A33 E‘EWESTEGATEQH {I}? EQS-flBLE VAR 313433-6335 ['34 EEEATQGENEC EE‘EZ‘“ {343233653 EV CYCLQEE-EQSEHAMEQE 334 C‘Fuf 333365 "'7 3%: SLEEVE-£4333 DAY 33F GEE? $3354 ‘3“; r. , ,c: {N . . T :1 4 JWSES L09 3310 #GQL’3U 3". Nu So Inwagmra : w “33,-“ “.7?" ’. 4'5"!" fifl‘vii “1:133. 532.11; 3.313253; Tit. :. 57mm Yea-n 9 6 *9“ “335‘s LIBRARY Michigan State University AN INVESTIGATION OF POSSIBLE VARIATION IN TERATOGENIC EFFECTS INDUCED BY CYCLOPHOSPHAMIDE IN CF-l MICE ON THE ELEVENTH DAY OF GESTATION by Joan Yoon A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy ACKNOWLEDGMENTS The researcher wishes to express her gratitude to Doctor James Blake Thomas for his constant guidance and encouragement which was always given unhesitatingly throughout the investigation. The author would also like to thank Doctors A. L. Foley, A. W. Stinson, D. L. Clayton and B. C. Walker who critically re— viewed the manuscript. Appreciation is also extended to Doctor A. E. Lewis for his statistical help and Doctor J. E. Gibson for his advice and consultation. The author is also indebted to Miss Bonnie Mollenk0pf for her typing of the manuscript. ii TABLE OF CONTENTS Page Introduction ........................... 1 Review of Literature ....................... 2 Materials and Methods ...................... 6 Results ............................. 8 General Results ....................... 8 Results Relating to Hypothesis ............... 9 Discussions and Conclusions ................... 18 General Considerations ................... 18 Specific Considerations Relating to Hypothesis ....... 18 Summary ............................. 22 Literature Cited ......................... 23 Appendix ............................. 25 iii LIST OF TABLES Table Page 1 Observations of all Embryos ............ 10 1A Appendix of Raw Data ............... 25 iv LIST OF FIGURES Figure Page 1 Open Eyes .................... 12 2 Cleft Palate .................. 13 3 Ectrodactyly .................. 14 4 Syndactyly ................... 15 5 .Adactyly .................... 16 6 Dorsal Lesion .................. 17 7 Examples of a control and deformed fetuses . . . 11 8 Cross section through the lower abdomen of a severely deformed fetus ............. 11 INTRODUCTION It has long been an established principle in experimental tera- tology that ”the susceptability of an embryo depends upon the develop- mental stage at whiCh an agent is applied" (Wilson). Thus, it has become common practice to investigate teratological agents in terms of effect on specific days of gestation. However, it is known that organisms are not uniform.in.terms of function and that they indeed fluctuate over time in circadian, lunar, seasonal and yearly cycles. Such rhythmicity of function must clearly also hold for those animal species commonly employed in experimental teratology research. FUrthermore, there is some evidence to indicate certain cyclic fluc- tuations during embryogenesis. In order to investigate the above considerations I prOpose the following Null hypothesis: That the frequency of specific terato- genic anomalies are evenly distributed over the twenty-four hour period and deviations at three hour intervals are due to chance alone. In order to test the hypothesis only certain teratological agents are appropriate, for these must satisfy two requirements. Specifically, the agent 1) must be effective with a single insult and 2) must act only during a Short period of time. This narrows the possible choice of teratogens essentially to radiation, radiomdmetic drugs, and reversible antimetabolites. The agent used in the present investigation is cyclophosphamide, a radiomimetic drug. REVIEW OF LITERATURE Cyclophosphamide (Cytotoxan* or Endotoxan*) is an antitumor agent of the nitrogen mustard group which is used clinically in the treatment of patients with certain neoplastic diseases. The drug is described chemically as N, N-bis (Q-chloroethy1)-N, O—prOpylene phosphoric acid ester diamide monohydrate. It is not active in XiEEQ.bUt requires activation by in viyg_biotransformation (Foley