P. 1



# OAR Box 

# 1846 Prepped by Ollie Stewart

Document

Number: 

# 88 ) IV-A-1 0
Docket Number: 

# A-90-0 7

Printed 11/18/20087:30:17

AM Sheet 88of 93P. 2 

# KJJJ J I I iRA^ggf^g y l98

7 

## -mVr-fl- ID

APPENDI X G

NATIONA L TOXICOLOG

YPROGRA

M
TECHNICA

L PROTOCOL FO

R

SPER M MORPHOLOG

YVAGINA

L CYTOLOG

YEVALUATIO

N I N TOXICITYTESTIN

G FO R RAT S AN

O MIC EP. 3

r w



### TABL E O F CONTENT S

Introductio n- SMVC E Protoco l Technica l Protoco l Sectio n1 : Mal e Orga nToxicit y Studie s inMic e and Rat s IA : Protoco l Co r Mal e Orga nToxicit y Studie s i nMic e an d Rat s IB : Lis t o f Material s Cor Sper m Assay s inMic e an d Rat s IC : BuCfe r an

d Stai nPreparation s (Mous e Studies ) 10 : Buffe r and Stai nPreparation s (Ra t Studies ) IE : Calculatio nProcedur e for Estimatio no f Sperm Densit y

- Mic e and Rat s IF : Summar y

o f Sperm

Evaluatio nProcedure s (Mouse ) 1G : Summar y

o f Sper

m Evaluatio nProcedure s (Rat )
Sectio n2 : Protoco l and Standar d Operatin g Procedure s for

th t Vagina l Cytolog y Assa y i nMie s 2A : Th e EstroCl a Cycl e 2B : Protoco l fo r Obtainin g a Vagina l Smea r from Mic e an d Rat s 2C : Lis t o f Material s fo r Vagina l Cytolog y Assay s 20 : Suggestion s for Improvin g Vagina l Cytolog y Qualit y Sectio n3 : Instruction s for Shippin g Slide s fro m NTPOesignate d Bioassa y

Laborator y

t o SMVC E Laborator y Sectio n4 : Instruction s fo r Shippin g Frose nTeste s fro m NTP Dssignate d Bioassa y

Laboratorie s t o SMVC E Laborator y Sectio nS s Retur no f Slide s fros t SMVCZ-Uborator y

t o EPL/NT P Archive sP. 4

INTRODUCTIO

N

Severa l short-ter

m bioassay s hav

e bee n recentl y

develope d to detec t suspected chemica l mutagens , teratogens , an d carcinogens . Amon g thes e ar e test s whichca n b e use d a s earl y indicator s o f compound s that ma y posses s activity

agains t reproductiv

e function . Studie s o f particula r interes t fo r thi s projec t ar e mal e an d femal e reproductiv e organ toxicity

studies . wyrobe k an

d Bruc e (1975 ) foun d tha t physica l an d chemica l mutagen s ca

n increas e th

e frequenc

y o f abnorma l sper

m i n mice . Simila r studie s hav e als o show

n tha t rat s exposed to mutagen s hav e increased frequencie s of abnorma l sperm . Ficsor an d Ginsber g (1980 ) proposed th e us e o f othe r sper m characteristics , sper m motility , an

d sper m count s in conjunction wit hth e sper m abnormalit y

tes t in mice . In th e presen t study , th

e mal e organ toxicity i s estimate d b

y th e followin

g parameters : sper m motility , sper

m count , an

d testicula r spermati d hea d counts . Th e existenc e o f a typica l estrou s cycl e i n th e guine a pi g
an d th e associate d change s i n vagina l cytolog

y wer e firs t reported b

y Stockard an

d Papanicolaou i

n 1917 . Shortl y thereafter , simila r phenomena wer e reported i n th e rat an

d
mouse . Vagina l smear s obtained fro m thes e animal s correlate d wel l wit hchange s occurrin g i n th e reproductiv

e trac t an

d wit h th e secretion o f th

e ovarian hormones . Th e mos t common cel l type s presen t i

n a vagina l smear ar e polymorphonuclea r leucocytes , nucleated epithelia l cells , an d larg e squamou s-Pi

epithelia l cell s (als o called cornified cells , nucleated , or non-nucleated) . The estrou s cycl e o f th e mous e an

d ra t i s complete d i

n fou r t o fiv e day s (polyestrous) , althoug hth e timin g o f th

e cycl e ma

y be influenced b

y exteroceptiv

e factor s (light , temperature , etc.) . The cycl e i s roughl y divisibl e int o fou r mai n phases : estrus , metestrus , diestrus , an d proestrus . Evaluation o f th

e stagin

g o f th

e estrou s cycl e ca n be use

d to determin e effect s o f toxi c chemical s o n reproductiv

e function i n mic e an

d rats .

REFERENCE S Wyrobek , A.J. , an

d H.R . Bruc e (1975) , Chemica l induction o f sper m abnormalitie s i

n mice , Proc . Nat . Acad . Sci. , 72(11):4425-4429 . Ficsor , G. , an

d L.C . Ginsber g (1980) , Th e effec t o f hydroxyure a an

d mitomyci n C o

n sper m motilit y

i n mice , Mut . Res. , 70:383-387 . Stockard , C.R. , an d G.N . Papanicolaou (1917) , Th e existenc e o f a typica l oesterou s cycl e in th e guine a pi g wit ha stud y o f it s historica l an d physiologica l changes , Am . J . Anat. , 22:225-283 .P. 6

TECHNICA

L PROTOCOL

A detaile d description o f technique s to b e use d b y th e NTP-designate d bioassa y laboratorie s an

d th e NTP-designate d SMVC E laborator

y t o conduc t th

e mal e an

d femal e toxicity , studie s properl y

an d uniforml y

i s provided below . Th e dos e level s fo

r thes e studie s wil l b e designated b

y th e NT P Projec t Office r Co

r th e bioassa

y laborator

y i n collaboration wit hth e NT P Chemica l Manager . Ten eachmal e an d femal e rat s o

r mic e i n thre e o f th

e fiv e dos e group s plu s contro l grou p wil l be utilized . Specifi c selectio n o f th

e thre e dos e group s wil l be based on th

e result s obtained durin g th e firs t 7 0 day s o f th e subchroni c studies . The objectiv e i s t o selec t dose s whic h
ar e not causin

g over t toxi c effects , i.e. , mortality , depresse d weigh t gain , etc . The protoco l to conduc t mal e an d femal e reproductiv e organ toxicity

studie s ha s bee

n divided int o th

e followin

g sections : Section 1 Mal e Organ Toxicity Studie s i

n Mic e an

d Rat s Section 2 Vagina l Cytolog y Assa y i n Mic e an

d Rat s Section 3 Instruction s fo r Shippin

g Slide s Sectio n 4 instruction s fo r Shippin

g Froze n Teste s Section 5 Return o f Slide sP. 7

SECTIO N 1 Protoco l fo

r Mal e Orga n Toxicit y Studie s i

n Mic e an

d Rat s (T o b e conducte d at NTP-designate d bioassa y laboratories )
Th e NT P designate d laboratorie s ar e urged to rea d th e materia l thoroughl y becaus e ther e ar e a fe w ke y difference s betwee n mic e an

d ra t studies . Furthermore , sper m motilit y

i n bot hmic e an

d rat s i s extremel y sensitiv

e t o pf l an d temperature . Reader s may , therefore , fin d tha t w

e have repeatedl y

stresse d tha t th

e correc t p

H an d temperatur e o f th e buffer s ca

n significantl y

improv e th e qualit y

o f the data . A

smal l waterbat hca n be used to keep al l buffer s a t th e correc t temperature . A smal l incubator ca n be used to kee p al l material s a t 3 7 C . A slide warme r shoul d be used to | kee p microscop e slide s an d coverslip s a t th e correc t temperatur e durin

g slid e preparation . A microscop e stag e
warme r shoul d be used durin g slide readin

g (see Sectio n I B Co r lis t o f equipmen t an d supplies) . Section I

A
Th e techniqu e t o conduc t mal e organ toxicity studie s i

n mic e an

d rat s i s stepwis e describe d below :
1 . Th e tes t anima l i s weighe d an d the n sacrifice d according t o the stud

y protoco l a t th

e en d o f th

e 90-da y study . Befor e sacrificing th e animal , b

e sur e tha t th

e buffe r solution , microscop e slides , coverslips , pipe t tips , petr i plates , an

d microscop e stag e hav e been warme d toP. 8

O th e appropriat e temperatur e (3 7 C) . Also , b e sur e tha t th e balanc e i s properl y calibrated . Chec k th e p H o f th

