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The teratogenic effect of vincaleukoblastine in the pregnant rat
T h e J o u r n a l of P E D I A T R I C S
541
The teratogenic effect of ri nca leu k oblast i ne in tbe pregnant rat Vincaleukoblastine ( V L B ) , a mitosis-inhibiting chemotherapeutic agent is teratogenie in the pregnant rat, producing a 9 per cent incidence of gross developmental malformations. Transplacentally acquired VLB inhibits cell mitosis in fetal tissues as evidenced by a sixfold increase in mitotic figures after a single injection of VLB. Glutamic acid which inhibits the effect of V L B in tissue culture does not protect the rat fetus from its teratogenic effect.
Sidney Q. Cohlan, M.D., * and David Kitay, M.D. NEW
YORK~
N.Y.
VINCALEUKOBLASTINE
(VLB)
and vincristine (VCR), alkaloids derived from the periwinkle plant ( V i n c a rosea Linn), are chemotherapeutic agents which produce remissions in generalized Hodgkin's disease, 1 lymphomas, 2 choriocarcinoma, 3 and leukemia. 4 These cytotoxic compounds are mitosis inhibitors, an'esting cell division in metaphase. This type of arrest is termed C-mitosis, which indicates an effect on the mitotic spindle. VLB mitotic inhibition can be reversed to some extent in vitro 5 and in vivo 6 by treatment with various amino acids. Glutamic acid appears to afford the most protectionfi From the Department of Pediatrics, New York University Medical Center. Supported by Grant HD 00187-11 from the United States Public Health Service. Read at the Fourth Annual Meeting of the Teratology Society, Arden House, N. Y., June lOth, 1964. *Address, Associate Professor of Pediatrics, New York University Medical Center, 566 First Ave., New York 16, N.Y.
This study was devised to explore (1) the teratogenic effect of VLB on the rat fetus, (2) the effect of transplacentally acquired VLB on fetal cell mitosis, and (3) the protective effect of glutamic acid.
MATERIAL AND METHODS VLB was administered intraperitoneally to 94 Wistar (CR) pregnant rats from the seventh t o the twelfth day of gestation. The morning after the night of mating is considered the first day of gestation. The drug was prepared in a 12.5 per cent saline solution and was administered in doses ranging from 0.12 to 0.50 mg. per kilogram per day. Another group of 45 pregnant rats similarly treated with 0.25 mg. per kilogram per day also received 50 mg. per kilogram glutamic acid intraperitoneally twice daily from the seventh to the fifteenth day of gestation. Control rats received aliquots of saline intraperitoneally. Animals were sacrificed on the twentieth day, examined for gross anomalies, and fixed and prepared for further study.
54 2
March 1965
Cohlan and Kitay
T a b l e I. F e t a l survival a n d gross m a l f o r m a t i o n s following i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of V L B a n d V L B + g l u t a m i c acid in the p r e g n a n t r a t Dose VLB Surviving litters/ Mortality No. fetuses (mg./Kg.) No. pregnant rats (%) surviving 0.12 8/10 20 78 0.25 30/74 40.5 286 0.25 + glutamic acid ~ 17/35 48 136 0.50 2/10 80 I1 ~Fifty milligramsper kilogramtwice a day from the seventh to the fifteenth day of gestation.