et_al,). Cyclophosphamide has been shown to be teratogenic in several experimental animals. Gerlinger gt El° (1963) reported the terato- genic effects of cyc10phosphamide in the chick, while the teratogenic effects of cyclophosphamide in mammals was first studied by Gerlinger (1964) and Gerlinger and Clavert (1965A) in rabbits. These latter studies found the drug to be teratogenic on days 10 through 13 of gestation at the dosage of 50 mg/rabbit. It was also determined that in rabbits cyclophosphamide interfered more with the gonadal differen— tiation of female offspring than with the male (Gerlinger and Clavert 1965B). The teratogenic effect of cyclophosphamide in rats was first noted by Brock and Kreybig. Kreybig (1965A) subsequently published a more detailed study of the gross malformations produced. Such malformations included exencephaly, microcephaly, syndactyly, polydactyly and missing ribs. Kreybig and Schmidt, using histological and cytological tech- niques, then investigated the cellular effects of cyclophosphamide on rats treated on day 13 (Kreybig and Schmidt 1966) and on days 15 and 16 (Kreybig and Schmidt 1967). Anticipating a possible difference in *Registered trade name. response among various strains of rats, Kreybig (1965B) investigated the teratogenic effects of 20-40 mg/kg doses of cyclophosphamide on days 4 and 11-15 of gestation in six different strains of rats, but was not able to demonstrate any differences in teratogenic effect among the strains. All fetuses were abnormal with the exception of those whose mothers were treated on day 11. In contrast, Chaube EE;213 reported that cyclophosphamide induced malformations in C. F. Wistar rats on day 11 but not on day 4. There has been one case report of human fetal abnormalities due to cyclophosphamide (Greenberg and Tanaka). In 1964 a child with multiple congenital abnormalities was born to a woman with Hodgkins disease. Cyclophosphamide therapy for the Hodgkins disease was begun at approximately the date of conception and continued throughout ges- tation. The normal dosage administered was 100 mg/day. However, the dosage was markedly increased during a six day period (total dose 1,810 mg) which corresponded to the seventh or eighth week of fetal life. There have been only a few studies of the teratologic effects of cyc10phosphamide in.mice. Hackenberger and Kreybig did a comparative study between rats and.mice, showing that the susceptibility of the two species was basically the same, although there were species Speci- fic differences in the type of malformation inducedv Another study used the DMVMK strain of mice (Shoji and tho). In this study pregnant females were divided into three dosage groups of 100 mg/kg, 50 mg/kg and 25 mg/kg. Each mouse was given the appr0priate dosage daily from day 11 through day 14. Dosages of 100 mg/kg and 50 mg/kg produced cleft palate and syndactyly at statistically significant levels. Gibson and Becker (1968A) have done the most extensive investiga- tion on the teratogenicity of cyclophosphamide in mice. Pregnant Swiss webster females were given a single dose of 5, 10 or 20 mg/kg on one specific day of gestation (9 through 14). Fetuses taken on day l8'were measured, weighed, examined for gross anomalies and divided into two groups for fixation. One group was fixed in Bouin's solution and subsequently hand sectioned at 2 to 3 mm for examination of soft tissue anomalies. The other group was fixed in 95% ethanol and sub- sequently stained with alizarin red for examination of skeletal abnor- malities. Gibson and Becker (1968A) reported statistically significant numbers of inlutgrg_deaths at dosage levels of 5, 10, and 20 mg/kg in all days studied. The body weight and measurement data indicated decreased fetal growth in response to increasing dosages of the drug. Except for two cases of exencephaly out of 250 fetuses examined, there ‘were no externally visual anomalies in fetuses of mothers who received the 5 or 10 mg/kg doses. .A variety of anomalies were Observed in fetuses from mothers who received the 20 mg/kg