170

A REVlEW OF SOME A P P L t E O HORHONE ASSAY METHODS

R, J. OEANS
........ MI C H I GAN S T A T E U N I V E R S 1 TY
o.o.ooo...o...ooooooo.oo....oo..oo...o.-oo.o.~o.

Toda;y much is being written and said about the use of hormonal
substances i n c m e r c i a l lamb and c a t t l e feeding.

Their use has been cleared in implanting lamb with cambination

progesterone-estradiol p e l l e t s and incorporating d i e t h y l s t i l b e s t r o l i n
cattle feeds.
With t h i s widespread ccermercial we of these physiologically
a c t i v e substances it is necessary t o determine residual hormonal substances
remaining i n meat from animals having been administered these hormonal
substances.
An assay is a technique either biological o r chemicd which is
used t o : (1)determine i f an unknown substance has hormonal character-
i s t i c s , and aP w h a t type hormonal substance it is; (2) t h e r e l a t i v e potency
of t h i s hormonal substance, i.e., 8, quantitative assay.
Biological assays are based on known e f f e c t s which these hormones
have on certain body f'unctiona or tissue6. It is based on the premise t h a t
c e r t a i n concentrations of unknown substances given t o s u f f i c i e n t numbers of
similar laboratory animals w i l l e l i c i t responses i n relationship t o potency.
This response is then compared t o that of a known s t a n b r d t o determine
r e l a t i v e potency.
Chemical methods are based on t h e a b i l i t y of t h e chemist t o
identify a corqpound and then t o determine quantitatively the amount of
hormone present in an unknown.
Because of the sizeable task of reviewing a l l types of hormone
assays, t h i s paper will deal with those most l i k e l y t o be encountered by
one interested in livestock and meats.
There are a number of problems t o be encountered i n assaying a
hormonal substance by a biological method.
1. The variation of laboratory animal not only between assays, but
within assay groups constitutes a major problex. Most bioassay
techniques have set f o r t h type of animals t o be used, size, age,
degree of maturit.y and quite often, s t r a i n . Certain inbred
s t r a i n s of laboratory animals have been developed for assay
purposes.

2. Environmental conditions must be kept constant. T h i s i s par-

t i c u l a r l y important when assaying substances such as thyroxin
where the assay is based on C02 output and O2 intake. Feeds
must be caref'ully studied so as not t o introduce endocrine
 171.

stimulating substances i n t h i s way. Temperature and l i g h t has

been shown t o have e f f e c t on thyroid a c t i v i t y . Since endocrine
interrelationships are so markedly important t h i s could cause
variation i n expected results.
3. Wen with highly inbred s t r a i n s of animals held under ideal con-
ditions, we m y get abnormal endocrine functions i n c e r t a i n
individuals.
4. The endocrine balance in l i v i n g organisms i s a very delicate r e -
lationship and when disturbed, a number of changes usually occur
i n other endocrine glands i n the same organism. A notable example
of t h i s is the thyroid anterior p i t u i t a r y balance.
5. Unknowns nzsy contain biologically a c t i v e substances which are not
i d e n t i c a l with t h e t r u e hormone substance. A crude extract w i l l
contain 8 mixture of estrogen and other substances and the potency
it exhibits w i l l depend on t h e method used and t h e nature of the
extract.
6. According t o E$;mens, d i f f e r e n t estimates can be obtained by alter-
ing such variables as: solvent used, spacing of injections, number
of injections, e t c , "The influence of such variables is so great
t h a t (except under special conditions) It is impossible t o a t t a c h
any precise meaning t o t h e r e s u l t s of an assay unless It is known
i n advance that the substance under t e s t is i n pure form and i s
i d e n t i c a l with the standard preparation i n chemical constitution.
At the present, t h e most sensitive and accurate assws available
a r e of t h e biological type, Even with these, only a few are considered t o
be reliably accurate from a quantitative standpoint. There is a strong
tendency f o r the most sensitive t e s t s t o be t h e l e a s t accurate. Combined
s e n s i t i v i t y and accuracy is yet t o be reached.
In order t o understand b&er t h e characteristics of the various
types of assays and t o r e s l i z e thelr limitations, specific assay methods
should be examined.
Estrogen - Characteristics
Estrogens a r e steroids having t h e basic steroid structure shown
below.
172.

