P.1 

OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

88) IV-A-10 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:17 AM Sheet 88 of 93 
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KJJJJ II 

iRA^ggf^gy l987 

-mVr-fl- ID 

APPENDIX G 

NATIONAL TOXICOLOGY PROGRAM 

TECHNICAL PROTOCOL FOR 

SPERM MORPHOLOGY VAGINAL CYTOLOGY EVALUATION 

IN TOXICITY TESTING FOR RATS ANO MICE 
P.3 

r

w 

TABLE OF CONTENTS 

Introduction - SMVCE Protocol 

Technical Protocol 

Section 1: Male Organ Toxicity Studies in Mice and Rats 

IA: Protocol Cor Male Organ Toxicity Studies in Mice and Rats 

IB: List of Materials Cor Sperm Assays in Mice and Rats 

IC: BuCfer and Stain Preparations (Mouse Studies) 

10: Buffer and Stain Preparations (Rat Studies) 

IE: Calculation Procedure for Estimation of Sperm Density -

Mice and Rats 

IF: Summary of Sperm Evaluation Procedures (Mouse) 

1G: Summary of Sperm Evaluation Procedures (Rat) 

Section 2: Protocol and Standard Operating Procedures for 

tht Vaginal Cytology Assay in Mies 

2A: The EstroCla Cycle 

2B: Protocol for Obtaining a Vaginal Smear from Mice and Rats 

2C: List of Materials for Vaginal Cytology Assays 

20: Suggestions for Improving Vaginal Cytology Quality 

Section 3: Instructions for Shipping Slides from NTPOesignated 

Bioassay Laboratory to SMVCE Laboratory 

Section 4: Instructions for Shipping Frosen Testes from NTPDssignated Bioassay Laboratories to SMVCE Laboratory 

Section Ss Return of Slides frost SMVCZ-Uboratory to EPL/NTP 

Archives 
P.4 

INTRODUCTION 

Several short-term bioassays have been recently developed 

to detect suspected chemical mutagens, teratogens, and 

carcinogens. Among these are tests which can be used as early 

indicators of compounds that may possess activity against 

reproductive function. Studies of particular interest for this 

project are male and female reproductive organ toxicity studies. 

wyrobek and Bruce (1975) found that physical and chemical 

mutagens can increase the frequency of abnormal sperm in mice. 

Similar studies have also shown that rats exposed to mutagens 

have increased frequencies of abnormal sperm. Ficsor and 

Ginsberg (1980) proposed the use of other sperm 

characteristics, sperm motility, and sperm counts in 

conjunction with the sperm abnormality test in mice. In the 

present study, the male organ toxicity is estimated by the 

following parameters: sperm motility, sperm count, and 

testicular spermatid head counts. 

The existence of a typical estrous cycle in the guinea pig 

and the associated changes in vaginal cytology were first 

reported by Stockard and Papanicolaou in 1917. Shortly 

thereafter, similar phenomena were reported in the rat and 

mouse. Vaginal smears obtained from these animals correlated 

well with changes occurring in the reproductive tract and with 

the secretion of the ovarian hormones. The most common cell 

types present in a vaginal smear are polymorphonuclear 

leucocytes, nucleated epithelial cells, and large squamous 
-Piepithelial cells (also called cornified cells, nucleated, or 

non-nucleated). 

The estrous cycle of the mouse and rat is completed in 

four to five days (polyestrous), although the timing of the 

cycle may be influenced by exteroceptive factors (light, 

temperature, etc.). The cycle is roughly divisible into four 

main phases: estrus, metestrus, diestrus, and proestrus. 

Evaluation of the staging of the estrous cycle can be used to 

determine effects of toxic chemicals on reproductive function 

in mice and rats. 

REFERENCES 

Wyrobek, A.J., and H.R. Bruce (1975), Chemical induction of 

sperm abnormalities in mice, Proc. Nat. Acad. Sci., 

72(11):4425-4429. 

Ficsor, G., and L.C. Ginsberg (1980), The effect of 

hydroxyurea and mitomycin C on sperm motility in mice, 

Mut. Res., 70:383-387. 

Stockard, C.R., and G.N. Papanicolaou (1917), The existence 

of a typical oesterous cycle in the guinea pig with a 

study of its historical and physiological changes, Am. 

J. Anat., 22:225-283. 
P.6 

TECHNICAL PROTOCOL 

A detailed description of techniques to be used by the 

NTP-designated bioassay laboratories and the NTP-designated 

SMVCE laboratory to conduct the male and female toxicity, 

studies properly and uniformly is provided below. The dose 

levels for these studies will be designated by the NTP Project 

Officer Cor the bioassay laboratory in collaboration with the 

NTP Chemical Manager. Ten each male and female rats or mice 

in three of the five dose groups plus control group will be 

utilized. Specific selection of the three dose groups will be 

based on the results obtained during the first 70 days of the 

subchronic studies. The objective is to select doses which 

are not causing overt toxic effects, i.e., mortality, 

depressed weight gain, etc. 

The protocol to conduct male and female reproductive 

organ toxicity studies has been divided into the following sections: 

Section 1 Male Organ Toxicity Studies in Mice and Rats 

Section 2 Vaginal Cytology Assay in Mice and Rats 

Section 3 Instructions for Shipping Slides 

Section 4 instructions for Shipping Frozen Testes 

Section 5 Return of Slides 
P.7 

SECTION 1 

Protocol for Male Organ Toxicity Studies in Mice and Rats 

(To be conducted at NTP-designated bioassay laboratories) 

The NTP designated laboratories are urged to read the 

material thoroughly because there are a few key differences 

between mice and rat studies. Furthermore, sperm motility in 

both mice and rats is extremely sensitive to pfl and 

temperature. Readers may, therefore, find that we have 

repeatedly stressed that the correct pH and temperature of 

the buffers can significantly improve the quality of the 

data. A small waterbath can be used to keep all buffers at 

the correct temperature. A small incubator can be used to 

keep all materials at 37 C. A slide warmer should be used to | 

keep microscope slides and coverslips at the correct 

temperature during slide preparation. A microscope stage 

warmer should be used during slide reading (see Section IB 

Cor list of equipment and supplies). 

Section IA 

The technique to conduct male organ toxicity studies in 

mice and rats is stepwise described below: 

1. The test animal is weighed and then sacrificed according 

to the study protocol at the end of the 90-day study. 

Before sacrificing the animal, be sure that the buffer 

solution, microscope slides, coverslips, pipet tips, 

petri plates, and microscope stage have been warmed to 
P.8 

O 

the appropriate temperature (37 C). Also, be sure that 

the balance is properly calibrated. Check the pH of the 

modified Tyrode's with egg yolk solution and adjust it 

. co 7.2 with HCl (IN) or NaOH (IN) (see Section IC Cor 

formula for mouse studies; 10 Cor rat studies). 

