Pattern of Anomalies Following Single Oral Doses of Ethylenethiourea to Pregnant Rats JOSEPH A. RUDDICK A N D K. S . K H E R A Ht>ctlth Protrctioii Brtriich, B i i w ( r c i of'Chr2mictilScifcty. Food Dirt,ctortrtt. Twcctology Sectio 1 1 , T i i i i > i r y ' sP t i s t i i r t ' , Otttrzvtr, O?tt(lrio K1 A O L 2 C~r?ctrrltr
ABSTRACT Single oral administration to rats of 240 mg/kg ethylenethiourea on days 10-21 of gestation produced visceral anomalies involving the nervous, urogenital, and ocular systems, and osseous anomalies affecting the axial and appendicular skeletons. The types of anomalies and organs affected were dependent on the stage of prenatal development at the time of treatment.
Ethylenethiourea (ETU) is a degradation product of the ethylenebis(dithi0carbamate) group of fungicides and has been identified in most commercial products of the fungicide (Bontoyan et al., '72). Two dithiocarbomates, maneb and zineb, were teratogenic a t dosages above 1 glkg in rats (Petrova-Vergieva and Ivanova-Tchemishanska, '73). ETU was reported to be carcinogenic (Innes et al., '69; Ulland et al., '72; Graham et al., '73) and to produce hyperplasia of the thyroid in rats (Graham and Hansen, '72). Cooking vegetables sprayed with ethylenebis(dithi0carbamates) promoted formation of ETU (Newsome and Laver, '73; Watts et al., '74). Awareness of ETU's carcinogenic potential and its presence in vegetables led to multiple-dose teratogenic studies in rats (Khera, '73). Subsequently studies were continued with single oral doses to establish a relation between the type of anomalies and day of treatment; results obtained form the subject of this report. MATERIALS AND METHODS
ETU was recrystalized from 2-imidazolidinethione (Eastman-Kodak, Rochester, New York); 500 g were dissolved in 800 ml of boiling water and decolorizing-carbon was added to the solution. The suspension was filtered (Whatman no. 1) while hot through a Buchner funnel and the filtrate was allowed to stand in the refrigerator overnight. The supernatant was discarded and the crystals were redissolved in boiling water. The above procedure was repeated, except for adding carbon. The supernatant
was discarded and the crystals were washed in absolute ethanol and dried a t 55" C. A melting point of 196" C (Allen et al., '46) and thin-layer chromatography on silica gel using ethanol as the solvent followed by development in an iodine chamber ( R F 0.76) indicated pure ETU. Virgin Wistar (Woodlyn Farms, Chatham, Ontario) female rats (175-200 g) were paired overnight with males, and the following morning was designated day 1 of gestation when a sperm-positive vaginal smear was observed. After mating, all females were caged individually and provided with food (Purina cubes) and water ad libitum. Females (5-l0/group) were each given a single oral administration, by intubation, of an aqueous solution of 40-480 mg/kg ETU on one of days &21 of gestation. The acute oral LD:,,, in rats was 1832 mg/kg (Graham and Hansen, '72). Results obtained with 240 mg/kg are reported principally and effects with other dosages briefly mentioned. The pregnant females were killed on day 22 of gestation and the number of live fetuses, and resorptions, and the individual litter weights determined. Two-thirds of the fetuses from each litter were processed for skeletal examination and the remainder were fixed in Bouin's fluid for visceral study. Visceral anomalies were determined by dissecting and razor-sectioning the fetuses. RESULTS
No maternal toxic effects were apparent following 240 mglkg ETU. The resorption rate (table 1) was increased following treatReceived Nov. 11, 1974 Accepted , J u l y 15, 1975.
277
4
3
2
1
Mean SE. No. x lOO/total implants Not determined. Empty cell denotes O V .
