 14 , 2 1 o f ag e an d 10 , 11 , 12 , an

d 1 3 veek s o f ag e 22 . Secon

d generatio n dail yfeed/vate r consumptio n Week s 10 , 11 , 12 , an d 1 3 o f ag e 23 . Th e patholog

yrepor t provide s a summar yo f th e result s o f th

e gros s an

d histopathologi c finding s an d an yconclusion s abou t th e effect s o f th

e tes t chemica l on th e animals ' organs . Th e follovin g table s ar e include d vit h thi s section : 1 . Correlatio n o f gros s an d histopatologi c change s fo r eac h anima l affected
. 2 . Th e incidenc e o f histopathologi c chang e i n th e organ s affecte d b y treatmen t group . Individua l Anima l Dat a 1 . Individua l initia l an d fina l bod

yveight s an

d litte r dat a fo r breeding pair s (Tas k 2 ) 2 . Reproductiv e performanc e o f individua l pair s afte r a mating tria l (Tas k 3 ) 3 . Individua l anima l necrops ydat a (mal e an d female ) (Tas k 3 an d 4 ) Bod y veight s Orga nveight s Sper m parameter s Vagina l cytolog

y(estrou s cycl e length ) 4 . Reproductiv e performanc e o f individua l pair s o f secon d generatio n mic e (Tas k 4 )P.4 4



### 4 1

CONCLUSIO N Th e RAC B protoco l provide s a comprehensiv e approac ht o the questio no f reproductiv e toxicity . Result s fro m studie s o f compound s teste da t tv o laboratorie s indicat e tha t the RAC B protoco l effectivel y identifie s toxicant s affectin g fertility , mating , prenata l viability , feta l grovthi nutero , gende r specifi c prenata l toxicity , toxicit y fo r bot hthe F Q an dF xgeneration , an d toxicit y

durin g th e preveanin g andpostlactationa l period s fo r F xoffspring , i nadditio nt o generalize dtoxicit y an dtreatment-specifi c toxicit y t o organ s
o f interes t i nbot hsexe s o f F Q an dF xgeneration . I naddition , the RAC B protoco l provide s the opportunit y t o observ e progressiv e change s i nfertilit y (vit hsuccessiv e litters ) afte r continuou s exposur e o f monogamou s breedin g pair s t o th e tes t compound . I nshort , throug hthe effort s o f the Nationa l Toxicolog y

Program

an dthei r contractin g laboratories , a valuabl e reproductiv e toxicit y

study desig nha s bee ndeveloped , refined , an dprove nsensitive , effectiv e anduseful . Th e basi c protoco l delineate di nthi s volum e ha s bee n utilize dvirtuall y

unmodifie dfo r the testin g o f ove r 5 0 chemicals . Hovever , thes e procedure s shoul dno t b e vieve da s absolute . A s the result s o f the initia l studie s ar e assessed , i t i s anticipate dtha t pitfalls , problem s and alternativ e o r supplementa l test s vil l become apparen t vhic hvil l resul t i n modification s bot hmino r andsubstantia l t o thi s protocol . Th e intereste dreade r i s urge dto contac t the Nationa l Toxicolog y Program' s Developmenta l an d Reproductiv e Toxicolog y Grou pfo r the lates t RAC B protocol . Severa l RAC B studie s hav e bee npublishe dandthes e ca nb e consulte d i norde r t o se e ho v the dat a i s actuall y organize dint o a publicatio n(Chapi n
e t al. , 1988 , Lam

be t al. , 1985 , Lam be t al. , 1987 , Heinde l e t al . 1989 , and Morrisse y

e t al. , 1988) . I naddition , copie s o f an y o f the fina l report s ca n b e obtaine d throug hNTI S (se e references ) fo r a nomina l fee .P.4 5