2. After sacrificing the animal, conduct the sperm 

collection as quickly as possible. Use the following 

procedure to remove the testis and epididymis: 

a) Pull the left tesits into the abdominal cavity by j 

grasping the fat surrounding the caput epididymis. 

Excise the left testis by cutting the vas deferens | 

approximately 0.5 cm from the point of attachment of 

the cauda epididymis. The caput epididymis should be 

located prjLor to excision so that it will not be 

clipped accidentally (see Figures 1, 2, and 3). 

b) The intact left epididymis is removed and weighed to | 

the nearest 0.1 mg. 

3. Clip the left cauda epididymis at the point of origin I 

of the ductus deferens at the distal end and at the 

boundary between the body and cauda epididymis of the 

- proximal end (See Figure 2). In rats, cauda epididymis 

can be seen as white tissue consisting of convoluted 

tubles. 

4. Weigh left cauda epididiymis (mice only) to the nearest | 

0.1 mg (0.0001 g). Record the weight on the data form 
P.9 

Figure 1 

inea of incieioa 

Figure Z 
P.10 

Photograph of a ret epididymis showing the sites nfmrremi to in the 

text for removal of sperm samples: 

Site 2: proximal caput 

Sites 2s and 2b: caput 

Site 3: 

Site 1: 

Site 5: 

Site I: 

Site 7: 

Site 8: 

distal caput 

mid-corpus 

distal corpus 

proximal cauda 

distal cauda 

ductus deferens 

Samples could not be removed from the initial segement (Site 1) 

FICURE 3 
P.11 

(Figure 4). In rats, the cauda epididymis is weighed as 

described in step 5b below. 

5. This step differs for mice and rats so please read these 

instructions carefully. 

a) Mice 

i Two independent slides are prepared to estimate 

sperm motility. Place 80 uL of prewarated 

Tyrode's Solution (modified) on each microscope 

slide. Slides shall be placed on a slide warmer 

o 

set at 37 C and retained at this temperature. 

ii Hold the left cauda epididymis with forceps and 

make a small incision near the center of the 

cauda with a sharp, clean surgical blade. 

iii Apply gently pressure on che caudal surface so 

that a small epididymal tubule is exposed or 

caudal fluid begins to oose out into the Tyrode's 

solution on the prewarmed slide (Caution: Do not 

apply too much pressure since there is an ample 

amount of sperm in a piece of cauda equivalent to 

1/2 of a pinhead. Use of excessive amounts of 

* caudal fluid for the sperm motility counts may 

significantly lower sperm density evaluations). 

Alternately, the cauda epididymis is placed into 

a petri plate (15 X 60 mm) containing 2 mL of 

prewarmed phosphate buffered saline (PBS). The 
P.12 

STUDY: 

OATA SHEET • MOUSE SPERM STUDIES 

STUDY*: 

"in 

KeviMU 

CODE/ANIMAL #: 

ANIMAL WEIGHT: 

CAUDAL WEIGHT (RIGHT): 

SPERM McrnuTY 

Held 

motile 

nonmotile 

± 

TREATMENT: 

DATE: 

LAB* 

j EPIDIDYMAL WEIGHT (RIGHT): 

j TESTICULAR WEIGHT (RIGHT): 

Total Motile: 

SIGNATURE: 

Observer A Data Total Nonmotile: 

field 

motile 

nonmotile 

6 7 

r-> 

a 9 10 

% Motility 

SIGNATURE: 

Observer a Date 

Sperm Count 

* Vol. Saline (cauda in 2 mL PBS) 

* * VoL Caudal Suio. (OJ mL in 2 mL) 

" "Soerm Concentration: XlO6 

Per g caudal tissue 

SIGNATURE: 

mL 

mL 

Count I Count! Mean 

| 

Date 

"Total motile/total motile * Noaaocik XlUOt 

^ Laava Mask tiakna utbar voiaaa ia uani. Oo aot dilute if the aaatnar uf »pcra pet 

^ Mcoedary square is lem tkae 15. . 

*X(J0000)Y (to tha aaarat 0.1 X 10°) (X - # sperm couaiwi Y - 4Uiuiua facuir. 

whicS ia IU cccaudal wtifhi ia graan) 

DILUTION FACTOR WILL CHANGE IF OIFFERE.NT VOLUME* OF DILUTENT ARE USED. 

Flgura 4 

9 
P.13 

cauda is then held in place with forceps and a 

very small piece (not more than 1/20 of the total 

caudal size) is removed with either a sharp 

scalpel or blade (see Figure 5). This tissue is 

then placed into the tyrodes solution on a 

prewarmed slide. Note: Surgical blades may be 

cleaned with saline^between animals. 

iv. If necessary, the caudal fluid is further chopped 

on the slides with a pair of needles or sharp 

blades. 

v. Place a warm coverslip (24 X 40 mm) on the slide. 

Maximum sperm motility is obtained 

if the removal of cauda, weighing, 

and preparation of sperm suspension 

is done expeditiously (approximately 

2-4 minutes). 

vi. Two slides are prepared simultaneously for 

estimation of sperm motility. 

vii. The sperm motility is estimated without any 

further delay as illustrated in Step 6. 

viii. Place remaining caudal tissue into a petri plate 

(IS X 60 mm) containing 2 mL of phosphate 

buffered saline (PBS). 

ix. The reaaining caudal tissue is gently chopped 

(only after the sperm motility counts have been 

performed) in the petri plate with two scalpels 

10 
P.14 

ECO.MMENOED PORTION OF 

CAUDA ADEQUATE FOR SPERM 

MOTILITY EVALUATIONS. 

REPRESENTATION OF DISTAL CAUOA IN .MOUSE 

(Sample Only) 

Figure 5 

11 
P.15 

until its contents are released. The petri plate 

is swirled several times and incubated for 15 

o 

minutes at 37 C. It allows aggregates of sperm, 

which can be seen as pieces of tissue in che 

suspension, to break up. Petri plates should 

remain covered during the incubation period. 

b. Rats Only 

i. The cauda epididymis in rats can be seen as white 

tissue consisting of convoluted tubules. After 

clipping the cauda at the point of origin of the 

ductus deferens (Figures 1, 2, and 3) from the 

cut end of the cauda (distal cauda), a very small 

sperm sample (not greater than the size of a 

pinhead) is removed with the tip of the scalpel 

blade and placed on a prewarmed slide with 80 ul 

o 

drops of test yolk buffer (37 C). Do not allow 

the sperm to dry on the scalpel blade. 

ii. A coverslip (24 X 40 mm) is placed on the slide 

after gentle mixing of sperm in the buffer 

solution. Two such slides are prepared for 

estimation of sperm motility as described in Step 

6 below. 

iii. After motility has been estimated, the remaining 

distal cauda is weighed to the nearest 0.1 mg. 

