TERATOGENESIS IN INBRED STRAINS OP MICE Major Professor V* y *r^ ^inor/Professox^. ^ • \ Director of the Department of ^Jology Deanl of the Graduate School
TERATOGENESIS IN INBRED STRAINS OP MICE
Major Professor
V* y *r^
^inor/Professox^. ^
• \
Director of the Department of ^Jology
Deanl of the Graduate School
TERATOGENESIS IN INBRED STRAINS OP MICE
THESIS
Presented to the Graduate Council of the
North Texas State University In Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF ARTS
By
Robert A. Morgan, E. A.
Denton, Texas
Augustt 1966
TABLE OP CONTENTS
Pag®
LIST OP TABLES lv
Chapter
I. INTRODUCTION 1 History of Teratogenesis Statement of Problem
II, MATERIALS AND METHODS 12
Selection of Mouse Strains Preparation of Thalidomide Experimental Procedure
III. RESULTS 21
IV. HIS CUSS IOM 31
APPENDIX A k2
APPENDIX B 50
BIBLIOGRAPHY 56
ill
Lic-p OF *A*-LES
Table Pace
I. Results of Administering Throe Dosage Levels of Thalidomide to Pour Wouse Strains 22
II. Results of Administering Distilled Water to Four Strains of Mice 23
III. Comparison of Per Cent Resorptions Due to Administration of Thalidomide to Pour Strains of Mice. . • . . . . . . 2k
IV. Scale for Rating Mice According to Extent of Deformity 26
V. Effect of Thalidomide Administration on Mean Deformity Score 27
VI. Mean Differences in Abnormality Scores in Pour Strains of wj.Ce 28
VII. Analysis of Vsrience in Mean Number of Mice Per Litter Due to Level of Thalidomide . . . . 2*>
VIII. Analysis of Variance in Mean Deformity Score Per Group Due to Level of Thalidomide . . . . 29
IX. Analysis of Variance Due to Route of Thalidomide Injection 30
iv
CHAPTER I
INTRODUCTION
History of Teratogenesis
The first experimental production of congenital ab-
normalities in manna la was accomplished by Altmann (1),
who utilised x-irradiatlon of the pregnant mother. Since
this work, which was performed in the first decade of this
century, various methods have been employed to bring about
deformations in the offspring of treated mothers. The fol-
lowing ia a review of some of the more Important work, and
la by no means exhaustive.
Diets deficient in the various vitamins have been proved
teratogenic in laboratory anlmala. The earliest study with
a vitamin deficient diet was conducted by Hale (26), who fed
a aow a regimen lacking In vitamin A, with the resultant
birth of a litter of eyeless offspring. Vitamin A deficiency
has since been found to be teratogenic in the rat (li|) and the
rabbit (39). Vfarkany and Nelson (8l, 82, 83, 8I4.) obtained
malformations in the progeny of female rats fed a fciboflavin-
deficlent diet. Other workers (21, 3k$ 53) have subsequently
corroborated these findings and expanded upon them somewhat.
The rat has been a popular animal for use in teratogenic
studies of vitamin deficiencies, as diets lacking in folic
acid (6I4.), pantothenic acid (55)» vitamin E (65), thiamin (5U),
X
•nd frltttaln D (80), have all caused congenital malform-
ations in the rat. Niacin antagonists have been shown to
causa developmental anomalies in the chick embryo (141, U2)
and In the mouse '62).
While moat of the above studies Involve diets deficient
in aorae particular vitamin, excesses of vitamin A can alao
cause gross deformities In the rat (85) and mouse (51).
Actadnistratlon of various other amenta to the present fe-
msle laboratory animal has* resulted In deformed offspring.
6-mercaptopurlne, a nucleic acid antagonist'(73)» and an ato
dye, trypan blue (77) ars teratogenic in the rat (7l|» 75# 76)
and In the mouae (33* 67* 66), while cortisone has been
found to eauae cleft palate in the mouse (11, 22, 26, 29,
30, 31# 32# 63) snd the rabbit (20).
In recent yeara, certain drugs used by humans have been
shown to be teratogenic in laboratory animals, a sulfon-
amide, aulphamoprine, causes skull abnormalities in mice
and rats (13)* Acetazolamide (k5)» Ancoloxan (6), and
phsnobarbltal (50) each cause developmental anomalies in
rats. Tetracyclines have been statiatioally implicated
as teratogenic in humans (9, 78) «nd definitely ahown to
cause limb and beak deformities in chick embryos (2, 10).
Even acetylsalicyllc acid, taken in large quantltiea, is
teratogenic in the mouse and the rat (57)*
In 1962 Somera (70) administered thalidomide, a tran-
quilizer, to pregnant white rabbits. There were deformed
fetuaei in three of the four litters delivered. This
tranquilizer had already been strongly suspected tc be the
eauaal agant of a rsssh of limb deformities in humans (lj.9, 60) .
Following Soners1 work, other experimenters produced skeletal
deformities In mice (17# 25, 56), monkeys (16, I4.fi), rats
(11, 37. 58), rabbits (1^, 72, 87), and the chick (3, 5,
6, 15, 19, 36, 52, 66) . Thalidomide has subsequently been
found to produce abnormal growth in a marine red alga (72),
but not in higher plants (59) .
Skeletal malformations due to ingestion of this tran-
quilizer by the pregnant human have been reported in West
Germany (U7, 61) , England (38, W>, 69, 71) , Scotland (22) ,
Taiwan (88) , Japan (28) , Africa (86) , and the Hetherlands
(18 ) . The drug was never placed on the American market;
thus the United States had little direct contact with the
trag«0y. nevertheless, the deformities due to thalidomide
have had the effect of emphasizing the fact that congenital
malformations do occur, and can be caused by seemingly
harmleaa agents.
With the above facts in mind, the Food and Drug Admin-
istration has established 8 program so that all new drugs
are now teated for effect on reproduction in the rat (35).
Both male and female rats are fed a diet containing sub-
toxic levels of the drug in question. These rats are mated,
and the offspring assayed for abnormalities. This procedure
haa three major shortcomings. The first is that the treated
fiaele rats are permitted to give birth to a litter of younp:,
tte Membera of whieh are then examined for abnormalitiee.
thla method of teetinr completely neglects the fact that
ptdmts oftm eat their dead or deformed offapring, thereby
leavinn no evidence of malformed young. The second fault ia
that, regardlass of the drug toeing tested, the pregnant rata
i n a uniform diet* Evidence of diet-teratogen inter-
action plays • major rola In level of malformation (1*0, l|3,
79) makee thia procedure fall aooiatfhat abort of the ideal,
the third fault la that the testing program, aa now estab-
lished, neglecta the likelihood that genetica may be an
important, if not the primary, factor in determining aua-
ce»tibillty to varloua teratogenic agents {?).
Statement of Problem
In view of the fact that the program now being utilised
to teat drugs for poeaible teratogenic effecta does not con-
sider genetic variability among teator atocka, it ia impor-
tant that a atudy te conducted to investigate the influence
that genotype may have on auaceptibility to thalidomide
teratogeneaia. The purpoae of this inveatigation ia to
determine if differencea exist between four inbred atraina
of *lee in auaceptibility to the drug thalidomide.
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lal of Experimental Zoology, CXXXVI (1Q57)»
35* Kelsey, Francis 0., "Problems Rsised for the PDA by the Occurrence of Thalidomide ttabryopathy in Germany, 1960-1961," American Journal of Pablic Health, LV (1965), 1*23-555":
36. Semper, P., "Thalidomide and Congenital Abnormalities," Lancet, II (1962), 836.
37* King, C. T. 0. and P. J. Kendriok, "Teratogenic Effects of Thalidomide in the Sprsgue Dawley Rat," Lancet, II (1962), 1116.