F o r the investigation of the effect of V L B on fetal mitosis, 4 e x p e r i m e n t a l 15-day-pregn a n t rats received a single intravenous injection of 1 rag. V L B in 2 ml. saline. Controls received 2 mh saline. F o u r hours post injection each a n i m a l was sacrificed. M a t e r nal bone marrow, fetuses, a n d placentas were f o r m e d into cellular suspensions in polyvinylpyrrolidone, smeared, fixed, stained with Giemsa, a n d e x a m i n e d for mitotic figures. T h e n u m b e r of mitotic figures p e r 1,000 cells was c o u n t e d for each p r e p a r a t i o n . A significant increase over the control rate of mitotic figures per 1,000 cells is the measure of mitotic arrest. RESULTS V L B administered in a dose of 0.25 mg. p e r k i l o g r a m p e r day from the seventh to the twelfth day of gestation p r o d u c e d a 9 per cent incidence of gross congenitaI m a l f o r m a tions in the surviving offspring ( T a b l e I ) . A t this dose level the fetal m o r t a l i t y rate was 40.5 per cent. Increasing the administered dose to 0.50 mg. per kilogram p e r day increased the fetal m o r t a l i t y rate to 80 p e r cent. T h o u g h the gross m a l f o r m a t i o n s varied, 60 p e r cent of the a b n o r m a l young displayed a characteristic constellation of anomalies including exencephaly, iniencephaly, rachischisis, gastroschisis, a n d bilateral clubbed feet posteriorly retroflexed (Fig. 1). As can be seen in T a b l e I the concurrent administration of glutamic acid h a d no effect on the embryocidal or m a l f o r m a t i o n rates. T h e fetal mortality rate was 48 p e r cent a n d the m a l f o r m a t i o n incidence was 10 per cent. T a b l e I I summarizes the results of the ob-
No. grossly malformed 2 26 (9.4%) 14 ( 10.2% ) 1
servations on the mitosis-inhibition effect on m a t e r n a l marrow, fetal cells, a n d p l a c e n t a of transplacentally acquired VLB. T h e average mitotic figure count in the m a t e r n a l bone m a r r o w of 4 p r e g n a n t rats four hours following a single 1 mg. intravenous dose of V L B was 21 per 1,000 as c o m p a r e d to 3.2 p e r 1,000 in the 4 saline-treated controls. T h e r e was a similar sixfold increase in mitotic figure count in the V L B - t r e a t e d fetuses (24 p e r 1,000) as c o m p a r e d to their controls (4.2 per 1,000). T h e effect on the p l a c e n t a was of m u c h lesser m a g n i t u d e . T h e mitotic count in the V L B - t r e a t e d placentas (5.9 p e r 1,000) was only 60 per cent higher t h a n in the controls (3.5 p e r 1,000). DISCUSSION
T h e mitotic inhibition of V L B has been studied clinically,z in the l a b o r a t o r y animal, s a n d in tissue culture. 9 I n bone m a r r o w 7 there is a m a r k e d increase in cells seen in rectaphase a n d a slight increase in prophase, while the postmetaphase mitotic figures are
T a b l e II. M i t o t i c figures per 1,000 cells counted in 15-day-old r a t fetus, p l a c e n t a , a n d m a t e r n a l m a r r o w , four hours following single intravenous injection of 1 mg. V L B
Control ~ VLB?
Maternal bone marrow 3.2 21
Fetal cell suspension 4.2 24.1
PEacental cell suspension 3.5 5.9
~Saline, 2 ml. intravenously.
"~VLB, 1 mg. in 2 ml. saline intravenously.
Volume 66 Number 3
Teratogenic effect o[ vincaleukoblastine in pregnancy
entirely absent, indicating an abrupt arrest at metaphase. This is followed by either pyknosis of the arrested nuclei or disrupted "exploded" metaphase forms. The chromosomes in many of the arrested cells remain in shortened clumped pairs. Forty-eight hours after VLB treatment, postmetaphase figures return, though many cell nuclei are still abnormal. Similar changes have been observed in tissue culture ~ and in autopsy specimens 7 of pancreas, liver, renal tubules, adrenals, and other parenchymatous organs from premortem VLB-treated patients. Though the teratogenicity of VLB may be "due to other drug-induced metabolic and/or cytotoxic effects on fetal development, the sixfold increase in metaphase arrest in the VLB-treated fetuses strongly suggests that mitosis inhibition probably plays an important role in VLB embryopathy. Some support for this belief derives from observations that colchicine, ~~ another mitosis-arresting agent, produces malformations in the pregnant hamster. It is noteworthy that in the 15 day VLB-treated placenta which is developing relatively slowly compared to its fetus, the mitotic arrest is of a much lesser magnitude. The reversal of the VLB effect by a variety of amino acids 6 has been studied in tissue cultures in J96 cell lines derived from peripheral blood of humans with monocytic leukemia and in cultures of embryonic connective tissue cells (LLC-HC1). Glutamic acid produced a complete reversal of the VLB effect, while glutamine, folic acid, tryptophane, and phenylalanine induced only varying degrees of partial reversal. Simultaneous administration of VLB and glutamic acid to the normal rat results in a temporary and incomplete inhibition of the VLB effect on rat bone marrow. It would appear from our observations that glutamic acid affords the rat fetus no protection from the teratogenie effect of transplacentally acquired VLB. Clinical experience with the use of cancer chemotherapeutic agents in human pregnancy is fortunately not c o m m o n Y It is not possible to predict the effect of VLB on human fetal development from our observations in
543
the rat. Its teratogenic potential is, however, apparently not limited to the rat, since during the course of our observations Ferm 12 reported on the embryocidal and teratogenic effect of VLB and V C R in the pregnant golden hamster. The intravenous administration of a single dose of VLB or V C R on the eighth day of gestation in the pregnant hamster resulted in a gross malformation rate of I0 per cent. AnornMies noted were
Fig. 1. Twenty day VLB malformed fetuses exhibiting iniencephaly, exencephaly, rachisehisis, gastroschisis, and retroflexed bilateral clubbed feet, compared to a control fetus.
