Mutation Research, 298 (1992) 71-79

71

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00

MUTGEN 01828

M u t a g e n i c i t y testing o f i m i d a z o l e and related c o m p o u n d s R. Forster a, S.D. Blowers b, S. Cinelli c, H. Marquardt d and J. Westendorf d ,z Italfarmaco Research Centre, 20092 Cinisello Balsamo, Milan, Italy, b BIBRA Toxicology International, Carshalton, Surrey SM5 4DS, UK, c Life Science Research Roma Toxicology Centre, 00040 Pomezia, Rome, Balv and a Department of Toxicology, Unit~'ersity of Hamburg Medieal School, 2000 Hamburg 13, Germany

(Received 26 March 1992) (Revision received 2 June 1992) (Accepted 12 June 1992)

Keywords. Imidazole; Ames test; Unscheduled DNA synthesis; Transformation assay

Summary Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite resulting from the action of intestinal bacteria, and histamine, a structurally related compound which is widely distributed in mammalian tissues, have also been tested. Imidazole and histamine were also tested in the UDS assay in primary rat hepatocytes, while imidazole alone was tested in the M2-C3H mouse fibroblast malignant transformation assay. Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave negative results in the Ames test. Histamine gave no evidence of mutagenic activity in the A m e s test or of genotoxicity in the UDS assay. These results indicate that imidazole and its metabolites are unlikely to present a mutagenic or carcinogenic hazard.

Imidazole is a simple heterocyclic molecule, which is a building block of many more complex organic compounds. It is readily soluble in water and after oral administration is rapidly absorbed from the gastrointestinal tract. In the body imidazole is metabolised to hydantoin and subsequently to hydantoic acid. Unchanged imidazole, hydantoin and hydantoic acid are eliminated in the urine. Bacterial metabolism within the G I tract can also result in the production of N-

Correspondence: Dr. R. Forster, Italfarmaco Research Centre, via dei Lavoratori 54, 20092 Cinisello Balsamo, Milan, Italy.

acetyl-imidazole. The pK~ value of imidazole is around 7, allowing it to function as both a proton donor and a proton acceptor at physiological p H levels. Imidazole has low acute toxicity and the mouse oral LDs0 value is reported to be 1.88 g / k g (Nishie et aI., 1969). Little published information is available about the general toxicology of imidazole and the potential long-term hazards of imidazole ingestion. In the present study we have undertaken a series of short-term tests for potential mutagenic and carcinogenic activity. The assay systems employed were the Ames test, unscheduled D N A synthesis in primary rat hepatocytes and the M2-C3H mouse fibroblast cell transformation assay.

72 histamine (CAS No. 51-45-6, structure V). The unscheduled DNA synthesis (UDS) assay in primary rat hepatocytes was performed with imidazole and histamine, while the transformation assay was performed with imidazole only.

I Imidazole

H

Materials and methods

II Hydantoin

HI Hydantoicacid

NH2CONHCH2COOH % ,CH3

cI

IV N-acetyl-imidazole

NI-I,CH,CH../N~ V Histamine

\\ L H N

VI Phcnytoin

C~Hs-\ o4

/ Nit

Fig. 1. Chemical structures of (1) imidazole, (II) hydantoin, (III) hydantoic acid, (IV) N-acetyl-imidazole,(V) histamine and (VI) phenytoin.

Ames tests were performed with imidazole (CAS No. 288-32-4, structure I: for structures refer to Fig. 1) and its principal metabolites, which are hydantoin (CAS No. 461-72-3, structure II) and hydantoic acid (CAS No. 462-60-2, structure III). N-Acetyl-imidazole (CAS No. 2466-76-4, structure IV), a potential metabotite resulting from intestinal bacterial metabolism, was also tested. In addition we selected for study a closely related imidazole derivative which is widely distributed in mammalian tissues, namely