38. Kohler, G. G., H. M. Fiacher, and P. M. Dunn, "Thalido-mide and Congenital Abnormalltiea," Lancet, I (1Q62), 326-327.
39* Laming, G. B., G. V. Salisbury, R. L. Hays, and K. A. Kendall, "The Effect of Incipient Vitamin A Defici-ency on Reproduction in the Rabbit," Journal of Nutrition, LII (1°5U), 217-236.
i|.0. Landauer, Walter, "The Effect of Nicotinamide and alpha-Ketoglutaric Acid on the Teratogenic Action of Insulin," Journal of Experimental Zoology, CIX (1948), 283-290.
, "niacin Antagoniats and Chick Devel-opment," journal of Experimental Zoology. CXXXVI (1957), 171*183. * "•
k2* , and E. M. Clark, "The Interaction in Teratogenic Activity of the Two Niacin Analoga 3-Acetylpyridine and 6-Aminonicotinamide,tt Journal of Experimental Zoology, CXIX (1952), 221-25T;
1)3* , and M. B. Rhode a, "Further Obser-vations on the Teratogenic Nature of Insulin and its Modification by Supplementary Treatment," Journal of Experimental Zoology, CLI (1962), 253-258.
Larson* Valdemar, M % e Teratogenic Effects of Thalido-mide, Imapr amine HC1 and laiApr amine N-Oxide HC1 an tihlte Danish Rabbits," Acta Physiologies Scandinavia, LIX (1963), 87. *
kS* Lay ton, W. M. and D. V. ffallesy, "Deformity of Pore-limb in Rata,* Science, CXXXXIX (1965), 306-307.
46* Leek, Ian and E. L. M. Millar, "Short-Term Changes in the Incidence of Malformations," British Journal of Preven-tive Medicine, XVII (1963), $TT"^
k7» Lens, W., "Thalidomide and CoagiMital Abnormalities," Lancet, I (1962), 1*5.
J|£. Lttoey, J. P. and R. Behnan, "Thalidomides Effect Upon Pregnancy in the Rheaua Monkey," Science, CXXXIX (1963).
J|.9. Mo Bride, P., "Thalidomide and Congenital Abnormalities," Lancet. II (1961), 1358.
50. Mo Ooll, J. D., M. Globus, and S. Robinson, "Drug-Indu4«d Skeletal Malformationa in the Rat," Experlentla. XIX (1963)* 323.
51. Murakami, U. and Y. Kameyama, "Malformations of the Mouse Caused by Hypervitaminosis A of the Mother During Pregnancy," Archives of Environmental Health. XI (1965), 732-7ET:
52. Haber, E. C. and E. J. Largent, "Malformations Induded by Thalidomide in the Developing Chick Embryo," Poultry Science, XXXXII (196^), 1293-12914..
53* Nelson, M. M.# C. D. Baird, H. V. Wright, and H. M. Evans, "Multiple Congenital Abnormalitiea in the Rat Resulting from Riboflavin Deficiency Induced by the Antimetabolite Galactoflavin," Journal of Nutrition. LVIII (1965), 125-145. '
54* » *nd H. M. Evans, "Relationship of Thiamine to production in the Rat/ Journal of Nutrition, LV (1955), 151-163.
55* « C. D. Baird, end H. M. Evans, "Terato-
fenic ffffeots of Pantothenic Acid Deficiency in the at» Journal of Nutrition. LXII (1957), 3^5-405.
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P p l T * r ' l t T — 1 H o t o - * 3 £ * m i
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Thalidomide on the Rat Foetus/ Experientla!CXIX°f (1963), 645*
59« Psrups, E. V, and X. Hoffman, "Ansence of Growth Effects of Thalidomide on Higher Plants," Nature. CCV (1965) . 1241.
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61. Pfeiffer, R. A . and W. Koaenow, "Thalidomide and Con-genital Abnormalities," Lancet. I (1962), 45-46.
62. Pin sky, L., "The Production of Sfcsleta* Malformalitie a in the Offapring of Pregnant Mice Treated with 6-Aminonicotinamide," Physiological Review, XXXIX (1959), 69-113.
m and Angelo Di George, "Cleft Palate in the Mouae: a Teratogenic Index of Glucocorticoid Potency." Solenoe. CXXXXVII (1965), 402-404.
64. Richardson, C. R. and A. 0. Hogan, "Diet of Mother and Hydrocephalus in Female Rata," Journal of Nutrition, XXXII (191*6), 459-465.
10
65- Shut*, I., "The Relation of Deficiency of Vitamin B to the Anti-Proteolytic Factor Pound in the Serure of the Aborting Woman," Journal of Obstetric® and Gynecology of the BrltlaE~5SpIre~xmHI (193RT
66. Smith, T. E., V. 0. Berndt, P. 0' Leary , and D. Cavanaugh, "The in vitro Inhibitory Effect of Thalidomide on the Chick~Ewfcryo7" -American Zoologist, II (1962), 559.
67* Sadthberg, M., "Teratogenic Effects of some Hypoglycemic Substances in Mice," Anatomical Record, CXXXVI (1960), 280.
68* , Ind H. M. Runner, "Teratogenic Effects of Hypoglycemic Treatment in Inbred Strains of Mice," American Journal of Anatomy, CXIII (1963), 172,
69* Smithella, R. V. tad M. B. Lend, "Thalidomide and Mal-formations in Liverpool," Lancet, I (1962), 1270.
70, 8oners, O. P.. "Thalidomide and Congenital Abnormalities," Lancet, I (1962), 912-913.
71* Speirs, A. L., "Thalidomide and Congenital Abnormalities," Lanoet. I (1962), 303.
72. Spencer, K. B. V., "Thalidomide and Congenital Abnormal-ities," Lanoet, II (1962), 100.
73* Thiersch, J. B., "Effect of 6-Mercaptopurine on Rat Fetus and on Reproduction of Rats," Annals New York Academy of Solence, LX,(1954), 220-227 •
74* Tuehmann-Dupleiss, H. and L. Merceir-Parot, '"Influence of Three Hypoglycemic Sulfamides, Carbutamlde BZ55, Tolbutamide Do60, and Chlorpropamide on the Pregnancy and Prenatal Development of the Rat," Anatomical Record, CXXXVI (I960), 294.
75. , "Oral Antl-ai acetic Druga ana Teratogenicity," Lancet, II (1963), 408.
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11
78. Walford, P., "Antibiotics and Congenital Malformations," Lancet, II (1963), 298-2Q9.
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CHAPTER II
"ATS:*.I ALS and methods
Selection of Mouae Strains
Inbred strains of nice are becoming increasingly popu-
lar as laboratory animals for use in genetic studies. In
order for mice to qualify as an inbred strain, they must be
the descendants of a minimum of twenty generations of inde-
pendent brother to sister matings. Parent to offspring matinps
may be substituted if the parent utilized is always the younger
of the two parents (13). Inbreeding has the effect of pro-
ducing genetic homogeneity; that is, Inbred mice are theoret-
ically homozygous at alnost every locus. Due to their high
degree of genetic homogeneity, mice of a particular strain
will usually exhibit much lass variation in response to a
given treatment than will randomly bred mice. Furthermore,
mloe of • particular strain may respond to a given treatment
in a markedly different manner than mice of seme other
strain* There are many well established inbred strains of
mice, whose responses to various parameters, such as irradia-
tion and contact with different viruses and carcinogens, are
well known. Practically nothing la known concerning the sus-
ceptibility of different mouse strains to teratogenic agenta.