544
Cohlan and Kitay
microphthalmia, a n o p h t h a l m i a , spina bifida, a n d m i n o r skeletal defects. I n the one clinical report is to date, a w o m a n with Hodgkin's disease received oral VLB t r e a t m e n t b e g i n n i n g two months prior to conception a n d c o n t i n u i n g to term, when a n o r m a l full-term i n f a n t was uneventfully delivered. At 3 months of age this i n f a n t was thriving without any a p p a r e n t congenital defect. SUMMARY
1. Vincaleukoblastine is teratogenic in the p r e g n a n t rat. T h e intraperitoneal administration of 0.25 mg. per kilogram per day from the seventh to the twelfth day of gestation produces a 9 per cent incidence of gross congenital malformations. Sixty per cent of the a b n o r m a l young exhibit a characteristic constellation of malformations, including exencephaly, iniencephaly, rachischisis, a n d gastroschisis. 2. T r a n s p l a c e n t a l l y acquired VLB induces a marked inhibition in fetal cell mitosis as evidenced by a sixfold increase in mitotic figures of fetal cell preparations four hours following the intravenous administration of 1 mg. VLB. 3. T h e simultaneous intraperitoneal administration of glutamic acid does not influence the teratogenic effect of VLB in the p r e g n a n t rat. The technical assistance of Mrs. Charlotte Violin is acknowledged. REFERENCES
1. Hodes, M. E., Rohn, R. J., and Bond, W. H.: Vincaleukoblastine. I. Preliminary clinical studies, Cancer Res. 20: 1041, 1960.
March 1965
2. Warwick, O. H., Darek, J. M. M., and Brown, T. C.: Some biological effects of vincaleukoblastine, an alkaloid of Vinca roses Linn, in patients with malignant disease, Cancer Res. 20: 1031, 1960. 3. Hertz, R., Lipsett, M. B., and Moy, R. H.: Effect of vincaleukoblastine on metastatic choriocarcinoma and related trophoblastic tumors in women, Cancer Res. 20: 1050, 1960. 4. Hill, J. M., and Loeb, E.: Treatment of leukemia, lymphoma and other malignant neoplasms with vinblastine, Cancer Chemother. Rep. 15: 41, 1961. 5. Johnson, I. S., Wright, Hi F., Svoboda, G. H., and Vlantis, J.: Antitumor principles derived from Vinca roses Linn: I. Vincaleukoblastine and leurosine, Cancer Res. 20: 1016, 1960. 6. Cutts, J. H.: Effect of vincaleukoblastine on dividing cells in vivo, Cancer Res. 21: 168, 1961. 7. Vaitkevicius, V. K., Talley, R. W., Tucker, J. L., and Brennan, M. J.: Cytological and clinical observations during vincaleukoblastine therapy of disseminated cancer, Cancer 15: 294, 1962. 8. Cutts, J. H., Beer, C. T . , and Noble, R. L.: Biological properties of vincaleukoblastine, alkaloid in Vinca roses Linn, with reference to its antitumor action, Cancer Res. 20: 1023, 1960. 9. Pahner, C. G., Livengood, D., Warren, A. K., Simpson, P. J., and Johnson, I. S.: Action of vincaleukoblastine on mitosis in vitro, Exper. Ceil Res. 20: 198, 1960. 10. Ferm, V. H.: Colchicine teratogenesis in hamster embryos, Proc. Soc. Exper. Biol. &Med. 112: 775, 1963. 1I. Sokal, S. E., and Lessman, E. M.: Effects of cancer chemotherapeutic agents on the human fetus, J. A. M. A. 172: 1765, 1960. 12. Ferm, V. H.: Congenital malformations in hamster embryos after treatment with vinbtastine and vincristine, Science 141: 426, 1963. 13. Armstrong, J. G., Dyke, R. W., and Fours, P. J.: Vinblastine sulfate treatment of Hodgkin's disease during a pregnancy, Science 143: 703, 1964.