Chemicals Imidazole (purity > 99%) was obtained from Fluka, Switzerland. Hydantoin, hydantoic acid, and N-acetyl-imidazole were obtained from Sigma. Histamine was obtained from Sigma (Ames test) and Merck (UDS assay). For the Ames tests, solutions of hydantoin and hydantoic acid were prepared freshly before use in dimethyl sulphoxide; solutions of imidazole, N-acetyl-imidazole and histamine were prepared freshly before use in distilled water. For the UDS and transformation assays solutions of imidazole (and histamine) were prepared directly in culture medium. Chemicals and media for cell culture (Williams Medium E, foetal calf serum, Hank's buffer, penicillin/streptomycin) were obtained from Grand Island Biochemicals, Karlsruhe. A m e s test The Ames test was performed essentially as described by Ames et al. (1975) and Maron and Ames (1983). The tester strains TA98, TA100, TA97 and TA102, obtained from Professor Ames, were used in this study. Results are presented from at least two independent experiments, with three replicate plates at each test point. Rat liver $9 homogenate was prepared from the livers of young male rats after pretreatment with /3-naphthoflavone and phenobarbital (Matsushima et al., 1976; experiments with imidazole) or Aroclor 1254 (Ames et al., 1975; experiments with all other compounds). UDS test in primary rat hepatocytes The methods employed were Williams et al. (1982).

based

on

Hepatocyte preparation and treatment, Hepatocytes were prepared freshly from untreated young male Wistar rats (body weight about 200 g)

73

by the in situ collagenase perfusion method. These animals were obtained from the Central Institute for Laboratory Animals, Hannover. A single animal was used for each experiment. The viability of the hepatocytes was estimated by Trypan blue exclusion, and was routinely greater than 70%. Incubations were at 3 7 ° C / 5 % CO 2. The hepatocytes were maintained in Williams E medium plus 10% foetal calf serum. About 2 × 105 viable hepatocytes were seeded in 30-ram petri dishes which contained plastic Thermanox cover slips. Two replicate dishes were p r e p a r e d for the evaluation of UDS at each test point. The cultures were placed in the incubator for 2 h to attach. The medium was then replaced with fresh medium containing 10 / z C i / m l tritiated thymidine. Appropriate amounts of the test material, positive control or vehicle treatment were added and the cells returned to the incubator for a treatment period of 18 h. Two positive control treatments were used, namely (1) 10 s U V treatment with a 4-W H a n a u U V lamp (254 nm) at a distance of 1 cm, as a direct-acting control, and (2) 7,12-dimethylbenzanthracene (10 p , g / m l ) as an indirect-acting agent.

Autoradiography and estimation of UDS. Following treatment the cover slips were removed, washed repeatedly in fresh medium, left to swell for 10 min in 1.0% sodium citrate solution and fixed in 3 : 1 ethanol:acetic acid. The cover slips were fixed on slides, dried and dipped in Kodak NTB2 emulsion and then maintained at - 3 0 ° C in a light-proof airtight container for an 8-day exposure period. The preparations were then developed in Kodak K10 developer, fixed and dried overnight. They were subsequently stained with haematoxylin and eosin. Evaluation at 1000-fold magnification was performed with the assistance of a Biotran II image analyser system. The number of nuclear and cytoplasmic grains was estimated for 20 representative cells at each test point. Concurrent toxicity. For the evaluation of concurrent toxicity a third petri dish was prepared and treated in the same way as the test cultures (but without the addition of tritiated thymidine). At the end of the treatment period the survival of these ceils was estimated by the Neutral red method.

TABLE 1 I M I D A Z O L E : I N D U C T I O N O F R E V E R S E M U T A T I O N IN Salmonella typhimurium Dose level (rag/plate)

TA97

TA98

TAI00

TAI02

131 143 131 147 138 142 422 9AA 50 izg

35 35 30 31 32 37 292 2NF 2 p~g

164 159 173 167 158 166 726 N a A Z 1/zg

35l 355 358 353 336 346 831 M M C 0.5/zg

158 161 154 162 154 127 1 950 5 ,ag

51 49 45 43 47 45 1245 1/zg

184 179 182 177 176 199 1531 1 #g

420 44 l 415 450 407 428 1816 10/xg

Absence of $9 metabolism Distilled water, 100 ~zl 0.625 1.25 2.5 5.0 10.0 Positive control

Presence of $9 metabolism Distilled water, 100 tzl 0.625 1.25 2.5 5.0 10.0 Positive control, 2-aminoanthracene

Average n u m b e r of revertants per plate (mean of two independent experiments, using three plates per test point). 9AA, 9-aminoacridine; 2NF, 2-nitrofluorene; N a A Z , sodium azide; MMC, mitomycin C.

