Ethyl Carbamate) As a Cosolvent of Drugs Commonly Used Parenterally in Humans

[CANCER RESEARCH 35, 2895-2899, October 1975]

Letter to the Editor

Urethan (Ethyl Carbamate) as a Cosolvent of Drugs Commonly Used Parenterally in Humans

Urethan, a potent teratogenic and carcinogenic agent in factured by Grelan Pharmaceutical Co., Ltd., Tokyo, experimental animals, has been used in Japan for the past 25 Japan, and have been sold by Takeda Pharmaceutical years as a cosolvent of water-insoluble analgesic and Ind., Co., Ltd., Osaka, Japan, since 1950 (a), 1956 (b and sedative drugs. Urethan is used also as a solubilizer and co- c), and 1958 (d). solvent for pesticides, fumigants, and cosmetics, because it For the detection of carcinogenicity, a single injection of is a good solvent for various organic materials (6). In the Grelan Injection (0.002 ml/g body weight) was given to past, urethan was used clinically as an antineoplastic agent 3-week-old ICR/Jcl mice. Lung tumors were induced in and a hypnotic (6). In experimental animals, urethan in- significantly high frequency (Table 1). From the tumor duces a high incidence of a variety of tumors (7, 13 15), malformations (8, 12), and chromosome aberrations (4). Carcinogenic effects of urethan via placenta (5, 9 11) and 123,24~ ~) via mother's milk (9) have also been reported. Furthermore, tumors and malformations were transmitted to the next Ilg 251 / IlO'lOt generation of mice subsequent to urethan treatment (11). As shown in Chart 1, small amounts of urethan (10 ug/g body weight) induce lung tumors in mice. Sixty-three drugs, which are commonly used parenterally in Japan, and 72 cosmetics were analyzed chromatographi-

cally. A thin-layer chromatogram (Silica Gel 60 F254, 0.25 10 % +,'I, .....- -4 mm thick, 20 x 20 cm; E. Merck, Darmstadt, West o_ t265D Germany) was developed in: Solvent System A, acetone:pe- cz:"-~ 0.5 I "GRELANINJECTION" ~lO 40~ lll! | troleum ether (b.p. 30-60"; 3:7, v/v); Solvent System B, /[1,9 33, , ethanol:benzene:aqueous 50% acetic acid (7:1:1, v/v/v); and , I Solvent System C, acetone:chloroform:ethyl ether:pe- I ! troleum ether (1:2:2:5, v/v/v/v). For the detection of car- 01 ~545~ I I bamates as red spots on chromatograms, p-dimethylamino- I ! cinnamaldehyde (1 g in 50 ml of 6 s HCI and 50 ml of I ! ethanol) was used (1). As authentic chemicals, methyl I carbamate, ethyl carbamate (urethan) (Wako Pure Chemi- 0.01 I O. 008 1,2 2431 l cal Ind., Ltd., Osaka, Japan), isopropyl carbamate, n-butyl | f.J0 72i / carbamate (Tokyo Chemical Ind., Co., Ltd., Tokyo, ..~r i i i f l i Japan), pyrabital (Grelan Pharmaceutical Co., Ltd., O 10 50 I00 150 500 1,000 Tokyo, Japan), and aminopyrine (Sumitomo Chemical Ind., Co., Ltd., Osaka, Japan) were used. Four products TRFATEb OO~I OF URETHAN,~q'q bodyweightl for parenteral use showed positive results following these Chart I. Changing urethan dose response of lCR/Jcl mice in relation to tumor induction. Various amounts of urethan (10, 50, 100, 450, and procedures and were chromatographically identical to 1000 pg/g body weight) were given s.c. to 3-week-old ICR/Jcl male (Q) urethan (Fig. 1). (a) Grelan Injection (pyrabitai, 200 rag; and female (O) mice. They were maintained on Mouse Diet CA-I (10) and and aminopyrine, 100 mg, in 2 ml), (b) Nobion-A In- were sacrificed on the 5th month after treatment. Controls were untreated jection (pyrabital, 200 mg; chlorpromazine hydrochloride, and were sacrificed 6 months after birth. Pathological lesions were 12.5 mg; and diphenhydramine, 20 mg, in 2 ml), (c) Noblon- examined especiall), for tumors. Lung tumors were counted again after B Injection (pyrabital, 300 mg; chlorpromazine hydro- fixation in 20~ neutral formaldehyde solution (mean • S.E.). Numbers in chloride, 25 mg; and diphenhydramine, 20 mg, in 2 ml), parentheses, ratio of tumor-bearing mice to survivors. Application of X2 and (d) C-Noblon Injection (pyrabital, 200 mg; surpyrine, test with Yates" correction and t test with approximation of Cochran-Cox 100 mg; chlorpromazine hydrochloride, 15 mg; prometha- yielded p value of 0.001 between 50-, 100-, 450-, and 1000-pg-treated zine hydrochloride, I0 mg; and 8-chlortheophylline, 25 groups and controls, and p value of 0.01 between 10-pg-treated group and controls. A, Lung tumor frequency following treatment with mg, in 2 ml) contain urethan as a cosolvent, because pyr- _ Grelan Injection (Table 1). From the tumor frequency, dose of urethan abital is insoluble in water. These products have been manu- given to mice was estimated to be 150 #g/g body weight. Consequently, concentration of urethan solution in Grelan Injection is 7.5%, because Received June 19, 1975; accepted July 25, 1975. 0.002 ml of this product was given per g body weight.