541
The teratogenic effect of ri nca leu k oblast i ne in tbe pregnant rat Vincaleukoblastine ( V L B ) , a mitosis-inhibiting chemotherapeutic agent is teratogenie in the pregnant rat, producing a 9 per cent incidence of gross developmental malformations. Transplacentally acquired VLB inhibits cell mitosis in fetal tissues as evidenced by a sixfold increase in mitotic figures after a single injection of VLB. Glutamic acid which inhibits the effect of V L B in tissue culture does not protect the rat fetus from its teratogenic effect.
Sidney Q. Cohlan, M.D., * and David Kitay, M.D. NEW
YORK~
N.Y.
VINCALEUKOBLASTINE
(VLB)
and vincristine (VCR), alkaloids derived from the periwinkle plant ( V i n c a rosea Linn), are chemotherapeutic agents which produce remissions in generalized Hodgkin's disease, 1 lymphomas, 2 choriocarcinoma, 3 and leukemia. 4 These cytotoxic compounds are mitosis inhibitors, an'esting cell division in metaphase. This type of arrest is termed C-mitosis, which indicates an effect on the mitotic spindle. VLB mitotic inhibition can be reversed to some extent in vitro 5 and in vivo 6 by treatment with various amino acids. Glutamic acid appears to afford the most protectionfi From the Department of Pediatrics, New York University Medical Center. Supported by Grant HD 00187-11 from the United States Public Health Service. Read at the Fourth Annual Meeting of the Teratology Society, Arden House, N. Y., June lOth, 1964. *Address, Associate Professor of Pediatrics, New York University Medical Center, 566 First Ave., New York 16, N.Y.
This study was devised to explore (1) the teratogenic effect of VLB on the rat fetus, (2) the effect of transplacentally acquired VLB on fetal cell mitosis, and (3) the protective effect of glutamic acid.
MATERIAL AND METHODS VLB was administered intraperitoneally to 94 Wistar (CR) pregnant rats from the seventh t o the twelfth day of gestation. The morning after the night of mating is considered the first day of gestation. The drug was prepared in a 12.5 per cent saline solution and was administered in doses ranging from 0.12 to 0.50 mg. per kilogram per day. Another group of 45 pregnant rats similarly treated with 0.25 mg. per kilogram per day also received 50 mg. per kilogram glutamic acid intraperitoneally twice daily from the seventh to the fifteenth day of gestation. Control rats received aliquots of saline intraperitoneally. Animals were sacrificed on the twentieth day, examined for gross anomalies, and fixed and prepared for further study.
54 2
March 1965
Cohlan and Kitay
T a b l e I. F e t a l survival a n d gross m a l f o r m a t i o n s following i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of V L B a n d V L B + g l u t a m i c acid in the p r e g n a n t r a t Dose VLB Surviving litters/ Mortality No. fetuses (mg./Kg.) No. pregnant rats (%) surviving 0.12 8/10 20 78 0.25 30/74 40.5 286 0.25 + glutamic acid ~ 17/35 48 136 0.50 2/10 80 I1 ~Fifty milligramsper kilogramtwice a day from the seventh to the fifteenth day of gestation.