12
13
With respect to the teratogenic effects of vita-nin
deficiencies on inbred mice, Kalter and Warkany (7) found
C57BL/6J mice to fee much ore resistant to a riboflavin-
deficient diet th*n ce of strains 12°/j and A/j. Experi-
ments reported by Praser et (2) indicate that A/J mice
•re very susceptible to niacin antagonists, while Ingalls
et si. (6) reported a difference between strains A/j and
C57BI/6J with respect to susceptibility to hypoxla-induced
teratogenesis.
Several workers have reported teratogenic effects of the
tranquilizer, thalidomide, In inbred strains of mice. Ha^en
(5) found that thalidomide Increased the resorption rate in
strain A/J but not In strain C57BL/6J. Di Paolo (1) reported
resorptions and skeletal deformities In strain A/J mice,
using thalidomide as the teratogenic agent. Glroud £t al.
(It) administered thalidomide to Swiss Albinos and the Black
and A/BC strains of mice and reported deformities In the pro-
geny, but did not report any differences between strains.
Silveatrini and Garau (11) reported nalformltlea in strains
CP} and Swiss upon administration of thalidomide. Again, no
differences between strains ware noted. Various other workers,
including Fratta et al. (3), Seller (10), Somers (lk), and
Woollaa ( 1 5 ) , failed to obtain abnormalities in offspring of
inbred nice administered thalidomide.
With the exception o f Hagen ( 5 ) , none of the above-
mentioned experimenters have reported any differences between
Ik
the Inbred strains utilized with reapect to susceptibility
to the teratogenic effects of thalidomide. Furthermore,
Hagen (5) reported a difference in resorption rate, not in
actual deformities.
Since different inbred strains of mice are known to
exhibit very different reactions to varioua treatments, it
seema feasible that some strains would show different sus-
ceptibilities to teratogenic apents. In order to test this
possibility, four inbred strains were chosen to be tested
for susceptibility to the teratogenic effects of thalidomide*
C57BL/6J and A'J were chosen because the work of Kalter and
Warkany (7) showed these two strains to have different sus-
ceptibilities to the teratogenic effects of a riboflavin-
deficient diet and also because of the results reported by
Hagen (5). 129/J ^ice were chosen for use in this study
beoauae of their high susceptibility to the riboflavin
deficient diet used by Kalter and Warkany (7)> C3HeB/PeJ,
not mentioned above, was chosen for this investigation
because they are phenotypically different from the other
three strains and hate been widely uaed in research.
Freparetion of Thalidomide
Thalidomide, 2, 3 dioxo-piperidyl phthalimide, for
use in this inveatigation was prepared according to the
wethod utilized by Chemie Grunthal In preparing the drug for
pharmaceutical use.
1*
Phthalylglutamic anhydride was prepared,using the
method of King and Kldd (0). Forty grams of phthalic an-
hydride find forty «$rams of L-?lutamlc acid were refluxed
In one hundred thirty mlliUlnars of dry pyridine for two
hours. Dry pyridine we. [repar;d by adding barium oxide
to stock pyridine «nd heating it gently. The clepr solution
was then evaporated under reduced pressure and ninety milli-
liters of acetic anhydride added. The solution was boiled
for three minutes and then concentrated under reduced pres-
sure. Anhydrous ether was added and the phthalylglutamic
anhydride, which precipitated at this point, dried under
reduced pressure.
Two grams of phthalylglutamic anhydride and two grams
of urea were placed in a live hundred milliliter round bot-
tom flask containing one hundred fifty milliliters of xylene.
The mixture was then refluxed for twenty-four hours, an
electric hotplate being used as the heat source. The heat
source was removed froT beneath the flask and the mixture
allowed to stop boiling. The still-hot liquid portion was
then decanted into a two hundred fifty milliliter Erlenmeyer
flask which was placed in an ice bath. Needles of thalido-
mide appeared upon cooling. The melting point of the
crystalline thalidomide was two hundred seventy degrees
Centigrade. This corresponds to the expected melting
point of two hundred seventy degrees Centigrade.
16
Experimental Procedure
Mice of four strains, CS7B1/6J, 12Q/J, A/j, snd C3HeE/PeJ
were caged separated, wit-' npprox4mateiy five females and one
male in each pan. e.-nfilea w»ir« inspected each morning for
the presence of " . r.rinai pi^g. Discovery of the vaginal
plug, a shiny mass o: mucous blocking the female's vagina,
was taken to be evidence of pregnancy. The date of discov-
ery of auch a vaginal flu? was considered to be the first
day of pregnancy, as mice normally breed between ten P. M.
and two A. M. (12)
On the sixth day, the pregnant females were weighed,
their ears punched for identification, and the mice of each
strain divided into eirht groups of approximately equal num-
ber. Three of the groups of mice from each strain were riven
intraperitoneal injections of a 0.5 per cent suspension of
thalidomide in sterile distilled water, at a dosae level of
twenty-five, one hundred, or one hundred fifty milligrams
of thalidomide per kilogrom tody weight. One group from
each strain was injected with sterile distilled water alone,
thus serving as a control group. Three other groups were
given a $ per cent suspension of thalidomide by oral intubation,
Again, the dosage levels were twenty-five, one hundred, or
one hundred fifty milligrams per kilogram body weight. The
final group of mice from each strain was administered dis-
tilled water by oral intubation, thus serving as an additional
17
control group. Intraperitoneal injection was accomplished
by use of e tuberculin syringe end sterile twenty-Fauge
needles. Oral intubation w a pccomplished uainp' a tuberculin
syringe and a blunt el^hteen-gauge needle. Treatments were
administered on the sixth through the *-enth days of pregnancy.
On the eighteenth day, the females were killed by
etheri?at'on and their uteri removed and examined for signs
of pregnancy. Fetuses present were eviscerated and stained
with Alizarin Red bone stain. The stainin? procedure is
given below. After staining, the fetuses were examined for
developmental abnormalities.
Alizarin Red Staining Technique
1. Fix fetuses in 95 per cent ethyl alcohol for
one week.
2. Place in 1 per cent potassium hydroxide for one
day.
3. Wash in tap water three times.
4. Place in 95 par cent ethyl alcohol. Change alcohol
twice. Leave fetuses in alcohol a total of six hours.
5. Place in diethyl ether for one to lour hours, or
until *11 fat is dissolved.
6. Place in <>5 P®r cent ethyl alcohol for one ni»ht.
7. Change the concentration of alcohol gradually
until the fetuses *re in tap water, "ftien add 1 per cent
18
potBsaium hydroxide. The fetuses should be in the potassium
hydroxide, for about on© week.
8. Wash in tap water three times find gradually add
ethyl alcohol until the concentration reaches 70 per cent.
9. Add enough Alizarin Red staining solution to make
the total stain concentration about 1 per cent.
10. When the bones are stained 8 dark red, place the
fetuses in 1 per cent potassium hydroxide.
11. Preserve the fetuses in 70 per cent ethyl alcohol
with a few drops of formalin added.
1. D1 Paolo, Joseph, "^onrenitsl Malformation in Strain A Mice." Journal o 1 the American Radical Associa-tion, C „ . X * V T T T , 13°-1U1.