dose. .All the following incidences of anomalies ‘were statistically significant at the .05 level unless otherwise noted. The incidence of Open eyes peaked on days 9 and 10. Digital anomalies showed a day dependent Character in that polydactyly occurred with greatest frequency from treatment on day 9, syndactyly on day 10, ectro- dactyly on day 11 and adactyly on days 11 and 12. Other anomalies were hindlimb dorsiflexion, exencephaly and kinky tails. On days 9 through 12 cleft palate was found. The soft tissue anomalies which were feund ‘were aphakia (days 9-11), mdcrOphakia (day 12) and external hydro- cephalus (day 11 and 12). Skeletal defects reported were limb bone fusion (days 10 and 11) , tibia and fibula fusion (day 10) and curvature of tibia and fibula (day 12) . Shortening of the long bones occurred on different days with different doses. On day 10 all doses induced shortening. Twenty mg/kg gave shortening on days 9-14. Some shorten- ing occurred with 10 mg/kg on days 10, ll, 12 and 13. Gibson (1969A) also noted what appeared to be hemorrhagic lesions on the body wall. From further studies using cyclophosphamide in mice Gibson and Becker (1968B) concluded that the form of cyclophosphamide which acts as a teratogen in mice is the umnetabolized cyclOphosphamide or a metabolite of it, formed by a nonenzymatic process. Gibson (1968B) further concluded that the teratogenic effects of the drug are corre- lated with the presence of unchanged cyclophosphamide, although these effects may be due to an unknown product of cyclophosphamide which is formed within the mother or embryo. MATERIALS AND METHODS Sprague Dawley CF-l mice (Carworth Farms, Kalamazoo, Michigan) were housed in plastic cages and given food and water in excess of requirements. The cageS'were kept in a light tight room which had a light-dark cycle regulated by an electric timer. The lights were on from 3:00 p.m. to 6:00 awm. and off from 6:00 a.m. to 3:00 p.m., thus giving a reversed day-night cycle with 15 hours of light and 9 hours of darkness. Young adult, 50 gram, male and female mice were entrained to the artificial light-dark cycle for a minimum of two weeks before being mated. The mating of nulliparous females was accomplished by pen breeding, three females being placed with each male from 8:00 a.m. to 3:00 p.m. on mating days. The females were Checked at 3:00 p.mv for the presence of vaginal plugs which were accepted as presumptive evidence for mating. All females with vaginal plugs were then caged together as an injection group. Eleven-thirty a.m. on the day of mating was desig- nated as hour 0. All pregnant females were injected on the eleventh day of gestation, each group being injected at a different hour of that day. There were nine injection groups, at hours 0, +3, +6, +9, +12, +15, +18, +21, and +24. The intraperitoneal injections were saline solutions of cyclOphos- phamide at a dose of 20 mg/kg. Control mice were injected at the same hours of day 11 as experimental mice with an equal volume of physio- logical saline. On the seventeenth day of gestation, pregnant females were sacri- ficed by cervical dislocation, the uterine horns were exposed and the number and position of resorption sites and fetuses were noted. The uterine horns were then removed and the fetuses were exposed. Each uterus with placentae and embryos still attached was fixed in Helly's fluid for five hours, then transferred to 2.5% potassium dichromate for 19 hours. .After this, the specimens were placed in running tap water for twentyefour hours and held in 70% alcohol. Each fetus was examined under the dissecting microsc0pe for gross anomalies. Selected fetuses were sectioned.by the method of Wilson and warkany for examination of soft tissue anomalies. Selected embryos were sectioned serially at 5 micra. The sections were then stained either with hematoxylin-Biebrich scarlet or a modification of Masson's triple stain (Masson). Statistical analysis of the frequencies of each anomaly over the twenty-four hour period was done by the chi square method (Lewis). RESULTS