*
Resorption rate 2 (‘,I 1 Cleft lip Cleft palatc Brachygn athia Coloboma or exophthalmos Exencephaly H ydroceph a lus H ydran encepha ly Hypoplastic cerebellum Scoliosis Fused ribs Sternoxhisis Absent or short tail Spina b f i d a Ectopic genitalia Hydronephrotic or hypoplastic kidney Hydroureter Edema Forelimb Micrornelia Ectrosyiidactyly Hindlimb Ec trosy ndactyl y Talipes or twisted tibia and fibula Sirenomelia
Fetal weight I ( g )
1
Day of gestation treated
N o litters Litter size
TABLE 1
20
6
20
4
~
2
75 75
100 30
100
2 2
*
100 ~
38
8
40
100
ND ND
30
100 100 14
70
25
12
+ -
0.9 3.8 2 0.04 7.6
9 10.6
15
40
100 75
90 100
ND 3 ND
100 67
100 100
100
2.4 3.1 f 0.1 14.8 34 34 74
f
5 9.2
14
98 100
100 100
100 75 60 37 100
90 100
50 58
0.9 3.8 f 0.03 4.8
100 100
100 100 88 88 85
0.08 8.9 70 68 68 15 91 94
f
0.01 13.3
0.2 6.3
*
1.7 4.2
+-
f
+1.7 4.2
f
1.7 4.9
15 10.4
6 11.8
4 13.0
5 10.6
13
12
11
10
-
21
100
6
100
0.8 4.5 f 0.6 7.5 6 100
+ -
8 10.8
16
100
100
5 10.2 f 1.1 4.4 f 0.02 7.3
17
100
100
100
0.04 6.9
f
5 13.4 f 0.7 3.5
18
20 100 100
100
100
100
0.04 3.0
f 0.2 13.2
*
f
1.2 5.2
1.1 4.5
5 12.0
+ -
8 10.8
ruts 20
111
19
Frequency of((nomaZzec (cfter si?zgIe orul trecrtmc’nt with 240 my/kg E T U o n g e s t u t z o n drrys 10-21
+-
12
6
0.02 5.2
0.4 5.1
f
5 14.4
21
+-
0.01 8.5
f
0.5 5.0
37 11.1
Control
ETHYLENETHIOUKEA I N RATS
2 79
ment on gestation days 11, 14, and 19 of Observed consistently after treatment on gestation ( P < 0.05, t test), and fetal weight day 16 were hydranencephaly, cleft palate, was reduced after treatment on days 13, and hydronephrosis or hypoplastic kidneys. 14, 15, and 18. Hydranencephaly was also present followNo malformations were observed after ing exposure on days 17-20 and declined treatment on days 6-9. Treatment on day to 6 % after treatment on day 21. 10 induced defective external genitalia The external morphology of hydranen(6 % ), hydronephrosis or hypoplastic kid- cephalic fetuses was indistinguishable from neys (20 % ), and absent or short tail (20 % ). that of controls but sectioning through the Administration on day 11 resulted in spina frontal region revealed cerebral hemibifida of the lumbar region in 30% of the spheres whose interiors were devoid of neufetuses, fused ribs, and a higher frequency ral tissue. Beginning with treatment on day of kidney and tail anomalies than induced 15 the cerebral hemispheres, cerebellum, by earlier treatment. Days 12-15 of gesta- and medulla oblongata were affected (fig. tion were the period of peak sensitivity and 1) but with later treatment hydranencephwere characterized by high frequencies of aly was restricted to the cerebral hemianomalies, prominent among which were spheres. hydrocephalus, exencephaly, brachygnaAll fetuses exposed on days 17-19 of thia, hydronephrosis, absent tail, and fore- gestation had hydronephrotic or hypoplastic limb micromelia (table 1). kidneys. The frequency of renal defects was Treatment with lower dosages on these 20 and 42%, respectively, following treatdays caused lower frequencies and reduced ment on days 20 and 21. Subcutaneous intensity of anomalies. On day 12, 200 mg/ edema of the head, neck and trunk was kg failed to produce fused ribs, sternoschi- noted in all fetuses exposed on days 18-20. sis, and absent ulna or digits, and 160 mg/ DISCUSSION kg did not induce cleft lip, cleft palate, and limb anomalies. The lowest dosage, 40 mg/ The earliest treatment causing teratogenkg, produced short or absent tail only ic effects was day 10 which coincides with the onset of organogenesis and the time of (37% ).
Fig. 1 Coronal section of the head of a hydranencephalic fetus revealing cerebral hemispheres and cerebellum deficient in neural tissue, from pregnant rats givcn 240 mg/kg ETU on day 17 of gestation.
