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Effects of Coumarin Following Perinatal and Chronic Exposure in Sprague–Dawley Rats and CD-1 Mice
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
30, 145–151 (1996)
0051
Effects of Coumarin Following Perinatal and Chronic Exposure in Sprague–Dawley Rats and CD-1 Mice BETSY D. CARLTON,*,1 JEAN-CLAUDE AUBRUN,†
AND
GLENN S. SIMON*
*Rhoˆne-Poulenc, 2 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709; and †Rhoˆne-Poulenc, 25 Quai Paul Doumer, 92408 Courbevoie Cedex, Paris, France Received June 29, 1995; accepted October 16, 1995
Effects of Coumarin Following Perinatal and Chronic Exposure in Sprague–Dawley Rats and CD-1 Mice. CARLTON, B. D., AUBRUN, J.-C., AND SIMON, G. S. (1996). Fundam. Appl. Toxicol. 30, 145–151. Coumarin, a naturally occurring substance most frequently used as a fragrance enhancer and stabilizer, was administered in the diet of Sprague–Dawley rats at dose levels of 0, 333, 1000, 2000, 3000, and 5000 ppm or in the diet of CD-1 mice at dose levels of 0, 300, 1000, or 3000 ppm. Rats receiving 333, 1000, and 2000 ppm coumarin were exposed to these dose levels in utero and during the lactational period, then chronically following weaning. Rats in the 3000- and 5000-ppm dose groups and all mice received only postweaning chronic exposure. All male rats were terminated after 104 weeks of postweaning exposure; female rats were terminated after 110 weeks. Male mice were terminated at Week 101 and female mice at Week 109. Among rats, survival was decreased at 333 ppm, but significantly increased among rats in the 3000and 5000-ppm dose groups. Dramatic dose-related decreases in body weight gain were recorded for rats receiving 2000, 3000, and 5000 ppm, clearly indicating that the MTD (maximum tolerated dose, as indicated by a body weight decrement of greater than 10– 15%) was exceeded. Food consumption also was decreased at the three highest dose levels, although body weight decrement was disproportionately large compared to changes in food consumption. Treatment-related decreases in hemoglobin were recorded from Week 6 onward. Minimal treatment-related changes in hematology and clinical chemistry were recorded. Increased liver weights were observed for male and female rats receiving 3000 or 5000 ppm and for females only at 1000 and 2000 ppm. Increased incidences of cholangiofibroma, cholangiocarcinoma, and parenchymal liver cell tumors were observed among male and female rats receiving 5000 ppm. One male rat receiving 3000 ppm developed a cholangiocarcinoma; no tumor increase was observed in males or females at 2000 ppm or below. Coumarin, at a dose clearly exceeding the MTD can, therefore, induce liver tumors in rats, although survival, relative to controls, was increased at the same dose levels. Among mice, a decrease in body weight gain was reported for males in the 1000- and 3000-ppm dose groups during the first 52 weeks of the study. No dose-related abnormalities in clinical signs, clinical pathology, hematology, or gross or microscopic pathology were noted. q 1996 Society of Toxicology
1
To whom correspondence and reprint requests should be addressed.
Coumarins, as a class, comprised numerous naturally occurring compounds with important and diverse physiological activities. The parent compound, coumarin, is used as an additive to perfumes, soaps, detergents, toothpaste, and some tobacco products and alcoholic beverages outside the United States. It is used clinically in the treatment of high-protein edema (Jamal et al., 1989), chronic brucellosis, and immune suppression (Moran et al., 1987). In isolation (Thornes et al., 1989) or in combination with cimetidine (Marshall et al., 1987a, b, c; Dexeus et al., 1990), coumarin is currently undergoing several clinical trials for the treatment of several types of malignancies in humans, including the treatment of lung and kidney carcinoma. Due to its use as a drug, there is information in the literature concerning its toxicity, metabolism, and pharmacokinetics in man. While several studies have been conducted to examine the chronic toxicity and carcinogenicity of coumarin, none were conducted in accordance with current scientific guidelines. A GLP-compliant study was therefore undertaken. (Since this study was completed, the U.S. National Toxicology Program has conducted a bioassay of coumarin in F344 rats and B6C3F1 mice.) MATERIALS AND METHODS Chemicals Coumarin (CAS 91-64-5) (ú98% pure) was supplied by Rhoˆne-Poulenc France as a white crystalline powder. Coumarin was admixed with Spratt’s Laboratory Animal Diet No. 2 to form a premix. The premix was then diluted with diet to the appropriate dose levels. Diets were prepared weekly. Animals and Treatments Rats. Rats were housed five per cage in suspended wire-mesh cages. Animal rooms were maintained at 227C and 50% relative humidity, with a 12-hr/12-hr light/dark cycle. Each cage was identified by cage card and each rat was individually identified by earmark and tattoo. Ad libitum access to food and tap water was provided. All animals for the chronic study were derived from Sprague–Dawley rats received from Charles River (Charles River, Manston, Kent, UK) and bred at Huntingdon Research Centre, Huntingdon, Cambridgeshire, England. A preliminary study (Hunter et al., 1980, unpublished data) conducted in pregnant Sprague–Dawley rats demonstrated that 3000 and 5000 ppm resulted in large weight gain decrements in the exposed dams and consequent decreases in the number of viable young. Therefore, in the main study described here, rats in the control, 3000-, and 5000-ppm dose groups
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0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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received control diet in utero and during the lactational phase until weaning at 21–28 days of age, when the main study phase dosing was initiated. Animals in the 333-, 1000-, and 2000-ppm dose groups received coumarindosed feed at those dose levels throughout the perinatal and chronic periods. A total of 390 male and 390 female rats were used in this main chronic phase of the study. Each rat was allocated to one of six treatment groups, each comprising a main group (50 rats/sex) and a satellite group (15 rats/ sex). Animals in the main group were given ophthalmoscopy evaluations and were used for complete chronic toxicity/carcinogenicity evaluation. The satellite groups were used for hematology, clinical chemistry, and urinalysis determinations. They were killed at Week 104 and received a gross necropsy and organ weight determinations. Only gross lesions were examined microscopically from the satellite animals, however. All male rats in the main groups were terminated after 104 weeks of postweaning exposure; female rats were terminated after 110 weeks. A complete gross necropsy with organ weights was conducted for all main group animals. The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid, and uterus. Tissues for a complete histopathologic evaluation were collected and preserved in neutral-buffered formalin. The following observations and measurements were recorded. Mortality and morbidity (recorded twice daily), clinical observations, body weight, food and water consumption, ophthalmoscopic evaluations, hematology, clinical chemistry, plasma cholinesterase, urinalysis, selected organ weights, and gross and microscopic pathology. Mice. Animals were housed four per cage in solid bottom polypropylene cages with autoclaved, sifted sawdust as bedding. Animal rooms were maintained at 227C and 50% relative humidity, with a 12-hr/12-hr light/ dark cycle. Each cage was identified by colored cage label and each mouse was individually identified by earmark. Ad libitum access to food and tap water was provided. A total of 416 male and female CD-1 mice (Charles River, Manston, Kent, UK) 28 { 2 days of age were randomly assigned to four coumarintreatment groups comprising 52 mice/sex/dose. Animals received 0, 300, 1000, or 3000 ppm coumarin in the diet. Male mice were terminated at Week 101 and female mice at Week 109. A complete gross necropsy, with organ weight determinations, was conducted for all animals. The following organs were weighed: brain, heart, kidneys, liver, testes. Tissues for a complete histopathologic evaluation were collected and preserved in neutral-buffered formalin. The following observations and measurements were recorded: mortality and morbidity (recorded twice daily), clinical observations, body weight, food consumption, hematology, selected organ weights, and gross and microscopic pathology.