2. Fraaer, F. C., M . Goldstein, and Merrille Feiner, "Teratogenic Effects of a '"wo Hour Inactivation of Nicotinamide Activity in the Mouse," American Journal of the Diseases of Children, CXIV (1061),
3. Pratta, I. <»>., E. f. Sicg, and K. Maiorena, "Terato-genic Effects of Thalidomide in Rabbits, Rats, Hams-ters, °nd vice,n Toxicology and Arplied fharmacology, VII (1^65), 26fi-2*5T
ij.. Giroud, A., H. Tuchmann-Dupleiss, and L. Merceir-Farot, "Thalidomide end Congenital Abnormalities," Lancet, I (1<>63), 298-299.
5. Hagen, Odette, "Drugs and Congenital Abnormalities," Lancet, I (1963), 501.
6. Ingalls, T. H., F. R. Avis, F. J. Curley, and H. M. Temin, "Genetic Determinants of Hypoxia-Induced Congenital Anomalies," Journal of Heredity, XXXXIV (1953). 185.
7. Ralter, Harold and Joseph Warkany. "Congenital Malfor-mations in Inbred Strains of Mioe Induced by Ribo-flavin Deficient, Galactoflavin-Contalnin^ Diets," Journal of Experimental Zoology, CXXXVI (1957)» 531-553."""
8. King, R. and J. Kidd, "Biochemical Preparations," Journal of the Chemical Society, WMCCCXV (19U9), T J I F :
9. Seller, Mary J., "^alidomide and Congenital Abnor-malities," Lancet, II (1962), 2^9.
10. Silveatrini, b. and A. Garau, "Malformazioni Feteli Frodotte Dalle Talidomide Nel Topo," Bollettino Chimico Farmaceutlco, CIII (196U), B04-81U.
19
20
11. Snell, 0. D., £. Fekette, K. P. Hummel, and L. W. Law, "The Relation of Mating, Ovulation, and the Eatroua Smear in the Houae Mou8e to Time of Day," Anatomical Record, LXXVI (19i*0), 39-5$.
12. , Joan Statts, M. P. Lyon, L. C. Dunn, H. Gruneberg, P.Herwlg, and W. £. Heston, "Standard-ized Nomenclature for Inbred Strains of Mice, Second Listing," Cancer Research, XX (1060), ll|5-l69.
13. Somers, G. F., "Thalidomide and Congenital Abnormalities," Lancet, I (1962), 912-913.
Ill* Woollam, D. H. M., "Thalidomide disaster Considered as an' Experiment in Mammallian Teratology," British Medical Journal, II (1962), 236-237*
CHAPTER I I I
RESULTS
During th« course of this investigation, a total of
three hundred fifty-six pregnant female mice of four strains
were administered either thalidomide or distilled water.
Routes of administration were divided equally between oral
intubation and intraperitoneal injection. Results of admin-
istering thalidomide to two hundred sixty-eight pregnant mice
is presented in Table I. This table shows the number of mice
given each treatment and the number of young born and resorb-
ed in each case. Table II lists the eighty-eight pregnant
females receiving water and indicates the mean number of
young recovered in each of the four strains investigated.
Examination of Table reveals that 86.2 per cent of the
thalidanldo-treated females failed to carry their fetusoa
full terra iind presumably resorted them. In comparison, Table
II shows that only 62.5 per cent of the control group females
resorbed their young. A statistical comparison of each treat*
ment group with its corresponding control is shown in Table
III.
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'"ABLE III
COMPARISON OF PER CENT RESORT TONS DUB TO ADMINISTRATION OF THALIDOMIDE TO POUR STRAINS OF MICE
Strain Thalidomide 3roup
Control Group
Chi-Squared
P
A/J 80.7 50.0 23.29 .001
C3HeB/FeJ 90.2 53.3 33.36 .001
C57BL/6J 85.2 81.1 1.29 .30
129/J 82.8 1*2.8
r 1+1.83 .001
Uae of the Chi-square teat revealed that thalidomide
increaaed the reaorption rate significantly in strains
C3HeE/FeJ, A / j , and 129/J, but not in strain C57&1/6J.
Examination of the fetuses after staining with Alizarin
led revealed various abnormalities in the treated group,
including stunting of growth, retardation of oasification,
shortening of long bones, absence of digits, anophthalmia,
microphthalmia, lateral curvature of the spine, exencephaly,
encephalocoel, hydrocephaly, cleft palate, and hare lip. Ey
far the most widely observed anomaly was general retardation
of development.
2?
The progeny of ••eh of the trtitcd mother mice are
11ated in Appendix A and th«lr abnormalities indicated.
A 11at of th« eontrol group progeny and their deformitiea
la given In Appendix B.
In order to facilitate eoniparlaon of the extent of ab-
normalltlea between the four atraina of mice, eaoh fetua
waa assigned a number between one and ten, with a larger
number corresponding to a greater degree of deformation.
9 » rating syatem uaed in arriving at theae numbera la given
ia Table I?.
The mean deformity aoore for eaoh atrain and within
atraina la ahown In Table Y. Thia table ahowa that atrain
129/J had higher deformity aoorea than the other atraina.
In order to teat the algnlfloanoe of thia difference, the
dlffereneea between atraina were compared statistically,
ualng the "atudent* a t" teat. The difference between the
M a n deformity aoorea for each of the atraina of mice ia
given in Table VI. The atatlatloal comparison indicatea
that atrain 129/J waa significantly more euaceptlble to
the teratogenic effeota of thalidomide than were any of the
other three atraina. the small dlffereneea between strains
C57BI/6J, C3SeB/PeJ"# and A/J were not atatlatieally signifi-
cant.
Table VII ahowa the reaults of an analyaia of variance
In the mean number of mice per litter due to the levels of
thalidomide adminletered. The variance due to dosage level
26
TABLE IV
SCALE FOR RATIHI *ICF ACCORDING TO EXTENT OF DEFORMITY
Condition of Mouse Score ^Iven
Normal . 1
Cleft Palate or Anophthalmia; Otherwise
Normal 2
Small Size but Normal In Other Reapecta 3
Retarded Ossification; Partially Reaorbed; Otherwise Normal U
Reterded Slcull Development; Otherwise Normal 5
Hydrocephalua; Curvature of the Spine;
Normal in Other Reapecta 6
Extreme Swill Size; Normal Ossification 7
Multiple Deformities; Vsrloua Combinetiona
of the Above 8
Extreme Shortening of Bonea; Missing Bones . . . . . 9
Grossly Malformed; Bonea Barely Vialble 10
27
TAFLE V
EFFECT OF THALIDOMIDE AD'THSTRATOK (T MEAN DS'r'OR1*!TY STORES
Strain Treatment Mean Deformity No. Mice tig /kg Score Recovered
C57BL/6J 25 2.3U 26 C57BL/6J 100 3.62 24
1.80 22. Total 2.^8 65
129/J 25 9.10 20 100 5.93 i 29 Igo 1.00 9 Total 6.26 35
C3H®B/P«J 25 1,00 9 100 1.00 17 l£0 2^6 il Total 1.73 k9
VJ 2$ 2.02 50 100 1.00 8 150 kill 27 Total 2.78 85
Orand Total 3.23 277
2fi
TAELE VI
MEAN DIFFERENCES IN AFNORMALITY SCORES IN FOUR STRAINS OF
Strain A/J C3Heh/Pe.T 12 9/J
C57BL/6J .30 .75 3.78*«*
129/J 3.48-tHH* lj.,53 ***
C3HeB/PeJ 1.05
««*lndlc<ites .001 level of significance by t test
is insignificant, indicating that increasing the level of
thalidomide administered from twenty-five milligrams per
kilogram boijr weight to one hundred fifty milligrams per
kilogram body weight had no significant affect on the number
of mioe per litter.
