Chronic toxicity and oncogenicity studies of Macrodantin in Sprague-Dawley rats

Fd Chem. Toxic. Vol. 28, No. 4, pp. 269-277, 1990 Printed in Great Britain.All rights reserved

0278-6915/90 $3.00+ 0.00 Copyright© 1990Pergamon Press plc

CHRONIC TOXICITY AND ONCOGENICITY STUDIES OF MACRODANTIN IN SPRAGUE-DAWLEY RATS W. H. BUTLER*, T. C. GRAHAM'["and M. L. SUTTON? *The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK and tNorwich Eaton Pharmaceuticals, PO Box 191, Norwich, New York, NY 13815, USA (Received 11 September 1989; revisions received 2 January 1990)

Abstract--Chronic toxicity and oncogenicity studies of nitrofurantoin formulated as Macrodantin have been undertaken. The doses ranged from 12 to 116 mg/kg body weight/day. At 116 mg/kg/day, female rats showed a decreased weight gain. In the high-dose groups in the chronic toxicity study (males, 81 mg/kg/day; females, 116 mg/kg/day) there was an increase in testicular degeneration, sciatic nerve degeneration and fibrosis in both males and females, and an increase in focal biliary proliferation in females. There was no evidence of renal toxicity. There was no compound-related effect upon neoplasms at any site. In the oncogenicity study, an increase in focal biliary proliferation was observed in females given 31 or 56 mg/kg/day. There was no treatment-related increase in the incidence of neoplasms at any rite. In particular there was no increase in the incidence of mammary or renal tumours. The observations in these studies indicate that therapeutic uses of Macrodantin would not present a carcinogenic hazard to man.

INTRODUCTION

MATERIALS AND METHODS

Nitrofurantoin is used extensively in the treatment of urinary tract infections. It is a broad spectrum antibacterial agent active against the majority of urinary pathogens. Macrocrystals of nitrofurantoin are formulated as Macrodantin. The crystal size is designed to control the speed of absorption and results in a somewhat reduced bioavailability of the drug without reduction in efficiency. Nitrofurantoin is rapidly absorbed and is primarily excreted unchanged in the bile and urine (Conklin, 1972). Since its introduction in 1953, nitrofurantoin, a 5-nitrofuran, has been the subject of many toxicological studies, a constant feature of which has been evidence of testicular toxicity (Butler et al., 1990; M. L. Sutton, unpublished observations, 1984) and, particularly in the mouse, ovarian toxicity (Butler et al., 1990; McConnell, 1987; Tracor-Jitco Inc., 1980). Carcinogenicity studies have been reported in the rat with varying results. Wang et al. (1984), in a small study, reported an increased incidence of mammary fibroadenomas in Sprague-Dawley rats given 0.19% nitrofurantoin in the diet. However, Morris et al. (1969) using the Holtzman rat and Cohen et al. (1973) using the Sprague-Dawley rat found no carcinogenic effects. None of these studies complies with current standards of carcinogenicity testing. Nitrofurantoin was investigated in the National Toxicology Program (NTP, 1987) at levels of 0, 1300 and 2500ppm in the diet. In this study no increase in mammary neoplasms was reported but three renal tumours were observed in the top-dose group and one in low-dose male rats. The present paper reports the results of chronic toxicity and oncogenicity studies and compares them with previously reported findings.

Test material Macrodantin, macrocrystals of nitrofurantoin, was manufactured by Norwich Eaton Pharmaceuticals, Inc. (Norwich, NY, USA). The identity of the nitrofurantoin was confirmed by infra-red and nuclear magnetic spectroscopy. The concentration of the drug in the feed was confirmed by high-performance liquid chromatography. Animals Male and female Charles River Sprague-Dawley CrI:CD (SD)BR rats supplied by Charles River Breeding Laboratories (Wilmington, MA, USA) were used. The rats were about 28 days old upon arrival at Norwich Eaton Pharmaceuticals Inc. and were acclimatized to the laboratory environment for 2 wk before use. The rats were uniquely marked and individually housed in metal suspended cages. They were kept under controlled conditions of temperature (22 +__2.7°C), relative humidity (30-70%) and light (12 hr light/12 hr dark). Experimental design Chronic toxicity study. Groups of 60 male and female rats were randomly assigned to each treatment group and fed ad lib. Purina Formulab Chow 5008 (Ralston Purina Co. Inc., St Louis, MO, USA). Macrodantin was added to the diet to provide target intakes of 0, 24, 48 or 96 mg/kg body weight/day (Table 1). Individual body weights and food consumption of the rats were determined weekly for the first 3 months and then every 2 wk. Clinical pathology assays were performed on 15 male and 15 female rats from each group at 3, 6, 12, 18 and 24 months and the rats were killed at 24 months. Blood