EstroGens induce estrus and stimulate secondary sex organs and sex
characteristics in normal and ovariectonized females. They stimulate growth
of the uterine muscle and endometrium, motility of the uterus and fallopian
tubes, increase capillary permeability in the uterus and vaeina, thickening
of the vaginal epithelium and lowering of the pH of the vaglnal secretion.
They are necessary for mammary gland development and prepare
rammary tissues for prolactin stimulation.

There are two general types, natural and synthetic.

The most potent natural type estrogen is
the main ovarin estrogen. Thep estradiol form is
urine an8 is much less potent:

-
Another natural estrogen, estrone is found in urine, ox adrenal
glands, and placentae. It is 1/4 '-active as estradiol and is formed
in vivo from estradiol. An international unit of estrogen activity is that
of .1 ug. of international standard preparation of estrone.
Estriol, a third natural estmgen is found in urine and is less
&ive than estrone. "The potency varies enormously with the test method."
VaSrues of 1:l to 250:l have been obtained for estrone/estriol ratio.

Like estrone, estriol is formed in vivo from estradiol.

Estriol is excreted in the urine as sulfates and glucuronides.
The most prominent synthetic estrogen at the present is
diwlstilbestrol

Diethyl stilbestrol
This subst>mceis highly potent orally as well as by injection.
The potency of diethylstilbestrol when injected, l i e s between that of
estradiol and estrone. Other types of synthetic estrogens are hexestrol,
and dienestrol, both of which have similar properties to diethylstilbestrol.
 173.

faanysynthetic estrogens and their estrogenic androgens are not themselves

estrogenic but exert their effect after metabolic transformation in the body.
Administration -
- of -
and Utilization - Estrogens
The mode of administration of estrogenic substances has lately
been of considerable significance .
The most useful vehicle for injection of natural or synthetic
estrogens is an -
oil. When injected, even large excessive doses of most
estrogens are rapidly absorbed and little prolongation of action is
attainable by increasing the amount injected.
Estrogens are readily absorbed in the intestine but when ingested,
they exert relatively little estrogenic activity, owing to oxidation and
conjue;ation in the liver. The liver can reversibly oxidize estradiol to
estrone and to other products and it secretes estrone into the bile.
The rate of uptake of an injected hormone substance is a vital
characteristic in its assay. Estrogens soluble enough in water or saline
to be injected in such are even more rapidly lost from the site of in-
jection and excreted or destroyed.
The characteristics of the hormonal substance itself offset the
rate of utilization. According to Meischer (1) the longer the aliphatic
chain the more greatly prolonged was the effect.
The minimum dose required to produce an estrogenic effect rises
with the length of the aliphatic chain or chains, as shown by Parkes ( 2 ) .
The mechanism by which esterification exerts its characteristic effects is
by delaying absorp5ion fromthe site of injection.
When an injection technique is used, the potencies of the natural
estrogens most closely approach one another if absorption is slow, but not
too slow. This occurs with hydrolyzed extracts when multiple injections of
an oil solution are given or by the addition of palmitic or other fatty acids.
Substances siTj.1.ar to these fatty acids are present in some ex-
tracts and may so equ.q,l.ize the potencies of the natural estrogens that use-
ful and reasonably consis5ent results may be obtained by a two injection
technique.
Estrogen -
A s s q Procedures

As previously stated, estrogen causes cornification in the vaginal

cells. This principle is used in the Allen-Daisey Vaginal Smear Assay
Dosage: Solutions made up so that .OS to . 2 m l . is in-
jected each time.
No. of
injections: For routine assap -2 injections are employed.
Smear: Smears are then taken 3rd and 4th d w . Three
smears are taken each time. Test is considered
174.

positive i f any of 3 smears is positive. All

smears a r e taken from dorsal vaginal wall.
Staining : Smears are then stained with methylene blue.

--- -- -
I n t e r - e t a t i o n --and Discussion
.-- of -
Data
Dosage expressed as logarithim should be plotted against per
cent positive slides

Sample data tabulation and graph i s shown below.