Table VIII shows an analysia of variance in mesh
deformity score due to the level of thalidomide administered.
Differences in thalidomide dosage level did not significantly
effect the variance, indicating that the three levels of
thalidomide were not different with reapect to the extent
to which they caused deformities.
29
TAPLE VII
ANALYSIS OF VARIANCE T'J MKAf! T'^ER OF V10L ISR LIFTER DT,E TC L:-;VSL 0Y HALIDOYIDE
Source 1 « 8utn of Squares Mean Square
Dose Levels 2 8.70 I+.35
Error 9 26.30 2.92
Total 11 35.00
F • • • I.I4.8 p • • . .20
TABLE VIII
ANALYSIS OP VARIANCE IN MEAN DEFORMITY SCORE PER GROUP DUE TO LEVEL OF THALIDOMIDE
Source df Sum of Squares Mean Square
Dose Levels 1
2 2.72 1.36
Error 9 66.38 .. . „ |
7.1*0
Total 11* 69.10
F . . . 0.018 P . . > • • 20
3C
An analysis of variance in mean deformity score due to
route of administration of thalidomide is shovm in Table IX.
Progeny born to nothers with thalidomide ora H y *«d
higher deformity scores than did the progeny of mothers p-iven
thalidomide by intrspsritonesl injection. The level of
significance of this difference is only .20, so that it is
doubtful that there is actually a real difference due to
route of administration. The RB an number of mice per litter
was not significantly effected by route of administration,
Indicating that raaorption rata is independent of adminis-
tration route.
TABLE IX
ANALYSIS OP VARIANCE DUE TO ROUTE OP THALIDOMIDE INJECTION
Souroa df Sun of Squares Mean Square
Routes 1 12.U9 12.1*9
Error 6 26.07 U.35
Total 7 3P.56
P . . . 2.Q8 P , a a * •20
CHAJThR IV
DISCUSSION
The primary mode of action by which thalidomide exerts
Its teratogenic effects la unknown. Several workera have
postulated possible mechanisms for the damage done the
developing fetus by thalidomide, but no explanation has
thus far been brought forward with enough supporting evidence
to warrant widespread acceptance.
7fte thalidomide syndrome in man and laboratory animals
resembles very closely the teratogenic effects of hypovita-
mlnosls B« For this reason, some workers feel that thalido-
mide may act as a vitamin B antagonist (9). T!here is some
evldeno* to support this opinion. Robertson (11*) reported
that adainlatratlon of thalidomide to the human interferes
with vitamin B metabolism la some cases. Kemper (8) , work-
ing with the chick embryo, reported striking aimllaritles
between the deformities obtained with vitamin B deficiencies
and with thalidomide.
More apecifioally, Roath (13) haa reported reaulta
indicating that thalidomide may act aa either a glutamlne
or nicotinic acid antagonist. Williams (15) haa shown that
soara metabolites of thalidomide will inhibit the enzyaw
glutamlne synthetase. Working with the ohiek, Peliaatl (3)
and Boylen (1) reported that glutamlne counteracted the
31
32
teratogenic effect® of thalidomide. Prank et al. (U) reported
reversal of the growth Inhibitory effects of thalidomide on
protozoans upon addition of either nicotinic acid or nicoti-
namide to the growth media.
All of the nbove results: point to the likelihood that
thalidomide exerts ita teratogenic effact by functioning as
either a vitamin 6 antagonist or a glutamine antagonist.
Again, although the evidence Indicates that this may be the
mode of action of thalidomide, this is by no means generally
accepted. The evidence is not sufficiently complete for a
definite conclusion to be reached.
Whatever the mechanism affected by thalidomide, it has
been well established that the drug does cause congenital
malformations. Accumulated evidence does not prove that
thalidomide alone brines on these malformations, otherwise
all Individuals or laboratory animals treated with thalido-
mide ahould react in the same manner, their diets pnd general
nutritional levela feeing equal. Since this is obviously not
the caae, then each treated individual or animal must possess
some mechanism which affects Its susceptibility to the tera-
togenic effects of thalidomide. Refering to the works cited
above* this mechanism might be vitamin B metabolism. What-
ever the mechanism Is, it is very likely under genetic
control (2). Demonstratlem of differences in susceptibility
to the teratogenic effects of thalidomide between genetically
h onto gene ou 11 inbred strains of mice would certainly support
33
tha likelihood that genetics playa • role in determination
Oi* reaponae to teratogenic agents.
The comparison shown in Table III indicates that there
la a difference existlnr between strains C57BL/6J and atraina
129/J# C3HeB/PeJ, and A/J with respect to increase in resorp-
tion rata due to the administration of thalidomide. The
control group mice of strain C57BL/6J had a higher resorption
rata than the other three 8traina. Administration of thali-
domide did not significantly increase the raaorptlon rate
In strain C57BL/6J, while the resorption rates of atraina
129/J, C|HeB/FeJ, and A/J were increased significantly.
lhaae results indicate that C57BL/6J mice are resistant to
the eabryocidal effecta of thalidomide. This la oonsiatent
with the reaulta reported by Hsgen ($), who found C57BL/6J
mloa to ba leas susceptible than A/j mice to increase of
resorption rate due to thalidomide.
Examination of Table VI raveala that strain 129/J mice
ara significantly more ausceptible to the actual terato-
genic affects of thslldomide than are atraina C57BL/6J,
C3HeB/FeJ, and A/j. In thla table the relative suacepti-
bl11tiea are shown aa mean differenoea in abnormality aeorea.
There era email dlfferencea in susceptibility between strains
C3HeB/PeJ# A/J, and C57BL/6J. These differences are too small
to ba statlatlcally significant and could be due to chance.
In this studyt varying the doaaga level by a faotor of
aix had no significant effect on either the embryoeidal or
3U
teratogenic effect® of thalidomide. This agrees with ^he
results obtained by MoCafferty et al. (12), who found no
differences in percentage of deformities in mice treated
with different dosage levels of thalidomide. MacKenrie and
McGrsth (10) rerorted that, in the rat, the amount of thal-
idomide absorbed is independent of the dosage level adminis-
tered. This would naturally rule out any dosage effects, as
long as the level is above the minimum necessary. Whether
limited absorption ability in the mouse Is the reason that
dotage level does not effect the embryopathic activity of
thalidomide has not been established.
In this investigation, route of administration of tha-
lidomide had no significant effect on the mean number of
mica par litter. The mice given the drug by oral Intubation
did give birth to progeny with higher mean deformity scores
than did the group to which the thalidomide was administered
lntraperltoneally. This difference in mean deformity scores
waa tested statistically, and waa of doubtful significance,
the probability lying between the .1 end .2. Additional
study, with larger number of mica, would be necessary to
determine if there ia a real effect in mean deformity score
due to the route of administration of thalidomide*
The results obtained in this study lead to the conclu-
aion that differences in susceptibility to thalidomide exist
between certain of the four strains of mice utilized, and
35
that susceptibility to embryocldal effects, reflected by
resorption rate, and to teratogenic effects, reflected by
abnormality scores, do not necessarily coincide. For example,
strain C3HeB/PeJ, which was susceptible to the increase in
resorption rate due to thalidomide, was least susceptible to
the teratogenic effects. On the other hand, mice of strain
129/J were highly susceptible to both effects of the drug.
The fact that susceptibility to teratogenic effects and
embryocldal effects do not felwaya coincide indicates that thee
genetic mechanism involved in resorption might differ from
the one Involved in teratogenesis.