## Reproductive Assessmen t B y Continuou s Breedin g Ro wDiagra m - Curren t Desig n

TAS K4 OlfapHn g Aaaaoamart

t P t QaoaTBtlon ) (Contro l * 1 doaoa : group) TASK S Ootonoiiutlo M o l AIIOBIO O 9mm (F , Oofwrotlo * TAS K4 Offsprin g AHOMfflon t P < Oanorallo n (Contra l 4 1 doao d groups )

NECROPS

Y Spor m Moipholog y Vagina l Cytolog y optiona l NECROPS Y Spor m Morphology Vagina l Cytolog y optiona l NECROPS Y Spor m Morpholog y Vagina l Cytolog y optiona l

HtatopatHoteg y Hormon o Aaaay

a Htatopatholog y Hormon o Aaaay

a Hlatopatholog y Hormon o Aaaay

a

FIGURE 1

## REPRODUCTIV E ASSESSMEN TB Y CONTINUOU S BREEDIN G POSITIV E STUDY : Contro l F- H |



# -*» 

# B E

Middl e B E Hig h Qb^ . 

# v

• v 

# ft

M


# V

I v » 

# M C 

# ^ r C C I | 1 TAS K 3 C » H | | 1 * * Creaaovo r

. * *

TAS K1 Dos e Range Findin g TA S K2

Continuou s Breedin g Holdin g Period ; TAS K4

Socon d Generatio n Rearin g

0 2 3 i I r — 1 7 2 0 2 3 i i i 3 1 3 4 

## NEGATIV E STUD

Y Wook a

Contro l ^ S ^ ^ - . * *

Lo wF=3P " 

### •^—*—*—*— r
Middl e R F • ^ * ^ S * 

### Hig h £3 E 

### •^— r M .* *
TASK J Ooa « RangeFindin g TASK 2

Continuou s Breedin g Holdin g TAS K4 Period ! Secon d Generatio n Rearin g

i i i 0 2 3 1 7 - r ~ 2 0 -r — 2 3 Week * 3 4 

### Fig . 2 . Time lin e schemati c o f th e RAC B protoco l fo r positiv e an d negativ e studies . * indicate s sacrifice . * * indicate s complet e necropsy .
'—- • indicate s male s an d female s house dseparatel y an d I— ' indicate s male s an dfemale s house da s breedin g pairs . Arrow s indicat e litter s bor nand M signifie s th e matin g perio dfo r Tas k 3 an d 4 .MILESTON E CHAR T

Majo r Task s i nth e RAC B Protoco l an d th e Associate d Sequenc e o f Event s fo r a Generi c Compound vit ha Theoretica l Star t Dat e o f Januar y 1 

### 4 2

MILESTONE S EVEN T DAT E
QUARANTINE : Animal s Arriv e Ea r Ta g Animal s (en d o f Vee k 1 ) Weig hAnimals , Randomize , an d Assig nRando

m Number s fo r Tas k 1 ( 1 to 2 day s befor e star t o f Task 1 )
TAS K1 : Januar y 1 Januar y

8 Januar y

1 3

Star t Dosing , Weig hAnimals , Weig hNe v Food an

d Wate r (da y 0 o f Tas k 1 ) Weig hAnimals , Weig hNe v an d Ol d Food an

d Wate r (en d Week 1 ) Weig hAnimals , Weigh t Ol d Food an d Wate r (en

d Wee k 2 ) Analyz e Tas k 1 Dat a an d Selec t Task 2 Dos e Group s (afte r Task 1 i s complete ) TAS K2 : Weig hAnimals , Randomiz e fo r Tas k 2 ( 1 to 2 day s befor e star t o f Task 2 ) Star t Tas k 2 - Weig hAnimals , Weig hNe v Food an

d Wate r (da y 0 o f Tas k 2 ) Pai r Animals , Weig hAnimals , Weig hNe v an d Ol d Food an d Wate r (en