The weight is recorded on the appropriate data 

sheet (Figure 6). 

12 
P.16 

DATA SHEET • RAT SPERM STUDIES 

STUDY: 

CODE/ANIMAL #: 

ANIMAL WEIGHT: 

'CAUDAL WEIGHT (RIGHT): 

STUDY #: 

TREATMENT: 

RaviMd 

DATE: 

LAB#: 

"Iff 

_g EPIDIDYMAL WEIGHT (RIGHT): 

a TESTICULAR WEIGHT (RIGHT): 

Held 

motile 

nonmotile 

SPERM MOTILITY 

t •y 3 4 -

Total Motile: 

.

m

8 

SIGNATURE: 

Observer A Date Total Nonmotile: 

field 

motile 

nonmotile 

6 7 a 9 10 

c

/t Motility 

SIGNATURE: 

Observer B" Date 

Sperm CQUOI 

" Vol. Saline (cauda in 10 mL PBS) 

* 'Vol. 2nd dilution (OJ mL in 2 mL) 

' * Sperm Concentralion: 

Per g caudal tissue 

SIGNATURE: 

Observer 

mL 

mL 

XlO* 

Date 

Count l Count*? Mean 

..Paul cauda oat* 

Total motile/total motile * auamutik X IUU*. 

^ m Laava Mask uaku otter voiaaa ia \g*4 

X(5O0UU)Y (to tha aeami 0.1 X til0) (X * • ipcnn uiuaictl Y • dilwiua lactor. 

whn.li ia 50 cccamial weight ia grama I 

DILUTION FACTOR WILL CHANGE IF DIFFERENT VOLUMES ARE USED 

Figura 6 

13 
P.17 

iv. This step is performed only after sperm motility 

has been estimated. The distal cauda is held in 

place with a scalpel and gently chopped with 

another sharp scalpel in 10 mL of 0.9% saline or 

o 

PBS in a petri plate incubated at 37 C for IS 

minutes. 

6. Estimation of Sperm Motility - this step is common for 

both rats and mice. 

i. To estimate sperm motility, the viewer determines 

the actual number of motile and nonmotile sperm 

for each field of vision. A small hand counter 

may be used to count the number of motile and 

nonmotile sperm. 

ii. The final motility is based on ten independent 

fields and observations made by two independent 

viewers. 

iii. Person A evaluates the motility in five 

independent fields and, similarly, Person B 

evaluates the motility for another five 

independent fields. Both viewers must use 

or 

independent slides and, when possible, 

independent microscopes. 

iv. Sperm are considered motile if they show any 

movement at all. A 40X objective is recommended 

14 
P.18 

to evaluate sperm motility. 

v. Percent motility decreases with time; therefore, 

evaluation is critical and must be accomplished 

as quickly as possible (2-4 minutes). 

o 

vi. A microscope stage warmer (37 C) is needed for 

sperm motility evaluation and to maintain the 

sperm suspension at optimum temperature. 

Sperm are very sensitive to temperature 

and pH changes. Extreme care must be 

taken to keep all materials at 37°C on 

a slide warmer and/or in an incubator. 

The pfl of the Tyrode solution should 

be checked routinely and adjusted when 

and if necessary. These factors are 

very important to obtain an accurate 

estimation of sperm motility. 

vii. Average sperm motility readings should be 

approximately 75% or higher in control mice and 

rats. If sperm motility is considerably lower 

than 75%, there is some problem and EHRT 

personnel should be consulted at (606) 278-0603 

or (606) 276-1436. 

Ouring the IS minute incubation period, the left testis 

is removed, weighed and frosen for testicular spermatid 

head-count as described below. 

i. Label each vial with the appropriate laboratory 

name, animal number, test article dose group, i,ft 

testes (or right testis), and date of collection. 

The following glass vials and racks are 

IS 
P.19 

recommended: 

Vendor: VWR 

Scintillation Vial Rack; Catalog No. 66022-525 

Scintillation Vials 28 X 61 mm; Catalog No. 66021-453 

or 

Sample Vials - 28 X 60 mm; Catalog No. 66011-405 

ii. Remove the left testes, weigh it, and record the 

weight. 

iii. Place the testes in the appropriately labelled vial 

and freeze on dry ice. 

iv; When convenient, transfer frosen samples to a 

o 

freezer (-20 C) for storage until ready for 

shipment to EHRT. 

8. Sperm Count - After the 15 minutes incubation period, a 

sperm count is performed but the sperm suspension must 

be further diluted. Due to a significant difference in 

the caudal weight for rats and mice, parts of the 

dilution procedures for the two species are different 

and should be noted. 

i. Swirl the petri plate to evenly distribute the sperm. 

Zn the event the sperm suspension is not uniform, 

gently chop the cauda until a uniform suspension is 

obtained, incubate further if necessary. Transfer 

0.5 mL (using a 1.0 mL pipet) to a test tube (16 X 

100 mm) containing 2 mt of the phosphate buffered 

saline. The rat suspension should be gently pipetted 

IS 
20 times to break up sperm aggregates. 

ii. Gently and thoroughly mix the suspension with a 

Vortex mixer for 2 seconds. If a vortex mixer is not 

available, gently agitate the sperm suspension then 

gently pipet (with a pasteur pipet) approximately 5 

times to thoroughly mix the suspension. 

iii. The volume of PBS may be varied according to the 

density of sperm suspension. The number of sperm per 

secondary square (see Step 8vii below) should not be 

less than 5 or more than IS. 

iv. The volume of sperm suspension and PBS solution used 

for the sperm count should be recorded on the data 

form if different from the suggested volumes. 

v. The sperm used for this assay should be killed by 

placing the test tube under hot, running water for 

one minute or an ice bath for 5 minutes. 

vi. The sperm suspension is gently mixed by pipetting 

with a Pasteur pipet 5 times and placed on a 

hemacytometer (Figure 7). 

vfri. The shiny surfaces in the middle of the hemacytometer 

have microscopic grids consisting of heavy lines 

which divide the field into 25 large squares. Within 

each of the large squares are 16 smaller squares. 

The coverslip does not touch the grid area when 

placed over the middle portion. 

17 
P.21 

Side View 

STANOARO HEMOCYTOMETER CHAMBER 

Secondary 

Square 

T 1 mm ( Tertiary 

Square 

_L 

CORNER 

SQUARE 

MIOOLE 

SQUARE 

Volume Occupied by a Primary Square • 2.S X 10*' mL 

Volume Occupied by a Secondary Square » * X io"5 mL 

Volume Occupied by a Tertiary Square * 1 X 10** mL 

Since sperm count is made in '5' secondary squares, 

it is equivalent to * S X 4 X 10"" mL or 2 X 10"rmL. 