Obviously, there is much to be learned concerning gene-
tic determinants in susceptibility to teratogenesis. At the
beginning of this thesis, it was hypothesized that the pro-
gram now being utilized by the Pood and Drug Administration
to screen new drugs for teratogenic effects is inadequate
and that one of the program,1* chief weak points 1s its fail-
ure to consider the effect of test animals• genetic back-
ground cm susceptibility to teratogenesis. There is a
definite need for more knowledge In this area. Inbred mice,
due to their genetic homogeneity, are ideal animals for
screening of possible teratogenic drugs. New drugs could
be tested for teratogenic effect using more than one strain
of miee. Naturally, if strains of mice were ohosen for
testing that ere known to be susceptible to teratogenic
agents, then the tests would be more senaitibe than if
realatant strains were chosen. This is assuming, of course,
36
that there are strains of mice which are hypersusceptible
to teratogenic agents In general. It is equally feasible
to postulate that different strains of mice will exhibit
different susceptlbi1'ties to different drugs, so that a
battery of mice strains, each suscept'ble to a particular
type of teratogenic agent, would have to be used In order
for such a testing program to be meaningful.
Kalter and Warkany f?) found that mice of strain
C57EL/6J were more resistant to the teratogenic effects
of a rlboflavln-deficlent diet than mice of strain A/j.
they then found that mice of strain C57BL/6J utilized
riboflavin more efficiently than those of strain A/j, SO
that the hypersusceptiblllty to a riboflavin deficient
diet's teratogenic effects waa probably due, In strain A/J,
to simple inability to utilize riboflavin efficiently.
Assuming that thalidomide exerta Its teratogenic
effeota by some mechanism akin to vitamin E antagonism, it
would be of value if strains of mice susoeptlble to thali-
domide teratogeneals, such aa 129/J, were tested for errors
in vitamin B metabolism. If It oould be establlahed that
the thalidomide-auaceptlble mice had faulty vitamin B
metabolism whereas resistant mice did not, then this would • *
serve as evidence that thalidomide functions aa a vitamin
B antagonist. The reverae experiment could alao be run,
In which miee known to have faulty metabolism of a parti-
cular vitamin, such as one of the B group, would be tested
37
This would not have to Involve a random choice of drug and
vitamins, as structural similarities between suspected
teratogens and vitamins coQld be noted. For example,
Pelisati (3) noted a similarity between riboflavin and
thalidomide. Riboflavin deficiency was subsequently found
to aceentuate the teratogenic effects of thalidomide, in-
dicating that thalidomide might function as a riboflavin,
vitamin Bg, antagonist. Metabolic studies failed to yield
evidence of any riboflavin antagonism by thalidomide.
Although these results were negative, this is an example of
the type of work whieh can be done in the study of terato-
gen! city.
Another worthwhile project would be to test several of
tha more videly used inbred strains of mice for susceptibility
to various teratogenic agents. Again, teratogenic effects
of diets deficient in the different vitamins could be con-
sidered* When enough is learned concerning the relative
sensitivities of different mouse strains to teratogens, then
much can be eluoldatod about the actual causes of teratogenic
•ffeota and the Inheritance of ausceptibllity to these ter-
atogenic effeots. To use the results obtslned in this study
aa an example, strains 129/J and C39eB/FeJ could be crossed.
Since 129/J mice are susceptible to the teratogenic aotion
of thalidomide, while C3HeB/FeJ mice are relatively resiatant,
and alnoe inbred mice are homozygoua at almost all loot,
than the progeny would presumably be heterozygous for whstever
38
genes control susceptibility to thalidomide teratoe-eneais.
The progeny could then t.•? *estsd for susceptibility *o the
teratogenic effects of
Assuminr th«t sus-ep t i t ill ty or resistance is determined
by a single t ene pair, if thf proeeny exhibited susceptibility
to thalidomide, then the f-ene causln* susceptibility would be
dominant to the allele causing resistance to the teratorenic
effects. Conversely, if the proeeny proved to be resistant
to the teratogenic effects, then the allele causing suscep-
tibility would be assumed to be recessive, if some of the
progeny were resistant and some susceptible, then the inheri-
tence of susceptibility to thalidomide teratogenesis would
probably be due to a dominant gene with incomplete penetrance.
Of course, the susceptibility to thalidomide terato-
genesis could be controlled by several gene pairs. If these
were additive genes, then the progeny would probaily be inter-
mediate between the parents. By backcrossing the hybrids, it
would be possible to estimate, statistically, the number of
genes Involved. It soon becomes obvious that studies con-
cerned with the inheritance of susceptibility or resistance
to teratogens will require large numbers of mice if they are
to be meaningful.
Another study suggested ty the results of this investi-
gation Involves the embryocidal rather than the teratogenic
action of thalidomide. It has been reported that the
39
chief cause of apparent resorptions in the armadillo is
damare to the preimplantation blastocyst (11). It could
be hypothesized that resistance to the erabryocidal effects of
thalidomide is due to early Implantation of the developing
blastocyst on the uterine wall. If it could be shown that
implantation in strains resistant to the embryocidal effects
of thalidomide, C57BL/6J for example, occurs earlier than in
more susceptible strains of mice, then this would indicate
that resistance to the embryocidal effects of thalidomide
could be due to early Implantation. This would be an example
of an apparent plelotropic effect; the genetic mechanism
controlling time of implantation of the uterine wall thus
controlling susceptibility or resistance to the increase of
resorption rate induced by thalidomide. It should be empha-
sised that this is merely a proposal of a study which could
be conducted. No evidence exists, at present, that time of
lmplanatlon has any effect on relative susceptibility of
different Inbred mice strains to thalidomide.
At this time, too little is known concerning the res-
ponses of sny of the mouse strains to any teratogenic agent.
Homburger et al. (6) have reported differences in suscepti-
bility to thalidomide teratogenesis between inbred strains
of hamsters; however, hamsters sre not used widely in
research as are inbred mice. For this reason, it seems prac-
tical that further study should be done concerning the effects
of various teratogens on different inbred mouse strains.
CHAPTER BIBLIOGRAPHY
1. BoyIon, J., H. K. Horn®, and W. J. Johnson, "Teratogenic Effects of 'ihalidomide and its Metabolites on the Developing Chick iimbryo," Caned 1 an Journa I of Biochemistry, XXXXII (196Jj.J, 55'2-553."
2. Carpener, Norman, "Teratogenssia," Lancet» II (1^63), 885«
3. Felisati, Dino. Wrperatogenio Action of Thalidomide," Lancet, II (I'M), 72^-725.
I±, frank, Oscar, H. baker, H. Ziffer, and C. M. Leevy, "Metabolic Lesions Induced by Thalidomide," Journal of Laboratory and Clinical Medicine, LX (1962), 89.
5. Hagen, Odette. "Drugs end Congenital Abnormalities," Lancet, I *1963), 501.
6. Horoburger, P., S. Chaube, M. Eppenberger, P. D. Bog-donoff, and C. W. Nixon, "Susceptibility of Certain Inbred Strains of Hamsters to Teratogenic Effects of Thalidomide," Toxicology and Applied Pharmacology* VII (1965), 686-693.
7. ICalter, Harold and Joseph Warkany, "Congenital Malfor-mations in Inbred Strains of Mice Induced by Ribo-flavin Deficient. GalactoflavIn-Containing Diets," Journal of Experimental Zoology, CXXXVI (1957)*
8. Kemper, Von Fritz, "Thalidamid und Entwicklung von Hunerembryoen, ArzneimitteIforachung, XII (1962), 836. ~
9. Leek, Ian °nd E. M. Millar, "Incidenoe of Malformations Since the Introduction of Thalidomide," British Medical Journal, II (1962), 16-20.