269

270

W, H. BUTLER et aL Table I. Consumption of Macrodantin by SpragueDawley rats in chronic toxicity and oncogenicity studies Consumption of Macrodantin (mg/kg body weight/day) Achieved Group

Proposed

Oneogenieity study 0 0 12 12.4 24 22.3 48 43.1

1 2 3 4 1 2 3 4

Males

Chronic toxicity 0 24 48 96

study 0 23.5 42.8 81.3

diagnoses for neoplastic and non-neoplastic lesions were subjected to statistical analysis by the methods of Tarone (1975), Thomas et al. (1977) and Grizzle et al. (1982). One-sided Fisher exact tests were also carried out between negative control and treated groups.

Females

0 16.8 31.0 55.8 0 31.7 57.3 116.1

was obtained by retro-orbital bleeding and used for haematological and biochemical investigations. A sub-group of 25 rats/sex/treatment group was designated for biochemical investigation. Five male and five female rats from each sub-group were bled by cardiac puncture following CO2 anaesthesia and then necropsied at 1, 3, 6, 12 and 24 months. Twenty male and twenty female rats were not subjected to investigation during the course of the study. The serum from rats in the biochemistry sub-groups was assayed for prolactin (Lactiquant Kit, Nuclear Medical Laboratories, Dallas, TX, USA) by radio-immunoassay, and for serum thyroxine, free thyroxine index and triiodothyronine uptake (Solidphase Component Kit, Beckton Dickenson, Orangeburg, NY, USA). All rats were monitored daily for morbidity and mortality. The rats were palpated monthly for abnormal masses and the incidence, size and location of such masses were recorded. Complete necropsies were performed on all rats that died or became moribund or were part of a scheduled kill. All surviving rats were killed at 24 months and the liver, kidneys, heart, brain, gonads, uterus or prostate, adrenal, thyroid and pituitary were weighed. A complete range of tissues was fixed in 10% neutral buffered formalin and subsequently evaluated histologically. Oncogenicity study. Groups of 50 male and 50 female rats were randomly assigned to each treatment group and were fed ad lib. pulverized Purina Formlab Rat Chow 5008 (Ralston Purina Co. Inc.). Macrodantin was added to the diet as given in Table 1. All rats were weighed and their food consumption was determined weekly for 3 months and then once every 2 wk for the rest of the study. All rats were monitored daily for signs of toxicity and those that died or were killed when moribund were necropsied. After 24 months the study was ended and all of the surviving rats were killed and necropsied. A complete range of tissues was fixed in 10% formal saline. The tissues were processed, sectioned and stained and examined histologically.

Statistical analysis All clinico-pathological data were analysed by a one-way analysis of variance and a comparison of mean values by the Dunnett method. The histological

RESULTS

Chronic toxicity study No effects on the condition of the rats were observed during the course of the study. There were no statistically significant differences in food consumption or body-weight gain (Fig. la,b) between the control and treated rats except for a decrease in weight gain in female rats in Group 4 (96 mg/kg/day). Similarly, there were no differences in mortality between the groups except for a slight increase of mortality among males in Group 4 (Fig. 2a,b). At no time during the study was a consistent change in haematological or biochemical parameters observed that might indicate a compound-related effect (data not shown). Serum prolactin and thyroid function was investigated after 1, 3, 6, 12 and 24 months, but at no time was a consistent change observed (Table 2). The normal range of non-neoplastic changes in senile rats was observed, and those that showed an apparent treatment-related increase are listed in Table 3. These included an increased incidence of testicular degeneration in the high-dose group (Group 4). The non-neoplastic changes observed in the mammary gland are listed in Table 4. There was no increase in the number of rats with mammary tumours, the number of mammary tumours or the incidence of malignant mammary tumours (Table 5). One renal adenoma was observed among Group 4 females, two renal lipomas occurred in males in Group 2 and a renal sarcoma was observed in the control males (Group 1). The only primary ovarian neoplasm observed was an adenocarcinoma in Group 3. At all other sites the normal range of neoplasms was observed and no compoundrelated effects were observed.