Per cent- Positive Respons

-1 2 hrs. 24 hrs. 36 hrs.
-I__

1 10 .5 I.U. 0 0 71
2 10 1.0 I.U. 14 42 89
3 10 2.0 I.U. 14 89 100
4 lo ---- 0 0 0

2.5 100

24 hour response plotted

0

-2.5

Log Total Dosage (expressed i n gama or I . U . )

Probably one of t h e most accurate and sensitive techniques f o r

estrogenic assay is t h a t set f o r t h by Rubin-Dorfman (3).
The assay I s based on the increase i n weight of t h e immature
mouse uterus and is conducted as follows:
 175.

1. Mice are obtained 23 t o 25 days of age.

2. Animals are indected subcutaneously once daily f o r 3 da,ys
with material suspended i n corn o i l . Daily dose was con-
tained .1 CC. o i l .
3. Twenty-four hours a f t e r injection, uterine weights and b o w
weights were determined.
4. The uterine weight/body weight r a t i o was determined.
5. Four equal sized groups of animals were injected, two with
a standard, two with an unknown.
The concentration of stambrd and unknown was chosen so that
t h e followindy relationship held:

High dose s t d . 1~ H i g h dose unknown

LQW dose s t d . Low dose unknown

With t h i s experimental design it is possible t o calculate:

1. Relative potency of EUI unknown preparation.

2. Standard error of potency r a t i o .
3. Mean slope ( )
4. Significance of difference between respective slopes.
5. Index of precision t h e smaller the value of , the
more t h e precision.
Most calculations were based on uterine r a t i o vs. log dose.
However, with estrone, calculations a r e based on the log of uterine r a t i o
vs. log of dosage.
This is necessary when dosages range i s wide (.04 t o .32 m i l l i -
grams). In general, it is better not t o assay t o o near t h e minimum or
maximum response levels, because chances of s e n s i t i v i t y threshold variation
may cause skewed results.
The mouse uterine weight method has t h e advantage over vaginal
smear methods of estrogen assay i n t h a t the r e s u l t s axe obdective.
Sensi t i v i t x
The s e n s i t i v i t y of the mouse uterus is such t h a t .4 micrograms
of e s t r i n , .02 micrograms of estradiol, 2 micrograms e s t r i o l c8n be
detected.
IntravaRinal Pellet Estrogen Assay
An assay technique by Albriewc. (3) has been established using a
pelleted type hormonal substance.
176.

Blood p e l l e t s were made using 10 per cent sesame o i l as a

"binder". Blood i s placed i n cellophane tubes and suspended i n front of an
e l e c t r i c fan a t roan temperature. After drying, it is mixed with sesame
oil and harmnered i n t o pellets weighing 40-80 mg.
Separate pellets =de from serum or c e l l s can be prepared by
centrifuging blood.
P e l l e t s are then inserted into t h e vagina with a trocar and are
completely dissolved i n 24 hours. This technique, while not widely used,
offers p o s s i b i l i t i e s f o r c e r t a i n phases of assqyrtng where t h e nature of t h e
unknown might make it d i f f i c u l t t o administer i n other ways.
ProaesteroE

-
Characteristics --
of t h e Hormone

T h i s hormone is secreted by t h e corpus luteum and has been iso-

lated only f r m t h i s gland. It is t h e only known, naturally occurring ccm-
pound, with full progestational a c t i v i t y . It can be produced i n c r y s t a l l i n e
form. It has been produced synthetic- from t h e vegetable s t e r o l stigmas-
t e r o l . It is excreted as pregnandiol.

.i
Progesterone) Pregnandiol Sodium Glucuronidate Pregnandiol has
no known hormonal action. However, it i s an anaesthetic. Cbsnges i n ex-
cretion of pregnandiol follow closely changes i n the production of
progesterone. However, pregmndiol and t h e excretory form of other related
compounds such as desoxycorticosterone. Thua, t h e detection of t h i s ma-
t e r i a l i n t h e urine cannot be taken as en accurate measure of the amount of
progesterone secreted