10, MacKensie, C. and W. R, McGrath, "Absorption of Thalidomide in the Rat," Proceedings of the Society of Experimental Biology and Medicine,"7lTTl%?), 511-515.
11. Marin-Padllla, Miguel and Kurt Benirschke, "Thalldomide-Induced Alterations in the Blastocyst and Flaoemta of the Armadillo, 'Dasypua novemcinctus mexiconos* including a Choriocarcinoma," American Journal of Pathology. XXXXIII (1963), 999-1009.
kl
12. MeCafferty, R. E., M. L. Wood, *nd W. H. Knisely, "Morphological and Physiological Effects of Thali-domide and Trypan Elue on Uteri and Concepti of Gravid Mice," American Journal of Obstetrics and Gynecology. LXXXU ( 1 % M , ^U-2gQ~
13. Roath, S., M. Elves, end M. C. G. Israels, "Effects of Thalidomide and its Derivatives on Human Leucocytes Cultured in vitro," Lancet, I (1°63), 2^9-2$0.
14. Robertson, W. P., "Thalidomide and Vitamin B Deficiency,' British Medical Journal, I (1962), 792-7^3.
15. Williams, R. T., "Teratogenic Effects of Thalidomide and Related Substances," Lancet, II (1963), 723-721+.
APPENDIX A
ABNORMALITIES OBSKHVrr TV wIOE TRSATED WITH THALIDOMIDE
Abnormality Symbol Uaed
HOD* A
Cleft Palate B
Anophthalmia C
Reduced Site D
Retarded Oaaification E
Partial Reaorption F
Underdeveloped Cranium G
Hydrocephaly H
Curvature of the Spine I
Shortened Bonea J
Kxtreae Reduced Size K
Mlsalng Bones L
1+3
Strain Littar No. Mousa !9o. Abnormalities Obsorvad
C3H«B/P«J C3HeB/P«J C3ReB/P®J C3HaB/F*J C3HeB/P«J C3HeB/P»J C3HeB/PeJ 2 C3HaB/PaJ 2 C3H©B/F»J 2 C3HaB/P»J 2 C3H«B/P«J 2 C3HaB/PaJ 2 C3H«B/P«J 2 C3H»B/P»J 3 C3H«B/P»J 3 C3H«B/P«J 3 C3HaB/PaJ 3 C3HeB/F«J 3 C3HaB/PaJ 3 C3HtB/P»J 3 C3HtB/FaJ 3 C3HaB/PaJ 3 C3H«B/P«J k C3H«B/P«J If C3HaB/PaJ 1+ C3H«B/F»J if C3HaB/PaJ ^ C3H6B/P«J k C3H«B/P«J if C3HaB/PaJ 4 C3H«B/P»J '+ C3H»B/P» J it C3B»B/P»J ^ C3H«B/PeJ 5 C3H»B/PeJ 5 C3H»B/P»J 5 C3H»B/P«J 5 C3H«B/F«J 5 C3HaB/PaJ 5 C3EaB/PaJ 6 C3B«B/P*J 6 C3H«B/P»J 6 C3H«B/P«J 6 C3H«B/P«J 6 C3H«B/F»J 6 C3HaB/PaJ 6 C3H#B/P«J 6 C3HaB/PaJ 6 C3HaB/PaJ 6
1 2 3
't 6 1 2 3 if 5 6 7 1 2 3
if 6
1 9 1 2 3
I 6
1 9 10 11 1 2
1 6 1 2 3
I 6
9 10
A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A h K K X K K A* A A A A A A A A A
1*4
Strain Litter No, Mouse No. Abnormalities Observed
129/ J 129/J 129/ J 129/J
ISA m 189/J J?9/J 129/J 129/J 129/J 129/J
HI; 129/J ^9/J R9/J [9/J h / j J9/J i9 /J
189/J
9/J
$
M S ;
19/J N / j ? / /
m
1 J,L
C H, J,L ** L
k D,L
5 D,L 2 l D,H,L 2 2 D,L 2 3 D,L 2 U D,L 2 5 D,L 2 6 D,H,L 2 7 S,H,L 3 1 D,H,L 3 2 D,L
k 1 D,H,L
k 2 D,H,L
k 3 D,H,L
k D,H,L
5 1 D,H,L
5 2 D,L
5 3 D,L 5 4 D,H,L
5 5 D,H,L 6 l D,H,K 6 2 D,H,K 6 3 D #H,K
7 1 A 7 2 A
7 3 A 7 A
7 A
7 6 A
7 7
7 e
A A
7 o / A
6 1 D,L 8 2 D,L 8 3 D,L 8 4 D,L 8 5 D,I,L 8 6 D,L 8 7 D,L 8 e D,L
kS
S t r a i n L i t t e r No. Mouse No. A b n o r m a l i t i e s Observed
129/J 9 1 D,L 1 2 9 / j 9 2 D,L 129/J 9 J D, J ,H,L 129/J 9 k Da JjHgXJ 129/J 9 5 D,L 12<VJ 9 6 D,L 129/J 10 1 A 129/J 10 2 A 129/J 10 3 A 129/J 10 k A 129/J 10 5 A 129/J 10 6 A 129/J 10 7 A 129/J 11 1 A 129/J 11 2 A 129/J 11 3 A
C57BL/6J C57BL/6J C57BL/6J C57BL/6J CS7BL/6J CS7BL/6J C57BI./6J
j 7 8 L / 6 J S7BL/6J
U / 6 J J L / f J
C ^ 7 B t / 6 j C57BL/6J C57BL/6J C57BI /6 J C5TBL/6J CgTBi./6J CS7BL/6J
f i t 6 J L / 6 J L / 6 J
1 1 A 1 2 A 1 3 A 1 k A 1 5 A 1 6 A 2 1 A 2 2 A 2 3 A 2 A 2 5 A 2 6 A 3 1 A 3 2 A 3 3 A 3 4 A 3 5 A 3 6 A 3 7 A 3 8 A 3 9 A 3 10 A
1*6
S t r a i n Litter No. WT ouae No. Abnomal lties Observed
C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57Bt/6j C57BL/6J CS7BL/6J C57BL/6J C57BL/6J C57?l/6J GS791/6J C|75t/6J C5TBI/6J C§7BI/6J <*5fBL/6J C57BL/6J C57BL/6J C5TPL/6J CSTet/6j
J# CS7B1./6J C§7fcl/6J (557BL/6J C579L/6J
if C57Bt/6j C57BL/6J C57BL/6J C57BL/6J C57BL/6J
§?P& C57PI/6J $ £ & C57BL/6J C57BL/6J C57BI/6J
W
U 1+ k k k I 5 5 5 5 5 5 6 6 6 6 6 6 6 6
f
I 8 8 8 8 8 8 8 8 9 9 9 9 9
1 2 3
i 6 7 1 2
A A C A A A A A A A A A C A A A A A A A A A A A A A A
G 0 a
47
Strain Litter No. Mouse No. Abnormalities Observed
C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J CS7BL/6J C57BL/6J G$7BL/6J CS7BL/6J C57BL/6J" C57BL/6J C57BL/6J CS7BL/6J C57Bi/6J
9 6 G, J 9 7
i G 9 P G 9 9 G 10 1 D,.T 10 2 D 10 3 D,G 1C k
**
> D
10 k **
> A 11 1 ? 11 2 G, J, K 11 G,J,K 12 1 K 12 2 K 12 3 G,K 12 k K 12 $ G,K 12 6 G,K 12 7 G,K 12 a G,K
A/J A/J A/J A/J
A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J $
A/J
1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3
it if
it
1 2 3
1 6 ?