General Results The gross malformations I found were essentially the same as those reported by other investigators. Those I observed were open eyes, cleft palate, ectrodactyly, syndactyly, adactyly, dorsal lesions, resorptions and amorphous embryonal masses. The incidences of each malformation for eadh test hour are summarized in Table l. The raw data of the number of fetuses having the malformations are tabulated according to litters in Table 1A of the appendix. .All control embryos 'were observed to be normal. An example of a control fetus, two moder- ately deformed fetuses and a severely defbrmed fetus are shown in Figure 7. The dorsal lesions noted grossly were found to be filled with unclotted blood on histological sections (Figure 8). The lesions 'were circumscribed and the spaces appeared to be lined by cells. It ‘was also noted that there were other cystic, cell lined spaces which ‘were not blood filled, but were rather filled with an acellular exudate. These spaces were particularly noticed around the central nervous system. In the most severely deformed fetuses there was a definite lack of cellular organization in the central nervous system. .Also, in the most severely deformed fetuses, internal structures with the exception of the heart and liver were either malformed or absent, while the liver was disproportionately large for the size of the embryo. Although no systematic evaluation of the skeletal system was included in the present investigation, sections of the severely deformed embryos revealed an absence of ribs and an absence or deformity of other bony structures. Results Relating to Hypothesis The incidence of each malformation was variable over the twenty- four hour period. Malformation frequencies were illustrated in bar graphs as the per cent of embryos with the defect against the time of day that they were injected (Figures 1-6). The variability of each anomaly (Open eyes, cleft palate, ectrodactyly, syndactyly, adactyly, and dorsal lesion) was analyzed to determine the probability that it was due to chance. The computed chi square values for each anomaly was given with its respective graph (Figures 1-6) . There were eight degrees of freedom. The level of statistical significance was arbitrarily chosen to be Ps0.0S. Tdflel. 10 Observations of all Embryos 0 +3 +6 +9 +12 +15 +18 +21 +24 NUmber of Litters 2 4 4 3 4 3 4 3 3 NUmber of Implantations 24 35 44 28 42 35 38 27 30 Number of Resorptions 1 4 l 5 0 0 0 0 0 Number of Amorphous Embryos 7 0 12 7 0 0 0 0 0 Number of Embryos 16 31 31 16 42 35 38 27 30 Open Eyes 11 8 21 12 38 26 28 27 21 Cleft Palate 12 24 29 10 38 18 34 25 30 Ectrodactyly 0 0 0 l 6 7 17 8 12 Syndactyly 10 1 16 10 34 6 ll 10 0 Adactyly 6 22 15 5 1 9 O 8 4 Dorsal Lesion 4 19 17 7 15 ll 4 18 0 11 Figure 7. .A. Control. B-C. Mbderately deformed fetuses from injec- tion times +6 hours and +18 hours respectively. D. Severely deformed fetus from injected time +3 hours. The arrow points to the dorsal hemorrhagic lesion. Figure 8. Cross section through the lower abdomen of a severely deformed fetus similar to embryo D in Figure 7. The arrow points to the dorsal hemorrhagic lesion. (C) spinal cord. (Magnification approximately 20X). 12 100- 9th- 8th- ()J O 3 6 9’ I2 15 IIIIIIIIIIIA Figure 1. Open Eyes Time Chi Square Iim§_ Chi Square 0 0.0261 +15 0.0216 +3 9.2396 +18 0.0118 +6 0.0850 +21 2.8937 +9 0.0175 +24 0.0195 +12 1.9453 Total Chi Square = 14.2601 P >0.05 13 'IOOq 9K)- 7o~ 60+ %so~ 404 304 20- Io-J OI 3 6+ 9’ 12 —§ Figure 2. Cleft Palate Iim§_ Chi Square Time Chi Square 0 0.1143 +15 4.1417 +3 0.1048 +18 0.2101 +6 0.4403 +21 0.3192 +9 0.7885 +24 1.0856 +12 0.3078 Total Chi Square = 7.5123 P >0.05 14 243 100W 90d 80- 704 60-4 96 50-1 40.. 30d 20... 10- 0L] 1 LI l I O 3 6 9 - Figure 3. Ectrodactyly Time Chi Square Time Chi Square 0 3.0700 +15 0.0125 +3 5.9400 +18 12.9778 +6 5.9400 +21 1.5352 +9 1.3957 +24 6.7934 +12 0.5220 Total Chi Square = 38.1866 P <0.001 15 1007 ONF‘ ll>4 70h+ 60-! 