.
There are synthetic substsnces which are of minor p r a c t i c a l
significance
--
E t h a l testosterone i s the most potent synthetic substance.
(1/10 t h e a c t i v i t y of progesterone). It i s as active by mouth as by in-
jection, but i s weak both estrogenically and androgenicaly. Ethinyl
androstenediol has about one-half the a c t i v i t y of ethiny:? testosterone.
Since progesterone is not effective t o any extent when given
orally, it cannot be incorporated i n t o feeds f o r laboratory animals. Thus,
t h e assay of animal t i s s u e s f o r residual progesterone is quite d i f f i c u l t .
In addition t o t h e problem of administration, we do not have such rela-
t i v e l y simple response mechanisms as uterine weights and vaginal smears i n
 177

assaying progesterone. Physiologically, progesterone is responsible for the

development of the estrogen primed uterus and the maintenance of pregnancy.
Within a few days after fertilization, or on artificial administration of
progesterone, there is a rapid increase in cell division of the surface
epithelial cells. This activity is accompanied by a downgrowth of glands
and an increase in strcinal cell proliferation.

A very sensitive sssrqy method for progesterone has been developed

by Hooker (4) This technique is based on the principle that the endometrium
of the mouse exhibits hypertrophy of the stromal nuclei when stinullated by
progesterone. This technique I s currently being used on samples of meat from
implanted steers on test at Michigan State.
Procedure :

The hormone is dissolved in sesame o i l . The standard mount ad-

ministered was .0006 ml. Undesirable distension was produced if
more W&S injected. In order to administer such small amounts, a
micrometer syringe technique must be established.
Test animals used were adult female mice which had been ovarecto-
mized 16 days before test. The technique of administration is
quite detailed. The animtrls are anesthesized, and then the mid-
ventral section opened to expose the uterine horn.
The test substances are carefully injected into the uterus with
the section being tied off to prevent leakage of the mterial.
The animals are then killed 48 hours after injection. The uterine
segment is removed snd fixed In Lavdowsky's fluid. Paraffin
sections 6 microns in thickness are stained with Harris'
hematoxylin and Eosin.
A positive test I s described as follows: Stromal nuclei are
enlarged and appear smooth and ovaJ. in outline as conpared to
shrunken nuclei and clumped chromatin granules, in non-treated
cells. This appearance in =one of the s t r d cells is con-
sidered positive.
The authors feel that the response obtained is specific for
progesterone since 6 microerams of desoxycorticosterone, 48 micrograms of
testosterone, .00075 microgram of estradiol or .6 ug. of estrone did not
produce similar results, although there was some enlargement of nuclei.
Sensitivity:
The minimal effective dose (least amount which induces a positive
response in any stromal nuclei) is said to be consistent3y ,0002 micrograms
of progesterone.

This method could be adapted to a quantal assay in which 100 per

cent positive responses could be expected with minimal effective dose.
178.

Progesterone - Chemical Assay:

Edgar (5) has developed a method by which micro-quantities of
progesterone can be estimated in blood from the ovarian vein of sheep and
from follicular fluid of Grmf'ian follicles and ovarian cysts. The method
involves extraction and partition between organic solvents and the semi-
quantitative estimation by chromatographic separation on filter pager.
Amounts varying from 1.5 to .3 micrograms per m l . of fluid could be detected.
The best method according to Ehraens is that of Venning (6). By
this technique, butmol extracts the sodium pregnanadiol glucuronidate from
the urine. After careful separation and filtration the Na P.G. residue is
dissolved in hot alcohol and dessicated. The amount present is determined
by weighing.
Testosterone:
This is a male sex hormone classified as an androgen. Testosterone
is the most potent androgen, being six times more potent than androsteroae.
Androgens affect the following biological activities:
1. Metabolism
2. muscle rnass
3. electrolyte metabolism

4. enzyme concentrations of various muscles and glands.

Androgens of course, cause the development of secondary sex
characteristics in males. There is evidence of some similarity in effects
of androgens and female sex hormones.
Transandrostenediol, dehydroisoandrostenediol, androstenedione,
and testosterone produce uterus growth in the immature rabbit. Trans-
androstenediol produces stratification and cornification of the vaginal
epithelium in the castrate rat.
Most of the androgen is derived from the testes. However, it
is possible that these are androgenic substances secreted by the adrenals.
Administration:
Androgens are ineffective when administered orally. Thus, making
an assay of their potency or presence in carcasses more difficat. When
crystalline androgen is implanted subcutaneously, slow absorption and pro-
longed effects are obtained.
Assay Methods:
There are a number of various a s s a y techniques for androgens.
The most successful are those using:
1. Comb growth response in fowl.
2. Seminar vesicle and prostate response in the rodent.
 179.