1 2 3
6 7 8 1 2 1 2 3
A A A A A E A B B A A A A A A A
4?
Strain Litter Ncu Mt^sa No* Abnormalities Observed
A/J I4. o A A/J k 7 A A/J J ft A A/J i* 9 A A / J £ 10 A A/J 5 1 S A/J 5 2 A A/J 5 3 A A/J 5 4 A A/J 5 5 E A/J 5 6 E A/J 5 7 A A/J 5 8 A A/J 5 9 S A/J 6 1 a A/J 6 2 E A/J 6 3 A A/J 6 4 A A/J 6 5 S A/J 6 6 K,3 A/J 7 1 S,K A/J 7 2 E,H,K A/J 7 3 E,K A/J Y k. E,K A/J 7 5 E,K A/J 7 6 E,K A/J 7 7 E,K A/J a 1 A A/J 8 2 A A/J 3 3 A A/J 3 k A A/J 3 $ A A/J <3 . 6 A A/J 9 1 E,I,K A/J 9 2 E,K A/J 9 3 E,K A/J 9 I E,H,I A/J 9 5 P A/J 10 1 B kft 10 2 A A/J 10 3 A A/J 10 h A A/J 10 5 A A/J 10 6 D,E
+9
Strain Litter No. Mouse Nc. Abnormalities Observed
A/j 1 A A/J 11 2 A A/J 11 3 A A/J 11 4 A A/J 11 5 A A/J 11 6 A A/j 11 7 A A/J 11 s A A/J 11 9 A A/J 11 10 D A/J 11 11 D A/J 12 1 D A/J 12 2 D A/J 12 3 D A/J 12 fc D A/J 12 5 D A/J 12 6 D A/J 12 7 D
AFP3NDIX h
ABNORMALITIES •VhSSRVEO T?3 COKTHOL MICE
Strain Litter No, Mouse Ho. Abnormalities Observed
129/J 129/j 129/J 129/J 129/j 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129^J
1 1 A 1 2 A 1 3 A 2 1 A 2 2 A 2 3 A 2 k A 2 5 A 2 6 A 3 1 A 3 2 A 3 3 A 3 1+ A 3 5 A k 1 A k 2 A 5 1 5 2 5 3 5 4 5 5 6 5 7 5 8 5 9 6 1 6 2 7 1 7 2 7 3 8 1 8 2 8 3 8 8 8 6 8 7
$0
51
Strain Litter No. Mouse Ho. Abnormalities Observed
A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A / J A/J A/J A/J
A/J A/J A// A/J A/J A/J A/J A/J A / J A/J A/J A / J A/J A/J A/J A/J A/J A / J A/J A/J
1 1 B 1 2 A 1 3 A 2 1 C,D 2 2 C,D 2 3 C,D 2 5 C,D 2 5 C,D 3 1 P 3 2 * 3 3 A 3 if A 3 A 3 6 A 3 1 A 3 B A 3 9 A it 1 A i* 2 A 4 3 A U if A
5 o
A 5 o A 7 A
If 8 A 9 A
5 1 A 5 2 A 5 3 A 5 k A 5 $ A 5 6 A 5 7 A 6 1 A 6 2 A 6 3 A 6 A 6 5 A 6 6 A 6 7 A 6 a A 6 9 A
?.
Strain Litter No. w>!se No. Abnormalities Observed
A / J 7 1 A A/J 7 £ A A / J 7 3 A A / J 7 & A A / J 7 5 A A / J 6 A A / J
8 1 A
A / J 8 2 A A / J 8 3
* J H
A / J 8 U A A / J 8 5 A A / J 8 6 A A / J 8 7 A A / J 8 8 A a/J 9 1 A A / J 9 2 A A / J 9 3 A A / J 9 $ A A / J 9 5 A A / J 9 6 A A / J 1 0 1 E A / J 1 0 2 A A / J 1 0 3 A A / J 1 0 k A A / J 1 0 5 A A / J 1 0 6 A A / J 1 0 7 A A / J 1 0 8 A A / J 1 1 1 A A / J 1 1 2 A A / J 1 1 3 A A / J 1 1 K A A / J 1 1 5 A A / J 1 1 6 A A / J 1 1 7 A
C3H«B/P«J 1 1 E C#H*B/FW 1 2 A C3H«B/P#J 1 3 A C3H«B/PeJ 1 4 A C3H«B/PeJ 1 5 A C3H«B/P«J 2 1 A C3H»B/P»J 2 2 A C3H«B/P«J 2 3 A C3B«B/P«J Z k A
Stra in L i t t e r No. Mouse No. Abnormalities Observed
A <3 A
A
C3H«B/PeJ 2 5 C3HeB/PeJ 2 6 C3HeB/FeJ 2 7 C3HeB/P«J 2 6 A C3HeB/PeJ 3 l A C3HeB/PeJ 3 2 a G3HeB/PeJ 3 3 a C3HeB/PeJ 3 k A C3H«B/PeJ 3 5 a C3H«B/PeJ 3 6 A C3HeB/P»J 3 7 a C3HeB/Pej 3 B A C3HeB/FeJ 4 1 A C3H«B/Pe J k 2 A C3HeB/PeJ 4 3 A C3HeB/P«J U k A C3H»B/PeJ k 5 a C3HeB/PeJ k 6 a C3HeB/PeJ 1+ 7 V C3HeB/PeJ 4 * J C3HeB/P*J I* 9 a C3HeB/p«j 5 1 a C3H«B/P«J 5 2 a C3H»B/FeJ 5 3 a C3HeB/PeJ 5 k a C3H«B/PeJ 5 5 a C3H«B/PeJ 5 6 a C3HeB/PfJ 5 7 A C3HeB/PeJ 5 6 A C3HeB/PeJ 5 9 J C3H«B/PeJ 5 10 ? C3H«B/F*J 5 11 A C3H«B/P*J 6 1 A C3HeB/P»J 6 2 a C3HeB/PeJ 6 3 A C33«B/PeJ 6 L 7 C3HeB/PeJ 6 5 A C3HeB/PeJ 6 6 . A C3HeB/Pej 6 7 V C3HeB/p*j 6 8 * C3HeB/Pej 6 9 A C3HeB/pej 7 x * C3HeB/PeJ 7 2 a C3HeB/P»J 7 3 A C3H»B/Pej 7 J
S t r a i n L i t t e r No. Mouse Mo. Abnormal i t ies Observed
C3KeB/PaJ 7 <5 A OH®b/PeJ 7 6 A C3H«B/PeJ 7 7 A C3HeB/P®J 7 p A
C5>7BL/6 J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C$7BL/6J C57BL/6J C57BL/6J C57BL/6 J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57Bl# t J C57BL/6J C57BL/6J C57BL/6J C57BL/6J CJ>7BL/6J
1 1 1 1 1 ?
2 2 2 2 2
! 5 5 5 5 5 5 5 6 6 6 6 6
1 2 3 *
1 2 3
1 6 7 1 2
i 6
1 2
1 6 1 2
I 6
c A A A A A A A A A A A C C A A A A A A A A
1 2 3 *
It y>
Strain Litter No. mOU3« NC . Abnormalitiea Observed
C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C573L/6J
1 ? 3 I* 5 6 I
A A A A A A A A
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