RESULTS
Rats The only clinical observation potentially dose-related was a temporary loss of fur among males and females receiving 5000 ppm and among females receiving 3000 ppm. Survival was decreased in animals receiving 333 ppm, but significantly increased among male and female rats receiving 3000 and 5000 ppm coumarin when compared with controls. These data are presented in Table 1. Body weight gains and food consumption were significantly reduced among male and female rats receiving 2000, 3000, and 5000 ppm. These data are presented in Tables 2 and 3. Growth of all groups of male rats was essentially similar between Weeks 52 and 78. From Weeks 78 through study termination, control rats lost weight, as commonly
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occurs for male Sprague–Dawley rats at this age. Body weights of male rats in the three highest dose groups remained relatively constant, reflecting the generally better condition of males receiving higher levels of coumarin. Female rats receiving 2000, 3000, or 5000 ppm continued to gain less weight than controls during Weeks 52–78. From Week 78 onward, bodyweight gain by all groups of female rats was essentially similar. The efficiency of food utilization by rats receiving 5000 ppm was impaired during Weeks 1–6. From Weeks 6–26, the efficiency of food utilization by these rats was superior to that of controls, indicating a degree of adaptation. From Weeks 13–26, efficiency of food utilization by males and females receiving 1000 or 2000 ppm was marginally impaired by treatment. Calculated group mean achieved intakes are described in Table 4. Significant clinical chemistry findings included a treatment-related anemia, principally characterized by low hemoglobin levels among males and females from Week 6 onward. This effect was noted primarily among rats in the three highest dose groups. Minimally elevated white cell and lymphocyte counts were recorded among 5000 ppm females from Week 25 to the end of the study. Treatment-related decreases in glucose and protein were found at Weeks 4 and 13 among 5000-ppm dose group animals, but the changes became less pronounced as the study progressed. Treatment-related increases in blood potassium levels, alkaline phosphatase, and glutamic–pyruvic transaminase were recorded throughout the study. Cholesterol levels of all groups of treated rats except for males in the highest dose group were increased throughout the study. Plasma cholinesterase, but not red blood cell cholinesterase, levels were decreased among females treated with coumarin, but not males. At necropsy, an increased incidence of liver masses was noted for male rats in the 5000-ppm dose group. Increased liver weights were recorded among males and females receiving 3000 and 5000 ppm and among females at 1000 and 2000 ppm. There was a twofold increase in relative liver weight observed at the highest doses. Decreased absolute brain, pituitary, heart, kidney, and adrenal weights were recorded for males in the 5000-ppm dose group. Heart weights were also decreased for males in the 2000- and 3000-ppm dose groups. Decreased absolute adrenal weights also were recorded for males receiving 1000, 2000, or 3000 ppm and among females receiving 3000 or 5000 ppm. Increased incidences of cholangiofibrosis, cholangiocarcinoma, and parenchymal liver cell tumors were recorded among male and female rats receiving 5000 ppm coumarin, although tumor incidence had no impact on survival. A single cholangiocarcinoma reported for a male rat receiving 3000 ppm was also considered to be potentially treatmentrelated. Rats receiving coumarin at 2000 ppm or less showed no evidence of increased hepatic tumor prevalence. No dose-
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TABLE 1 Survival of Male and Female Sprague–Dawley Rats Receiving Coumarin in the Diet for 104–110 Weeks Group
Males Weeks 0–104 Females Weeks 0–110
0 ppm
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
21/50 42%
11/50* 22%
17/50 34%
22/50 44%
27/50* 54%
33/50** 66%
13/50 26%
11/50 22%
17/50 34%
19/50 38%
25/50* 50%
32/50** 64%
* p £ 0.05. ** p £ 0.001.
related increases in renal tumors or tumors at other tissue sites were observed. Histopathology results are presented in Table 5.
among males or females receiving coumarin in the diet. Females receiving 3000 ppm coumarin showed marginally, but statistically significant, increases in liver weight, although there were no associated changes at the cellular level. Males were not similarly affected. No other differences in organ weights were observed across dose groups. No increases in tumors were observed in liver, lung, or other tissues (Table 7). Gross and microscopic pathology was unchanged by dose exposure.
Mice Body weight gain by males receiving 3000 ppm was significantly reduced compared to controls, demonstrating an 18% reduction over the first 52 weeks of the study. Body weight gain was reduced by 10% relative to controls among males in the 1000-ppm dose group. Body weights were similar across all female dose groups. Food utilization by males, but not females, in the 3000-ppm dose group was marginally inferior to that of controls. Achieved average coumarin intakes were calculated and are shown in Table 6. No treatment-related changes in survival, clinical observations, hematology, or clinical chemistry were observed
DISCUSSION
Concerns about coumarin’s carcinogenic potential arose from a series of studies which started in 1967 with reports by Ba¨r and Griepentrog (1967). These authors reported that lifetime exposure of rats to doses of 5000 ppm or higher
TABLE 2 Body Weight Gain for Male and Female Sprague–Dawley Rats during the Chronic Bioassay of Coumarin (Weight Gain in Grams { SD) Week
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
Males 0–13 13–26 26–52 52–78 78–term
455 129 156 52 064
{ { { { {
51 33 47 82 137
464 132 151 75 071
{ { { { {
62 34 85 60* 132
435 113 110 50 043
{ { { { {
54* 30** 59*** 68 131
372 99 118 56 05
{ { { { {
46*** 33*** 39*** 53 71*
329 114 132 62 6
{ { { { {
41*** 27** 38* 44 69*
189 115 118 42 12
{ { { { {
33*** 23** 30*** 25 48**
216 40 71 56 39
{ { { { {
25** 16*** 35*** 38** 57
197 47 68 52 40
{ { { { {
23*** 18* 37*** 44** 56
128 53 41 20 26
{ { { { {
21*** 16 18*** 16*** 36
Females 0–13 13–26 26–52 52–78 78–term
230 56 113 80 18
{ { { { {
34 29 58 45 106
234 56 108 68 33
{ { { { {
31 24 47 63 102
228 46 93 71 42
{ { { { {
24 18** 41** 43 78
* p £ 0.05. ** p £ 0.01. *** p £ 0.001.
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TABLE 3 Food Consumption (g/Rat) for Male and Female Sprague–Dawley Rats in the Chronic Bioassay of Coumarin Week
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
1883*** 82 84% 2103*** 73 87% 8225*** 376 89 4492** 242 89%
1757*** 68 79% 2069*** 88 86% 8209*** 357 89% 4369*** 183 86%
1209*** 103 54% 1602*** 113 66% 6772*** 415 73% 3644*** 243 72%
1487*** 46 91% 1544*** 51 93% 6379*** 198 92% 4409* 271 91%
1394*** 63 85% 1507*** 79 90% 6229*** 392 90% 4354** 414 90%
1027*** 84 63% 1249*** 69 75% 5104*** 263 73% 3550*** 344 73%
Males 0–13 SD % Control 13–26 SD % Control 27–78 SD % Control 79–term SD % Control
2236 88 — 2419 132 — 9246 626 — 5056 283 —
2272 65 102% 2459 89 102% 9452 369 102% 5096 394 101%
2176 68 97% 2362 48 98% 8922 265 96% 5094 461 101% Females
0–13 SD % Control 13–26 SD % Control 27–78 SD % Control 79-term SD % Control
1637 76 — 1669 75 — 6959 425 — 4849 500 —
1666 77 102% 1693 112 101 6973 571 100% 4674 217 96%
1626 62 99% 1629 78 98% 6734 287 97% 4894 326 101%
* p £ 0.05. ** p £ 0.01. *** p £ 0.001.