Figure 7 

18 
P.22 

viii. Microhematocrit capillary tubes are used to place 

suspension on the hemacytometer. The sperm 

suspension is placed on the hemacytometer by touching 

the tip of the capillary tube to the angle formed by 

the coverslip and counting chamber of the 

hemacytometer. The area under the coverslip should 

not be overfilled. 

ix. Two counts are performed using separate samples of 

the diluted suspension. Both slides for sperm count 

must be prepared after thorough mixing of the sperm 

suspension. Consistency in the preparation of the 

sperm samples for density determinations is essential 

to obtain^accurate results. 

x. To perform the counts, the counting area is located 

under low power (10X objective) and then switched to 

the 20X objective for counting. It is not necessary 

to count every cell in every square. The number of 

sperm in five secondary squares are counted. Sperm 

lying on the lines on the bottom and right sides of 

the large squares are included so that they will not 

be counted twice. 

xi. In case there are less than five (5) sperm per 

secondary square and it is not possible to make a new 

(less diluted) suspension, count sperm in two (2) 

tertiary squares. This change must be recorded on 

13 
P.23 

Appendix G 

MODIFICATION TO THE APRIL, 1987 SOW 

Revised June 1989 

the data sneet and taken into consideration wruia 

estimating sperm density. 

xii. Calculations for determining sperm density also vary 

for mice and rats as explained below: 

3 

a) The viewer has counted the sperm in 1/50 nm 

volume; 

b) The dilution factor for the caudal sperm is the 

final dilution of eaudal weight (normally for 

mice it is 10 cc/eaudal weight, and for rats SO 

cc/caudal weight). These values apply only when 

tht suggested volumes are used. 

c) The number of cells counted multiplied by the 

3 

volume fraction (1/50 mm ) csunted multiplied by 

the dilution factor should equal the number of 

cells per cubic millimeter. 

d) Sperm concentration is normally expressed in 

cubic centimeters or millimeters of gram tissue 

weight. Sperm density in the control sice is 

6 6 

generally between SOO X 10 and 1000 x 10 sperm 

per gram caudal tissue, 

q. Tj*>e>4e*Q 

* Sperm Head Staining Procedure: was completely deleted 12/C7 

Slide Labelling, coding and shipment was deleted from this section 12/87. 

** The requirement for collection of plasma samples for hormone determination 

was deleted 5/89. 

2C 
P.24 

iO. A person from the NTP-designated SMVCE laboratory will 

visit all bioassay laboratories conducting these studies 

to assess their ability to perform these bioassays and 

provide technical assistance, if necessary. Contact Dr. 

Gulati for technical questions (606) 276-1436. 

21 
P.25 

Section IB 

List of Materials for Sperm Assays in Mice and Rats 

(To be supplied by the bioassay laboratory) 

1. Scalpels, scissors, and forceps 

2. Petri plates (15 X 60 mm) 

3. Pasteur pipets (5-3/4") 

4. Pre-cleaned microscope slides (3" X 1") 

5. Coverslips (24 X 40 mm) 

6. Hemacytometers and coverslips (improved Neubaur, 0.1 mn 

deep) 

7. Pipets (1.0 and 5.0 mL) and pipetters 

8. Test tubes (16 X 100 mm) 

9. Modified Tyrode's Solution (See Section IC) 

10. Test Yolk (See Section ID) 

11. Phosphate Buffered Saline 

12. Protective Clothing 

13. Incubator or slide warmer 

14. Microhematocrit capillary tubes 

15. Band tally counter (ENM-4290 or Clay Adams 4300) 

16. Stage warmer for the microscope 

17.. Pipet tips 

18. Micro-pipettor 

19. Scintillation Vials 28 X 61 mm or Sample Vials 28 X 60 mm 

20. Scintillation Vials Racks 

22 
P.26 

Section IC 

Buffer for Sperm Motility Preparations (Mouse Studies) 

1. The diluent (Modified Tyrode's Solution) can be prepared 

(See "a" below) or purchased from Environmental Health 

Research and Testing, Inc., Lexington, Kentucky (See "b" 

below). 

a) To make 1200 mL .of modified Tyrode's solution, bring 

the following up to 1000 mL with distilled deionized 

water: 

Ingredients 

NaCl 

KC1 

CaCl .2B 0 

2 2 

Nafl PO .H 0 

2 4 2 

MgCl .6B 0 

2 2 

NaflCO 

3 

Na lactate 

Na pyruvate 

Glucoae 

Pinal 

Concentration 

99.23 

2.68 

1.80 

0.36 

0.49 

25.00 

25.00 

O.SO 

5.56 

mM 

mM 

mN 

ON 

mN 

mN 

mN 

mN 

mN 

Amount/Liter 

5.801 g/l 

0.200 g/l 

0.265 g/l 

0.050 g/l 

0.100 g/l 

2.1 g/l 

2.801 g/l 

0.055 g/l 

1.001 g/l 

Nix thoroughly until all chemicals are properly 

dissolved. Then add 200 mL of egg yolk without the 

yolk membranes. Shake until yolk is in solution. 

Thermoprecipitate the solution by boiling for 3 

o 

minutes at 92-95 C. Filter through 4 layers of 

cheesecloth. Centrifuge the filtrate at 10,000 rpm 

23 
for 20 minutes. Filter through Whatman 11 paper. 

Add 0.7 g/100 mL of percent of D-anhydrous glucose to 

the solution to raise the osmolarity. Measure the 

osmotic pressure and it should be at 325 mOsm/L 

(accuracy within 5 mOSm/L). Adjust the pH to 7.2 

with IN HCl or IN NaOB. At this time add penicillin 

(100 u/mL) and streptomycin (50 ug/mL). Aliquot the 

buffer into 5 or 10 mL volumes. Since this buffer is 

easily contaminated, it is recommended that all steps 

after thermoprecipitation be conducted under asceptic 

atmosphere when possible. 

o 

Store at -20 C. Prior to each usage, the buffer's pB 

should be taken and adjusted to 7.2 if needed. If 

o 

the buffer is kept at 37 C (incubator temperature) 

for long periods of time (1-2 hours) then again the 

pB should be taken and adjusted if necessary. 

b) The Modified Tyrode's Buffer to suspend mouse 

spermatozoa can be purchased from Environmental 

Health Research and Testing, Inc. 

Size Unit Price* 

5 mL $5.00 

10 mL $7.00 

Each lot of modified Tyrode's Buffer will be 

pretested for various parameters of male organ 

•This price does not include the shipping and handling 

charges. 