d Week 1 ) Weig hAnimals , Weig hOl d Food an d Wate r (en

d Week 2 ) Weig hAnimals , Ne v Food an

d Wate r (star t Wee k 5 ) Weig hAnimals , Weig hOl d Food an d Wate r (en

d Wee k 5 ) Weig hAnimals , Ne v Food an

d Wate r (star t Wee k 9 ) Weig hAnimals , Weig hOl d Food an d Vate r (en

d Wee k 9 ) Weig hAnimals , Ne v Food an

d Wate r (star t Week 13 ) Weig hAnimals , Weig hOl d Food an d Wate r (en

d Wee k 13 ) Separat e Pair s (en d Vee k 15 ) Weig hAnimals , Ne v Food an

d Wate r (star t Week 18 ) Weig hAnimals , Weig hOl d Food an d Wate r (en

d Week 18 ) Wea nF 1 Animal s (veek s 3 to 6 afte r separatio no f pairs ) Januar y 1 5 Januar y 2 2 Januar y 2 9 Januar y 30 - Februar y

2

Februar y

3 Februar y

5 Februar y

1 2 Februar y

1 9 Marc h5 Marc h1 2 Apri l 2 . Apri l 9 Apri l 3 0 May 7 May 2 1 Jun e 4 Jun e 1 1 Jun e 11 - Jul y 1

### 44

REFERENCE S Armitage, P . (1971 ) Statistica l Method s n i Medica l Research . Joh nVile y & Sons , Ne v

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o f Tricresy l Phosphat e i na Continuou s Breedin g Proto l i nSvis s (CD-I ) Mice . Fund , an

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47 , 583-621 . Lamb , J.C , IV , Chapin , R.E. , Teague , J. , Lavton , A.D. , an d Reel , J.R . (1987 ) Reproductiv e Toxicit y o f Fou r Phthalat e Aci d Esters : Structur e Activit y Relationships . Toxicol . Appl . Pharmacol . 88 , 255-269 . Lamb , J.C , I V

and Chapin , R.E . (1985 ) Experimenta l Model s o f Mal e Reproductiv e Toxicology . In : Endocrin e Toxicology , J.A . Thomas , K.S . Korac han

d J.A . McLachlan , eds , Rave nPress , Ne v York , NY , p

p 85-115 .P.4 9



### 4 5

Lamb , J.C , IV , Maronpot , R.R. , Gulati , D.K. , Russell , V.S. , Hommel-Barnes , L. , an d Sabharval , P.S . (1985 ) Reproductiv e an d Developmenta l Toxicit y
o f Ethylen e Glyco l i nthe Mouse . Toxicol . Appl . Pharmacol . 81 , 100-112 . Morrison , D.F . (1976 ) Multivariat e Statistica l Methods . McGraw Hill , Ne v York . Morrissey , R.E. , Collins , J.J. , Lamb , J.C , IV , Manus , A.G. , and Gulati , D.K . (1988 ) Reproductiv e Effect s o f Theophylline i nMic e and Rats . Fund , and Appl . Toxicol . 10 , 525-536 . Nationa l Technica l Informatio nService , U.S . Departmen t o f Commerce , 528 5 Por t Royal Road , Springfield , V A 2216 1 (703-487-4650) . Neter , J . and Vasserman , V . (1974 ) Applie d Linea r Statistica l Models . Richar d Irvin , Inc. , Homevood , IL . Shirley , E . (1977 ) A Non-Parametri c Equivalen t o f Williams' s Tes t fo r Contrastin g Increasin g Dos e Level s o f a Treatment . Biometric s 33 , 386-389 . Soares , E.R. , Sheridan , V. , Haseman , J.K. , an d Segal , M . (1979 ) Increase d Frequencie s o f Aberran t Sper m a s Indicator s o f Mutageni c Damag e i nMice . Mutatio nRes . 64 . 27*35 . Williams , D.A . (196% ) A HgU t o nShirley' s Nonparametri c Tes t fo r Comparin g Severa l Dos e Level s wit ha Zero-Dos e Control . Biometric s 42 , 183-186 . Vyrobek , A.J . and Bruce , V.R . (1975 ) Chemica l Inductio no f Sper m Abnormalitie s i nMice . Proc . Nationa l Academ y o f Sciences , USA , 72 , 4425-4429 .