induced bile duct carcinoma. Cohen (1979) reviewed these slides and cast doubts on the interpretation of the histopathology and the reality of these carcinomas. Reevaluation of the original histopathology slides indicated that cholangiofibrosis and not cholangiocarcinoma was present (Evans et al., 1989). Evans reported that cholangiofibrosis is not a prerequisite for hepatocellular carcinoma and is not part of the carcinogenic process. Hagan et al. (1967) reported that groups of five to seven Osborne–Mendel rats given up to
TABLE 4 Achieved Intake (mg/kg/Day) Dose group 333 1000 2000 3000 5000
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5000 ppm coumarin in the diet were unaffected at 1000 ppm. Hepatotoxicity was noted at 2500 ppm. Animals fed 5000 ppm had decreased hemoglobin levels; enlarged livers with fatty metamorphosis, masses, and pale foci; and cholangiofibrosis. No cholangiocarcinomas were reported. The National Toxicology Program (NTP) (1993) conducted 2-year bioassays of coumarin in F344 rats and B6C3F1 mice. Dose levels for these corn oil gavage studies were 0, 25, 50, and 100 mg/kg/day for rats and 0, 50, 100, and 200 mg/kg/day for mice, doses which are comparable to, or slightly lower than, the doses used in our studies. In the NTP study, survival of the male rats was severely compromised. Some hepatocellular necrosis and bile duct hyperplasia was found, but no increase in hepatocellular neoplasia was reported at any dose. The highest dose level in the NTP rat study was approximately equivalent to the 2000-ppm dose group reported here (achieved intake 87 mg/ kg/day in males; 107 mg/kg/day in females). Hepatocellular findings were similar at this dose level, where no increase in tumor incidence was observed. In contrast to findings of
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TABLE 5 Liver Weights and Histopathologic Findings for Male and Female Sprague–Dawley Rats during the Chronic Bioassay of Coumarin (65 male and 65 Female per Group) Animals (65/sex/group) Males
Females
Finding
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
Liver weight (grams { SD) Cholangiocarcinoma (nonmetastasizing) Cholangiocarcinoma (metastasizing) Parenchymal tumors (benign / malignant)
28.3 { 5.91 0
29.4 { 7.03 0
29.3 { 5.37 0
29.7 { 3.87 0
34.3 { 10.55 1
44.6 { 18.85 31
0
0
0
0
0
6
2
2
1
1
6
29
Liver weight (g { SD) Cholangiocarcinoma (nonmetastasizing) Cholangiocarcinoma (metastasizing) Parenchymal tumors (benign / malignant)
18.8 { 4.8 0
21.7 { 4.1 0
23.4 { 4.3 0
20.6 { 4.5 0
2.2 { 4.6 0
22.9 { 5.5 22
0
0
0
0
0
1
0
0
0
0
1
12
marginal increases in renal adenoma in the NTP study, no increase was observed in the Rhoˆne-Poulenc (RP) study. No renal tumors were reported in females and only 1 renal adenoma was observed in a 2000-ppm dose group males. The NTP bioassay in B6C3F1 mice reported an increase in alveolar/bronchiolar adenoma and carcinoma among male and female mice (14/50, 9/50, 15/50, 25/51 males; 2/51, 5/ 49, 7/49, 27/50 females). Although an increase in pulmonary tumors was noted in the current study, all tumor incidences were within laboratory historic control ranges for the age and strain of mouse. There were no significant dose-related increases in tumors of any type in the CD-1 mice receiving coumarin in the diet at doses slightly higher than those given the mice treated by gavage in the NTP study (achieved high doses 280 mg/kg/day males; 271 mg/kg/day females). Chronic toxicity/carcinogenicity studies conducted in numerous species indicate that the liver is the main target organ for coumarin toxicity, but the severity of the effect appears to be dependent on the prevalent metabolic pathway. The 7hydroxylation pathway appears to be a more efficient detoxification/elimination pathway. Chronic toxicity/carcinogenicity studies conducted in species with significant 7-hydroxylating capacity such as baboons (Gangolli et al., 1974; Evans
TABLE 6 Achieved Intake (mg/kg/Day) Dose group 300 ppm 1000 ppm 3000 ppm
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et al., 1979), hamsters (Ueno and Hirono, 1980), and DBA/ 2J mice (Wood and Conney, 1974) have demonstrated significantly less hepatotoxicity and, in the bioassays, no increases in tumors at any site. DBA/2J mice are one of the few nonprimate species so far shown to possess a significant 7-hydroxylating capability (Wood and Conney, 1974). They appear to be more resistant to the hepatotoxic action of coumarin than is the CH3/HeJ mouse strain which possesses only a limited 7-hydroxylating potential (Endell and Seidel, 1978). Similarly, a bioassay study by Ueno and Hirono (1980) suggests that hamsters, which possess some 7-hydroxycoumarin (7-OHC) capacity, are refractory to coumarininduced hepatotoxicity, although limited conclusions can be drawn due to the small number of animals. In addition, no carcinogenic or adverse toxicologic effects were found in this study when groups of 10–13 Syrian Golden hamsters were fed diet containing 0, 1000, and 5000 ppm coumarin for 2 years. When administered as a single high dose to the rat, coumarin has been shown to produce hepatic centrilobular necrosis. Lake (Lake, 1984; Lake et al., 1989) demonstrated that coumarin-induced hepatotoxicity in rats is likely to be mediated via one or more reactive metabolites generated by cytochrome P450-dependent enzymes. Hepatotoxicity of coumarin was decreased by pretreatment with cobaltous chloride, ellypticine, and metyrapone and potentiated by pretreatment with diethyl maleate (known to reduce hepatic-reduced glutathione concentration). In in vitro studies, reactive [3-14C]coumarin metabolite(s) generated by cytochrome P450-dependent enzyme was found to bind covalently to microsomal proteins. The authors suggest that the toxic compound may be a coumarin 3-4 epoxide intermediate. Lake et al. (1989) compared the metabolism of [3-14C]-
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TABLE 7 Liver and Lung Histopathologic Findings for Male and Female CD-1 Mice during the Chronic Bioassay of Coumarin Tissue
Findinga
Control
300 ppm
1000 ppm
3000 ppm
Liver—male Liver—female Lung—male
Benign / malignant parenchymal tumors Benign / malignant parenchymal tumors Pulmonary adenoma Pulmonary adenocarcinoma Pulmonary adenoma Pulmonary adenocarcinoma
20 0 0 11 2 10
22 8 1 12 1 11
19 4 2 10 2 11
12 3 0 20 5 14
Lung—female
a
All tumor incidences were within laboratory historic control values for this age and strain of mice.