8 50h+ 4N0- 30%- 201 10-1 04 0 3 6 9 I2 15 18 21 24 IIIIIIIIIIIE;_ HOURS Figure 4. Syndactyly Time Chi Square Time Chi Square 0 2.8679 +15 3.6828 +3 9.5075 +18 0.6428 +6 1.8368 +21 0.0002 +9 2.8679 +24 11.0500 +12 22.1952 Total Chi Square = 54.6511 P <0.001 l6 100-! 90-3 30.4 70- 60- 8. 50.. 40- 30- 20.. 10- l L 0- 6 9 12 18 21 24 _ HOURS Figure 5 . Adactyly Time Chi Square Time Chi Square 0 0.7924 +15 0.0024 +3 23.8530 +18 9.9300 +6 5.8777 +21 0.1251 +9 0.1608 +24 1.8808 +12 9.0611 Total Chi Square = 51.6833 P <0.001 100% 904 80+-I 70%| «60H ENO- IUOJ :30H 120H 10H Figure 6. Time 0 +3 +6 +9 +12 17 Dorsal Lesion Chi Square 0.5121 5.6806 3.1765 0.2914 0.0000 Total Chi Square 34.5495 Time +15 +18 +21 +24 P <0.001 Chi Square 0.1800 6.7490 7.2499 10.7100 DISCUSSIONS AND CONCLUSIONS General Considerations Cyclophosphamide at a dose of 20 mg/kg on day 11 induces essen- tially the same spectrum of malformations in the Sprague Dawley mice as those reported for Swiss webster (Gibson 8 Becker, 1968A). Both studies showed cleft palate, however, the present study showed open eyes on day 11 where Gibson's did not. Digital anomalies also appeared to be the same with the exception of polydactyly which was not induced in the Sprague Dawley strain on day 11. The dorsal hemorrhagic lesions were present in both strains but appeared to be more severe and more frequent in the Sprague Dawley mice. Skeletal anomalies also appeared to be similar; the absence of ribs being induced in both strains. Beyond the confirmation of Gibson's data in a different strain of mice, the present investigation adds the histological examination of the dorsal hemorrhagic lesions. This examination revealed the lesions to be circumscribed and blood filled. There were also cell lined spaces, primarily located around the central nervous system, filled with an acellular exudate. Although the location of the majority of hemorrhagic lesions was dorsal and midline, occasional similar lesions were noted on the extremities. Since both the hemorrhagic lesions and cystic spaces are circumscribed and cell lined, the question should be raised as to whether these are related in any way to congenital hemangiomas to lymphangiomas. Specific Considerations Relating to the Hypothesis The chi square values of the malformations Open eyes and cleft palate were not significant at the 0.05 level. Therefore, for these 18 19 anomalies, the hypothesis that the variations in frequency found over the twenty-four hour period are due to chance, cannot be rejected. The remaining anomalies of ectrodactyly, syndactyly, adactyly and dorsal lesion all had statistically significant chi square values. In all four of these malformations the prObability of getting the amount of fluctuation by chance was less than 0.001. In computing the Chi square value at 0 and +9 hours for ectrodactyly and adactyly the expected figures were less than 5, thus questioning the validity of the values at these hours. But the values of chi square at all of these hours added only small amounts to the summed Chi square value for the defect. Thus, for the defects of ectrodactyly, syndactyly, adactyly and dorsal lesion the Null hypothesis can be rejected. The nonsignificance of the fluctuations in two of the four defects investigated does not necessarily mean that there is no meaningful variability in the incidence of these anomalies. There could be a dampening of this due to other influencing factors. The deveIOpmental stage of the embryo at the time of injection is a principal influence in determining the frequency of teratogenic effect. Because of this influence, the developmental scale would be the baseline for any further fluctuations which occur. Another influencing factor is the mother's metabolism, This could have the effect of the embryo receiv- ing the drug at only those periods when the mother's metabolism was the greatest. These two factors working at the same time could damp out their respective variations if they were out of phase with each other. On the other hand, if they were both working at the same time and in phase‘with each other, then their effect could be additive. 