74 TABLE 2 H Y D A N T O I N : I N D U C T I O N O F R E V E R S E M U T A T I O N IN Salmonella typhimurium Dose level ( m g / p l a t e )

TA97

TA98

TA100

TA102

139 143 135 146 140 143 139 1 126 N O P D 50/xg

30 34 30 30 30 27 27 529 2NF 5/xg

121 125 125 121 121 126 114 950 N a A Z 5/xg

196 202 206 212 203 201 188 550 M M C 0.5 ,ag

Absence of $9 metabolism Untreated DMSO, 100/xl 0.1 0.33 1.0 3.3 10.00 Positive control

Presence of $9 metabolism Untreated DMSO, 100/xl 0.1 0.33 1.0 3.3 10.00 Positive control, 2-aminoanthracene, 4/xg

170 158 165 156 161 156 153

31 32 32 35 38 32 27

134 133 129 130 134 130 116

223 227 220 228 225 230 212

1315

1384

2061

664

Average n u m b e r of revertants per plate (mean of two independent experiments, using three plates per test point). NOPD, nitro-ortho-phenylenediamine; 2NF, 2-nitrofluorene, N a A Z , sodium azide; MMC, mitomycin C.

TABLE 3 H Y D A N T O I C ACID: I N D U C T I O N OF R E V E R S E M U T A T I O N IN Salmonella typhimurium Dose level ( m g / p l a t e )

TA97

TA98

TA100

TA102

160 157 172 163 158 148 141 1192 N O P D 50/xg

33 33 31 32 31 34 26 585 2NF 5/xg

124 119 107 113 110 116 112 955 N a A Z 5/xg

181 186 183 185 189 184 175 585 M M C 0.5/xg

Absence of $9 metabolism Untreated DMSO, 100/xl 0.1 0.33 1.0 3.3 10.00 Positive control

Presence of $9 metabolism Untreated DMSO, 10(J/xl 0.10 0.33 1.0 3.3 10.00 Positive control 2-aminoanthracene, 4 ,ag

175 173 162 174 160 177 160

36 34 30 33 32 33 32

140 131 138 131 129 128 134

216 213 222 203 209 210 192

1491

1557

2117

600

Average n u m b e r of revertants per plate (mean of two independent experiments, using three plates per test point). NOPD, nitro-ortho-phenylenediamine; 2NF~ 2-nitrofiuorene, NaAZ, sodium azide; MMC, mitomycin C.

75 TABLE 4 N-ACETYL-IMIDAZOLE: INDUCTION OF REVERSE MUTATION IN Salmonella typhimurium Dose level (mg/plate)

TA97

TA98

TA100

TA102

145 146 156 135 142 136 128 1 116 NOPD 50 ~g

26 28 29 29 26 29 25 645 2NF 5/zg

117 123 120 114 125 128 107 1039 NaAZ 5/xg

219 219 222 227 231 216 207 782 MMC 0.5/zg

Absence of $9 metabolism Untreated Distilled water, 100/xl 0.1 0.33 1.0 3.3 10.00 Positive control

Presence of $9 metabolism Untreated Distilled water, 100 tzl 0.10 0.33 1.0 3.3 10.00 Positive control, 2-aminoanthracene, 4/xg

158 156 158 147 162 158 142

32 40 35 37 31 32 36

138 133 138 137 134 124 133

256 259 266 250 249 242 228

1521

1348

1758

665

Average number of revertants per plate (mean of two independent experiments, using three plates per test point). NOPD, nitro-ortho-phenylenediamine; 2NF, 2-nitrofluorene, NaAZ, sodium azide; MMC, mitomycin C.

TABLE 5 HISTAMINE: INDUCTION OF REVERSE MUTATION IN Salmonella typhimurium Dose level (rag/plate)

TA97

TA98

TA100

TA102

145 139 135 131 134 126 141 955 NOPD 50/zg

29 28 28 29 28 28 28 552 2NF 5 Izg

119 126 124 114 120 124 102 839 NaAZ 5 #g

207 212 204 187 186 193 172 648 MMC 0.5/zg

Absence of $9 metabolism Untreated Distilled water, 100/zl 0.1 0.33 1.0 3.3 10.00 Positive control

Presence of $9 metabolism Untreated Distilled water, 100 tzl 0.10 0.33 1.0 3.3 10.00 Positive control, 2-aminoanthracene, 4/zg

162 171 169 162 169 170 160

34 34 35 35 37 32 37

135 128 128 137 136 137 134

249 255 264 241 250 246 237

1325

146l

2 064

647

Average number of revertants per plate (mean of two independent experiments, using three plates per test point). NOPD, nitro-ortho-phenylenediamine; 2NF, 2-nitrofluorene, NaAZ, sodium azide; MMC, mitomycin-C.