*» 4 
P.28 

toxicity studies in mice before shipping to 

individual laboratories. Modified Tyrode's buffer 

should be frozen upon receipt, thawed, and the pH 

adjusted immediately before use (pH 7.2). All 

requests for obtaining Tyrode's Buffer should be 

addressed to: 

Dr. O.K. Gulati 

Environmental Health Research and Testing, Inc. 

2514 Regency Road 

Lexington, KY 40S03 

(606) 276-1436 

25 
P.29 

Section ID 

Buffer and Stain Preparation (Rat Studies) 

1. The diluent (Test Yolk Buffer) to suspend rat 

spermatozoa can be purchased from Environmental Health 

Research and Testing, Inc. 

Size Unit Price* 

5 mL $5.00 

10 mL $7.00 

Each lot of Test Yolk buffer will be pretested for 

varioua parameters of the male organ toxicity studies in 

rats before shipping to the individual laboratories. 

Test Yolk Buffer should be frosen upon receipt and 

thawed immediately before use. All requests for 

obtaining Test Yolk Buffer should be addressed to: 

Dr. O.K. Gulati 

Environmental Health Research and Testing, Inc. 

2514 Regency Road 

Lexington, KY 40503 

(606) 276-1436 

2. Test Yolk Buffer can be prepared uaing the following 

ingredients per liter: 

TES** 14.5 g 

TRIS*** 3.5 g 

Glucose 2.0 g 

*This price does not include the shipping and handling charges 

**(N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid) 

* * *Tr is(hydroxymethy1)aminomathane 

25 
P.30 

Skim Milk Powder 33.0 g 

Sodium Citrate 6.0 g 

Mix the ingredients in 700 mL of distilled deionized 

water. Adjust the final volume to 800 mL. Add 200 oL 

of egg yolk without the yolk membranes. 

o 

Thermoprecipitate the solution by boiling at 92-95 C for 

3 minutes. Filter through 4 layers of cheesecloth. 

Centrifuge the filtrate at 10,000 rpm for 20 minutes. 

Filter through Whatman #1 paper. Aliquot into 5 or 10 

o 

mL volumes. Store at -20 C until further use. pB will 

be 7.1-7.2. 

3. Phosphate Buffered Saline is prepared by using the 

following ingredients per 1000 mL: 

NaCL 

KC1 

KH PO 

2 4 

Na BPO 7H O 

2 4 2 

CaCl 

2 

Ng CI 6H 

2 2 

Final pa • 7.2 

8.0 g 

0.2 g 

0.2 g 

2.1 g 

0.1 g 

0.1 g 

Nlx'the contents in 950 mL of distilled deionized water. 

Adjust the pH to 7.2. Bring the final volume to 1000 

mL. Filter sterilization is recommended. 

27 
P.31 

Section IS 

Calculation Procedure for Estimation of Sperm Density 

Mice 

Suppose the mean number number of sperm in "5" secondary 

-5 

squares in mice * 20. The number of sperm in 2 X 10 mL of 

diluted sperm suspension > 20. The number of sperm per mL of 

diluted suspension * 

20 

• 1 X 106/mL 

-5 

2 X 10 

Since thr diluted suspension represents one-fifth of the 

original suspension* if the standard dilution of 0.5 mL in 2 

mL was used, the number of sperm per mL of the original 

6 6 

suspension » 5 X (1 X 10 ) » 5 X 10 . 

e* 

Volume of the original suspension * 2 mL. 

6 

Total sperm in the original suspension «2X(5X10.)«1 X 

7 

10 /suspension » number sperm/cauda. 

If cauda weight * 0.030 g, total sperm in 0.030 g of eaudal 

7 

tissue » 1 X 10 . 

Total sperm per "g" of caudal tissue » 

- 7 

1 X 10 

— — — a 3.33 x 108 sperm/mg cauda. 

0.030 

This value can also be estimated by the following formula: 

Mean No. of sperm in 5 secondary squares 

— — ...... ....... . x 50,000 X 10 (dilution facter 

caudal weight in g 

23 
Calculation Procedure for Estimation of Sperm Density 

Rats 

Suppose the mean number number of sperm in "5" secondary 

-5 

squares in rat * 20. The number of sperm in 2 X 10 mL of 

diluted sperm suspension » 20. The number of sperm per mL of 

diluted suspension » 

20 

» 1 X 106/mL 

-5 

2 X 10 

Since the diluted suspension represents one-fifth of the 

original suspension, if the standard dilution of 0.5 mL in 2 

mL waa used, the number of sperm per mL of the original 

6 6 

suspension »SX(1X10)*5X10 . 

Volume of the original suspension * 10 mL. 

6 

Total sperm in the original suspension * 10 X (5 X 10 ) » 5 X 

7 

10 /suspension • number sperm/cauda. 

If cauda weight » 0.150 g, total sperm in 0.150 g of caudal 

7 

tissue • 5 X 10 . 

Total sperm per "g" of caudal tissue * 

7 

5 X-10 

——— — «"3.33 X 108 sperm/mg cauda. 

0.150 

This value can also be estimated by the following formula: 

Mean No. of sperm in 5 secondary squares 

....................... — - x 50,000 X 50 (dilution fact 

caudal weight in g 

29 
P.33 

SECTION l.G 

Summary of Sperm Evaluation Procedure - Mouse 

o 

1. Place instruments and glassware in oven at 37 C. (TT) 

o 

2. Put Tyrode's in water bath at 37 C. (TT) 

3. pH of Tyrode's should be 7.2. (TT) 

4. Weigh animal (NT), record weight (NT), bleed animal (TT), kill animal 

(NT), and bleed again when required (TT). 

5. Excise left testis and epididymis. Remove intact epididymis, weigh ( 

and record to nearest 0.1 mg. (NT) 

6. Cut off left cauda, weigh to 0.1 mg and record. (NT) | 

7. Place 80 ul warm Tyrode's on each of 2 slides. Place sperm the size 

of half a pinhead on the slides and gently mix with Tyrode's, 

coverslip and do motility (40X). (TT) 

8. weigh left testis and record. (NT) I 

9. Place remaining cauda in petri dish with 2 ml PBS, gently chop, 

swirl, cover, and incubate for IS minutee at 37oC. (TT) 

10. Remove petri dish, ensure homogeneity, and transfer 0.5 ml 

sample to test tube containing 2 ml PBS. (TT) 

11. Kill sperm by holding test tube under hot water for 1 minute. (TT) 

12. Pipette S times and place sample on hemacytometer for count 

(20X). Repipette and place a second sample on another hemacytometer 

for a second count. (TT) Note: Count sperm in 5 secondary squares 

and if less than S per square, then count sperm in 2 tertiary squares 

and record change. 