F o r the investigation of the effect of V L B on fetal mitosis, 4 e x p e r i m e n t a l 15-day-pregn a n t rats received a single intravenous injection of 1 rag. V L B in 2 ml. saline. Controls received 2 mh saline. F o u r hours post injection each a n i m a l was sacrificed. M a t e r nal bone marrow, fetuses, a n d placentas were f o r m e d into cellular suspensions in polyvinylpyrrolidone, smeared, fixed, stained with Giemsa, a n d e x a m i n e d for mitotic figures. T h e n u m b e r of mitotic figures p e r 1,000 cells was c o u n t e d for each p r e p a r a t i o n . A significant increase over the control rate of mitotic figures per 1,000 cells is the measure of mitotic arrest. RESULTS V L B administered in a dose of 0.25 mg. p e r k i l o g r a m p e r day from the seventh to the twelfth day of gestation p r o d u c e d a 9 per cent incidence of gross congenitaI m a l f o r m a tions in the surviving offspring ( T a b l e I ) . A t this dose level the fetal m o r t a l i t y rate was 40.5 per cent. Increasing the administered dose to 0.50 mg. per kilogram p e r day increased the fetal m o r t a l i t y rate to 80 p e r cent. T h o u g h the gross m a l f o r m a t i o n s varied, 60 p e r cent of the a b n o r m a l young displayed a characteristic constellation of anomalies including exencephaly, iniencephaly, rachischisis, gastroschisis, a n d bilateral clubbed feet posteriorly retroflexed (Fig. 1). As can be seen in T a b l e I the concurrent administration of glutamic acid h a d no effect on the embryocidal or m a l f o r m a t i o n rates. T h e fetal mortality rate was 48 p e r cent a n d the m a l f o r m a t i o n incidence was 10 per cent. T a b l e I I summarizes the results of the ob-
No. grossly malformed 2 26 (9.4%) 14 ( 10.2% ) 1
servations on the mitosis-inhibition effect on m a t e r n a l marrow, fetal cells, a n d p l a c e n t a of transplacentally acquired VLB. T h e average mitotic figure count in the m a t e r n a l bone m a r r o w of 4 p r e g n a n t rats four hours following a single 1 mg. intravenous dose of V L B was 21 per 1,000 as c o m p a r e d to 3.2 p e r 1,000 in the 4 saline-treated controls. T h e r e was a similar sixfold increase in mitotic figure count in the V L B - t r e a t e d fetuses (24 p e r 1,000) as c o m p a r e d to their controls (4.2 per 1,000). T h e effect on the p l a c e n t a was of m u c h lesser m a g n i t u d e . T h e mitotic count in the V L B - t r e a t e d placentas (5.9 p e r 1,000) was only 60 per cent higher t h a n in the controls (3.5 p e r 1,000). DISCUSSION
T h e mitotic inhibition of V L B has been studied clinically,z in the l a b o r a t o r y animal, s a n d in tissue culture. 9 I n bone m a r r o w 7 there is a m a r k e d increase in cells seen in rectaphase a n d a slight increase in prophase, while the postmetaphase mitotic figures are
T a b l e II. M i t o t i c figures per 1,000 cells counted in 15-day-old r a t fetus, p l a c e n t a , a n d m a t e r n a l m a r r o w , four hours following single intravenous injection of 1 mg. V L B
Control ~ VLB?
Maternal bone marrow 3.2 21
Fetal cell suspension 4.2 24.1
PEacental cell suspension 3.5 5.9
~Saline, 2 ml. intravenously.
"~VLB, 1 mg. in 2 ml. saline intravenously.
Volume 66 Number 3
Teratogenic effect o[ vincaleukoblastine in pregnancy
entirely absent, indicating an abrupt arrest at metaphase. This is followed by either pyknosis of the arrested nuclei or disrupted "exploded" metaphase forms. The chromosomes in many of the arrested cells remain in shortened clumped pairs. Forty-eight hours after VLB treatment, postmetaphase figures return, though many cell nuclei are still abnormal. Similar changes have been observed in tissue culture ~ and in autopsy specimens 7 of pancreas, liver, renal tubules, adrenals, and other parenchymatous organs from premortem VLB-treated patients. Though the teratogenicity of VLB may be "due to other drug-induced metabolic and/or cytotoxic effects on fetal development, the sixfold increase in metaphase arrest in the VLB-treated fetuses strongly suggests that mitosis inhibition probably plays an important role in VLB embryopathy. Some support for this belief derives from observations that colchicine, ~~ another mitosis-arresting agent, produces malformations in the pregnant hamster. It is noteworthy that in the 15 day VLB-treated placenta which is developing relatively slowly compared to its fetus, the mitotic arrest is of a much lesser magnitude. The reversal of the VLB effect by a variety of amino acids 6 has been studied in tissue cultures in J96 cell lines derived from peripheral blood of humans with monocytic leukemia and in cultures of embryonic connective tissue cells (LLC-HC1). Glutamic acid produced a complete reversal of the VLB effect, while glutamine, folic acid, tryptophane, and phenylalanine induced only varying degrees of partial reversal. Simultaneous administration of VLB and glutamic acid to the normal rat results in a temporary and incomplete inhibition of the VLB effect on rat bone marrow. It would appear from our observations that glutamic acid affords the rat fetus no protection from the teratogenie effect of transplacentally acquired VLB. Clinical experience with the use of cancer chemotherapeutic agents in human pregnancy is fortunately not c o m m o n Y It is not possible to predict the effect of VLB on human fetal development from our observations in
543
the rat. Its teratogenic potential is, however, apparently not limited to the rat, since during the course of our observations Ferm 12 reported on the embryocidal and teratogenic effect of VLB and V C R in the pregnant golden hamster. The intravenous administration of a single dose of VLB or V C R on the eighth day of gestation in the pregnant hamster resulted in a gross malformation rate of I0 per cent. AnornMies noted were
Fig. 1. Twenty day VLB malformed fetuses exhibiting iniencephaly, exencephaly, rachisehisis, gastroschisis, and retroflexed bilateral clubbed feet, compared to a control fetus.