OCTOBER 1975 2895

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1975 American Association for Cancer Research. Letter to the Editor frequency, the concentration of urethan solution in Grelan the Japanese Ministry of Health and Welfare), Ed. 7, that Injection was estimated to be about 7.5% (Chart 1). 15% urethan solution can be used to dissolve pyrabital (3). Significant embryotoxic and teratogenic effects of Grelan Consequently, the dose commonly used as a single injection Injection were also detected, but since these were not in humans [1 to 2 ampuls (0.3 to 0.6 g as pyrabital) to the elicited by urethan only (10), synergism or other combined adult] (3) corresponds to 5 to 10 /ag/g body weight as action with pyrabital and aminopyrine must be considered urethan, which is equivalent to the carcinogenic dose of (Table 2; Fig. 2). urethan in mice [10 •g/g body weight (Fig. 1)]. The Quantitative determination of urethan contained in these maximum dose of these injections (3 g as pyrabital) in 1 day products was conducted following the microdiffusion (3) corresponds to 50 ug of urethan per g body weight. From method (2) after isolation of urethan by thin-layer chroma- the clinical experience of the author (surgeon in Osaka tography. Doses of urethan used to dissolve pyrabital University Hospital from 1967 to 1972), these injections (average of 6 experiments) were 0.254 g/ampul (2 ml) have been used commonly as a nonalkaloid analgesic (12.7%) in Noblon-A Injection, 0.246 g/ampul (12.3%) in several times at 3- to 5-hr intervals after operation. Conse- Noblon-B Injection, 0.210 g/ampul (10.5%) in C-Noblon quently, urcthan, at up to 5 times the carcinogenic dose in Injection, and 0.154 g/ampul (7.7%) in Grelan Injection. mice, has been used in almost all patients after operation. In It is described in Japanese Pharmacopoeia (Regulations of Japan, 100 million ampuls of these 4 injections have been

Table 1 Tumor induction in mice following treatment with Grelan Injection ICR/Jcl mice (3 weeks old) received a single s.c. injection of 0.002 ml of Grelan Injection per g body weight. They were maintained on Mouse Diet CA-I (10) and sacrificed on the 5th month after treatment. Pathological lesions were examined especially for tumors. Histologically, lung tumors were papillary adenomas. Controls were untreated and were sacrificed 6 months after birth.

No. of Lung tumors mice at Experimental groups Sex start Incidence % • test = (p) Tumors/lung t test ~ (p)

Grelan Injection M 31 11/27 40.7 <0.001 0.667 • 0.177" <0.001 F 26 15/24 62.5 <<0.001 1.250 • 0.290 <<0.001 57 <<0.001 0.941 ~: 0.169 <<0.001 26/51 51.0 Controls M 72 0.0 F 244 0/72 0.0 0.008 • 0.006 2/243 0.8 316 2/315 0.6 0.006 + 0.005

Applied with Yates' correction. b Applied with approximation of Cochran-Cox, because variance ratio was over F value at 1%. c Mean :e S.E.

Table 2 Embrvotoxic and teratogenic effects Of Grelan Injection on the developing mouse embryo Pregnant mice received 3 s.c. injections of Grelan Injection (0.002 ml/g body weight) on Days 9, 10, and 11 of gestation and were sacrificed on Day 19. Equivalent dose of pyrabital (200 ~tg/g), aminopyrine (I00/~g/g), or urethan (150 ug/g) to Grelan Injection was also given to pregnant mice in the same time schedule. Detailed methods for the detection of malformations were given elsewhere (10). Controls were untreated during pregnancy.

Early deaths Late deaths Living fetuses Malformations

% of % of % of % of Experimental lm- im- im- ira- living groups plants No. plants X2 test '~ No. plants X2 test No. plants X2 test No. fetuses X2test Details

Grelan Injection 104 6 5.8 NS ~ 47 45.2 <<0.001 51 49.0 <<0.001 25 49.0 <<0.001 22 O, c 9 C 9K, 2P Pyrabital 41 0 0.0 NS 3 7.3 NS 38 92.7 NS 4 10.5 <0.01 4 O, 2 C, 2K Aminopyrin 50 3 6.0 NS 4 8.0 NS 43 86.0 NS 6 14.0 <0.01 3 O, 2 C, IK Urethan 41 2 4.9 NS 3 7.3 NS 36 87.8 NS 2 5.5 <0.05 2 K Controls 351 18 5.1 13 3.7 320 91.2 1 0.3 1 E