Note: NT • Necropsy Technician TT * Toxicology technician 

30 
P.34 

EQCJIPMEOT NEEDED 

Sharps 

Tyrode's 

Petri Oishes 

Scalpels 

Slides 

Coverslips 

Oven 

PBS 

Paper towels 

1 N NaOH 

Slide Warmer 

Pipettors 

Pipette tips 

Pasteur Pipettes 

Garbage Bags 

Hemacytometers 

Test Tube Rack 

Test Tubes 

Timers 

Waterbath 

Pencils 

1 N HCl 

Test Tube Labels 

Stage Warmer 

Counters 

Syringes (1, 3, and 5 ml; 

Data Sheets 

Pipettes (5 and 1 ml) 

Eraser 

Paper 

Test Tube Brush 

Kim Wipes 

pH Paper 

Thermometers 

pH Meter 

Parafilm 

31 
SECTION I.H 

Summary of Sperm Evaluation Procedures - Rat 

o 

1. Place instruments and glassware in oven at 37 C. (TT) 

o 

2. Put Yolk in water bath at 37 C. (TT) 

3. Weigh animal (NT), record weight (NT), bleed animal (TT), kill 

animal (NT), and bleed again when required (TT). 

4. Excise left testis and epididymis. Remove intact epididymis, 

weigh and record to nearest 0.1 mg. (NT) 

5. Cut off left cauda, weigh to 0.1 mg and record. (NT) 

6. Place 80 ul of warm yolk on each of 2 slides. Place sperm 

sample the size of a pinhead on the slides and gently mix with 

yolk, coverslip, and do motility (40X). (TT) 

Note: There should be 3X as many motile compared to nonmotile 

sperm. 

7. Weigh left testis and record. (NT) 

8. Place remaining cauda in petri dish with 10 ml PBS, gently chop, 

swirl, cover, and incubate for IS minutes at 37oC. (TT) 

9. Remove suspension from oven and pipette 20 times and remove 

connective tissue. (TT) 

10. Place 0.S ml sample in test tube containing 2 ml PBS. (TT) 11. 

Kill sperm by holding test tube under hot water for 1 minute. (TT) 

12. Pipette S times and place sample on hemacytometer for count (20X). 

Repipette and place a second sample on another hemacytometer for a 

second count. (TT) 

Note: Count sperm in 5 secondary squares and if less than 5 per 

square* then count sperm in 2 tertiary squares and record change. 

Note: NT * Necropsy Technician TT » Toxicology Technician 
EQUIPMENT NEEDED 

P.36 

Sharpe 

Yolk 

Petri Dishes 

Scalpels 

Slides 

Coverslips 

Oven 

PBS 

Paper Towels 

Slide Warmer 

Pipettors 

Pipette tips 

Pasteur Pipettes 

Garbage Bags 

Hemacytometers 

Test Tube Rack 

Test Tubes 

Timers 

Waterbath 

Pencils 

1 N HCl 

Test Tube Labels 

Stage Warmer 

Counters 

Syringes (3 and 20 ml) 

Data Sheets 

Pipettes 

Eraser 

Paper 

Test Tube Brush 

Kim Wipes 

pH Paper 

Thermometers 

pH Meter 

Parafilm 

33 
P.37 

Section 2A 

Protocol and Standard Operating Procedure for the 

Vaginal Cytology Assay in Mice 

(To be conducted at NTP-designated bioassay laboratories) 

The following information provides a quick overview of 

the estrous cycle and describes the technical protocol for 

the vaginal cytology assay. Also provided are explanatory 

figures and a list of materials. 

The Estrous Cycle 

The existence of a typical estrous cycle in the guinea 

pig and the associated changes in the vaginal cytology were 

first reported by Stockard and Papanicolaou in 1917. Shortly 

thereafter, similar phenomena were reported in the rat and 

mouse. Vaginal smears obtained from these animals were found 

to correlate well with changes occuring in the reproductive 

tract and with the secretion of the ovarian hormones. The 

most common types of cells present in a vaginal smear are: 

1. Polymorphonuclear leucocytes 

2. Nucleated epithelial cells 

3. Large squamous epithelial cells, also called cornified 

• cells (nucleated or non-nucleated). 

The estrous cycle of the mouse and rat is completed in 

four to five days (polyestrous) although the timing of the 

cycle may be influenced by exteroceptive factors (light, 

temperature, etc.). The cycle is roughly divisible into four 

34 
P.38 

main phases (see Figure 8): 

1. Estrus - This is the period of heat and copulation is 

possible only at this time. This phase lasts from 9 to 

IS hours and is characterized by a high rate of running 

activity. Many mitoses occur in the vaginal mucosa and 

as new cells accumulate, the superficial layers become 

squamous and cornified. The latter cells are exfoliated 

into the vaginal lumen and their presence in vaginal 

smears is indicative of estrus. During late estrus 

there are cheesy masses of cornified cells with 

degenerate nuclei present in the vaginal lumen, but few, 

if any, leucocytes are found during estrus. Much of the 

luminal fluid in the uteri is lost before ovulation. 

2. Metestrus: This occurs shortly after ovulation and is 

intermediate between estrus and diestrus. This phase 

lasts for 10 to 24 hours and mating is usually not 

permitted. Many leucocytes appear in the vaginal lumen 

along with a few cornified cells. 

3. Diestrus: This phase lasts for 60 to 70 hours, during 

which functional regression of the corpora leutea 

occurs. The vaginal mucosa is thin, and leucocytes 

" migrate through it, giving a vaginal smear consisting 

almost entirely of these cells. 

4. Proestrus: This heralds the next heat and is 

characterized by functional involution of the corpora 

lutea and preovulatory swelling of the follicles. Fluid 

35 
P.39 

collects in the uteri and they become highly 

contractile. The vaginal smear is dominated by 

nucleated epithelial cells which occur singly or in 

sheets. 

35 
P.40 

Figure 8. Sections through the vaginal wall of the rst 

during different stages of the estrous cycle 

showing the corresponding types of cells 

which appear in smears obtained from the 

vaginal lumen. A, Diestrus; B, Proestrus; 

C, Estrus; 0, Metestrus; and 6, adult 

animal which had been oophorectomized fbr 

six months. 

37 
P.41 

Section 2B 

Protocol for Obtaining a Vaginal Smear from Mice and Rats 

1. Two vaginal smears should be prepared from each animal 

between 8-10 a.m. for twelve consecutive days in the 

last twelve days of the 90-day study. Dose levels at 

which these are prepared will be designated by the NTP 

project officer for the bioaasay laboratory in 

collaboration with the NTP chemical manager. 