544
Cohlan and Kitay
microphthalmia, a n o p h t h a l m i a , spina bifida, a n d m i n o r skeletal defects. I n the one clinical report is to date, a w o m a n with Hodgkin's disease received oral VLB t r e a t m e n t b e g i n n i n g two months prior to conception a n d c o n t i n u i n g to term, when a n o r m a l full-term i n f a n t was uneventfully delivered. At 3 months of age this i n f a n t was thriving without any a p p a r e n t congenital defect. SUMMARY
1. Vincaleukoblastine is teratogenic in the p r e g n a n t rat. T h e intraperitoneal administration of 0.25 mg. per kilogram per day from the seventh to the twelfth day of gestation produces a 9 per cent incidence of gross congenital malformations. Sixty per cent of the a b n o r m a l young exhibit a characteristic constellation of malformations, including exencephaly, iniencephaly, rachischisis, a n d gastroschisis. 2. T r a n s p l a c e n t a l l y acquired VLB induces a marked inhibition in fetal cell mitosis as evidenced by a sixfold increase in mitotic figures of fetal cell preparations four hours following the intravenous administration of 1 mg. VLB. 3. T h e simultaneous intraperitoneal administration of glutamic acid does not influence the teratogenic effect of VLB in the p r e g n a n t rat. The technical assistance of Mrs. Charlotte Violin is acknowledged. REFERENCES
1. Hodes, M. E., Rohn, R. J., and Bond, W. H.: Vincaleukoblastine. I. Preliminary clinical studies, Cancer Res. 20: 1041, 1960.
March 1965
2. Warwick, O. H., Darek, J. M. M., and Brown, T. C.: Some biological effects of vincaleukoblastine, an alkaloid of Vinca roses Linn, in patients with malignant disease, Cancer Res. 20: 1031, 1960. 3. Hertz, R., Lipsett, M. B., and Moy, R. H.: Effect of vincaleukoblastine on metastatic choriocarcinoma and related trophoblastic tumors in women, Cancer Res. 20: 1050, 1960. 4. Hill, J. M., and Loeb, E.: Treatment of leukemia, lymphoma and other malignant neoplasms with vinblastine, Cancer Chemother. Rep. 15: 41, 1961. 5. Johnson, I. S., Wright, Hi F., Svoboda, G. H., and Vlantis, J.: Antitumor principles derived from Vinca roses Linn: I. Vincaleukoblastine and leurosine, Cancer Res. 20: 1016, 1960. 6. Cutts, J. H.: Effect of vincaleukoblastine on dividing cells in vivo, Cancer Res. 21: 168, 1961. 7. Vaitkevicius, V. K., Talley, R. W., Tucker, J. L., and Brennan, M. J.: Cytological and clinical observations during vincaleukoblastine therapy of disseminated cancer, Cancer 15: 294, 1962. 8. Cutts, J. H., Beer, C. T . , and Noble, R. L.: Biological properties of vincaleukoblastine, alkaloid in Vinca roses Linn, with reference to its antitumor action, Cancer Res. 20: 1023, 1960. 9. Pahner, C. G., Livengood, D., Warren, A. K., Simpson, P. J., and Johnson, I. S.: Action of vincaleukoblastine on mitosis in vitro, Exper. Ceil Res. 20: 198, 1960. 10. Ferm, V. H.: Colchicine teratogenesis in hamster embryos, Proc. Soc. Exper. Biol. &Med. 112: 775, 1963. 1I. Sokal, S. E., and Lessman, E. M.: Effects of cancer chemotherapeutic agents on the human fetus, J. A. M. A. 172: 1765, 1960. 12. Ferm, V. H.: Congenital malformations in hamster embryos after treatment with vinbtastine and vincristine, Science 141: 426, 1963. 13. Armstrong, J. G., Dyke, R. W., and Fours, P. J.: Vinblastine sulfate treatment of Hodgkin's disease during a pregnancy, Science 143: 703, 1964.