e modifie d Tyrode' s witheg g yol k solutio

n an d adjus t it

. c o 7. 2 wit hHC l (IN ) o r NaO H (IN ) (se

e Sectio

n I C Co r formul a fo

r mous e studies ; 1 0 Co r ra t studies) . 2 . Afte r sacrificin g th e animal , conduc t th e sper m collection a s quickl y a s possible . Us e th e followin

g procedur e t

o remov e th e testi s an

d epididymis :

a ) Pul l th

e lef t tesit s int o th

e abdomina l cavit y b y j graspin g th e fa t surroundin

g th e capu t epididymis . Excis e th

e lef t testi s b

y cuttin g th e va s deferen s | approximatel y

0. 5 c m fro m th e poin t o f attachmen t o f th e cauda epididymis . The capu t epididymi s shoul d be locate d prjLo r to excisio

n s o that it wil l not be clipped accidentall y (se e Figure s 1 , 2 , an d 3) . b ) The intac t lef

t epididymi s i s remove d an d weighe d t

o | th e neares t 0. 1 mg .
3 . Cli p th

e left cauda epididymi s a t th e poin t o f origi n I
o f th e ductu s deferen s at th e dista l en

d an d a t th

e boundar y between th

e bod y an d cauda epididymi s o f th e

- proxima l en

d (Se e Figur e 2) . In rats , cauda epididymi s
can b e seen a s whit e tissue consistin g o f convolute d tubles .
4 . Weig

hleft cauda epididiymi s (mic e only ) t o th e neares t | 0. 1 m g (0.000 1 g) . Recor d th e weigh t o

n th e dat a for

mP. 9

Figur e 1

ine ao f

incieio a

Figur e ZP.1 0

Photograp h o f a re t epididymi s showin g th e site s nfmrremi t o i n th e tex t fo r remova l o f sper m samples : Sit e 2 : proxima l capu t Site s 2s an d 2b : capu t Sit e 3 : Sit e 1 : Sit e 5 : Sit e I : Sit e 7 : Sit e 8 : dista l capu t

mid-corpu s dista l corpu s proxima l caud a dista l caud a ductu s deferen s Sample s coul d no

t b e removed fro m th e initia l segemen t (Sit e 1 )

FICUR E 3P.1 1

(Figur e 4) . I

n rats , th

e cauda epididymi s i s weighe d a s describe d i

n step 5

bbelow .
5 . Thi s ste p differ s fo r mic e an

d rat s s o pleas e rea d thes e instruction s carefully .

a ) Mic e
i Tw o independen t slide s ar e prepared t o estimat e sper m motility . Plac e 8 0 u L o f prewarate

d Tyrode' s Solution (modified ) o n eac hmicroscop e slide . Slide s shal l be placed o n a slid e warme r o se t at 3 7 C an

d retaine

d at thi s temperature . i i Hol d th

e left cauda epididymi s wit hforcep s an d mak e a smal l incisio n near th

e cente r o f th

e cauda wit ha sharp , clean surgica l blade . ii i Appl y

gentl y

pressur e o

n ch e cauda l surfac e s o tha t a smal l epididyma l tubul e i s expose d o r cauda l flui d begin s to oos e out int o th e Tyrode' s solution o

n th e prewarme d slid e (Caution : D o not appl y too muchpressur e sinc e ther e i s a n ampl e
amoun t o f sper

m i n a piec e o f cauda equivalen t to 1/ 2 o f a pinhead . Us e o f excessive amount s o f

* cauda l flui d fo r th e sper m motilit y

count s ma

y significantl y

lower sper

m densit y

evaluations) . Alternately , th

e cauda epididymi s i s place d int o
a petr i plat e (1 5 X6 0 mm ) containing 2 m L o f prewarme d phosphat e buffere d salin e (PBS) . Th eP.1 2

STUDY : OAT

A SHEE T • MOUS E SPER M STUDIE S

STUDY* : 

### "in KeviM U

CODE/ANIMA L # : ANIMA L WEIGHT : CAUDA L WEIGH T (RIGHT) :

SPER M McrnuT Y Hel d motil e nonmotil e 

# ± TREATMENT : DATE : LAB *
j EPIDIDYMA L WEIGHT (RIGHT) : j TESTICULA RWEIGH T (RIGHT) :

Tota l Motile :

SIGNATURE : ObserverA Dat aTota

l Nonmotile :

fiel d motil e nonmotil e 

### 6 7

r-> 

## a 9 1 0 % Motilit y

SIGNATURE : Observera Dat e
Sper m Coun t
* Vol . Salin e (caudain 2 m L PBS )
* * Vo L Cauda l Suio . (O J m L in 2 mL)

" "Soerm Concentration : XlO 6 Pe rg cauda l tissu e SIGNATURE : 

### m L
m L Coun

tI Count ! Mea

n |

Dat e "Tota l motile/total motil e * Noaaoci k XlUO t ^ Laav aMask tiakn

autba rvoiaa ai

auani . Oo aotdilute i

f the aaatna

ru f »pcr apet ^ Mcoedar

y squar e i s le m tka e 15 . . *X(J0000) Y (t o th

aaaara t0. 1 X 10° ) ( X - #sper m couaiw i Y - 4Uiuiu afacuir . whic S i aI U cccauda l wtifh i i agraan ) DILUTIO N FACTO RWILL CHANG E I F OIFFERE.NT VOLUME * O F DILUTEN T AR E USED . Flgur a4 9P.1 3



### caud a i s the nhel d inplac e wit hforcep s and a very smal l piec e (no t mor e than1/2 0 o f th e tota l cauda l size ) i s remove d wit heithe r a shar p scalpe l or blad e (se e Figur e 5) . Thi s tissu e i s the nplace d int o the tyrode s solutio no na prewarme d slide . Note : Surgica l blade s ma y b e cleane d wit hsaline^betwee nanimals . iv . I f necessary , the cauda l flui d i s furthe r choppe d
o nthe slide s wit ha pai r o f needle s o r shar p blades .

v . Plac e a warm

coversli p (2 4 X4 0 mm ) o nth e slide . Maximu m sperm motilit y

i s obtaine d i f th e remova l o f cauda , weighing , and preparatio no f sperm suspensio n i s don e expeditiousl y (approximatel y 2- 4 minutes) . vi . Two slide s ar e prepare d simultaneousl y fo r estimatio no f sper

m motility . vii . Th e sper

m motilit y

i s estimate d withou t an y furthe r dela y

a s illustrate d i nSte p 6 . viii . Plac e remainin g cauda l tissu e int o a petr i plat e (I S X6 0 mm ) containin g 2 mL o f phosphat e buffere d salin e (PBS) . ix . Th e reaainin g cauda l tissu e i s gentl y choppe d (onl y afte r th e sperm

motilit y

count s hav e bee n performed ) i nthe petr i plat e wit htw o scalpel s

1 0P.1 4



### ECO.MMENOE

D PORTION

O F CAUDA ADEQUAT E FO

RSPER M MOTILIT YEVALUATIONS .

REPRESENTATIO N

O F DISTA L CAUOA

I N .MOUS E (Sampl e Only ) Figur e 5

11P.1 5

unti l it s content s ar e released . Th e petr i plat e i s swirled severa l time s an d incubated fo

r 1 5 o minute s at 3 7 C . I t allows aggregate s o f sperm , whic hca

n b e see n a s piece s of tissue i n ch e suspension , to brea k up . Petr i plate s shoul d remai n covered durin g th e incubation period . b . Rat s Onl y
i . Th e cauda epididymi s i n rat s ca

n be seen a s whit e tissu e consisting o f convolute d tubules . Afte r clippin g th e cauda a t th

e poin t o f origi n o f the ductu s deferen s (Figure s 1 , 2 , an d 3 ) fro

m the
cut en d o f th

e cauda (dista l cauda) , a ver y smal l sper m sampl e (not greate r tha n th e siz e o f a pinhead ) i s removed wit hth e ti p o f th

e scalpe l blad e an

d placed on a prewarme d slid e wit h8 0 u l o drop s o f tes t yol k buffe r (3 7 C) . D o no t allo

w th e sper m t o dr y on th e scalpe l blade . ii . A coversli p (2 4 X4 0 mm ) i s placed o n th e slid e afte r gentl e mixin g o f sper

m i n th e buffe r solution . Tw

o suchslide s ar e prepare d fo r estimatio n o f sper m motilit y

a s describe d i

n Ste p 6 below . iii . Afte r motilit y ha s been estimated , th e remaining dista l cauda i s weighe d to th e neares t 0. 1 mg . Th e weigh t i s recorded o n th e appropriat e dat a shee t (Figur e 6) .