76

Transformation assay

transformation plates were coded and scored ' b l i n d ' for type I l l foci; t h e s e w e r e r e c o g n i s e d as p i l e d up, m u l t i l a y e r e d foci with criss-cross patt e r n at the e d g e s arid p r e d o m i n a n c e of n u c l e a r material.

T h e t r a n s f o r m a t i o n assay was p e r f o r m e d as d e s c r i b e d in M a r q u a r d t et al. (1976). Briefly, M2C 3 H m o u s e f i b r o b l a s t s (12th to 15th p a s s a g e ) w e r e c u l t u r e d in b a s a l E a g l e ' s m e d i u m plus 10% foetal calf serum. Cells h a r v e s t e d from logarithmically growing c u l t u r e s w e r e p l a t e d in 60-mm p e t r i d i s h e s at 100 c e l l s / p l a t e for the d e t e r m i n a tion of cytotoxicity a n d 1000 c e l l s / p l a t e for t h e d e t e r m i n a t i o n o f t r a n s f o r m a t i o n rate, F o r e a c h t r e a t m e n t two r e p l i c a t e p l a t e s w e r e p r e p a r e d for cytotoxicity a n d e i g h t r e p l i c a t e p l a t e s for the det e r m i n a t i o n of t r a n s f o r m a t i o n rate. A f t e r 24 h the cells w e r e t r e a t e d with the test substance, positive c o n t r o l ( N-methyl-N '-nitro-N-nitrosoguanidine; M N N G ) o r vehicle t r e a t m e n t . T h e t r e a t m e n t p e riod was 24 h. T h e m e d i u m was t h e n c h a n g e d a n d the cells w e r e a l l o w e d to grow for 2 w e e k s (for the d e t e r m i n a t i o n of p l a t i n g efficiency) or 8 w e e k s (for t h e d e t e r m i n a t i o n of t r a n s f o r m a t i o n rate). D u r i n g t h i s - p e r i o d t h e m e d i u m was c h a n g e d twice weekly. T h e c u l t u r e s w e r e t h e n fixed with m e t h a n o l , s t a i n e d with G i e m s a a n d scored. T h e

Results A m e s test T h e results o f the A m e s tests are p r e s e n t e d in T a b l e s 1 - 5 . N o n e of t h e c o m p o u n d s was toxic to t h e S a l m o n e l l a t e s t e r strains a n d in all cases the test m a t e r i a l s w e r e t e s t e d up to 10 r a g / p l a t e w i t h o u t a p p r e c i a b l e toxicity. N o e v i d e n c e of mut a g e n i c activity was o b s e r v e d in any of t h e experim e n t s p e r f o r m e d , in t h e a b s e n c e or p r e s e n c e of rat liver $9 m e t a b o l i c activation, using Salmonella typhimurium t e s t e r strains T A 9 8 , TA100, T A 9 7 a n d TA102.

UDS test in primary rat hepatocytes The compounds imidazole and histamine were t e s t e d for possible D N A r e p a i r i n d u c t i o n in pri-

TABLE 6 UNSCHEDULED DNA SYNTHESIS IN PRIMARY RAT HEPATOCYTES Treatment

Expt. 1

Expt. 2

NNG (%pos)

Control

Expt. 3

%S

NNG (%pos)

%S

NNG (%pos)

%S

(0)

100

-2.1

(5)

100

-0.2 (20)

100

0.0 (15) 1.3 (45) -0.5 (25) NT NT

100 100 81

0~2 (20) -1.0 (30) 0.4 (15) 2.1 (35) Too toxic

100 79 48 25 2

0.9 (10) 1.6 (25) 0.6 (20) 1.9 (30) Too toxic

95 73 60 61 5

0.9 (35) -1.9 (15) -0.6 (15) NT NT

100 100 100

-1.0 -3.1 0.3 -2.2 0.1

1.0

lrnidazole 0.25 mg/ml 0.50mg/ml 1.00mg/ml 2.00 mg/ml 4.00 mg/ml

Histamine 0.25 mg/ml 0.50mg/ml 1.00mg/ml 2.00 mg/ml 4.00 rng/ml

(35) (5) (15) (10) (15)

88 88 81 70 33

0.5 0.2 0.9 1.6 2.0

(15) (15) (15) (20) (45)

91 93 89 80 57

Positit,e controls DMBA, 10/zg/ml UV, 10 s

13.7 (100) * 52.2 (100) *

29 32

25.0 (100) * 38.8 (100) *

52 32

12.5 (100) * 48.4 (100) *

82 45

NNG, net nuclear grains; %pos, percentage of cells with NNG > 3.0; %S, percentage viability as measured by the Neutral red assay; NT, not tested. * Positive results according to criteria stated in text.