Capon Comb Growth

IC

Gallagher and Koch (7) developed a method using as a response

t o o l the comb growth of capons. Five daily injections of testosterone
unknown i n 1 ml. o i l are used i n a group of capons, while inJections of
testosterone standard in lml, o i l a r e made i n another group of capons.
From t h i s a characteristic curve was developed, which used a dose-response
relationship.
Bliss (8) elaborated on t h i s technique by running l i v e concentra-
t i o n s of unknown and two concentrations of standard and then determining
potency r a t i o . The t o t a l concentration of testosterone propionate used f o r
each animal should be i n the range of 20 t o 160 micrograms. A t least 32
animals should be used on standard and unknown. A l i n e a r response i s
obtained when log dose plotted against l o g response.
Enslens (9) applied testosterone d i r e c t l y t o combs. Obtained
s t r a i g h t l i n e f o r log dose response.

--
Chick Comb Method

Dorknan (10) injected chicks with hormone suspended i n o i l . ;&e

-
obtained a s t r a i g h t l i n e log dose response relationship.
-
Seminal Vesicle - Prostate Response Method
Principle: -
Testosterone w i l l repair t h e damages t o seminal
vesicles and prostate caused by castration.
Procedure:
Testosterone standard and unknown a r e dissolved i n corn o i l of
which .1 t o .2 ml. are injected d a i l y (each animEll receiving
e q w amounts of o i l . )
T o t a l dosage with testosterone should f a l l i n range of 0-5 mg.
Androsterone has been administered up t o 30 mg. Variation i n
length of treatment ranges from 8-10 days. Rats a r e killed,
body weights taken and seminal. vesicles and prostate removed.
The seminal vesicles are then weighed (with prostate o f f ) . The
log of the dose is then plotted against the gland weights.
Thyroxin
T h i s hormone secreted by the thyroid gland is of v i t a l importance
i n regulating t h e rate of energy exchange i n t h e body. It can be observed
by i t s e f f e c t on basal metabolic rate and i n oxygen uptake of t i s s u e s l i c e s .
Among the physiological characteristics with which thyroxin i s
associated are:
180

1. amount of adipose tissue

2. rate of glucogeneisis
3. rate of excretion of urinary nitrogen

4. rate of i n t e s t i n a l absorption of oxygen and i n t e s t i n a l

peristalsis.
5. respiratory quotient
6. l e v e l s of blood l i p i d s
7. s t o r e of body proteins
8. storage of l i v e r glycogen
9. l e v e l of blood sugar

10. rate of secretion of urinary H20

ll. rate of excretion of urinary calcium and phosphorus.
On the endocrine system, thyroxin a f f e c t s :
1. secretion of thyrotropic hormone from t h e a n t e r i o r p i t u i t a r y
2. secretion by adrenal cortex by ACTH stlmulation
3. conditioning of reactive parts of body epinepherine
4. appears necessary f o r adequate secretion of p r o l a d i n and
consequent milk secretion.
There are a number of asssy methods available which apply some
of the above Characteristics in response characteristics.
Goiter Prevention Method
-
Principle when a goitrogenic substance (thiouracil) which
inhibits thyroxin production is given, we get increased thyrotropic hormone
secretion and resultant hypertrophy of t h e thyroid,
This thyroid hypertrophy can be prevented by administering
thyroxin t o restore n o d belance.
Procedure :
100-200 gram male rats a r e grouped (8-10). Five groups 4re usually
sufficient t o establish a response curve All groups are given
. l p e r cent t h i o u r a c i l i n feed during treatment period. (Dosage
should be given t o f a l l In middle of response curve i f possible.)
AU. but one group of t h i o u r a c i l t r e a t e d rats are then injected
with 1-4 microgrems/100 g. body weight of thyroxin.
 181.