coumarin (concentration 0.19 mM) by hepatic microsomes of different species including human. In the rat, and in most strains of mice, 3-hydroxylation predominates while 4-, 5-, 6-, and 8-hydroxylation are possible but minor routes of metabolism. The 3-hydroxycoumarin is then extensively degraded to ring-opened metabolites. Recent studies have shown that in rat liver microsomes, coumarin is a substrate for isoenzymes in both P450 IA and P450 IIB subfamilies (Peters et al., 1991), whereas a member of the P450 IIA subfamily is responsible for the majority of coumarin 7hydroxylase activity in human liver microsomes (Miles et al., 1990) and the main metabolism pathway goes through 7-OHC. This difference of metabolism leads also to a difference in pharmacokinetics. In the rat, within 24 hr of an oral or intraperitoneal (ip) dose of 50 mg/kg coumarin, 50% of the dose was excreted in the bile of rats as unidentified ringopened compounds. When 50 mg/kg 7-OHC was administered ip, only 0.2% was excreted in the bile unchanged or as glucuronide conjugate (Williams et al., 1965). It is also important to note that the main feature in chronic liver toxicity is bile duct alteration, which may correspond to the biliary excretion of the coumarin metabolites in the rat. No such toxicity is observed with species metabolizing through 7OHC, which is not significantly excreted through the bile. In humans, coumarin is rapidly eliminated after oral administration, with 83% of the absorbed dose (200 mg/kg) found in the urine within 24 hr (79% as 7-OHC), while only 35% is found in the urine of rats given an equivalent dose (õ0.1% as 7-OHC) (Shilling et al., 1969; Feuer et al., 1966). This is consistent with a rapid absorption by the gut in man and with an absence of enterohepatic circulation of the metabolites. Given body weight decrements up to 60% among highdose male rats, clearly indicating that the maximum tolerated dose was exceeded, the carcinogenic response seen in the current Sprague–Dawley rat study is likely due to the hepatotoxicity incurred following high-dose exposure. Tumors were not metastatic and survival was significantly increased among rats in the two highest dose groups. While hepatic responses were comparable at similar doses in the NTP and
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RP rat studies, findings of minimal increases in renal adenomas only in the NTP study may indicate a strain susceptibility to renal abnormalities among F344 rats. Similarly, the increase in lung tumors observed among B6C3F1 mice in the NTP study was not confirmed in the RP study. This may reflect a dose-route-dependent phenomenon (NTP corn oil gavage vs RP dietary exposure) or a strain-related difference in metabolism or susceptibility. The significance of any of these positive carcinogenic results to man is highly questionable in view of man’s much lower exposure potential and the large metabolic differences between species. REFERENCES Ba¨r, F. U., and Griepentrog, F. (1967). Die Situation in der gesundheitlichen Beurteilung der aromatisierungsmittel fu¨r lebensmittel. Med. Erna¨rhr. 8, 244. Cohen, A. J. (1979). Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food Cosmet. Toxicol. 17, 277–289. Dexeus, F. H., Logothetis, C. J., Sella, A., Fitz, K., Amato, R., Reuben, J. M., and Dozier, N. (1990). Phase II study of coumarin and cimetidine in patients with metastatic renal cell carcinoma. J. Clin. Oncol. 8, 325– 329. Endell, W., and Seidel, G. (1978). Coumarin toxicity in different strains of mice. Agents Actions 8, 299–302. Evans, J. G., Gaunt, I. F, and Lake, B. G. (1979). Two-year toxicity study on coumarin in the baboon. Food Cosmet. Toxicol. 17, 187–193. Evans, J. G., Appleby, E. C., Lake, B. G., and Conning, D. M. (1989). Studies on the induction of cholangiofibrosis by coumarin the rat. Toxicology 55, 207–224. Feuer, G., Golberg, L., and Gibson, K. I. (1966). Liver response tests. VII. Coumarin metabolism in relation to the inhibition of rat-liver glucose 6phosphatase. Food Cosmet. Toxicol. 4, 157–167. Gangolli, S. D., Shilling, W. H., Grasso, P., and Gaunt, L. F. (1974). Studies on the metabolism and hepatotoxicity of coumarin in the baboon. Biochem. Soc. Trans. 2, 310–312. Hagan, E. C., Hansen, W. H., Fitzhugh, O. G., Jenner, P. M., Jones, W. I., Taylor, J. M., Long, E. L., Nelson, A. M., and Brouwer, J. B. (1967). Food flavorings and compounds of related structure. II. Subacute and chronic toxicity. Food Cosmet. Toxicol. 5(2), 141–157. Hunter, B., Warren, S., Heywood, R. C., and Gibson, W. (1980). Huntingdon Research Centre, unpublished Rhoˆne-Poulenc Rep. RNP 173/81137.
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CHRONIC EFFECTS OF COUMARIN IN RODENTS Jamal, S., Casley-Smith, J. R., and Casley-Smith, J. R. (1989). The effects of 5,6-benzo-[a]-pyrone (coumarin) and DEC on filaritic lymphoedema and elephantiasis in India. Ann. Trop. Med. Parasitol. 83(3), 287–290. Lake, B. G. (1984). Investigations into the mechanism of coumarin-induced hepatoxicity in the rat. Arch. Toxicol. (Suppl. 7), 16–29. Lake, B. G., Gray, T. J., Evans, J. G., Lewis, D. F. V., Beamand, J. A., and Hue, K. L. (1989). Studies on the mechanism of coumarin-indiced toxicity in rat hepatocytes: Comparison with dihydrocoumarin and other coumarin metabolites. Toxicol. Appl. Pharmacol. 97, 311–323. Lake, B. G., Gaudin, H., Price, R. J., and Walters, D. G. (1992). Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans. Food Chem. Toxicol. 30(2), 105–115. Marshall, M. E., Mendelsohn, L., Butler, K., Riley, L., Cantrell, J., Wiseman, C., Taylor, R., and MacDonald, J. S. (1987a). Treatment of metastatic renal cell carcinoma with coumarin (1,2-benzopyrone) and cimetidine: A pilot study. J. Clin. Oncol. 5(6), 862–866. Marshall, M. E., Mendelsohn, L., Butler, K., Cantrell, J., Harvey, J., and MacDonald, J. S. (1987b). Treatment of non-small cell lung cancer with coumarin and cimetidine 1,2. Cancer Treat. Rep. 71(1), 91–92. Marshall, L., Butler, K., Cantrell, J., Wiseman, C., and Mendelsohn, L. (1987c). Treatment of advanced malignant melanoma with coumarin and cimetidine: A pilot study. Cancer Chemother. Pharmacol. 24, 65–66. Miles, J. S., McLaren, A. W., Forrester, L. M., Glancey, M. J., Lang, M. A., and Wolf, C. R. (1990). Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity. Biochem. J. 267, 365–371.
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151
Moran, E., O’Kennedy, R., and Thornes, R. D. (1987). Analysis of coumarin and its urinary metabolites by high-performance liquid chromatography. J. Chromatogr. 416, 165–169. National Toxicology Program (1993). Toxicology and carcinogenesis studies of coumarin (CAS No. 91-64-5) in F344/N rats and B6C3F1 mice (gavage studies). U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Technical Rep. Series No. 422, NIH Publication No. 92-3153. Peters, M. M., Walters, D. G., van Ommen, B., van Bladeren, P. J., and Lake, B. G. (1991). Effect of inducers of cytochrome P450 on the metabolism of [3-14C] coumarin by rat hepatic microsomes. Xenobiotica 21, 499–514. Shilling, W. H., Crampton, R. F., and Longland, R. C. (1969). Metabolism of coumarin in man. Nature 221, 664–665. Thornes, D., Daly, L., Lynch, G., Browne, H., Tanner, A., Keane, F., O’Loughlin, S., Corrigan, T., Daly, P., Edwards, G., Breslin, B., Brown, H., Shine, M., Lennon, F., Hanley, J., McMurray, N., and Gaffrey, E. (1989). Prevention of early recurrence of high risk malignant melanoma by coumarin. Eur. J. Surg. Oncol. 15, 431–435. Ueno, I., and Hirono, I. (1980). Non-carcinogenic response to coumarin in Syrian golden hamsters. Food Cosmet. Toxicol. 19, 353–355. Williams, R. T., Millburn, P., and Smith, R. L. (1965). The influence of enterohepatic circulation on toxicity of drugs. Ann. N. Y. Acad. Sci. 123, 110. Wood, A. W., and Conney, A. H. (1974). Genetic variation in coumarin hydroxylase activity in the mouse (mus musculus). Science 185 (4151), 612–614.