77 mary rat hepatocytes (Table 6). Both compounds were tested into toxic dose ranges and three independent experiments were performed. Slight differences in the cytotoxicity were observed from experiment to experiment. T h e criterion adopted for the evaluation of the results was that a test treatment was considered positive if the net silver grain rate showed a dose-related increase and values of greater than +3.0 silver grains were achieved. The increase should also be statistically significant (comparing the test and control data using Student's t-test) and more than 50% of the treated nuclei should achieve a net silver grain rate of greater than + 3.0. Applying this criterion to the data obtained, it can be concluded that neither imidazole nor histamine induced UDS in these experiments. Positive results were obtained with the positive control treatments D M B A and U V light.

Transformation assay In the transformation assay, treatment with imidazole was toxic to the M2-C3H fibroblasts at the higher dose levels used, reducing the plating efficiency to about one third of the control value at 4 m g / m l . No transformed foci were observed at any imidazole treatment level in two independent experiments (Table 7), and it was concluded that imidazole does not induce cell transformation in this system. Marked increases in transformed foci were observed after treatment with the positive control compound M N N G .

Discussion We have examined the potential mutagenicity and tumorigenicity of imidazole and its principal metabolites in three assay systems: the Ames test, the induction of unscheduled D N A synthesis in primary rat hepatocytes, and the M2-C3H mouse fibroblast malignant transformation assay. The A m e s test was performed using the most sensitive battery of tester strains currently available (TA97, TA98, TA100 and TA102). This test was performed in both the absence and the presence of a rat liver metabolising system with imidazole and with its principal metabolites, thus providing an exhaustive and very sensitive study of potentially mutagenic pathways for the metabolism of this material. The test for unscheduled D N A synthesis was performed using primary rat hepatocytes, which are rich in xenobiotic metabolising enzymes, thus providing an additional stringent test for potentially mutagenic metabolites. Finally, the cell transformation assay was performed using M2-C3H cells, which retain an endogenous metabolising capacity (Marquardt et al., 1974). Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. Negative mutagenicity data have previously been reported for imidazole and several alkyl-substituted imidazoles using a mutant Klebsiella pneumoniae tester strain in an adaptation of the fluctuation test (Voogd et aI., 1979). It

TABLE 7 IMIDAZOLE: MALIGNANT TRANSFORMATION OF C3H MOUSE M2 FIBROBLASTS Treatment

Control Imidazole

MNNG

Concentration (/zg/ml)

Plating efficiency Expt. 1 Expt. 2

Transformed foci per treated culture Expt. 1 Expt. 2 0/5 0/7

-

28

19

100 1000 2000 4000

33 28 20 8

22 18 13 8

0/6 0/8 0/8 0/8

19 9

12 5

10/6 6/6

0.25 0.5

Plating efficiency (summary)

Transformed loci/culture (summary)

23

0/12

0/5 0/6 0/6 0/8

27 23 16 8

0/11 0/14

8/7 5/6

15 7

18/13

0/14

0/16 11/12

78

has also been shown that nitrosation of imidazole at acid pH values in the presence of nitrite, as may occur in the stomach, does not result in mutagenic nitroso derivatives (Gatehouse and Wedd, 1983). The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave entirely negative results in the Ames test. Hydantoin is occasionally confused with the anticonvulsant product phenytoin (CAS No. 5741-0, structure VI), and 'hydantoin' is sometimes used as a common name for phenytoin. For example, the syndrome of craniofacial deformity and mental defects which can afflict infants born to mothers taking phenytoin, is referred to as the 'foetal hydantoin syndrome'. It has been suggested that the teratogenic and carcinogenic properties of phenytoin (IARC, 1987) result from metabolism via reactive arene oxides on the phenyl substituen~s (Martz et al., 1977). This view has been contested and data presented to support a role for the generation of reactive radicals of the hydantoin nucleus by prostaglandin synthase (Wells et al., 1989). It has proved difficult, however, to demonstrate reproducible mutagenic activity of phenytoin. The available data have recently been summarised by McFee et al. (1992). We have not studied phenytoin in the work described here. Our negative data with hydantoin in Salmonella, including the tester strain TA102 which is a sensitive indicator of radical-induced DNA damage, argue against a mutagenic role for hydantoin radicals. The negative results obtained in the Ames test with N-acetyl-imidazole conflict with the report of Stoner et a|. (1975) that this compound displayed tumorigenic action in a short-term bioassay. These authors reported that intraperitoneal injection of N-acetyl-imidazole into strain A / H e mice at total doses of 20, 50 and 100 mg/kg resulted in increases in lung adenomas. These increases were statistically significant when compared with the vehicle control group. Histamine, which is a substituted imidazolc compound, also gave entirely negative results in the Ames test and in the UDS assay. It should be noted, however, that the metabolism of histamine differs from that of imidazole. The two principal