### 1 2P.1 6

DAT A SHEE T • RA

T SPER M STUDIE S

STUDY : CODE/ANIMA L # : ANIMA L WEIGHT : 'CAUDA L WEIGH T (RIGHT) : STUD Y # : TREATMENT : RaviM d DATE : LAB# : "If f

_ g EPIDIDYMA L WEIGH T (RIGHT) :
aTESTICULA RWEIGH T (RIGHT) :

Hel d motil e nonmotil e SPER

M MOTILIT

Y 

## t •y 

### 3 4

- Total Motile : 

### . m 

### 8

SIGNATURE : Observer A Date Total Nonmotile :

fiel d motil e nonmotil e 6 7 

### a 9 1 0 c /t Motilit y

SIGNATURE : Observer B " Dat e
Sper m CQUOI

" Vol . Salin e (caud ai

n 1 0 m L PBS )
* 'Vol . 2nd dilutio n (O

J m L i n 2 mL )

' * Sper m Concentralion : Pe

r g caudal tissu

e SIGNATURE : Observe r m L
m L XlO *

Dat e Coun

tl Count* ? Mea

n

..Pau l caud

aoat * Tota l motile/tota l motil e * auamuti k X IUU* . ^ m Laav a Mas k uak u otte r voiaa a i a \g*4 X(5O0UU)Y (t o th a aeam i 0. 1 X til 0 ) ( X * •ipcn n uiuaict l Y •dilwiu a lactor . whn.l i ia 5 0 cccamia l weigh t i a gram a I DILUTIO N FACTORWIL L CHANG E I

F DIFFEREN T VOLUME S AR E USE D Figur a6 1 3P.1 7



### iv . Thi s ste p i s performe d onl y afte r sperm

motilit y ha s bee nestimated . Th e dista l caud a i s hel d i n plac e wit ha scalpe l and gentl y choppe d wit h anothe r shar p scalpe l i n1 0 mL o f 0.9 % salin e o r o PB S ina petr i plat e incubate d a t 3 7 C fo r I S minutes . 6 . Estimatio no f Sperm Motilit y

- thi s ste p i s commo nfo r bot hrat s and mice . i . T o estimat e sperm motility , th e viewe r determine s th e actua l numbe r o f motil e and nonmotil e sper m fo r eac hfiel d o f vision . A smal l han d counte r ma y b e use d t o coun t the numbe r o f motil e an d nonmotil e sperm . ii . Th e fina l motilit y i s base d o ntenindependen t field s an d observation s mad e b y tw o independen t viewers . iii . Perso nA

evaluate s th e motilit y i nfiv e independen t field s and , similarly , Perso nB evaluate s th e motilit y fo r anothe r fiv e independen t fields . Bot hviewer s mus t us e

or 

### independen t slide s and , whe npossible , independen t microscopes . iv . Sperm

ar e considere d motil e i f they show an y movemen t a t all . A 40 Xobjectiv e i s recommende d

1 4P.1 8

t o evaluat e sper

m motility .

v . Percen t motility decrease s wit htime ; therefore , evaluatio

n i s critica l an

d mus t b

e accomplished
a s quickl y

a s possibl e (2- 4 minutes) . o vi . A microscope stage warme r (3 7 C ) i s neede d fo r sper m motility

evaluatio

n an d to maintai n th

e sper m suspensio

n a t optimu

m temperature . Sper m ar e ver

y sensitiv

e to temperatur e an d p H changes . Extrem e car e mus t b

e take n t o keep al l material s at 37° C o n a slide warme r and/or in a n incubator . Th e pf l o f th e Tyrod e solution shoul d b e checked routinel y an d adjusted whe n an d if necessary . Thes e factor s ar e ver y important t

o obtai n a

n accurat e estimatio

n o f sper

m motility . vii . Averag e sper

m motility

reading s shoul d be approximatel y

75 % o r highe r i

n contro l mic e an

d rats . I f sper

m motility

i s considerabl y

lower tha n 75% , ther e i s som e proble

m an d EHR T personne l shoul d be consulted a t (606 ) 278-060 3
o r (606 ) 276-1436 . Ourin g the I S minut e incubatio n period , th

e lef t testi s i s removed , weighe d an d frose n fo r testicula r spermati d head-coun t a s described below . i . Labe l eac hvia l wit hth e appropriat e laborator y
name , anima l number , tes t articl e dos e group , i,f t teste s (o

r righ t testis) , an

d dat e o f collection . Th e followin

g glas s vial s an

d rack s ar e



### I SP.1 9



### recommended : Vendor : VW

R Scintillatio nVia l Rack ; Catalo g No . 66022-52 5 Scintillatio nVial s 2 8 X6 1 mm ; Catalo g No . 66021-45 3 o r Sampl e Vial s - 2 8 X6 0 mm ; Catalo g No . 66011-40 5 ii . Remov e th e lef t testes , weig hit , an drecor dth e weight . 

### iii . Plac e th e teste s i nth e appropriatel y labelle dvia l
an dfreez e o ndr y

ice . iv ; Whe nconvenient , transfe r frose nsample s t o a

o freeze r (-2 0 C ) fo r storag e unti l read y fo r shipmen t t o EHRT . 8 . Sper m

Coun t - Afte r th e 1 5 minute s incubatio nperiod , a sper m coun t i s performe dbu t th e sper m suspensio nmus t b e furthe r diluted . Du e t o a significan t differenc e i n th e cauda l weigh t fo r rat s an dmice , part s o f th e dilutio nprocedure s fo r th e tw o specie s ar e differen t
an dshoul db e noted . i . Swir l th e petr i plat e t o evenl y distribut e th e sperm . Z nth e even t th e sper m suspensio ni s no t uniform , gentl y cho pth e caud a unti l a unifor m suspensio ni s obtained , incubat e furthe r i f necessary . Transfe r 0. 5 m L (usin g a 1. 0 m L pipet ) t o a tes t tub e (1 6 X 10 0 mm ) containin g 2 m t o f th e phosphat e buffere d saline . Th e ra t suspensio nshoul db e gentl y pipette d

I S2 0 time s t o brea k u p sper m aggregates . ii . Gentl y

an d thoroughl y

mi xth

e suspension wit ha Vortexmixe r fo

r 2 seconds . I f a vorte xmixe r i s no t available , gentl y

agitat e th

e sper m suspension the

n gentl y pipe t (wit

ha pasteu

r pipet ) approximatel y 5 time s t o thoroughl y

mi xth

e suspension . iii . The volum

e o f PB S ma

y b e varied accordin

g t o th e density o f sper

m suspension . The number o f sper m pe r secondar

y squar e (see Step 8vi i below ) shoul d not b e les s tha

n 5 o r mor e tha

n IS . iv . Th e volum

e o f sper

m suspensio

n an d PB S solution used fo r th e sper m count shoul d b

e recorde

d on th

e dat a for m i f differen t fro

m th e suggested volumes .

v . The sper

m use d fo r thi s assa

y shoul d b

e killed b

y placin g th e tes t tub

e unde r hot , runnin g wate r fo

r
on e minut e o

r a n ice bat hfo

r 5 minutes . vi . The sper

m suspensio

n i s gentl y

mixed b

y pipettin

g wit ha Pasteu

r pipe t 5 time s an d placed o

n a hemacytomete r (Figur e 7) . vfri . Th e shin

y surface s i

n th e middl e o f th

e hemacytomete r have microscopi c grid s consistin g o f heav

y line s whic hdivid e th

e fiel d int o 2 5 large squares . Withi n
eac ho f th

e large square s ar e 1 6 smalle r squares . Th e coversli p doe s not touc hth e gri d are a whe n placed ove r th

e middl e portion .


### 1 7P.2 1

Sid e Vie w

STANOAR

O HEMOCYTOMETE R CHAMBE R

Secondar

y Square 

# T
1 m m (

Tertiar y Squar e



# _ L
CORNE R SQUAR E

MIOOL E SQUAR E

Volum e Occupie d b y a Primar y Squar e •2. S X 10* ' m L Volum e Occupie d b y a Secondar y Squar e » * X io" 5 m L Volum e Occupie d b y a Tertiar y Square * 1 X 10* * m L Since sper m coun t i s made i n '5 ' secondary squares , i t i s equivalen t to * S X 4 X 10" " m L o r 2 X 10" r mL .