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0051
Effects of Coumarin Following Perinatal and Chronic Exposure in Sprague–Dawley Rats and CD-1 Mice BETSY D. CARLTON,*,1 JEAN-CLAUDE AUBRUN,†
AND
GLENN S. SIMON*
*Rhoˆne-Poulenc, 2 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709; and †Rhoˆne-Poulenc, 25 Quai Paul Doumer, 92408 Courbevoie Cedex, Paris, France Received June 29, 1995; accepted October 16, 1995
Effects of Coumarin Following Perinatal and Chronic Exposure in Sprague–Dawley Rats and CD-1 Mice. CARLTON, B. D., AUBRUN, J.-C., AND SIMON, G. S. (1996). Fundam. Appl. Toxicol. 30, 145–151. Coumarin, a naturally occurring substance most frequently used as a fragrance enhancer and stabilizer, was administered in the diet of Sprague–Dawley rats at dose levels of 0, 333, 1000, 2000, 3000, and 5000 ppm or in the diet of CD-1 mice at dose levels of 0, 300, 1000, or 3000 ppm. Rats receiving 333, 1000, and 2000 ppm coumarin were exposed to these dose levels in utero and during the lactational period, then chronically following weaning. Rats in the 3000- and 5000-ppm dose groups and all mice received only postweaning chronic exposure. All male rats were terminated after 104 weeks of postweaning exposure; female rats were terminated after 110 weeks. Male mice were terminated at Week 101 and female mice at Week 109. Among rats, survival was decreased at 333 ppm, but significantly increased among rats in the 3000and 5000-ppm dose groups. Dramatic dose-related decreases in body weight gain were recorded for rats receiving 2000, 3000, and 5000 ppm, clearly indicating that the MTD (maximum tolerated dose, as indicated by a body weight decrement of greater than 10– 15%) was exceeded. Food consumption also was decreased at the three highest dose levels, although body weight decrement was disproportionately large compared to changes in food consumption. Treatment-related decreases in hemoglobin were recorded from Week 6 onward. Minimal treatment-related changes in hematology and clinical chemistry were recorded. Increased liver weights were observed for male and female rats receiving 3000 or 5000 ppm and for females only at 1000 and 2000 ppm. Increased incidences of cholangiofibroma, cholangiocarcinoma, and parenchymal liver cell tumors were observed among male and female rats receiving 5000 ppm. One male rat receiving 3000 ppm developed a cholangiocarcinoma; no tumor increase was observed in males or females at 2000 ppm or below. Coumarin, at a dose clearly exceeding the MTD can, therefore, induce liver tumors in rats, although survival, relative to controls, was increased at the same dose levels. Among mice, a decrease in body weight gain was reported for males in the 1000- and 3000-ppm dose groups during the first 52 weeks of the study. No dose-related abnormalities in clinical signs, clinical pathology, hematology, or gross or microscopic pathology were noted. q 1996 Society of Toxicology
1
To whom correspondence and reprint requests should be addressed.
Coumarins, as a class, comprised numerous naturally occurring compounds with important and diverse physiological activities. The parent compound, coumarin, is used as an additive to perfumes, soaps, detergents, toothpaste, and some tobacco products and alcoholic beverages outside the United States. It is used clinically in the treatment of high-protein edema (Jamal et al., 1989), chronic brucellosis, and immune suppression (Moran et al., 1987). In isolation (Thornes et al., 1989) or in combination with cimetidine (Marshall et al., 1987a, b, c; Dexeus et al., 1990), coumarin is currently undergoing several clinical trials for the treatment of several types of malignancies in humans, including the treatment of lung and kidney carcinoma. Due to its use as a drug, there is information in the literature concerning its toxicity, metabolism, and pharmacokinetics in man. While several studies have been conducted to examine the chronic toxicity and carcinogenicity of coumarin, none were conducted in accordance with current scientific guidelines. A GLP-compliant study was therefore undertaken. (Since this study was completed, the U.S. National Toxicology Program has conducted a bioassay of coumarin in F344 rats and B6C3F1 mice.) MATERIALS AND METHODS Chemicals Coumarin (CAS 91-64-5) (ú98% pure) was supplied by Rhoˆne-Poulenc France as a white crystalline powder. Coumarin was admixed with Spratt’s Laboratory Animal Diet No. 2 to form a premix. The premix was then diluted with diet to the appropriate dose levels. Diets were prepared weekly. Animals and Treatments Rats. Rats were housed five per cage in suspended wire-mesh cages. Animal rooms were maintained at 227C and 50% relative humidity, with a 12-hr/12-hr light/dark cycle. Each cage was identified by cage card and each rat was individually identified by earmark and tattoo. Ad libitum access to food and tap water was provided. All animals for the chronic study were derived from Sprague–Dawley rats received from Charles River (Charles River, Manston, Kent, UK) and bred at Huntingdon Research Centre, Huntingdon, Cambridgeshire, England. A preliminary study (Hunter et al., 1980, unpublished data) conducted in pregnant Sprague–Dawley rats demonstrated that 3000 and 5000 ppm resulted in large weight gain decrements in the exposed dams and consequent decreases in the number of viable young. Therefore, in the main study described here, rats in the control, 3000-, and 5000-ppm dose groups
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received control diet in utero and during the lactational phase until weaning at 21–28 days of age, when the main study phase dosing was initiated. Animals in the 333-, 1000-, and 2000-ppm dose groups received coumarindosed feed at those dose levels throughout the perinatal and chronic periods. A total of 390 male and 390 female rats were used in this main chronic phase of the study. Each rat was allocated to one of six treatment groups, each comprising a main group (50 rats/sex) and a satellite group (15 rats/ sex). Animals in the main group were given ophthalmoscopy evaluations and were used for complete chronic toxicity/carcinogenicity evaluation. The satellite groups were used for hematology, clinical chemistry, and urinalysis determinations. They were killed at Week 104 and received a gross necropsy and organ weight determinations. Only gross lesions were examined microscopically from the satellite animals, however. All male rats in the main groups were terminated after 104 weeks of postweaning exposure; female rats were terminated after 110 weeks. A complete gross necropsy with organ weights was conducted for all main group animals. The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid, and uterus. Tissues for a complete histopathologic evaluation were collected and preserved in neutral-buffered formalin. The following observations and measurements were recorded. Mortality and morbidity (recorded twice daily), clinical observations, body weight, food and water consumption, ophthalmoscopic evaluations, hematology, clinical chemistry, plasma cholinesterase, urinalysis, selected organ weights, and gross and microscopic pathology. Mice. Animals were housed four per cage in solid bottom polypropylene cages with autoclaved, sifted sawdust as bedding. Animal rooms were maintained at 227C and 50% relative humidity, with a 12-hr/12-hr light/ dark cycle. Each cage was identified by colored cage label and each mouse was individually identified by earmark. Ad libitum access to food and tap water was provided. A total of 416 male and female CD-1 mice (Charles River, Manston, Kent, UK) 28 { 2 days of age were randomly assigned to four coumarintreatment groups comprising 52 mice/sex/dose. Animals received 0, 300, 1000, or 3000 ppm coumarin in the diet. Male mice were terminated at Week 101 and female mice at Week 109. A complete gross necropsy, with organ weight determinations, was conducted for all animals. The following organs were weighed: brain, heart, kidneys, liver, testes. Tissues for a complete histopathologic evaluation were collected and preserved in neutral-buffered formalin. The following observations and measurements were recorded: mortality and morbidity (recorded twice daily), clinical observations, body weight, food consumption, hematology, selected organ weights, and gross and microscopic pathology.