20 There are several ways of viewing the data after inspection of the graphs (Figures 1-6). .Although each graph has very different fluctuations there is a definite tendency for the frequency of each anomaly to decrease at the end of the light period (between +12 and +15 hours), followed by an increase in frequency at the beginning of the dark period. There was also a tendency for the frequency of the anomaly to increase (Figures 1, 2, 4, and 6) at the beginning of the light period. If one looks at the digital defects (Figures 3, 4 and 5) as a group it can be Observed that each has its maximal incidence at a separate time. .Adactyly peaks first at +3 hours, syndactyly follows at +12 hours and then ectrodactyly at +18 hours. All these peaks occur very close to or within.the light period (from +3.5 to +18.5 hours). Inasmuch as digital anomalies are also induced by cyclophos- phamide on days 9, 10, and 12 then it would be interesting to test whether or not a light-dark differential would also hold for those days. Even though four of the six anomalies studied showed statistically significant variability on day 11, there is no means of determining if these variations are circadian in nature. But a circadian nature might be expected because there are many physiological functions which have this type of fluctuation. .Also, if one compares times of the greatest chi square values with the same times on the graphs, it can be noted that these large variations oscillate above the mean, below the mean, then above it again. This is the type of oscillation expected in a circadian cycle. This type of fluctuation certainly would not be expected if the susceptability of the embryo were influenced only by developmental stage. 21 The importance of this investigation is twofold. The immediate value is that in teratological work of the past, it has been assumed that there would be no significant variation in the effects of a teratogen during a specific day of gestation. Therefore, the time of the administration of the teratogen was never noted. It must be assumed that each investigator was probably consistent in his own experiments, but others doing similar studies could be getting very different results simply because they were administering the teratogen at a different time of the same day of gestation. The statistically significant variability found in this study raises reasonable doubts as to the wisdom of not noting the time at which the teratogen is administered. The second important result of the study is the number of questions it raises which should be investigated further. The first investigation would be to test other days of gestation using the same experimental design. In this case it would be wise to shorten the time intervals between injections. Ideally, it would be best to use a drug which caused only one anomaly. .Another follow-up study would be to determine if these fluctuations are circadian. An approach to this problem would be to manipulate the twenty-four hour cycle. This could be accomplished by lengthening or shortening the light-dark cycle. It could also be done by housing the mother in constant light or constant darkness, although this could impose serious fertility problems in experimental animals. SUMMARY The present investigation tests the Null hypothesis that the frequency of specific teratogenic anomalies are evenly distributed over the twenty-four hour period and deviations at each three hour interval are due to chance alone. Pregnant Sprague Dawley mice were injected with 20 mg/kg of cyclOphosphamide at various times on the eleventh day of gestation. The malformations noted in the experi- mental fetuses were essentially the same as those noted by Gibson and Becker (1968) in Swiss webster mice. The fluctuations observed 'were analyzed by the chi square method. In four of the six anomalies found the variation was statistically significant at the 0.05 level. In these four cases the Null hypothesis was rejected. The data were discussed. Possible reasons for the insignifi- cant variations of two anomalies were noted. Different ways of viewing the data were presented. The investigation was discussed in terms of its implications in the study of teratology and possible further investigation. 