Figur e 7 

### 18P.2 2

viii . Microhematocri t capillar y tube s ar e use

d t o plac e suspensio

n o n th e hemacytometer . The sper m suspensio

n i s place

d o n th e hemacytomete r b

y touchin

g th e ti p o f th

e capillar

y tub e to th e angl e formed b

y th e coversli p an

d countin

g chambe r o f th

e hemacytometer . The are a unde r th e coversli p shoul d
no t b e overfilled . ix . Tw o count s ar e performed usin g separat e sample s o f th e diluted suspension . Bothslide s fo r sper m coun t
mus t b e prepared afte r thoroug hmixin g o f th

e sper m suspension . Consistency i n th e preparation o f the sper m sample s fo

r densit y

determination s i s essentia l to obtain^accurat e results .

x . T o perfor

m th e counts , th

e countin

g are a i s locate

d unde r lo

w powe r (10 Xobjective ) an d the n switched to th e 20 Xobjective fo r counting . It i s not necessar y t o coun t ever

y cel l i n ever y square . The number o f sper m i n five secondar

y square s ar e counted . Sper m lyin g o n th e line s o

n th e botto m an d righ t side s o f th e larg e square s ar e included s o tha t the y wil l no t b e counted twice . xi . In cas e ther e ar e les s tha n fiv e (5 ) sper

m per secondar

y squar e an

d i t i s not possibl e to mak e a ne w (les s diluted ) suspension , coun t sper m in tw o (2 ) tertiar y squares . Thi s chang e mus t be recorded o n



### 13P.2 3

Appendi x G MODIFICATIO N T O TH E APRIL , 198 7 SO

W Revise d Jun e 198 9



### th e dat a snee tan

d take n int o consideratio n wrui a estimatin g sper

m density . xii . Calculation s fo rdeterminin g sper m densit y als o vary fo rmic e an

d rat s a s explaine d below : 3 a ) Th e viewe r ha s counte dth e sperm i n 1/5 0 n

m volume ; b ) Th e dilutio nfacto r fo r th e cauda l sper m i s th e fina l dilutio no f eauda l weigh t (normall y fo r mic e i t i s 1 0 cc/eauda l weight , an d fo r rat s S O cc/cauda l weight) . Thes e value s appl y onl y whe n th t suggeste dvolume s ar e used .

c ) Th e numbe r o f cell s counte dmultiplie db y th e 3 volum e fractio n(1/5 0 m m ) csunte dmultiplie db y th e dilutio nfacto r shoul dequa l th e numbe r o f cell s pe r cubi c millimeter . d ) Sper m

concentratio ni s normall y expresse di

n cubi c centimeter s o r millimeter s o f gra m tissu e weight . Sper m

densit y

i n th e contro l sic e i s 6 6 generall y

betwee nSOO X 1 0 an d 100 0 x1 0 sper m
pe r gra m

cauda l tissue ,
q . Tj*>e>4e*

Q
* Sper m Hea dStainin

g Procedure : wa s completel y delete d12/C 7 Slide Labelling , codin g an d shipment wa s deletedfro m thi s sectio

n12/87 . * * Th e requiremen t fo

r collectio

no f plasm

a sample s fo

r hormone determinatio n wa s deleted5/89 .

2 CP.2 4



### iO . A perso nfro

m the NTP-designate dSMVC E laborator y wil l visi t al l bioassa y

laboratorie s conductin g thes e studie s t o asses s thei r abilit y t o perform

thes e bioassay s and provid e technica l assistance , i f necessary . Contac t Dr . Gulat i for technica l question s (606 ) 276-1436 .

2 1P.2 5



### Sectio nI B
Lis t o f Material s for Sper m Assay s i

nMic e and Rat s (To be supplie d b

y the bioassa y

laboratory )
1 . Scalpels , scissors , an d forcep s 2 . Petr i plate s (1 5 X6 0 mm ) 3 . Pasteu r pipet s (5-3/4" ) 4 . Pre-cleane d microscop e slide s (3 " X1" ) 5 . Coverslip s (2 4 X4 0 mm ) 6 . Hemacytometer s and coverslip s (improve d Neubaur , 0. 1 mn deep ) 7 . Pipet s (1. 0 and 5. 0 mL ) an d pipetter s 8 . Tes t tube s (1 6 X10 0 mm ) 9 . Modifie d Tyrode' s Solutio n(Se e Sectio nIC ) 10 . Tes t Yol k (Se e Sectio nID ) 11 . Phosphat e Buffere d Salin e 12 . Protectiv e Clothin g 13 . Incubato r o r slid e warme r 14 . Microhematocri t capillar y tube s 15 . Ban d tall y

counte r (ENM-429 0 or Cla y Adam s 4300 ) 16 . Stag e warme r for th e microscop e 17. . Pipe t tip s 18 . Micro-pipetto r 19 . Scintillatio nVial s 2 8 X6 1 m m o r Sampl e Vial s 2 8 X6 0 m m 20 . Scintillatio nVial s Rack s

2 2P.2 6

Sectio n I C
Buffe r fo

r Sper m Motility

Preparation s (Mous e Studies )
1 . The diluen t (Modifie d Tyrode' s Solution ) ca n b e prepare d (Se e "a " below ) o

r purchased fro

m Environmenta l Healt h Researchan

d Testing , Inc. , Lexington , Kentuck y (Se e "b " below) .

a ) T o mak e 120 0 m

L .o f modified Tyrode' s solution , brin g th e followin

g u p to 100 0 m

L wit hdistille d deionize d water : Ingredient s NaC l 

### KC 1 CaC l .2B 0 2 2 Naf l P O . H 0 2 4 2 MgC l .6 B 0 2 2 NaflC O 3 N a lactat e N a pyruvat e Glucoa e Pina l Concentration 99.2 3 2.6 8 1.8 0 0.3 6 0.4 9 25.0 0 25.0 0 O.S O 5.5 6 

### m M
m M
m N O N
m N
m N 

### m N
m N m N Amount/Lite r

5.80 1 g/ l 0.20 0 g/ l 0.26 5 g/ l 0.05 0 g/ l 0.10 0 g/ l 2. 1 g/ l 2.80 1 g/ l 0.05 5 g/ l 1.00 1 g/ l
Ni xthoroughl y

unti l al l chemical s ar e properl y dissolved . Then ad d 20 0 m L o f eg g yol k withou t the yol k membranes . Shake unti l yol k i s i n solution . Thermoprecipitat e th e solution b

y boilin g fo r 3 o minute s at 92-9 5 C . Filte r throug h4 layer s o f cheesecloth . Centrifug e th e filtrat e a t 10,00 0 rp m



### 2 3fo r 2 0 minutes . Filte r throug hWhatman 1 1 paper . Ad d 0. 7 g/10 0 m

L o f percen t o f D-anhydrou s glucos e t o th e solution t

o rais e th

e osmolarity . Measur e th e osmoti c pressur e an d i t shoul d b

e a t 32 5 mOsm/ L (accurac

y withi n 5 mOSm/L) . Adjus t th e p H t o 7. 2 wit hI N HC l o r I N NaOB . A

t thi s tim

e ad d penicilli n (10 0 u/mL ) an

d streptomyci n (5 0 ug/mL) . Aliquo t th e buffe r int o 5 o

r 1 0 m L volumes . Sinc e thi s buffe r i s easil y contaminated , it i s recommended tha t al l step s afte r thermoprecipitation b e conducte d unde r ascepti c atmospher e when possible .

o Stor e a t -2 0 C . Prior t o eachusage , th e buffer' s p B shoul d be taken an d adjusted t

o 7. 2 i f needed . I f o th e buffe r i s kep t at 3 7 C (incubator temperature ) fo r lon g period s o f tim

e (1- 2 hours ) then agai n the p B shoul d be take

n an d adjusted i f necessary . b ) Th e Modifie d Tyrode' s Buffe r t o suspen

d mous e spermatozo a ca

n be purchase d fro m Environmenta l Healt hResearc han d Testing , Inc . Siz e Uni t Price * 5 m L $5.0 0 1 0 m L $7.0 0

Eac hlot o f modifie d Tyrode' s Buffe r wil l b e preteste d fo

r variou s parameter s o f mal e organ •Thi s pric e doe s no t include th e shippin

g an d handling charges .