RESULTS
Rats The only clinical observation potentially dose-related was a temporary loss of fur among males and females receiving 5000 ppm and among females receiving 3000 ppm. Survival was decreased in animals receiving 333 ppm, but significantly increased among male and female rats receiving 3000 and 5000 ppm coumarin when compared with controls. These data are presented in Table 1. Body weight gains and food consumption were significantly reduced among male and female rats receiving 2000, 3000, and 5000 ppm. These data are presented in Tables 2 and 3. Growth of all groups of male rats was essentially similar between Weeks 52 and 78. From Weeks 78 through study termination, control rats lost weight, as commonly
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occurs for male Sprague–Dawley rats at this age. Body weights of male rats in the three highest dose groups remained relatively constant, reflecting the generally better condition of males receiving higher levels of coumarin. Female rats receiving 2000, 3000, or 5000 ppm continued to gain less weight than controls during Weeks 52–78. From Week 78 onward, bodyweight gain by all groups of female rats was essentially similar. The efficiency of food utilization by rats receiving 5000 ppm was impaired during Weeks 1–6. From Weeks 6–26, the efficiency of food utilization by these rats was superior to that of controls, indicating a degree of adaptation. From Weeks 13–26, efficiency of food utilization by males and females receiving 1000 or 2000 ppm was marginally impaired by treatment. Calculated group mean achieved intakes are described in Table 4. Significant clinical chemistry findings included a treatment-related anemia, principally characterized by low hemoglobin levels among males and females from Week 6 onward. This effect was noted primarily among rats in the three highest dose groups. Minimally elevated white cell and lymphocyte counts were recorded among 5000 ppm females from Week 25 to the end of the study. Treatment-related decreases in glucose and protein were found at Weeks 4 and 13 among 5000-ppm dose group animals, but the changes became less pronounced as the study progressed. Treatment-related increases in blood potassium levels, alkaline phosphatase, and glutamic–pyruvic transaminase were recorded throughout the study. Cholesterol levels of all groups of treated rats except for males in the highest dose group were increased throughout the study. Plasma cholinesterase, but not red blood cell cholinesterase, levels were decreased among females treated with coumarin, but not males. At necropsy, an increased incidence of liver masses was noted for male rats in the 5000-ppm dose group. Increased liver weights were recorded among males and females receiving 3000 and 5000 ppm and among females at 1000 and 2000 ppm. There was a twofold increase in relative liver weight observed at the highest doses. Decreased absolute brain, pituitary, heart, kidney, and adrenal weights were recorded for males in the 5000-ppm dose group. Heart weights were also decreased for males in the 2000- and 3000-ppm dose groups. Decreased absolute adrenal weights also were recorded for males receiving 1000, 2000, or 3000 ppm and among females receiving 3000 or 5000 ppm. Increased incidences of cholangiofibrosis, cholangiocarcinoma, and parenchymal liver cell tumors were recorded among male and female rats receiving 5000 ppm coumarin, although tumor incidence had no impact on survival. A single cholangiocarcinoma reported for a male rat receiving 3000 ppm was also considered to be potentially treatmentrelated. Rats receiving coumarin at 2000 ppm or less showed no evidence of increased hepatic tumor prevalence. No dose-
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TABLE 1 Survival of Male and Female Sprague–Dawley Rats Receiving Coumarin in the Diet for 104–110 Weeks Group
Males Weeks 0–104 Females Weeks 0–110
0 ppm
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
21/50 42%
11/50* 22%
17/50 34%
22/50 44%
27/50* 54%
33/50** 66%
13/50 26%
11/50 22%
17/50 34%
19/50 38%
25/50* 50%
32/50** 64%
* p £ 0.05. ** p £ 0.001.
related increases in renal tumors or tumors at other tissue sites were observed. Histopathology results are presented in Table 5.
among males or females receiving coumarin in the diet. Females receiving 3000 ppm coumarin showed marginally, but statistically significant, increases in liver weight, although there were no associated changes at the cellular level. Males were not similarly affected. No other differences in organ weights were observed across dose groups. No increases in tumors were observed in liver, lung, or other tissues (Table 7). Gross and microscopic pathology was unchanged by dose exposure.
Mice Body weight gain by males receiving 3000 ppm was significantly reduced compared to controls, demonstrating an 18% reduction over the first 52 weeks of the study. Body weight gain was reduced by 10% relative to controls among males in the 1000-ppm dose group. Body weights were similar across all female dose groups. Food utilization by males, but not females, in the 3000-ppm dose group was marginally inferior to that of controls. Achieved average coumarin intakes were calculated and are shown in Table 6. No treatment-related changes in survival, clinical observations, hematology, or clinical chemistry were observed
DISCUSSION
Concerns about coumarin’s carcinogenic potential arose from a series of studies which started in 1967 with reports by Ba¨r and Griepentrog (1967). These authors reported that lifetime exposure of rats to doses of 5000 ppm or higher
TABLE 2 Body Weight Gain for Male and Female Sprague–Dawley Rats during the Chronic Bioassay of Coumarin (Weight Gain in Grams { SD) Week
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
Males 0–13 13–26 26–52 52–78 78–term
455 129 156 52 064
{ { { { {
51 33 47 82 137
464 132 151 75 071
{ { { { {
62 34 85 60* 132
435 113 110 50 043
{ { { { {
54* 30** 59*** 68 131
372 99 118 56 05
{ { { { {
46*** 33*** 39*** 53 71*
329 114 132 62 6
{ { { { {
41*** 27** 38* 44 69*
189 115 118 42 12
{ { { { {
33*** 23** 30*** 25 48**
216 40 71 56 39
{ { { { {
25** 16*** 35*** 38** 57
197 47 68 52 40
{ { { { {
23*** 18* 37*** 44** 56
128 53 41 20 26
{ { { { {
21*** 16 18*** 16*** 36
Females 0–13 13–26 26–52 52–78 78–term
230 56 113 80 18
{ { { { {
34 29 58 45 106
234 56 108 68 33
{ { { { {
31 24 47 63 102
228 46 93 71 42
{ { { { {
24 18** 41** 43 78
* p £ 0.05. ** p £ 0.01. *** p £ 0.001.
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TABLE 3 Food Consumption (g/Rat) for Male and Female Sprague–Dawley Rats in the Chronic Bioassay of Coumarin Week
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
1883*** 82 84% 2103*** 73 87% 8225*** 376 89 4492** 242 89%
1757*** 68 79% 2069*** 88 86% 8209*** 357 89% 4369*** 183 86%
1209*** 103 54% 1602*** 113 66% 6772*** 415 73% 3644*** 243 72%
1487*** 46 91% 1544*** 51 93% 6379*** 198 92% 4409* 271 91%
1394*** 63 85% 1507*** 79 90% 6229*** 392 90% 4354** 414 90%
1027*** 84 63% 1249*** 69 75% 5104*** 263 73% 3550*** 344 73%
Males 0–13 SD % Control 13–26 SD % Control 27–78 SD % Control 79–term SD % Control
2236 88 — 2419 132 — 9246 626 — 5056 283 —
2272 65 102% 2459 89 102% 9452 369 102% 5096 394 101%
2176 68 97% 2362 48 98% 8922 265 96% 5094 461 101% Females
0–13 SD % Control 13–26 SD % Control 27–78 SD % Control 79-term SD % Control
1637 76 — 1669 75 — 6959 425 — 4849 500 —
1666 77 102% 1693 112 101 6973 571 100% 4674 217 96%
1626 62 99% 1629 78 98% 6734 287 97% 4894 326 101%
* p £ 0.05. ** p £ 0.01. *** p £ 0.001.