After 14 days, thyroids are removed and weighed. Response i s

reported as micrograms thyroxin/100 grams body weight. An i l l u s t r a t i o n of
graphic presentation i s as follows:

m$100 g. body w t . l6 \
14
f
\
l2

10
\\
-----t-- -
Normal thyroid weight

8 -V

-
Note: when curve intercepts normal weight l i n e can be taken
as measure of thyroid secretion rate since it represents t h e amount of DL-
thyroxin required t o maintain normal thyroid p i t u i t a r y balance.
Metabolic Rate Method
L-

Dressler and Halling (11)reported a method of assaying thyroxin

by i t s e f f e c t on the metabolic rate of guinea pigs.
Procedure :
_I_-

Feed and water are withheld from guinea pigs f o r 18 hours before
determination. Oxygen consumption and CO2 output is then deter-
mined with metabolism being expressed as ml. 02 o r C02 exchanged
per 100 g. body weight per hour. These control readings a r e taken
on 3 separate days.
Thyroid preparation i s administered o r a l l x for 4 consecutive
days. One hundred hours after t h e f i r s t dosage B.M.R. deter-
mination is made and percent increase noted f o r each animal
over control value.
A reference curve i s estimated by graded dosage with standard
thyroid powder. The potency of t h e test preparation i s then
computed i n terms of t h e standard.
The thyroid equivalent can be established by reference t o standard
slopes
The percent increase in oxygen consumption is substituted f o r y
-
i n the equation. Then the equation is solved f o r X t o get
logarithm of thyroxin dosage that must be given o r a l l y t o give
t h e same response.
 182.

42

36
$ increase
30
in
24
oxygen consumption
18

12
6
0
Thyroxin dosage ug/day/lOO g. body w t .

In the use of this type of assay, the reference curve should be

established for a standard substance 'similar to those being tested.

BIBLIOGRAPBY
1. Meischer, K. Wettstein, A. Tschoppe, 1936. Biochemical J. 30, 1977.
2. Parkes, A. S. 1937. Biochemical J. 31, 579.
3. U e r i e u x , A. S. J. Clin. Endocrinology 1. pp. 889.
4. Hooker, C. W. 1940. Proc. SOC. Exptl. Biol. Xed. 45, 270.
5 . Edg8r, P. G. Prog. in Bcdy Fluids. Nature 170:543. 1952.
6. Venning, E. H. Gravimetric method for the determination of sodium pre-
gnandiol glucuronidate. J. Biol. Chem. ll9:473. 1937.
7. Gallagher, T. F., Koch. F. E. J. Pharm. Exptl. Therapy. 55: 97. 1935.
8. Bliss, B. I. 1944, J. h r . Statis, Assoc. 39: 479.
9. Emmens, C. W. 1929. Med. Research Council. (Brit.) Special Rept. Ses,
234,l
10. Dorflnsn, I. Endocrinology 42, 7 1948.
11. DmeBLer, E., and U h g , K. 1940. Arch. Exptl. hth. Phamcology
196, 266.

12 EhPPens, "Hormone Assay"

 183

MR. A U " : We sure enJoyed the topic t h a t you presented,

and certainly when you cam t o some of the chemical compounds it was
more enjoyable.
A t t h i s time we w i l l ask H. D. Naumann t o lead the discussion.

IVAUMA": Thank you, M r . Chairman.

The recent popularity of the use of hormones has caused
quite a good deal of concern in the glace I am working. The physi-
ologists and the endocrinologists are p r e t t y much up i n the a i r about
it becauae from the standpoint of public health it could be a r e d
problem if soll~ethinggot out of control. They are p a r t i c u l a r l y ques-
tioning i n t h e i r own minds the v a l i d i t y of many of t h e assay technics
that are being used today framthe standpoint t h a t a l o t of them are
b i o l o g i c s t assqys and as a result they are used on t e s t animals and
there isn't d e f i n i t e proof that the response of the t e s t animal is
analogous t o the response of t h e human with whom we are ultimately
concerned. The other question i s perhaps j u s t as important i n that
i n many of these products t h e hormones specifically seem t o have a
cumulative e f f e c t or a t least t h a t i s a strong p o s s i b i l i t y . So t h e
reviews of hormone assw methods i n qy opinion are pertinent a t the
moment.
Do you have any questions t o d i r e c t t o the speaker?