22 LITERATURE CITED LITERATURE CITED Brock, N. and T. von Kreybig. 1964. Experimenteller Beitrag zur Prugung Teratogener Wirkungen von Arzoneimitteln an der Laboratoiumsratte. Naunyn- Schmiedebergs Arch. exp Path. u. Pharmak. 243117-145. Chaube, Shakuntala, G. Kury and M. L. Murphy. 1967. Teratogenic Effects of Cyclophosphamide in the Rat. Cancer Chemotherapy Reports. 51:363-376. Foley, G. E., O. M. Friedman and B. P. Drolet. 1961. Studies of the Mechanism of Action of Cytoxan--Evidence of Activation _i_n_ vivo and in vitro. Cancer Research. _2_l_:57-63. Gerlinger, P., J. V. Ruch and J. Clavert. 1963. Action du CyclOphosphamide sur la Formation de l'embryon. Compt. Rend. Soc. Biol. 1573173476. . 1964. Action du CyclOphosphamide Injecté 3 la Mére sur la Réalisation de la Forme du Corps des Embryons de Lapin. Compt. Rend. Soc. Biol. 1_5_8: 2154-2157. and J. Clavert. 1965A. Anomalies Observées chez de Lapins Issus de fires Traitées au Cyclophosphamide. Compt. Rend. Soc. Biol. 1_5_9_:1462-1466. and J. Clavert. 19658. Etude de 1"évolution des cellules sexuelles d'embryons issus de lapines traitees au cyclOphosphamide. Compt. Rend. Soc. Biol. 1_59_: 1386-1389. Gibson, J. E. and B. A. Becker. 1968A. The Teratogenicity of CyclOphosphamide in Mice. Cancer Research. _2_8_:475—480. and B. A. Becker. 1968B. Effect of Phenobarbital and SKF 525-A on the Teratogenicity of Cyclophosphamide in Mice. Teratology. _1_: 393-398. 1969A. Private commmication. 1969B. Unpublished data. Greenberg, L. H. and K. R. Tanaka. 1964. Congenital Anomalies Probably Induced by Cyclophosphamide. Journal American Medical Association. 18_8_:423-426. Hackenberger, I. and T. von Kreybig. 1965. Comparative Teratologic Studies in Mouse and Rat. Arzneimittel—Forschung. _l_5_:l456—l460. 23 24 Kreybig von, Thomas. 1965A. Extremity Malformations in Animal Experiments. Arzneimittel-Forschung. 15:1213-1217. . 1965B. Die Taratogene Wirkung von CyclOphosphamide wahrend der Embryonalen Entwicklungsphase bei der Tatte. Naunyn Schmiedebergs.Arch. Exptl. Pathol Pharmakol. 2523173-195. and walter Schmidt. 1966. Zur Chemischem Teratogenese bie der Ratte. .Arzneimittel-Forschung. 195989—1001. and walter Schmidt. 1967. Chemisch Induzierte FetOpathien bie der Ratte. .Arzneimittel-Forschung. 12:1094-1100. Lewis, A. 1966. Biostatistics. New York, Reinhold Publishing Co., pp. 110—124. Masson, P. J. 1957. ZManual pleistologic and Special Staining Techniques. Washington, D.C. Armed Forces Institute 0 PathOIOgy, pp. 67-68. Shoji, R. and E. thu. 1965. Some Aspects of the Effect of Endotoxan on Developing Mbuse Embryos. Journ. Faculty of Science, kukaido University, Series VI. 15:662-665. ‘Wilson, J. G. 1959. Experimental Studies on Congenital Malformations. Journal of Chronic Diseases. 10:111-130. and J.'Warkany. 1965. Teratolo : Principles and TeEhniques. Chicago, Illin01s. The University of Chitago Press, pp. 262-277. APPENDIX O O O O O N O N O N O HH O H N n m O O O O O n O O O u n O O :onOH Hmmhom O O O m H O O O O O O O O O O O H O O O H O OH O O O n O O O .JNHNwomO< O O O O O N H m H N O O O O OH OH O OH O O HH O O O O O O H OH O .wamumvexw O O n H N O H O m N O O N m H O O H O O O O O O O O O O O O szuomOOHuum HH O OH O O O m HH 5 HH O OH O OH NH O n O N O HH O OH O O O O O O O OpmHmO HHOHO HH O OH O O HH H HH m HH O OH HH O NH O O n O O NH O O O O O m O O m moan ammo HH O OH O O HH O HH 5 HH O OH HH OH NH OH OH HH O O NH O OH O O n O OH OH O moxmaem mo honesz O O O O O O O O O O O O O O O O O O O n O O O NH O O O O O n moxuOEm msogmuos< mo uwmezz, O O O O O O O O O O O O O O O O O O m N O O H O O O H O O H mcoHHmwomom mo HOOESZ HH O OH O O HH O HH 5 HH O OH HH OH NH OH OH HH O O NH O HH NH O N O OH OH OH mcoHpmuemHOEH (mo MOOESZ m O O O. O O O O N whouuHH Ho HOOESZ ON+ HN+ OH+ mH+ NH+ O+ O+ m+ O+ muse: 5 2E. mumm.3mm mo XHOOOQQ< 25 MICHIGAN STATE UNIVERSITY LIBRARIES II IIIIIIIIIII l 3 1233 03175 2631