280
JOSEPH A . K U D D I C K AND K . S . K H E R A
Fig. 2 Frontal section through the optic region ( 0 ) of a 22-day-old fetus exposed to ETU o n day 12 of gestation, showing combined hydrocephalus ( H ) and exencephaly ( E l . x 20.
posterior-neuropore closure in rats (Edwards, '68). Malformations induced a t this time, such as failure of coccygeal growth, spina bifida, ectopic genitalia, and nephrosis, were restricted to the pelvic region. Various organs and tissues were the targets of ETU activity on days 12-15. The period of sensitivity was relatively long for many anomalies (tail defect, cleft palate, and sternoschisis), whereas for others (spin a bifida, exencephaly) i t was restricted to a relatively shorter period perhaps reflecting the time and duration of development of the structures involved. Cranial defects of the types observed by us are thought to arise through interference with anterior-neuropore closure (Nakano, '73). However, those in the present study (table 1) were produced by treatment after the anterior neuropore had closed, suggesting that the mode of action of ETU is different from that of agents inducing such conditions by interfering with neuralplate closure. The uniqueness of ETU was further illustrated by the simultaneous appearance of anomalies that are generally considered
to be mutually exclusive. Generally, hydrocephalus and exencephaly do not occur together; but ETU caused them concomitantly (fig. 2). Production of these combined anomalies was not due to disturbance of ancestral cells of the organ or the organ anlage undergoing differentiation (Murakami, '63) since the time of treatment was well past the differentiation stage. Pressure from a n expanding hydrocyphalic brain through a weaken skull (ex. necrosis) probably resulted in the combined anomalies. After day 16 of gestation, which marks the end of organogenesis, defects noted were hydranencephaly, hydronephrosis, and subcutaneous edema. These hydropic anomalies could have resulted from local disturbances in vascular permeability, intravascular pressure, or normal movement of tissue fluids. Thiourea and its derivatives are known to produce edematous changes in adult mammals (Combs and Giri, '73). Anomalies produced by multiple treatment with ETU on days 6-15 of gestation (Khera, '73) can now be ascribed to interference with prenatal development at specific stages. The effects observed in this
ETHYLENETHIOUREA I N RATS
study, although not of the same intensity or frequency as caused by single dosages, it now appears resulted primarily from treatment on days 12-15 of gestation. Furthermore, comparison of multiple and single treatment indicates the usefulness of both procedures for teratological assessment of ETU. Single treatment was useful for isolating specific anomalies and relating them to the stage of intrauterine development, Some of the malformations provide good model systems for examining biochemical and histological events associated with various defects. ACKNOWLEDGMENT
We would like to thank R. Tanner, L. Hierlihy, G. Trivett, R. Oliver, and G. Terry for their assistance. LITERATURE CITED Allen, C. F. H., C. 0. Edens and J. Von Allen 1946 Ethylenethiourea. Org. Synth., 26: 34-35. Bontoyan, W. R., and J. B. Looker 1972 Survey of ethylenethiourea in commercial ethylenebisdithiocarbamate formulation. J. Ass. Anal. Chem., 55: 923-925. Combs, A. B., and S. N. Giri 1973 Trichloroacetic acid-induced binding of phenylthiourea to eryth. rocytes. J. Pharmaceut. Sci., 62: 631-633. Edwards, J. A. 1968 The external development of rabbit and rat embryo. Adv. Terat., 3: 239263.
28 1
Graham, S. L., and W. H. Hansen 1972 Effects of short-term administration of ethylenethiourea upon thyroid function of the rat. Bull. Env. Cont. Toxic., 7: 19-25. Graham, S. L., W. H. Hansen, K. J. Davis and C. H. Perry 1973 Effects of one-year administration of ethylenethiourea upon the thyroid of the rat. J. Agr. Food Chem., 21 : 324-329. Innes, J . R. M., B. M. Ulland, M. G. Valerio, L. Petrucelli, L. Fisbien, E. R. Hart, A. J. Pallotta, I. Mitchell and J. Peters 1969 Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary report. J. Nat. Cancer Inst., 42: 1101-1104. Khera, K. S. 1973 Ethylenethiourea: teratogenicity study in rats and rabbits. Teratology, 7: 243252. Murakami, U. 1963 Studies on mechanismsmanifesting anomalies. Jap. J. Human Genet., 8: 202-226. Nakano, K. K. 1973 Anencephaly: a review. Dev. Med. Child Neur., 15: 383-400. Newsome, W. H.,and G. W. Laver 1973 Effect of boiling on the formulation of ethylenethiourea in zineb-treated foods. Bull. Env. Cont. Toxic., 10: 151-154. Petrova-Vergieva, T., and L. Ivanova-Tchemishanska 1973 Assessment of the teratogenic activity of dithiocarbamate fungicides. Food Cosm. Toxic., 1 I : 239-244. Watts, R. R.,R. W. Storherr and J. H. Onley 1974 Effects of cooking on ethylenebisdithiocarbamate degradation to ethylenethiourea. Bull. Env. Cont. Toxic., 12:224-226. Ulland, B. M., J. H. Weisburger, E. K. Weisburger, J . M. Rice and R. Cypher 1972 Thyroid cancer in rats from ethylenethiourea intake. J. Nat. Cancer Inst., 49: 583-584. '
ABSTRACT Single oral administration to rats of 240 mg/kg ethylenethiourea on days 10-21 of gestation produced visceral anomalies involving the nervous, urogenital, and ocular systems, and osseous anomalies affecting the axial and appendicular skeletons. The types of anomalies and organs affected were dependent on the stage of prenatal development at the time of treatment.