* » 

### 4P.2 8

toxicity

studie s i

n mic e befor e shippin g t o individua l laboratories . Modified Tyrode' s buffe r shoul d be froze

n upo n receipt , thawed , an d th e p H adjusted immediatel y befor e us e (p

H 7.2) . Al l request s fo

r obtainin

g Tyrode' s Buffe r shoul d b e addressed to :
Dr . O.K . Gulat i Environmenta l Healt hResearchan d Testing , Inc . 251 4 Regenc

y Roa d Lexington , K

Y40S0 3 (606 ) 276-143 6



### 2 5P.2 9

Sectio n I D
Buffe r an

d Stai n Preparation (Ra t Studies )
1 . Th e diluen t (Tes t Yol k Buffer ) t o suspen

d ra t spermatozo a ca

n b e purchased fro

m Environmenta l Healt h Researc han

d Testing , Inc . Siz e Uni t Price * 5 m L $5.0 0 1 0 m L $7.0 0

Eachlot o f Tes t Yol k buffe r wil l b e preteste d fo

r variou a parameter s o f th e mal e organ toxicit y studie s i

n rat s befor e shippin g to th

e individua l laboratories . Tes t Yol k Buffe r shoul d be frosen upon receip t an d thawed immediatel y befor e use . Al l request s fo r obtaining Tes t Yol k Buffe r shoul d be addresse d to :

Dr . O.K . Gulat i Environmenta l Healt hResearc han d Testing , Inc . 251 4 Regency

Roa d Lexington , K

Y4050 3 (606 ) 276-143 6
2 . Tes t Yol k Buffe r ca n b e prepared uain

g th e followin

g ingredient s pe r liter :
TES* * 14. 5 g TRIS** * 3. 5 g Glucos e 2. 0 g *Thi s pric e doe s no t include th e shippin

g an d handling charge s **(N-tris(hydroxymethyl)methyl-2-aminoethan e sulfoni c acid ) * * *Tr is(hydroxymethy1)aminomathan e



### 2 5P.3 0

Skim Mil k Powder 33. 0 g Sodiu m Citrat e 6. 0 g
Mi xth e ingredient s i

n 70 0 m L o f distilled deionize d water . Adjus t th

e fina l volum

e to 80 0 mL . Ad

d 20 0 o L
o f eg g yol k withou t th

e yol k membranes . o Thermoprecipitat e th e solutio

n b y boilin g at 92-9 5 C fo

r 3 minutes . Filte r throug h4 layer s o f cheesecloth . Centrifug e th

e filtrat e a t 10,00 0 rp m fo r 2 0 minutes . Filte r throug

hWhatma

n # 1 paper . Aliquo t int o 5 o r 1 0 o m L volumes . Stor e at -2 0 C unti l furthe r use . p B wil l be 7.1-7.2 . 3 . Phosphat e Buffered Saline i s prepared b y usin g th e followin

g ingredient s per 100 0 mL :
NaC L 

### KC 1 K H P O 2 4 N a BP O 7 H O 2 4 2 CaC l 

### 2 N g C I 6 H 2 2 Fina l pa • 7. 2 8. 0 g 0. 2 g 0. 2 g 2. 1 g 0. 1 g 0. 1 g

Nlx'th e content s i n 95 0 m L o f distille d deionize d water . Adjus t th

e pH t o 7.2 . Brin g th e fina l volum

e t o 100 0 mL . Filte r sterilization i s recommended .



### 27P.3 1

Sectio n I S Calculation Procedur e fo r Estimation o f Sper m Densit y
Mic e Suppos e th

e mean number number of sper m i n "5 " secondary

- 5 square s i

n mic e * 20 . The number o f sper m i n 2 X1 0 m

L o f dilute d sper

m suspension > 20 . Th e number o f sper m per m L o f dilute d suspension * 2 0 • 1 X106/m

L

- 5 2 X1 0 Sinc e th r dilute d suspension represent s one-fift ho f th e origina l suspension * i f th e standard dilution o f 0. 5 m L i n 2
m L wa s used , th

e number o f sper

m per m L o f th

e origina l 6 6 suspension » 5 X( 1 X1 0 ) » 5 X1 0 .
e* Volum e o f th

e origina l suspension * 2 mL . 6 Tota l sper

m i n th e origina l suspension «2X(5X10.)«1 X 7 1 0 /suspensio n » number sperm/cauda .
I f cauda weigh t * 0.03 0 g , tota l sper m i n 0.03 0 g o f eauda l 7 tissu e » 1 X1 0 .
Tota l sper

m pe r "g " o f cauda l tissue »

- 7 1 X1 0

— — —

a 3.3 3 x10 8 sperm/m g cauda . 0.03 0 Thi s valu e ca

n als o be estimate d b y th e followin

g formula :
Mea n No . o f sper

m in 5 secondar

y square s

— — ..... . ...... . . x50,00 0 X1 0 (dilution facte r cauda l weigh t i

n g



### 2 3Calculation Procedur e fo r Estimatio

n o f Sper

m Density

Rat s Suppos e th

e mea n number number o f sper m i n "5 " secondar

y

- 5 square s i

n ra t * 20 . Th

e number o f sper

m i n 2 X1 0 m

L o f diluted sper

m suspensio

n » 20 . The number o f sper m per m L o f diluted suspensio

n » 2 0 » 1 X106/m

L

- 5 2 X1 0
Sinc e th

e diluted suspensio n represent s one-fift ho f th e origina l suspension , i f th e standar

d dilution o f 0. 5 m L in 2
m L wa a used , th

e number of sper

m per m L o f th e origina l 6 6 suspension »SX(1X10)*5X10 . Volum e o f th

e origina l suspensio n * 1 0 mL . 6 Tota l sper

m in th e origina l suspensio n * 1 0 X( 5 X1 0 ) » 5 X 7 1 0 /suspension • number sperm/cauda .
I f cauda weigh t » 0.15 0 g , tota l sper m i n 0.15 0 g o f cauda l 7 tissue • 5 X1 0 .
Tota l sper

m per "g " o f cauda l tissue * 7 5 X-1 0

——— — «"3.3 3 X10 8 sperm/m g cauda . 0.15 0 Thi s valu e ca

n als o be estimated b y th e followin

g formula :
Mean No . o f sper

m i n 5 secondar

y square s

...................... . — - x50,00 0 X5 0 (dilution fac t cauda l weigh t i

n g



### 2 9P.3 3

SECTIO N l. G Summar y o f Sper

m Evaluation Procedur e - Mous e
o 1 . Plac e instrument s an d glasswar e i

n oven a

t 3 7 C . (TT ) o 2 . Put Tyrode' s i n wate r bathat 3 7 C . (TT ) 3 . p H o f Tyrode' s shoul d b e 7.2 . (TT ) 4 . Weighanima l (NT) , recor d weigh t (NT) , bleed anima l (TT) , kil l anima l (NT) , an

d bleed agai n when required (TT) . 5 . Excis e lef

t testi s an

d epididymis . Remove intac t epididymis , weigh( an d recor

d t o neares t 0. 1 mg . (NT ) 6 . Cut of f left cauda , weig ht o 0. 1 m g an d record . (NT ) | 7 . Plac e 8 0 u l war m Tyrode' s o

n eacho f 2 slides . Plac e sper m th e siz e o f hal f a pinhead o n th e slide s an

d gentl y

mi xwit hTyrode's , coversli p an

d do motilit y

(40X) . (TT ) 8 . weighleft testi s an d record . (NT ) I 9 . Plac e remainin g cauda i

n petr i dis hwit h2 m l PBS , gentl y chop , swirl , cover , an

d incubat e fo

r I S minute e a t 37oC . (TT ) 10 . Remove petr i dish , ensur e homogeneity , an d transfe r 0. 5 m l sampl e t

o tes t tub

e containing 2 m l PBS . (TT ) 11 . Kil l sper

m b y holdin g tes t tub

e unde r ho t wate r fo r 1 minute . (TT ) 12 . Pipett e S time s an d plac e sampl e o

n hemacytomete r fo r coun t (20X) . Repipett e an d plac e a secon

d sampl e o

n anothe r hemacytomete r fo r a secon

d count . (TT ) Note : Coun t sper m i n 5 secondar

y square s an d i f les s tha

n S pe r square , then coun t sper m in 2 tertiar

y square s an d recor

d change .