2. Fully frosted slides will be used to make vaginal smears 

(Dakln-Erie 2958). All smears are made on the frosted 

front of the slide. 

3. Microscope slides are marked with a lead pencil into a 

grid consisting of six squares per slide on the frosted 

eside of the slide. The squares are labelled 1 through 6 

and 7 through 12 in the upper right hand corner. This 

is convenient as it allows the incorporation of six days 

of smears on one slide which can be stained at the same 

time. 

4. A 3-4" medicine dropper is moistened by aspirating 0.9% 

saline or PBS solution. A very small amount of saline 

is left in the medicine dropper then placed in the 

vagina and the vaginal fluids aspirated several times. 

5. The contents of the medicine dropper are transferred 

onto the slide. The use of excessive saline should be 

avoided to prevent the flow onto surrounding squares. 

33 
6. At the end of each day, the smears are fixed by lightly 

1 

spraying fixative (Spray-Cyte II by Clay Adams 7180 ). 

In order to fix only the specific square of the slide, 

use a 2" X 4" file card with a window. A cardboard 

window can also be used. The slides should be stored 

in closed boxes or in a dust-free atmosphere between 

collection dates. 

7. On the 12th day after smears have been made, the slides 

are allowed to dry completely. To stain the smears, the 

slides can be stained the same day or within the next 

few days. 

8. Slides are then loaded in glass racks and dipped in the 

following solutions: 

a) 95% Ethanol - 30 minutes 

b) 80% Ethanol - 1 minute with gentle shaking. 

c) 70% Ethanol - 1 minute with gentle shaking. 

d) 50% Ethanol - 1 minute with gentle shaking. 

e) 20% Ethanol - 1 minute with gentle shaking. 

f) 0.5% Toluidine Blue in 20% Ethanol - 30 - 45 

seconds. Personal judgement should be used 

because Toluidine Blue improves with age. 

g) Running tap water rinse - until tap water runs clear 

(at this point if slides aren't dark enough, dip in 

1 

Spray-Cyte II can be purchased from American Scientific 

Products, Obetz, OH, Catalog No. S-7788-2. 

39 
P.43 

20% Ethanol and put back in the stain; this process 

can be repeated until desired color is obtained). 

9. The stained slides are blotted gently under bibulous 

paper. Wet papers are thrown away because material on a 

slide can be transferred to other slides. Slides are 

allowed to dry overnight and the coverslipped using 

Permount or suitable mounting medium and xylene. If 

slides cannot be coverslipped the following day, store 

in a dust-free atmosphere and coverslip as soon as 

possible. 

10.fr Slide* fcmfceHlinq sad CodLame Each laboratory 

participating in this project will be assigned an 

identification number by the NTP-designated 

laboratory. Please contact the SMVCE laboratory to 

find out assigned identification number. Furthermore, 

each laboratory will randomly designate a serial number 

for each experimental mouse (M-l through M-n) and R-l 

through R-n for rats. An example of the code is as 

follows: 01-MIA will designate the first slide, 

laboratory 1, and animal one; second slide will be 

labelled as 01-MlB*. The date when the first smear was 

mader is recorded on each label. In addition to the code 

number and date, 01-6 and 07-12 are also added to the 

two separate slides. This indicates smears from days 1 

through 6 are on one slide and smears from days 7 

through 12 are on another slide (See Figure 9). 

1lw%> stxJrt©i\ f^visc a 

40 
P.44 

I* 

e g l 

ago 

i 

4 

2 

S 

3 

6 

Dale 

Cod

e I 

D7I2 

a 

7 

10 

8 

11 

9 

12 

Figure 9. Preparation of slide for observation 

of the estrous cycle by means of 

vaginal changes. 

41 
P.45 

MODIFICATION TO THE APRIL 1987 SOW 

Appendix G Revised June 1989 

11. All eodes are to remain with tne individual bioassay 

laboratories and a espy of tne code sent to the SMVCE 

laboratory to be opened only after completing all 

evaluations and in the presence of the Quality Assurance 

Officer wno will date and sign wnen opened. Codes must 

include (i) treatment levels, (ii) venicle, (iii) 

route of edainiatration, (iv) date(s) of sacrifice, 

(v) first and last day of vaginal smears and (vi) 

animal strain. Terminal body weights for the females 

should also be included. All slidee will be shipped 

according to attached instructions. 

\aXfl 

• The requirement to collect plasma samples for possible hormone determination 

was deleted 8/88. 

e>. 

••The requirement for processing the vagina from rats and mice for histopathologic 

examination was deleted 5/89. 

12. A person froa the NTP-designated SMVCE laboratory, will 

visit al l bioassay laboratories conducting these tests 

to assess their ability to perform these bioassays. 

4* 
P.46 

Section 2C 

List of Materials for Vaginal Cytology Assays 

(To be supplied by the bioassay laboratory) 

1. Medicine droppers 

2. Pre-cleaned microscope slides (3 X 1")' fully frosted 

(Dakin) 

3. Coverslips (24 X 60 mm) 

4. 95, 80, 70, SO, and 20% ethanol. These grades of 

alcohol can be prepared from 95% ethanol stock. 

5. Spray Cyte II (Clay Adams 7180) 

6. Permount mounting medium and xylene 

7. 100-slot slide boxes 

8. 0.9% saline solution or phosphate buffered saline 

9. Microscope slide labels 

10. Bibulous paper 

11. Toluidine Blue 0 (can be purchased from J.T. Baker 

Chemical Co., Catalog No. W144; if some other source is 

utilised, ensure that the stain is certified for use in 

histology). 

Beakers 

Traye 

3 X 5" cards or cardboard with small window 

1 N BC1 

IN NaOH 

Parafilm 

43 
P.47 

Preparation of Toluidine Blue Stain 

2.5 g Toluidine Blue 

500 mL 20% ethanol 

Mix and allow to sit for one hour before filtering 

through Whatman Grade filter paper. 

44 
P.48 

Section 2D 

Suggestions for Improving Vaginal Cytology Quality 

General instructions to make vaginal smears are 

described in detail in the protocol. A few simple 

precautions, however, can significantly increase the quality 

of the slides. These are discussed in this section. 

Under each category there are suggestions and/or 

possible explanations for correcting the potential problems 

which may be encountered during the preparation of a vaginal 

cytology smear. 

1. Crystallization: This is a frequent problem since most 

staining solutions easily crystallize upon drying. 0.5% 

Toluidine Blue in 20% ethanol solution is optimal for 

staining vaginal smears. We have not noticed any 

excessive crystallization with this concentration. 

Mucous crystallises easily in the presence of stain. 

Since the mucous is darkly stained, that make the smears 

hard to evaluate and, furthermore, the presence of 

crystals distorts the morphology of the cells. Also, 

debri on the slide will provide crystallization sites. 