Ethylenethiourea (ETU) is a degradation product of the ethylenebis(dithi0carbamate) group of fungicides and has been identified in most commercial products of the fungicide (Bontoyan et al., '72). Two dithiocarbomates, maneb and zineb, were teratogenic a t dosages above 1 glkg in rats (Petrova-Vergieva and Ivanova-Tchemishanska, '73). ETU was reported to be carcinogenic (Innes et al., '69; Ulland et al., '72; Graham et al., '73) and to produce hyperplasia of the thyroid in rats (Graham and Hansen, '72). Cooking vegetables sprayed with ethylenebis(dithi0carbamates) promoted formation of ETU (Newsome and Laver, '73; Watts et al., '74). Awareness of ETU's carcinogenic potential and its presence in vegetables led to multiple-dose teratogenic studies in rats (Khera, '73). Subsequently studies were continued with single oral doses to establish a relation between the type of anomalies and day of treatment; results obtained form the subject of this report. MATERIALS AND METHODS
ETU was recrystalized from 2-imidazolidinethione (Eastman-Kodak, Rochester, New York); 500 g were dissolved in 800 ml of boiling water and decolorizing-carbon was added to the solution. The suspension was filtered (Whatman no. 1) while hot through a Buchner funnel and the filtrate was allowed to stand in the refrigerator overnight. The supernatant was discarded and the crystals were redissolved in boiling water. The above procedure was repeated, except for adding carbon. The supernatant
was discarded and the crystals were washed in absolute ethanol and dried a t 55" C. A melting point of 196" C (Allen et al., '46) and thin-layer chromatography on silica gel using ethanol as the solvent followed by development in an iodine chamber ( R F 0.76) indicated pure ETU. Virgin Wistar (Woodlyn Farms, Chatham, Ontario) female rats (175-200 g) were paired overnight with males, and the following morning was designated day 1 of gestation when a sperm-positive vaginal smear was observed. After mating, all females were caged individually and provided with food (Purina cubes) and water ad libitum. Females (5-l0/group) were each given a single oral administration, by intubation, of an aqueous solution of 40-480 mg/kg ETU on one of days &21 of gestation. The acute oral LD:,,, in rats was 1832 mg/kg (Graham and Hansen, '72). Results obtained with 240 mg/kg are reported principally and effects with other dosages briefly mentioned. The pregnant females were killed on day 22 of gestation and the number of live fetuses, and resorptions, and the individual litter weights determined. Two-thirds of the fetuses from each litter were processed for skeletal examination and the remainder were fixed in Bouin's fluid for visceral study. Visceral anomalies were determined by dissecting and razor-sectioning the fetuses. RESULTS
No maternal toxic effects were apparent following 240 mglkg ETU. The resorption rate (table 1) was increased following treatReceived Nov. 11, 1974 Accepted , J u l y 15, 1975.
277
4
3
2
1
Mean SE. No. x lOO/total implants Not determined. Empty cell denotes O V .