Note : N T • Necrops y Technicia n T T * Toxicolog y technician



### 3 0P.3 4

EQCJIPMEO

T NEEDE D 

### Sharp s Tyrode' s Petr i Oishe s Scalpel s Slide s Coverslip s Oven PB S Pape r towel s 1 N NaOH Slide Warme r Pipettor s Pipett e tip s Pasteu r Pipette s Garbag e Bag s Hemacytometer s Tes t Tube Rack Tes t Tube s Timer s Waterbat h Pencil s 1 N HC l Tes t Tub

e Label s Stag e Warme r Counter s Syringe s (1 , 3 , an d 5 ml ; Dat a Sheet s Pipette s ( 5 an

d 1 ml ) Erase r Paper Tes t Tube Brus h Ki m Wipe s p H Pape r Thermometer s p H Mete r Parafilm



### 3 1SECTIO N I. H

Summar y o f Sper

m Evaluation Procedure s - Ra t

o 1 . Plac e instrument s an d glasswar e i

n oven a t 3 7 C . (TT ) o 2 . Put Yol k i

n wate r bathat 3 7 C . (TT ) 3 . Weig

hanima l (NT) , recor d weigh t (NT) , bleed anima l (TT) , kil l anima l (NT) , an

d bleed agai n when required (TT) . 4 . Excis e lef

t testi s an

d epididymis . Remov e intac t epididymis , weighan

d recor d t o neares t 0. 1 mg . (NT ) 5 . Cu t of f left cauda , weig ht o 0. 1 m

g an d record . (NT ) 6 . Plac e 8 0 u l o f war m yol k o

n eacho f 2 slides . Plac e sper m sampl e th

e siz e o f a pinhead o n th e slide s an

d gentl y

mi xwit h yolk , coverslip , an d do motilit y

(40X) . (TT ) Note : Ther e shoul d be 3 Xa s man

y motil e compare d t o nonmotil e sperm . 7 . Weighleft testi s an d record . (NT ) 8 . Plac e remainin g cauda i

n petr i dis hwit h1 0 m l PBS , gentl y chop , swirl , cover , an

d incubat e fo

r I S minute s a t 37oC . (TT ) 9 . Remove suspension fro m oven an

d pipett e 2 0 time s an d remove connective tissue . (TT ) 10 . Plac e 0. S m l sampl e i n tes t tub

e containing 2 m l PBS . (TT ) 11 . Kil l sper

m b y holdin g tes t tub

e unde r ho t wate r fo r 1 minute . (TT ) 12 . Pipett e S time s an d plac e sampl e o

n hemacytomete r fo r coun t (20X) . Repipett e an

d plac e a secon

d sampl e o

n anothe r hemacytomete r fo r a secon d count . (TT ) Note : Coun t sper

m i n 5 secondar

y square s an

d i f les s than 5 pe r square * then coun t sper m i n 2 tertiar

y square s an

d record change .

Note : NT * Necrops y Technicia n T T » Toxicolog y Technicia nEQUIPMENT NEEDED P.3 6

Sharp e Yol k Petr i Dishe s Scalpel s Slide s Coverslip s Ove n PB S Pape r Towel s Slide Warme r Pipettor s Pipett e tip s Pasteu r Pipette s Garbag e Bag s Hemacytometer s Tes t Tube Rack Tes t Tube s Timer s Waterbat h Pencil s 1 N HC l Tes t Tube Label s Stag e Warme r Counter s Syringe s ( 3 an

d 2 0 ml ) Dat a Sheet s Pipette s Eraser Paper Tes t Tube Brus h Ki m Wipe s p H Paper Thermometer s p H Mete r Parafilm



### 3 3P.3 7

Sectio n 2 A

Protoco l an

d Standar

d Operatin

g Procedur e fo

r th e Vagina l Cytolog

y Assa y i n Mic e (T o b e conducted at NTP-designated bioassa y laboratories )
Th e followin

g informatio

n provide s a quic k overvie w o f th e estrou s cycl e an d describe s th

e technica l protoco l fo r th e vagina l cytolog

y assay . Als o provided ar e explanatory figure s an

d a lis t o f materials .
Th e Estrou s Cycl e Th e existenc e o f a typica l estrou s cycl e i n th e guine a pi g an d th e associate d change s i n th e vagina l cytolog

y wer e firs t reported b

y Stockard an

d Papanicolaou i

n 1917 . Shortl y thereafter , simila r phenomena wer e reported i n the rat an

d
mouse . Vagina l smear s obtaine d fro m thes e animal s wer e foun d to correlat e wel l wit hchange s occurin g i n th e reproductive trac t an

d wit hth

e secretion o f th

e ovarian hormones . The
mos t common type s o f cell s presen t i n a vagina l smea r are :
1 . Polymorphonuclea r leucocyte s 2 . Nucleate d epithelia l cell s 3 . Larg e squamou s epithelia l cells , als o calle d cornifie d

• cell s (nucleated o r non-nucleated) . Th e estrou s cycl e o f th e mous e an

d rat i s complete d in fou r t o fiv

e day s (polyestrous ) althoug hth e timin g o f th

e cycl e ma

y be influenced b

y exteroceptive factor s (light , temperature , etc.) . Th e cycl e i s roughl y divisibl e int o fou r



### 3 4P.3 8

mai n phase s (see Figur e 8) : 1 . Estru s - Thi s i s th e perio d o f hea t an

d copulation i s possibl e onl y

a t thi s time . Thi s phas e last s fro m 9 to I S hour s an

d i s characterized b y a hig hrat e o f runnin

g activity . Man

y mitose s occu

r i n th e vagina l mucos a an d
a s ne w cell s accumulate , th e superficia l layer s becom e squamou s an

d cornified . The latter cell s ar e exfoliate d int o th e vagina l lume

n an d thei r presenc e i n vagina l
smear s i s indicativ e o f estrus . Durin g lat e estru s ther e ar e chees y

masse s o f cornified cell s wit h degenerat e nucle i presen t i n th e vagina l lumen , but few , i f any , leucocyte s ar e foun d durin g estrus . Muc ho f the lumina l flui d i

n th e uter i i s los t befor e ovulation .
2 . Metestrus : Thi s occur s shortl y afte r ovulation an d i s intermediat e between estru s an d diestrus . Thi s phas e last s fo

r 1 0 to 2 4 hour s an d matin g i s usuall y

not permitted . Man

y leucocyte s appea r i n th e vagina l lume

n alon g wit ha fe

w cornified cells .
3 . Diestrus : Thi s phas e last s fo r 6 0 t o 7 0 hours , durin

g whic hfunctiona l regressio n o f th

e corpor a leute a occurs . Th e vagina l mucos a i s thin , an d leucocyte s

" migrat e throug

hit , givin

g a vagina l smear consisting almos t entirel y

o f thes e cells .
4 . Proestrus : Thi s herald s th e nex t hea t an

d i s characterize d b

y functiona l involutio n o f th

e corpor a lute a an

d preovulator

y swellin

g o f th

e follicles . Flui d



### 3 5P.3 9

collect s i

n th e uter i an

d the y becom e highl y contractile . The vagina l smear i s dominated b y nucleate d epithelia l cell s whichoccu r singl y

o r i n sheets .



### 3 5P.4 0

Figur e 8 . Section s throug h th e vagina l wal l o f th e rs t

durin g differen t stages o f th e estrou s cycl e showin g th e correspondin g type s o f cell s whic h appea r i n smear s obtaine d fro m th e vagina l lumen . A , Diestrus ; B , Proestrus ; C , Estrus ; 0 , Metestrus ; an d 6 , adul t anima l whic h had bee n oophorectomized fb r si x months .

3

7P.4 1



### Sectio n2 B Protoco l for Obtainin g a Vagina l Smea r fro m Mic e and Rat s
1 . Tw o vagina l smear s shoul d be prepare d fro m eac hanima l betwee n8-1 0 a.m . for twelv e consecutiv e day s inthe las t twelv e day s o f the 90-day study . Dos e level s a t whic hthes e ar e prepare d wil l b e designate d b y th e NT P projec t office r for the bioaasa y laborator y

i n collaboratio nwit hthe NT P chemica l manager . 2 . Full y

froste d slide s wil l b e use d t o mak e vagina l smear s (Dakln-Eri e 2958) . Al l smear s ar e mad e o nth e froste d fron t o f th e slide . 3 . Microscop e slide s ar e marke d wit ha lea d penci l int o a gri d consistin g o f si xsquare s pe r slid e o nth e froste d

e

### sid e o f th e slide . Th e square s ar e labelle d 1 throug h6
and 7 throug h1 2 i nthe uppe r righ t hand corner . Thi s i s convenien t a s i t allow s th e incorporatio no f si xday s o f smear s o non e slid e whic hca nb e staine d a t th e same time . 4 . A 3-4 " medicin e droppe r i s moistene d b y aspiratin g 0.9 % salin e o r PB S solution . A ver y smal l amoun t o f salin e i s lef t i nth e medicin e droppe r the nplace d i nth e vagin a an d th e vagina l fluid s aspirate d severa l times . 5 . Th e content s o f th e medicin e droppe r ar e transferre d ont o th e slide . Th e us e o f excessiv e salin e shoul d b e avoide d t o preven t the flo w ont o surroundin g squares .