induced bile duct carcinoma. Cohen (1979) reviewed these slides and cast doubts on the interpretation of the histopathology and the reality of these carcinomas. Reevaluation of the original histopathology slides indicated that cholangiofibrosis and not cholangiocarcinoma was present (Evans et al., 1989). Evans reported that cholangiofibrosis is not a prerequisite for hepatocellular carcinoma and is not part of the carcinogenic process. Hagan et al. (1967) reported that groups of five to seven Osborne–Mendel rats given up to
TABLE 4 Achieved Intake (mg/kg/Day) Dose group 333 1000 2000 3000 5000
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5000 ppm coumarin in the diet were unaffected at 1000 ppm. Hepatotoxicity was noted at 2500 ppm. Animals fed 5000 ppm had decreased hemoglobin levels; enlarged livers with fatty metamorphosis, masses, and pale foci; and cholangiofibrosis. No cholangiocarcinomas were reported. The National Toxicology Program (NTP) (1993) conducted 2-year bioassays of coumarin in F344 rats and B6C3F1 mice. Dose levels for these corn oil gavage studies were 0, 25, 50, and 100 mg/kg/day for rats and 0, 50, 100, and 200 mg/kg/day for mice, doses which are comparable to, or slightly lower than, the doses used in our studies. In the NTP study, survival of the male rats was severely compromised. Some hepatocellular necrosis and bile duct hyperplasia was found, but no increase in hepatocellular neoplasia was reported at any dose. The highest dose level in the NTP rat study was approximately equivalent to the 2000-ppm dose group reported here (achieved intake 87 mg/ kg/day in males; 107 mg/kg/day in females). Hepatocellular findings were similar at this dose level, where no increase in tumor incidence was observed. In contrast to findings of
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TABLE 5 Liver Weights and Histopathologic Findings for Male and Female Sprague–Dawley Rats during the Chronic Bioassay of Coumarin (65 male and 65 Female per Group) Animals (65/sex/group) Males
Females
Finding
Control
333 ppm
1000 ppm
2000 ppm
3000 ppm
5000 ppm
Liver weight (grams { SD) Cholangiocarcinoma (nonmetastasizing) Cholangiocarcinoma (metastasizing) Parenchymal tumors (benign / malignant)
28.3 { 5.91 0
29.4 { 7.03 0
29.3 { 5.37 0
29.7 { 3.87 0
34.3 { 10.55 1
44.6 { 18.85 31
0
0
0
0
0
6
2
2
1
1
6
29
Liver weight (g { SD) Cholangiocarcinoma (nonmetastasizing) Cholangiocarcinoma (metastasizing) Parenchymal tumors (benign / malignant)
18.8 { 4.8 0
21.7 { 4.1 0
23.4 { 4.3 0
20.6 { 4.5 0
2.2 { 4.6 0
22.9 { 5.5 22
0
0
0
0
0
1
0
0
0
0
1
12
marginal increases in renal adenoma in the NTP study, no increase was observed in the Rhoˆne-Poulenc (RP) study. No renal tumors were reported in females and only 1 renal adenoma was observed in a 2000-ppm dose group males. The NTP bioassay in B6C3F1 mice reported an increase in alveolar/bronchiolar adenoma and carcinoma among male and female mice (14/50, 9/50, 15/50, 25/51 males; 2/51, 5/ 49, 7/49, 27/50 females). Although an increase in pulmonary tumors was noted in the current study, all tumor incidences were within laboratory historic control ranges for the age and strain of mouse. There were no significant dose-related increases in tumors of any type in the CD-1 mice receiving coumarin in the diet at doses slightly higher than those given the mice treated by gavage in the NTP study (achieved high doses 280 mg/kg/day males; 271 mg/kg/day females). Chronic toxicity/carcinogenicity studies conducted in numerous species indicate that the liver is the main target organ for coumarin toxicity, but the severity of the effect appears to be dependent on the prevalent metabolic pathway. The 7hydroxylation pathway appears to be a more efficient detoxification/elimination pathway. Chronic toxicity/carcinogenicity studies conducted in species with significant 7-hydroxylating capacity such as baboons (Gangolli et al., 1974; Evans
TABLE 6 Achieved Intake (mg/kg/Day) Dose group 300 ppm 1000 ppm 3000 ppm
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et al., 1979), hamsters (Ueno and Hirono, 1980), and DBA/ 2J mice (Wood and Conney, 1974) have demonstrated significantly less hepatotoxicity and, in the bioassays, no increases in tumors at any site. DBA/2J mice are one of the few nonprimate species so far shown to possess a significant 7-hydroxylating capability (Wood and Conney, 1974). They appear to be more resistant to the hepatotoxic action of coumarin than is the CH3/HeJ mouse strain which possesses only a limited 7-hydroxylating potential (Endell and Seidel, 1978). Similarly, a bioassay study by Ueno and Hirono (1980) suggests that hamsters, which possess some 7-hydroxycoumarin (7-OHC) capacity, are refractory to coumarininduced hepatotoxicity, although limited conclusions can be drawn due to the small number of animals. In addition, no carcinogenic or adverse toxicologic effects were found in this study when groups of 10–13 Syrian Golden hamsters were fed diet containing 0, 1000, and 5000 ppm coumarin for 2 years. When administered as a single high dose to the rat, coumarin has been shown to produce hepatic centrilobular necrosis. Lake (Lake, 1984; Lake et al., 1989) demonstrated that coumarin-induced hepatotoxicity in rats is likely to be mediated via one or more reactive metabolites generated by cytochrome P450-dependent enzymes. Hepatotoxicity of coumarin was decreased by pretreatment with cobaltous chloride, ellypticine, and metyrapone and potentiated by pretreatment with diethyl maleate (known to reduce hepatic-reduced glutathione concentration). In in vitro studies, reactive [3-14C]coumarin metabolite(s) generated by cytochrome P450-dependent enzyme was found to bind covalently to microsomal proteins. The authors suggest that the toxic compound may be a coumarin 3-4 epoxide intermediate. Lake et al. (1989) compared the metabolism of [3-14C]-
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TABLE 7 Liver and Lung Histopathologic Findings for Male and Female CD-1 Mice during the Chronic Bioassay of Coumarin Tissue
Findinga
Control
300 ppm
1000 ppm
3000 ppm
Liver—male Liver—female Lung—male
Benign / malignant parenchymal tumors Benign / malignant parenchymal tumors Pulmonary adenoma Pulmonary adenocarcinoma Pulmonary adenoma Pulmonary adenocarcinoma
20 0 0 11 2 10
22 8 1 12 1 11
19 4 2 10 2 11
12 3 0 20 5 14
Lung—female
a
All tumor incidences were within laboratory historic control values for this age and strain of mice.