*
Resorption rate 2 (‘,I 1 Cleft lip Cleft palatc Brachygn athia Coloboma or exophthalmos Exencephaly H ydroceph a lus H ydran encepha ly Hypoplastic cerebellum Scoliosis Fused ribs Sternoxhisis Absent or short tail Spina b f i d a Ectopic genitalia Hydronephrotic or hypoplastic kidney Hydroureter Edema Forelimb Micrornelia Ectrosyiidactyly Hindlimb Ec trosy ndactyl y Talipes or twisted tibia and fibula Sirenomelia
Fetal weight I ( g )
1
Day of gestation treated
N o litters Litter size
TABLE 1
20
6
20
4
~
2
75 75
100 30
100
2 2
*
100 ~
38
8
40
100
ND ND
30
100 100 14
70
25
12
+ -
0.9 3.8 2 0.04 7.6
9 10.6
15
40
100 75
90 100
ND 3 ND
100 67
100 100
100
2.4 3.1 f 0.1 14.8 34 34 74
f
5 9.2
14
98 100
100 100
100 75 60 37 100
90 100
50 58
0.9 3.8 f 0.03 4.8
100 100
100 100 88 88 85
0.08 8.9 70 68 68 15 91 94
f
0.01 13.3
0.2 6.3
*
1.7 4.2
+-
f
+1.7 4.2
f
1.7 4.9
15 10.4
6 11.8
4 13.0
5 10.6
13
12
11
10
-
21
100
6
100
0.8 4.5 f 0.6 7.5 6 100
+ -
8 10.8
16
100
100
5 10.2 f 1.1 4.4 f 0.02 7.3
17
100
100
100
0.04 6.9
f
5 13.4 f 0.7 3.5
18
20 100 100
100
100
100
0.04 3.0
f 0.2 13.2
*
f
1.2 5.2
1.1 4.5
5 12.0
+ -
8 10.8
ruts 20
111
19
Frequency of((nomaZzec (cfter si?zgIe orul trecrtmc’nt with 240 my/kg E T U o n g e s t u t z o n drrys 10-21
+-
12
6
0.02 5.2
0.4 5.1
f
5 14.4
21
+-
0.01 8.5
f
0.5 5.0
37 11.1
Control
ETHYLENETHIOUKEA I N RATS
2 79
ment on gestation days 11, 14, and 19 of Observed consistently after treatment on gestation ( P < 0.05, t test), and fetal weight day 16 were hydranencephaly, cleft palate, was reduced after treatment on days 13, and hydronephrosis or hypoplastic kidneys. 14, 15, and 18. Hydranencephaly was also present followNo malformations were observed after ing exposure on days 17-20 and declined treatment on days 6-9. Treatment on day to 6 % after treatment on day 21. 10 induced defective external genitalia The external morphology of hydranen(6 % ), hydronephrosis or hypoplastic kid- cephalic fetuses was indistinguishable from neys (20 % ), and absent or short tail (20 % ). that of controls but sectioning through the Administration on day 11 resulted in spina frontal region revealed cerebral hemibifida of the lumbar region in 30% of the spheres whose interiors were devoid of neufetuses, fused ribs, and a higher frequency ral tissue. Beginning with treatment on day of kidney and tail anomalies than induced 15 the cerebral hemispheres, cerebellum, by earlier treatment. Days 12-15 of gesta- and medulla oblongata were affected (fig. tion were the period of peak sensitivity and 1) but with later treatment hydranencephwere characterized by high frequencies of aly was restricted to the cerebral hemianomalies, prominent among which were spheres. hydrocephalus, exencephaly, brachygnaAll fetuses exposed on days 17-19 of thia, hydronephrosis, absent tail, and fore- gestation had hydronephrotic or hypoplastic limb micromelia (table 1). kidneys. The frequency of renal defects was Treatment with lower dosages on these 20 and 42%, respectively, following treatdays caused lower frequencies and reduced ment on days 20 and 21. Subcutaneous intensity of anomalies. On day 12, 200 mg/ edema of the head, neck and trunk was kg failed to produce fused ribs, sternoschi- noted in all fetuses exposed on days 18-20. sis, and absent ulna or digits, and 160 mg/ DISCUSSION kg did not induce cleft lip, cleft palate, and limb anomalies. The lowest dosage, 40 mg/ The earliest treatment causing teratogenkg, produced short or absent tail only ic effects was day 10 which coincides with the onset of organogenesis and the time of (37% ).
Fig. 1 Coronal section of the head of a hydranencephalic fetus revealing cerebral hemispheres and cerebellum deficient in neural tissue, from pregnant rats givcn 240 mg/kg ETU on day 17 of gestation.
280
JOSEPH A . K U D D I C K AND K . S . K H E R A
Fig. 2 Frontal section through the optic region ( 0 ) of a 22-day-old fetus exposed to ETU o n day 12 of gestation, showing combined hydrocephalus ( H ) and exencephaly ( E l . x 20.