3 36 . A t th e en d o f eac

hday , th

e smear s ar e fixe

d b y lightl y 1 sprayin g fixativ

e (Spray-Cyt e I I b y Cla y Adams 718 0 ) . I n orde r t

o fixonly

th e specifi c squar e o f th e slide , us e a 2 " X4 " fil e car d wit ha window . A cardboard windo w ca n als o b

e used . The slide s shoul d b e stored i n closed boxe s o

r i n a dust-fre e atmospher e between collection dates .
7 . O n th e 12thda

y afte r smear s hav

e been made , th

e slide s
ar e allowed t

o dr y completely . T o stai n th e smears , th

e slide s ca

n b e staine

d th e sam e da y o r withi n th

e next fe w days . 8 . Slide s ar e the n loaded i

n glas s rack s an

d dippe d i

n th e followin

g solutions :

a ) 95 % Ethano l - 3 0 minute s b ) 80 % Ethano l - 1 minut e wit hgentl e shaking . c ) 70 % Ethano l - 1 minut e wit hgentl e shaking . d ) 50 % Ethano l - 1 minut e wit hgentl e shaking . e ) 20 % Ethano l - 1 minut e wit hgentl e shaking . f ) 0.5 % Toluidin e Blue i n 20 % Ethano l - 3 0 - 4 5 seconds . Persona l judgemen t shoul d be use d becaus e Toluidin e Blue improve s wit hage . g ) Runnin

g ta p wate r rins e - unti l ta p wate r run s clea r (a t thi s poin t i f slide s aren' t dark enough , di p i n 

### 1 Spray-Cyt e I I ca

n b e purchased fro

m America

n Scientifi c Products , Obetz , OH , Catalog No . S-7788-2 .



### 3 9P.4 3

20 % Ethano l an

d put bac

k i n th e stain ; thi s proces s
can be repeated unti l desire d color i s obtained) . 9 . The stained slide s ar e blotted gentl y unde r bibulou s paper . We t paper s ar e throw n awa y becaus e materia l on a slid e ca

n be transferred t o othe r slides . Slide s ar e allowe d to dr

y overnigh t an

d th e coverslippe d usin g Permoun t o

r suitabl e mountin g mediu m an d xylene . I f slide s canno t b

e coverslippe d th

e followin

g day , stor e i n a dust-fre e atmospher e an d coversli p a s soon a s possible . 10.f r Slide * fcmfceHlinq sa d CodLam e Eac hlaborator

y participating in thi s projec t wil l be assigne d an identification number b y th e NTP-designate d laboratory . Pleas e contac t th e SMVC E laborator

y t o fin d ou t assigne d identification number . Furthermore , eachlaborator

y wil l randoml y

designat e a seria l numbe r fo r eac hexperimenta l mous e (M- l throug hM-n ) an

d R- l throug hR- n fo

r rats . A

n exampl e o f th

e cod e i s a s follows : 01-MI A

wil l designat e th e firs t slide , laboratory

1 , an d anima l one ; secon d slid e wil l be labelle d a s 01-MlB* . Th e dat e when th e firs t smea r wa s made r i s recorded o

n eachlabel . I

n addition to th

e cod e numbe r an

d date , 01- 6 an

d 07-1 2 ar e als o adde d t o the tw o separat e slides . Thi s indicate s smear s fro m day s 1 throug h6 ar e o

n on e slid e an

d smear s fro

m day s 7 throug h1 2 ar e o

n anothe r slid e (See Figur e 9) .


### 1lw% > stxJrt©i \

f^visc a 40P.4 4

I * 

### e g l ag o i

4 2

S 3

6DaleCodeID7I2a 7
1 0 8

1 1 9

1 2

Figur e 9 . Preparatio n o f slid e fo r observatio n

o f th e estrou s cycl e by mean s o f vagina l changes .



### 4 1P.4 5



### MODIFICATIO

N T O TH E APRI L 198 7 SO

W Appendi xG Revise dJun e 198 9

11 . Al l eode s ar e t o remai nwit htn e individua l bioassa y laboratorie s and a espy o f tn e cod e sen t t o th e SMVC E laborator y

t o b e opene donl y

afte r completin g al l evaluation s and i n th e presenc e o f th e Qualit y Assuranc e Office r wn o wil l dat e an d sig nwne nopened . Code s mus t includ e (i ) treatmen t levels , (ii ) venicle , (iii ) rout e o f edainiatration , (iv ) date(s ) o f sacrifice , (v ) firs t an d las t da y o f vagina l smear s an d (vi ) anima l strain . Termina l bod y weight s fo r th e female s shoul d als o b e included . Al l slide e wil l b e shippe d accordin g t o attache dinstructions . 

## \aXfl

• Th e requirement t

o collec t plasm

a sample s fo

r possibl e hormone determinatio n wa s delete

d8/88 . e>. 

### ••Th e requiremen t fo rprocessin g th e vagin a fro m rat s an d mic e fo rhistopathologi c examinatio n wa

s delete d 5/89

. 12 . A perso n fro a th e NTP-designate d SMVC E laboratory , wil l visi t al l bioassa y laboratorie s conductin g thes e test s t o asses s thei r abilit y t o perfor m thes e bioassays .

4 *P.4 6

Sectio n 2 C Lis t o f Material s fo r Vagina l Cytolog

y Assay s (T o b e supplie

d b y th e bioassa

y laboratory )
1 . Medicin e dropper s 2 . Pre-cleaned microscope slide s ( 3 X1") ' fully froste d (Dakin ) 3 . Coverslip s (2 4 X6 0 mm ) 4 . 95 , 80 , 70 , SO , an d 20 % ethanol . Thes e grade s o f alcoho l ca

n b e prepared fro

m 95 % ethano l stock . 5 . Spra

y Cyt e I I (Cla

y Adams 7180 ) 6 . Permoun t mountin g mediu m an d xylene 7 . 100-slo t slide boxe s 8 . 0.9 % saline solutio n o r phosphat e buffered saline 9 . Microscop e slide label s 10 . Bibulou s pape r 11 . Toluidin e Blue 0 (ca n b e purchased fro

m J.T . Bake r Chemica l Co. , Catalog No . W144 ; i f som e othe r sourc e i s utilised , ensur e that th e stai n i s certified fo r us e i n histology) . Beaker s Traye 3 X5 " card s o

r cardboar

d wit hsmal l windo w 1 N BC 1 I N NaOH Parafilm



### 4 3P.4 7



### Preparatio no f Toluidin e Blu e Stai n 2. 5 g Toluidin e Blu e 50 0 mL 20 % ethano l
Mixan dallo w

t o si t fo r on e hou r befor e filterin g throug hWhatma nGrad e filte r paper .

4 4P.4 8

Sectio n 2 D Suggestion s fo

r Improvin

g Vagina l Cytolog

y Qualit y
Genera l instruction s t o mak e vagina l smear s ar e described i

n detai l i

n th e protocol . A

fe w simpl e precautions , however , ca n significantl y

increas e th

e qualit y
o f th e slides . Thes e ar e discussed i n thi s section . Unde r eac

hcategor

y ther e ar e suggestion s and/o r possibl e explanation s fo r correctin

g th e potentia l problems whic hma

y b e encountered durin g th e preparation o f a vagina l cytolog y smear . 1 . Crystallization : Thi s i s a frequen t proble m sinc e mos t stainin g solution s easil y crystalliz e upo

n drying . 0.5 % Toluidin e Blue i

n 20 % ethano l solution i s optima l fo r stainin g vagina l smears . W e hav e not noticed an

y excessive crystallization wit hthi s concentration . Mucou s crystallise s easil y i n th e presenc e o f stain . Sinc e th

e mucou s i s darkl y stained , that mak e th e smear s har d to evaluat e and , furthermore , th e presenc e o f crystal s distort s th e morpholog

y of th e cells . Also , debr i on th e slide wil l provide crystallization sites . Saesr s aus t be mad e o n clea n slide s an

d slide s shoul d alway s be stored i n a dust-fre e atmosphere .
2 . Debri : Presenc e o f debr i i n vagina l smear s wil l interfe r durin