Saesrs aust be made on clean slides and slides should 

always be stored in a dust-free atmosphere. 

2. Debri: Presence of debri in vaginal smears will 

interfer during the evaluation. Debri usually comes 

froa dirty slides, tissue fragments, or dust in the air. 

Therefore, all slides should be precleaned by wiping 

45 
P.49 

them with a clean gauze or Kim Wipe. Most of the tissue 

fragments can be eliminated by carefully aspirating 

vaginal fluids. It is also important to place all 

uncoverslipped smears in a dust-free environment so that 

dust settling on the slides is minimized. Coverslips, 

if dusty, should be cleaned prior to use. 

3. Staining: The major problem encountered in this 

category is the nonuniformity of the stain. This 

results from the different densities of cells in each 

smear. By scanning the slide, it is possible to 

determine if all the smears are adequately stained. If 

they are not, place the slides back in the alcohol 

series and then in Toluidine Blue to restain them. Be 

careful not to stain cells too dark because it severely 

e* 

interferes with evaluation. 

4. Clumping and Cell Density: The clumping of cells is 

mainly due to an excess number of cells on the slide. 

This can be remedied by not placing too many cells on 

the slide. If the smear is too dense, it will stain 

heavily and clump. If too much saline is used in making 

the saear, the cell density of the saear will be very 

low.* This causes the saear to have too few cells to 

allow accurate evaluation. It aust be added that some 

of the stages of the estrous cycle have a higher density 

of cells than others. This adds to the problem of cell 

density. 

46 
P.50 

5. Air Bubbles: Air bubbles are a major problem especially 

when they occupy a large portion of the slide. They are 

mainly a technical problem caused by faulty technique 

and/or carelessness. More mounting medium may be needed 

to cover the entire slide or better pressing of the 

coverslip to squeeze out air bubbles is required. The 

use of Flo-Tex or similar medium may also alleviate this 

problem. 

47 
P.51 

VAGINAL CYTOLOGY QDALITY CODE 

Code 

00 Excellent 

01 Good 

02 Moderate crystallization 

03 Heavy crystallization 

04 Moderate debris 

05 Heavy debris 

06 Debri surrounding cells 

07 Staining too light 

08 Cornified*cells stained lightly 

09 Staining too dark 

0 Non-uniform staining 

1 Cells heavily clumped 

2 Few cells 

3 No cells 

4 Air bubbles (>5% <20% slide) 

5 Air bubbles (>20% slide) 

6 Slides prepared on non-frosted 

side of slide 

Subcode 

-1 

-2 

-3 

-4 

-5 

-6 

-7 

-8 

-9 

-10 

-11 

-12 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

Refers 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

to 

Day 

Day 

Day 

Day 

Day 

Day 

Day 

Day 

Oay 

Day 

Day 

Day 

1 

2 

3 

4 

5 

6 

7 

8 

9 

only 

only 

only 

only 

only 

only 

only 

only 

only 

10 only 

11 only 

12 only 

Subcodes are used in conjunction with code when that remark 

applies to a particular day (e.g., 13-2 means no cells on Day 2) 

48 
P.52 

SECTION 3 

Instructions for Shipping Slides from NTP Testing 

Laboratory to SMVCE-Laboratory 

1. Shipping and packaging cost will be incurred by the NTP testing 

laboratory conducting the toxicity studies. 

2. Stained and coverslipped slides will be randomly organized and 

shipped to the NTP SMVCE laboratory within 2 weeks 'zt necropsy. 

3. A copy of the code for all slides will be placed in a sealed 

envelope and sent to the SMVCE laboratory along with the coded 

slides. 

4. All vaginal cytology slides will be placed in plastic slide 

boxes, keeping slides from mice and rats separate. 

5. These slide boxes will be placed in 350 lb. test cardboard 

boxes, separated by abundant packaging material for shipment. 

6. An appropriate listing of slides will be packed in the slide 

boxes. 

7. Slide boxes will be packed to avoid any breakage. 

8. Shipping cartons will be sealed and bound with filament tape 

prior to shipment. 

9. Slides sent separately in slide set will be counted as present 

in the inventory; a copy of the slide set inventory will 

accompany the major inventory document. 

10. Each plastic shipping box will be marked to show the range of 

chemical, animal numbers, participating laboratory and address 

and the chemical number or Cl. 

11. Shipping cartons containing slides and the appropriate 

• information will be directly mailed to the NTP designated SMVCE 

laboratory. 

49 
P.53 

SECTION 4 M*uJ S^ehcn 

Instructions for Shipping Frozen Testes from NTP-Designated 

Bioassay Laboratories to SMVCE-Laboratory 

1. Shipping and packaging cost will be incurred by the NTP-designated 

bioassay laboratory conducting the toxicity studies. 

2. The frozen testes will be shipped to the NTP-designated SMVCE 

laboratory along with the corresponding raw data sheets within two 

weeks of necropsy. 

3. All vials will be placed in vial racks and wrapped with bubble 

packing to avoid breakage during shipment. 

4. The wrapped vial racks will be placed in 350 lb. styrofoam-lined 

boxes filled with dry ice for shipment. 

5. An inventory of the vials will be sent with the cover letter. 

6. Shipping cartons will be sealed and bound with filament tape prior 

to shipment and labelled "freeze upon arrival". 

7. Shipping will be expedited so that the testes do not thaw during 

transit. Overnight service by Federal Express is recommended. 

8. If Federal Express is used, the following information should be 

written on the box: ORM-A, UN 1345, Medical Purposes Only! 

Mail to: Environmental Health Research & Testing, inc. 

2514 Regency Road 

Lexington, KY 40503 

Attention: Dr. O.K. Gulati 

SO 
P.54 

SECTION 5 RCvJl%€i „|97 

Return of Slides £:•-. SMVCE-laboratory to the EPL/NTP Archives 

1. Shipping and packaging cost will be incurred by tne SMVCElaboratory. 

2. All slider will be returned ta the EPL/NTP Archives. A cover 

letter wich the following information will be included: 

a) Chemical Name 

b) Participating Laboratory and Address 

c) SMVCE Study No. 

d) Chemical No. (Cl) 

e) Type of Study 

f) Date Slides Received ac EHRT 

g) Date Final Report Submitted 

Also enclosed will be a ccpy of the slide authorization return form 

3. All slides will be placed in the original plastic slide boxes. 

4. These slide boxes will be placed in 350 lb. test cardboard boxes, 

separated by abundant packaging material for shipment. 

5. Slide boxes will be packed co avoid any breakage. 

6. Shipping cartons will be sealed and bound with filament tape prior 

to shipment. 

51