posterior-neuropore closure in rats (Edwards, '68). Malformations induced a t this time, such as failure of coccygeal growth, spina bifida, ectopic genitalia, and nephrosis, were restricted to the pelvic region. Various organs and tissues were the targets of ETU activity on days 12-15. The period of sensitivity was relatively long for many anomalies (tail defect, cleft palate, and sternoschisis), whereas for others (spin a bifida, exencephaly) i t was restricted to a relatively shorter period perhaps reflecting the time and duration of development of the structures involved. Cranial defects of the types observed by us are thought to arise through interference with anterior-neuropore closure (Nakano, '73). However, those in the present study (table 1) were produced by treatment after the anterior neuropore had closed, suggesting that the mode of action of ETU is different from that of agents inducing such conditions by interfering with neuralplate closure. The uniqueness of ETU was further illustrated by the simultaneous appearance of anomalies that are generally considered
to be mutually exclusive. Generally, hydrocephalus and exencephaly do not occur together; but ETU caused them concomitantly (fig. 2). Production of these combined anomalies was not due to disturbance of ancestral cells of the organ or the organ anlage undergoing differentiation (Murakami, '63) since the time of treatment was well past the differentiation stage. Pressure from a n expanding hydrocyphalic brain through a weaken skull (ex. necrosis) probably resulted in the combined anomalies. After day 16 of gestation, which marks the end of organogenesis, defects noted were hydranencephaly, hydronephrosis, and subcutaneous edema. These hydropic anomalies could have resulted from local disturbances in vascular permeability, intravascular pressure, or normal movement of tissue fluids. Thiourea and its derivatives are known to produce edematous changes in adult mammals (Combs and Giri, '73). Anomalies produced by multiple treatment with ETU on days 6-15 of gestation (Khera, '73) can now be ascribed to interference with prenatal development at specific stages. The effects observed in this
ETHYLENETHIOUREA I N RATS
study, although not of the same intensity or frequency as caused by single dosages, it now appears resulted primarily from treatment on days 12-15 of gestation. Furthermore, comparison of multiple and single treatment indicates the usefulness of both procedures for teratological assessment of ETU. Single treatment was useful for isolating specific anomalies and relating them to the stage of intrauterine development, Some of the malformations provide good model systems for examining biochemical and histological events associated with various defects. ACKNOWLEDGMENT
We would like to thank R. Tanner, L. Hierlihy, G. Trivett, R. Oliver, and G. Terry for their assistance. LITERATURE CITED Allen, C. F. H., C. 0. Edens and J. Von Allen 1946 Ethylenethiourea. Org. Synth., 26: 34-35. Bontoyan, W. R., and J. B. Looker 1972 Survey of ethylenethiourea in commercial ethylenebisdithiocarbamate formulation. J. Ass. Anal. Chem., 55: 923-925. Combs, A. B., and S. N. Giri 1973 Trichloroacetic acid-induced binding of phenylthiourea to eryth. rocytes. J. Pharmaceut. Sci., 62: 631-633. Edwards, J. A. 1968 The external development of rabbit and rat embryo. Adv. Terat., 3: 239263.
28 1
Graham, S. L., and W. H. Hansen 1972 Effects of short-term administration of ethylenethiourea upon thyroid function of the rat. Bull. Env. Cont. Toxic., 7: 19-25. Graham, S. L., W. H. Hansen, K. J. Davis and C. H. Perry 1973 Effects of one-year administration of ethylenethiourea upon the thyroid of the rat. J. Agr. Food Chem., 21 : 324-329. Innes, J . R. M., B. M. Ulland, M. G. Valerio, L. Petrucelli, L. Fisbien, E. R. Hart, A. J. Pallotta, I. Mitchell and J. Peters 1969 Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary report. J. Nat. Cancer Inst., 42: 1101-1104. Khera, K. S. 1973 Ethylenethiourea: teratogenicity study in rats and rabbits. Teratology, 7: 243252. Murakami, U. 1963 Studies on mechanismsmanifesting anomalies. Jap. J. Human Genet., 8: 202-226. Nakano, K. K. 1973 Anencephaly: a review. Dev. Med. Child Neur., 15: 383-400. Newsome, W. H.,and G. W. Laver 1973 Effect of boiling on the formulation of ethylenethiourea in zineb-treated foods. Bull. Env. Cont. Toxic., 10: 151-154. Petrova-Vergieva, T., and L. Ivanova-Tchemishanska 1973 Assessment of the teratogenic activity of dithiocarbamate fungicides. Food Cosm. Toxic., 1 I : 239-244. Watts, R. R.,R. W. Storherr and J. H. Onley 1974 Effects of cooking on ethylenebisdithiocarbamate degradation to ethylenethiourea. Bull. Env. Cont. Toxic., 12:224-226. Ulland, B. M., J. H. Weisburger, E. K. Weisburger, J . M. Rice and R. Cypher 1972 Thyroid cancer in rats from ethylenethiourea intake. J. Nat. Cancer Inst., 49: 583-584. '