coumarin (concentration 0.19 mM) by hepatic microsomes of different species including human. In the rat, and in most strains of mice, 3-hydroxylation predominates while 4-, 5-, 6-, and 8-hydroxylation are possible but minor routes of metabolism. The 3-hydroxycoumarin is then extensively degraded to ring-opened metabolites. Recent studies have shown that in rat liver microsomes, coumarin is a substrate for isoenzymes in both P450 IA and P450 IIB subfamilies (Peters et al., 1991), whereas a member of the P450 IIA subfamily is responsible for the majority of coumarin 7hydroxylase activity in human liver microsomes (Miles et al., 1990) and the main metabolism pathway goes through 7-OHC. This difference of metabolism leads also to a difference in pharmacokinetics. In the rat, within 24 hr of an oral or intraperitoneal (ip) dose of 50 mg/kg coumarin, 50% of the dose was excreted in the bile of rats as unidentified ringopened compounds. When 50 mg/kg 7-OHC was administered ip, only 0.2% was excreted in the bile unchanged or as glucuronide conjugate (Williams et al., 1965). It is also important to note that the main feature in chronic liver toxicity is bile duct alteration, which may correspond to the biliary excretion of the coumarin metabolites in the rat. No such toxicity is observed with species metabolizing through 7OHC, which is not significantly excreted through the bile. In humans, coumarin is rapidly eliminated after oral administration, with 83% of the absorbed dose (200 mg/kg) found in the urine within 24 hr (79% as 7-OHC), while only 35% is found in the urine of rats given an equivalent dose (õ0.1% as 7-OHC) (Shilling et al., 1969; Feuer et al., 1966). This is consistent with a rapid absorption by the gut in man and with an absence of enterohepatic circulation of the metabolites. Given body weight decrements up to 60% among highdose male rats, clearly indicating that the maximum tolerated dose was exceeded, the carcinogenic response seen in the current Sprague–Dawley rat study is likely due to the hepatotoxicity incurred following high-dose exposure. Tumors were not metastatic and survival was significantly increased among rats in the two highest dose groups. While hepatic responses were comparable at similar doses in the NTP and
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RP rat studies, findings of minimal increases in renal adenomas only in the NTP study may indicate a strain susceptibility to renal abnormalities among F344 rats. Similarly, the increase in lung tumors observed among B6C3F1 mice in the NTP study was not confirmed in the RP study. This may reflect a dose-route-dependent phenomenon (NTP corn oil gavage vs RP dietary exposure) or a strain-related difference in metabolism or susceptibility. The significance of any of these positive carcinogenic results to man is highly questionable in view of man’s much lower exposure potential and the large metabolic differences between species. REFERENCES Ba¨r, F. U., and Griepentrog, F. (1967). Die Situation in der gesundheitlichen Beurteilung der aromatisierungsmittel fu¨r lebensmittel. Med. Erna¨rhr. 8, 244. Cohen, A. J. (1979). Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food Cosmet. Toxicol. 17, 277–289. Dexeus, F. H., Logothetis, C. J., Sella, A., Fitz, K., Amato, R., Reuben, J. M., and Dozier, N. (1990). Phase II study of coumarin and cimetidine in patients with metastatic renal cell carcinoma. J. Clin. Oncol. 8, 325– 329. Endell, W., and Seidel, G. (1978). Coumarin toxicity in different strains of mice. Agents Actions 8, 299–302. Evans, J. G., Gaunt, I. F, and Lake, B. G. (1979). Two-year toxicity study on coumarin in the baboon. Food Cosmet. Toxicol. 17, 187–193. Evans, J. G., Appleby, E. C., Lake, B. G., and Conning, D. M. (1989). Studies on the induction of cholangiofibrosis by coumarin the rat. Toxicology 55, 207–224. Feuer, G., Golberg, L., and Gibson, K. I. (1966). Liver response tests. VII. Coumarin metabolism in relation to the inhibition of rat-liver glucose 6phosphatase. Food Cosmet. Toxicol. 4, 157–167. Gangolli, S. D., Shilling, W. H., Grasso, P., and Gaunt, L. F. (1974). Studies on the metabolism and hepatotoxicity of coumarin in the baboon. Biochem. Soc. Trans. 2, 310–312. Hagan, E. C., Hansen, W. H., Fitzhugh, O. G., Jenner, P. M., Jones, W. I., Taylor, J. M., Long, E. L., Nelson, A. M., and Brouwer, J. B. (1967). Food flavorings and compounds of related structure. II. Subacute and chronic toxicity. Food Cosmet. Toxicol. 5(2), 141–157. Hunter, B., Warren, S., Heywood, R. C., and Gibson, W. (1980). Huntingdon Research Centre, unpublished Rhoˆne-Poulenc Rep. RNP 173/81137.
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CHRONIC EFFECTS OF COUMARIN IN RODENTS Jamal, S., Casley-Smith, J. R., and Casley-Smith, J. R. (1989). The effects of 5,6-benzo-[a]-pyrone (coumarin) and DEC on filaritic lymphoedema and elephantiasis in India. Ann. Trop. Med. Parasitol. 83(3), 287–290. Lake, B. G. (1984). Investigations into the mechanism of coumarin-induced hepatoxicity in the rat. Arch. Toxicol. (Suppl. 7), 16–29. Lake, B. G., Gray, T. J., Evans, J. G., Lewis, D. F. V., Beamand, J. A., and Hue, K. L. (1989). Studies on the mechanism of coumarin-indiced toxicity in rat hepatocytes: Comparison with dihydrocoumarin and other coumarin metabolites. Toxicol. Appl. Pharmacol. 97, 311–323. Lake, B. G., Gaudin, H., Price, R. J., and Walters, D. G. (1992). Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans. Food Chem. Toxicol. 30(2), 105–115. Marshall, M. E., Mendelsohn, L., Butler, K., Riley, L., Cantrell, J., Wiseman, C., Taylor, R., and MacDonald, J. S. (1987a). Treatment of metastatic renal cell carcinoma with coumarin (1,2-benzopyrone) and cimetidine: A pilot study. J. Clin. Oncol. 5(6), 862–866. Marshall, M. E., Mendelsohn, L., Butler, K., Cantrell, J., Harvey, J., and MacDonald, J. S. (1987b). Treatment of non-small cell lung cancer with coumarin and cimetidine 1,2. Cancer Treat. Rep. 71(1), 91–92. Marshall, L., Butler, K., Cantrell, J., Wiseman, C., and Mendelsohn, L. (1987c). Treatment of advanced malignant melanoma with coumarin and cimetidine: A pilot study. Cancer Chemother. Pharmacol. 24, 65–66. Miles, J. S., McLaren, A. W., Forrester, L. M., Glancey, M. J., Lang, M. A., and Wolf, C. R. (1990). Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity. Biochem. J. 267, 365–371.
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Moran, E., O’Kennedy, R., and Thornes, R. D. (1987). Analysis of coumarin and its urinary metabolites by high-performance liquid chromatography. J. Chromatogr. 416, 165–169. National Toxicology Program (1993). Toxicology and carcinogenesis studies of coumarin (CAS No. 91-64-5) in F344/N rats and B6C3F1 mice (gavage studies). U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Technical Rep. Series No. 422, NIH Publication No. 92-3153. Peters, M. M., Walters, D. G., van Ommen, B., van Bladeren, P. J., and Lake, B. G. (1991). Effect of inducers of cytochrome P450 on the metabolism of [3-14C] coumarin by rat hepatic microsomes. Xenobiotica 21, 499–514. Shilling, W. H., Crampton, R. F., and Longland, R. C. (1969). Metabolism of coumarin in man. Nature 221, 664–665. Thornes, D., Daly, L., Lynch, G., Browne, H., Tanner, A., Keane, F., O’Loughlin, S., Corrigan, T., Daly, P., Edwards, G., Breslin, B., Brown, H., Shine, M., Lennon, F., Hanley, J., McMurray, N., and Gaffrey, E. (1989). Prevention of early recurrence of high risk malignant melanoma by coumarin. Eur. J. Surg. Oncol. 15, 431–435. Ueno, I., and Hirono, I. (1980). Non-carcinogenic response to coumarin in Syrian golden hamsters. Food Cosmet. Toxicol. 19, 353–355. Williams, R. T., Millburn, P., and Smith, R. L. (1965). The influence of enterohepatic circulation on toxicity of drugs. Ann. N. Y. Acad. Sci. 123, 110. Wood, A. W., and Conney, A. H. (1974). Genetic variation in coumarin hydroxylase activity in the mouse (mus musculus). Science 185 (4151), 612–614.
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