,2 Applied with Yates' correction. b NS, not significant; O, ruptured omphaiocele (eventration of the abdominal viscera): C, cleft palate; K, kinky tail; P, polydactyly; E, exencephalus. c Thirteen of 22 fetuses with omphaloceles had cleft palates or kinky tails.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1975 American Association for Cancer Research. Letter to the Editor used for the past 25 years. 5. Larsen, C. D. Pulmonary-tumor Induction by Transplacental Expo- This letter was written in order to warn of the possible sure to Urethan, J. Natl, Cancer Inst., 8:63 69, 1947. hazards of using products that contain urethan. Further- 6. Merck Index, Ed. 8, p. 1095. Rahway, N. J.: Merck & Co., Inc., 1968. more, it is hoped that all drugs for parenteral use will be 7. Nettleship, A., and Henshaw, P. S. Induction of Pulmonary Tumors in examined, because urethan may be (or may have been) used Mice with Ethyl Carbamate (Urethane). J. Natl. Cancer Inst., 4. 309-319, 1943. as a cosolvent of water-insoluble drugs in other countries. 8. Nishimura, H., and Kugimuki, M. Congenital Malformations Induced Long-term follow-up of patients who have been exposed to by Ethyl-urethan in Mouse Embryos. Okajima Folia Anat. Japan., urethan-containing medicinal preparations is mandatory to 31: 1-10, 1958. ascertain the possible carcinogenic effects of urethan in 9. Nomura, T. Carcinogenesis by Urethan via Mother's Milk and Its humans. Such studies are in progress. Enhancement of Transplacental Carcinogenesis in Mice. Cancer Res., 33." 1677 1683, 1973. 10. Nomura, T. An Analysis of the Changing Urethan Response of the ACKNOWLEDGMENTS Developing Mouse Embryo in Relation to Mortality, Malformation, and Neoplasm. Cancer Res., 34. 2217--2231, 1974. 11. Nomura, T. Transmission of Tumors and Malformations to the Next ! wish to thank Y. lsa and S. Kimura for their assistance and Dr. J. Generation of Mice Subsequent to Urethan Treatment. Cancer Res., Kamahora, President of Osaka University, for his encouragement to 35." 264 266, 1975. publish this letter. 12. Sinclair, J. G. A Specific Transplacental Effect of Urethane in Mice. Texas Rept. Biol. Med., 8. 623 632, 1950. 13. Tannenbaum, A., Vesselinovitch, S. D., Maltoni, C., and Mitchell, D. REFERENCES S. Multipotential Carcinogenicity of Urethan in the Sprague-Dawley Rat. Cancer Res.,22.' 1362 1371, 1962. 1. Boyland, E., and Nery, R. The Metabolism of Urethane and Related 14. Toth, B. Multipotential Carcinogenesis with Urethan in the Syrian Compounds. Biochem. J., 94. 198-208, 1965. Golden Hamster. Cancer Res., 21. 1537-1541, 1961. 2. Boyland, E., and Rhoden E. The Distribution of Urethane in Animal 15. Vesselinovitch, S. D., and Mihailovich, N. The Development of Tissues, as Determined by a Microdiffusion Method, and Effect of Neurogenic Neoplasms, Embryonal Kidney Tumors, Harderian Urethane Treatment on Enzymes. Biochem. J., 44:528 531, 1949. Gland Adenomas, Anitschkow Cell Sarcomas of the Heart, and Other 3. Japanese Society of Official Publications. Commentary of the Japa- Neoplasms in Urethan-treated Newborn Rats. Cancer Res., 28." nese Pharmacopoeia, Ed. 7, pp. 1148 1149. Tokyo: Hirokawa Pub- 888 897, 1968. lishing Co., 1966. Taisei Nomura 4. Kurita, Y., Shisa, H., Matsuyama, M., Nishizuka, Y., Turuta- Institute.for Cancer Research, Osaka University Tanaka, R., and Yosida, T. H. Carcinogen-Induced Chromosome Medical School, Dbiima-hamadbri, Fukushima-Ku, Aberrations in Hematopoietic Cells of Mice. Gann, 60." 91 95, 1969. Osaka 553, Japan

Fig. I. Chromatograms of 4 products containing urethan as a cosolvent. Chromatograms were developed in Solvent System C (see text). Carbamates gave red spots with p-aminocinnamaldehyde (1). A, Noblon-A Injection; B, Noblon-B Injection: C, C-Noblon Injection; G~, Grelan Injection, manufactured in August 1974 (128YHE); G2, Grelan Injection, manufactured in October 1974 (I 33YJY): Pv, pyrabital: Ap, aminopyrine; M, methyl carbamate; U, urethan (ethyl carbamate); P, isopropyl carbamate; BC, n-butyl carbamatc: Gt v /~', mixture of G1 and U. Fig. 2. Ruptured omphaloceles (eventration of the abdominal viscera). Six late deaths and 4 living fetuses were observed in I litter. Three of 4 fetuses developed ruptured omphaloceles. Liver and intestine were found outside the abdominal cavity. One dead fetus (arrowhead) also developed this defect. One fetus without omphalocele developed polydactyly.

OCTOBER 1975 2897

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1975 American Association for Cancer Research. Urethan (Ethyl Carbamate) as a Cosolvent of Drugs Commonly Used Parenterally in Humans

Taisei Nomura

Cancer Res 1975;35:2895-2899.

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