W E L I C : I should l i k e t o make a comment about the

point of the amount of a substance, whatever it may be, that may be
present i n t i s s u e . I have seen reference i n several reports, as you
a l l have recently, concerning how l i t t l e there may be of a given sub-
stance in meat. I t h i n k we should &e it absolutely clear t h a t the
quantitative amount is not the most important thing. The most im-
portant thing i s what amount will produce physiological effects.
I should l i k e t o drive home t h i s point by pointing out
that it mtters l i t t l e i f it takes a f r a c t i o n of a gram of a biologic
substance t o kill a person and it takes several pounds of sodium
chloride. If t h e end result is fatal, it matters not how much it took.
That is one point I should l i k e t o put across.

I think, too, there has been a dangerous point i n the con-

clusions drawn i n some of these experiments. That concerns the e f f e c t
that one observes when he deals with a short-term experiment o r an
acute t o x i c i t y test. Personally I am far more concerned about chronic
e f f e c t s than I am about acute, because i f we can produce e f f e c t s immedi-
ately that are syqptomaticslly obvious, we a r e warned, but there are
instances *ere it may take years, indeed, before t h e results of t h e
ingestion or the use of acme rtmterialmight come about and be recog-
nizable as such. It is i n t h i s l a t t e r category t h a t I t h i n k we should
express the most concern; y e t I hear people with p r a c t i c a l l y no temerity
whatsoever saying, as the r e s u l t of t h e i r assays, t h a t there are not
l i k e l y t o be any e f f e c t s . I t h i n k t h a t i s naive, and I am glad that i n
the remarks Mr. D e w made he brought forward same of these things
184.

Another t h i n g t h a t I t h i n k we should be concerned about

( t h i s i s my t h i r d point) i s that i f by one c r i t e r i o n you produce no
evidence of e f f e c t t h a t does not preclude t h e p o s s i b i l i t y that there
a r e no e f f e c t s . I n the case of the s t i l b e s t r o l assay the uterine
weight of the mouse i s frequently used t o establish whether or not the
material my be present. That I s one thing, but the animal organism
is extraordinarily complex and there may be other effects.

I am not an alarmist. I want t o make t h a t clear, but l e t ' s

be a l i t t l e thoughtful about some of these assays before we write some
of t h e things t h a t have been written.
EIR, BULL: M r . Chairman, is it possible t o hook a hot carbon
on these estrogens that a r e fed the animal? If so, has it ever been
done?
MR. BVTLER: The question?

MR. Can you use a t r a c e r technic on these hormones?

Who would care t o answer t h a t question?
MR. KASTELIC: There are some papers concerned with the tagging
of carbon i n d i e t h y l s t i l b e s t r o l . The carbon t a g i n these instances has
been t h e e t h y l groups. We a l s o have two-ring structures, and while it
has been indicated from such studies t h a t t h e material is not metabo-
l i z e d I would not be satisfied with such information u n t i l t h e r e -
maining part of the molecule had been tagged and u n t i l the degradation
products, if any, a r e carefully mapped out. Tagging one piece of a
molecule i s not s u f f i c i e n t .
MR. BULL: In connection with D r . Kastelic's remarks about
long-time effects, there i s rather prevailing sentiment among medical
men that the use of estrogen by the ladies i n face cream, bust devel--
opers, etc., causes cancer. The same is t r u e about the use of t e s t o s -
terone among you old fellows.
MR. "N: Do we have other comments o r questions?

Well, I want t o take the l i b e r t y , since Deans has mentioned

t h i s i n h i s paper, of directing a question t o him, although perhaps
it i s n ' t r e a l i y within the scope of the t i t l e . .What were the results
of these progesterones on meat t i s s u e s ?

MR. DEANS: On our l a s t bunch we did not get any a t a l l . We

have d i f f e r e n t hormone treatments a t t h i s t i m e . The t h i n g we are com-
paring here is an implant as w e l l . O f course, we have e s t r a d i o l . We
have d i f f e r e n t hornone dosage. So we don't want t o go out on a limb
and predict w h a t we are going t o get. W e didn't get any the last time.

Any other questions? Then I will t u r n it back t o the

Chairman.
MR. AUWW: A t t h i s time we w i l l hear from D r . Breidenstein
of the University of I l l i n o i s .
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