g th e evaluation . Debr i usuall y come s fro a dirt y

slides , tissue fragments , o r dus t i

n th e air . Therefore , al l slide s shoul d b e precleane d b

y wiping



### 4 5P.4 9

the m wit ha clean gauz e o r Ki m Wipe . Mos t o f th e tissu e fragment s ca

n b e eliminated b

y carefull y

aspirating vagina l fluids . I t i s als o important t o plac e al l uncoverslippe d smear s i n a dust-fre e environmen t s o tha t dus t settlin

g o n th e slide s i s minimized . Coverslips , i f dusty , shoul d be cleaned prior t o use . 3 . Staining : Th e majo r proble m encountered i

n thi s categor y i s th e nonuniformit y

o f th e stain . Thi s result s fro

m th e differen t densitie s o f cell s i n eac h
smear . B

y scannin

g th e slide , i t i s possibl e t o determin e i f al l th e smear s ar e adequatel y stained . I f the y ar e not , plac e th e slide s bac k in th e alcoho l serie s an

d the n in Toluidin e Blu e t o restai n them . B e carefu l no t to stai n cell s to o dar k becaus e i t severel y e* interfere s wit hevaluation .
4 . Clumpin

g an d Cel l Density : Th e clumpin g o f cell s i s mainl y du e t o a

n exces s numbe r o f cell s on the slide . Thi s can b e remedied b y no t placin

g too man

y cell s on th e slide . I f th

e smear i s too dense , i t wil l stai n heavil y an d clump . I f to

o muc hsalin e i s used i n makin g the saear , th

e cel l densit y

o f th e saea r wil l be ver y low. * Thi s cause s th e saea r t o have to o fe w cell s t o allo w accurat e evaluation . I t aus t be adde d tha t som e
o f th e stage s o f th

e estrou s cycl e hav e a highe r densit y
o f cell s tha

n others . Thi s add s t o the proble m o f cel l density .



### 4 6P.5 0

5 . Ai r Bubbles : Ai r bubble s ar e a major proble m especiall y when the

y occup y a large portio

n o f th

e slide . The

y ar e mainl y a technica l proble m cause d b y fault y technique and/o r carelessness . Mor e mountin g mediu m ma y b e needed t o cove r th

e entir e slid

e o r bette r pressin

g of th

e coversli p t

o squeez e out ai r bubble s i s required . Th e
us e o f Flo-Te

xo r similar mediu

m ma y als o alleviat e thi s problem .



### 4 7P.5 1

VAGINA L CYTOLOG

YQDALIT YCOD E
Cod e 0 0 Excellen t 0 1 Good 0 2 Moderat e crystallization 0 3 Heav

y crystallization 0 4 Moderat e debri s 0 5 Heav

y debri s 0 6 Debr i surrounding cell s 0 7 Staining too light 0 8 Cornified*cell s stained lightl y 0 9 Staining too dark 0 Non-unifor

m stainin

g 1 Cell s heavil y

clumped 2 Fe w cell s 3 N o cell s 4 Ai r bubble s (>5 % <20 % slide ) 5 Ai r bubble s (>20 % slide ) 6 Slide s prepare d on non-frosted sid e o f slid e Subcod e



### - 1

- 2

- 3

- 4

- 5

- 6

- 7

- 8

- 9 -1 0 -1 1 -1 2 Refer s Refer s Refer s Refer s Refer s Refer s Refer s Refer s Refer s Refer s Refer s Refer s 

### t o t o t o t o t o t o t o t o t o t o t o t o Da y Da y Da y Da y Da y Da y Da y Da y Oa y Da y Da y Da y 1 2 3 4 5 6 7 8 9 onl y onl y onl y onl y onl y onl y onl y onl y onl y 1 0 onl y 1 1 onl y 1 2 onl y

Subcode s ar e used i n conjunction wit hcod e when tha t remark applie s t o a particula r da y (e.g. , 13- 2 mean s n o cell s o

n Da y 2 )



### 48P.5 2

SECTIO N 3
Instruction s fo

r Shippin

g Slide s fro

m NT P Testin g Laborator

y t o SMVCE-Laboratory
1 . Shippin

g an d packagin

g cos t wil l b

e incurred b

y th e NT P testing laborator

y conducting th

e toxicity

studies . 2 . Staine d an

d coverslippe d slide s wil l b e randoml y

organize d an

d shippe d to th

e NT P SMVC E laborator y withi n 2 week s 'zt necropsy . 3 . A cop y o f th e cod e fo

r al l slide s wil l be place d i n a seale d envelop e an

d sent to th

e SMVC E laborator

y alon g wit hth

e code d slides . 4 . Al l vagina l cytolog y slide s wil l b

e place d i

n plasti c slid e boxes , keepin

g slide s fro

m mic e an

d rat s separate . 5 . Thes e slid e boxe s wil l be place d i n 35 0 lb . tes t cardboar d boxes , separate d b y abundan t packaging materia l fo r shipment . 6 . A n appropriat e listin g o f slide s wil l be packe d i n the slid e boxes . 7 . Slid e boxe s wil l b e packed to avoi d an y breakage . 8 . Shipping carton s wil l b e sealed an

d boun d wit hfilamen t tap e prio r to shipment . 9 . Slide s sent separatel y i n slid e se t wil l be counte d a s presen t in the inventory ; a cop y o f th e slid e se t inventor y wil l accompan

y the majo r inventor y document . 10 . Eac hplasti c shipping bo xwil l be marke d t o sho w the rang e o f chemical , anima l numbers , participating laboratory an d addres s an d the chemical numbe r o r Cl . 11 . Shipping carton s containing slide s an d th e appropriat e • information wil l b e directl y

maile d t

o th e NT P designate d SMVC E laboratory .



### 4 9P.5 3

SECTIO N 4 M*u J S^ehc n Instruction s fo

r Shipping Frozen Teste s fro m NTP-Designate d Bioassa y Laboratorie s t

o SMVCE-Laboratory
1 . Shipping an

d packaging cos t wil l b e incurred b

y th e NTP-designate d bioassay

laborator

y conducting th

e toxicit y

studies . 2 . Th e frozen teste s wil l b e shippe d to th

e NTP-designate d SMVC E laboratory

alon g wit hth

e corresponding ra w dat a sheet s withi n tw o week s o f necropsy . 3 . Al l vial s wil l be place d i n via l rack s an

d wrappe d wit hbubbl e packin g to avoi d breakag e durin g shipment . 4 . Th e wrappe d via l rack s wil l b e place d i

n 35 0 lb . styrofoam-line d boxe s fille d wit hdr y ic e fo

r shipment . 5 . A n inventor

y o f th e vial s wil l be sent wit hth e cove r letter . 6 . Shippin g carton s wil l be seale d an d boun d wit hfilamen t tap e prio r t o shipmen t an

d labelled "freez e upon arrival" . 7 . Shippin g wil l b e expedite d s o tha t th e teste s d o no t tha w durin g transit . Overnigh t servic e b y Federa l Expres s i s recommended . 8 . I f Federa l Expres s i s used , th e followin

g informatio n shoul d b e writte n on the box : ORM-A , U N 1345 , Medica l Purpose s Only ! Mai l to : Environmenta l Healt hResearc h& Testing , inc .

251 4 Regenc y

Roa d Lexington , K

Y4050 3 Attention : Dr . O.K . Gulat i



### S OP.5 4

SECTIO N 5 R CvJl%€ i „| 9 7
Return of Slide s £:•- . SMVCE-laborator y t o th e EPL/NT P Archive s
1 . Shippin

g an d packagin

g cos t wil l b

e incurred b

y tn e SMVCE laboratory .

2 . Al l slide r wil l b e returned t

a th e EPL/NT P Archives . A cove r lette r wic hth

e followin

g informatio

n wil l b

e included :
a ) Chemica l Nam

e b ) Participating Laborator y an d Addres s
c ) SMVC

E Stud y No . d ) Chemical No . (Cl )


### e ) Typ e o f

Stud y f ) Dat e Slide s Receive d a c EHR T g ) Dat e Fina l Repor t Submitte d Als o enclose d wil l b e a ccp y o f th e slide authorizatio n return for m 3 . Al l slide s wil l b e place d i

n th e origina l plasti c slid e boxes . 4 . Thes e slid e boxe s wil l b e place d i

n 35 0 lb . tes t cardboar d boxes , separate d b

y abundan t packaging materia l fo r shipment . 5 . Slid e boxe s wil l b e packed c

o avoi d an

y breakage . 6 . Shipping carton s wil l b e sealed an

d boun d wit hfilamen t tap e prio r t o shipment .



### 5 1