pathways for histamine metabolism are through (i) the action of N-methyltransferase followed by oxidation to give methylimidazolacetic acid and (ii) the action of diamine oxidase (histaminase) followed by ribose conjugation to give imidazolacetic acid riboside. The M2-C3H mouse fibroblast transformation assay is able to detect some apparently epigenetic carcinogens such as TCDD, arsenic and polychlorinated hydrocarbons. Thus, the findings obtained in the present study also suggest that imidaz01e is unlikely to act through so-called 'nongenotoxic' mechanisms of tumour induction (as for example appears to be the case with ethylenethiourea). These results do not represent an exhaustive study of imidazole and related compounds. We have not performed a comprehensive study of different genetic end-points and no assay for the induction of numerical or structural chromosomal abnormalities has been included in the present work. In the present study, however, imidazole and related compounds gave uniformly negative results in the assays performed and presented a reassuring safety profile.

Acknowledgements Our thanks are due to Eileen Waters for performing EMIC searches.

References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Gatehouse, D., and D. Wedd (1983) The bacterial mutagenicity of three naturally occurring indoles after reaction with nitrous acid, Mutation Res., 124, 35-51. IARC (1987) Monographs on the Evaluation of Carcinogenic Risks to Humans, Suppl. 7. IARC, Lyon. Maron, D.R. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Marquardt, H., F.S. Phillips and S.S. Sternberg (1976) Tumorigenicity in vivo and induction of malignant transformation and mutagenicity in cell cultures by adriamycin and daunomycin, Cancer Res., 36, 2065-2079. Marquardt, H., J.E. Sodergren, P. Sims and P.L. Grover (1974) Transformation in vitro of mouse fibroblasts by 7,12-dimethylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene and by their K-region derivatives, Int. J. Cancer, 13, 304-310.

79 Martz, F., C. Failinger and D.A. Blake (1977) Phenytoin teratogenesis: correlation between embryopathic effect and covalent binding of putative arene oxide metabolite in gestational tissue, J. Pharmacol. Exp. Ther., 203, 231-239. Matsushima, T., M. Sawamura, K. Hara and T. Sugimura (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation systems, in: F.J. de Serres et al. (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier/North Holland, Amsterdam, pp. 85-88. McFee, A.F., R.R. Tice and M.D. Shelby (1992) In vivo cytogenetic activity of diphenylhydantoin in mice, Mutation Res., 278, 61-68. Nishie, K., A.C. Waiss and A.C. Keyl (1969) Toxicity of methylimidazoles, Toxicol. Appl. Pharmacol. 14, 301-307. Stoner G.D., E.K. Weisburger and M.B. Shimkin (1975) Tumor response in strain A mice exposed to silylating corn-

pounds used for gas-liquid chromatography, J. Natl. Cancer Inst. 54, 495-497. Voogd, C.E., J.J. van der Stel and J.J.J.A.A. Jacobs (1979) Mutagenic action of nitroimidazoles. IV: Comparison of the mutagenic action of several nitroimidazoles and some imidazoles, Mutation Res., 66, 207-221. Wells, P.G., M.K. Nagai and G. Spano Greco (1989) Inhibition of trimethadione and dimethadione teratogenicity by the cycloxygenase inhibitor acetylsalicylic acid: a unifying hypothesis of the teratologic effects of hydantoin anticonvulsants and structurally related compounds, Toxicol. Appl. Pharmacol., 97, 406-414. Williams, G., M. Laspia and V. Dunkel (1982) Reliability of the hepatocyte primary culture/DNA repair test in testing coded carcinogens and non-carcinogens, Mutation Res., 97, 359-370.

Mutagenicity testing of imidazole and related compounds.

Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite ...
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