Oncogenicity study No effects were observed on the condition of the rats. There were no statistically significant differences in food consumption or body-weight gain (Fig. 3a,b) or mortality (Fig. 4a,b) between the control and treated groups. The percentage of rats bearing i> 1 mammary tumours, as confirmed by histological examination, is given in Table 6. The histological types of mammary tumours are given in Table 7. There was no observable effect on incidence, time to tumour onset, or in the number of multiple tumours, nor an increase in the malignancy of the mammary tumours (Tables 6 and 7; IARC Working Group, 1980). The time to the observation of the first tumour was similar in all groups. The non-neoplastic changes observed in the mammary gland are listed in Table 4. The most common non-neoplastic lesion observed was chronic progressive nephropathy and associated lesions. However the incidence was not modified by treatment. Both male and female rats showed an

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Fig. I. Chronic toxicity study: body weights of (a) male and (b) female rats fed nitrofurantoin in the diet to provide target doses of 0 (©, Group I), 24 (O, Group 2), 48 (A, Group 3) or 96 (11, Group 4) mg/kg body weight/day. increase of fatty change in the liver in all dose groups in comparison with the controls but there was no evidence of a dose response (Table 8). In female Groups 3 and 4 there was an increase in focal biliary proliferation. This lesion is commonly observed in senile rats. DISCUSSION

A chronic toxicity study and an oncogenicity study of Macrodantin have been carded out according to

the guidelines of the National Cancer Institute (1975) and other national and international agencies (Committee on Safety of Medicines, 1979; WHO, 1969). The highest dose selected in the chronic toxicity study is about 20 times the highest recommended human dose of 5.0 mg/kg body weight/day. At this level the female rats showed a > 10% reduction in weight gain and there was an increased early mortality among male rats, indicating that a maximum tolerated dose had been achieved. In the oncogenicity study the highest dose of 48 mg/kg/day was chosen

272

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Fig. 2. Chronic toxicity study: survival curves for(a) male and (b) female rats fed nitrofurantoin in the diet to provide target doses of 0 (--, Group 1), 24 (O, Group 2), 48 (m, Group 3) or 96 (O, Group 4) mg/kg body weight/day. in order to ensure good survival throughout the study. In the chronic toxicity study there was an increased incidence of degenerative changes in the testes, while no compound-related effects were observed in the ovaries. These findings correspond to those of the

NTP (1987) study, and indicate that the ovaries of both Sprague-Dawley and F344 rats are much less susceptible to nitrofurantoin toxicity than are those of either Swiss (Butler et aL, 1990) or B6C3F~ mice (NTP, 1987). In both the chronic toxicity study and oncogenicity study reported here, the livers of female

O n c o g e n i c i t y s t u d y o f n i t r o f u r a n t o i n in r a t s

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Table 2. Summary of determinations of serum prolactin, serum thyroxine and triiodothyronine uptake in female control rats and those given Macrodantin in the chronic toxicity study Levels on treatment day: Treatment group

Parameter

15

80

170

349

709t

1

Serum prolaetin (ng/ml) T3 uptake (%) Serum T4 (#g/100 ml)

2.2 63.9 5.8

2.5 61.1 3.0

1.1 59.1 3.5

3.0 58.3 3.0

4.4 64.8 2.4

2

Serum prolactin (ng/ml) T3 uptake (%) Serum T4 (#g/100ml)

1.9 64.5 4.6*

2.3 60.5 3.3

1.3 56.7* 3.3

2.3 59.0 3.5

3.0 60.3 2.0

3

Serum prolactin (ng/ml) T3 uptake (%) Serum "1"4(#g/100 ml)

2.7 60.3* 3.8*

2.7 63.0 3.1

2.0* 57.9* 2.9

3.0 63.1" 3.4

3.4 60.8 1.1

4

Serum prolactin (ng/ml) T3 uptake (%) Serum T4 (gg/100 ml)

2.3 61.1" 3.9*

2.2 63.4 3.6

2.0* 58.8 3.5

3.2 63.3* 2.7

3.7 61.7 2.5

T3 = triiodothyronine T4 = thyroxine t O n day 709 no statistical analysis was done as too few animals remained for the results to be considered valid. Values are means for groups of five rats and those marked with asterisks differ significantly from the corresponding values for the control group (1) as determined by a one-way analysis of variance and a comparison of mean values by the Dunnett method (*P < 0.05).

Table 3. Summary of treatment-related non-neoplastic lesions observed in Sprague-Dawley rats fed Macrodantin in the chronic toxicity study No. of rats with lesion in Group*: 1 Lesion

Sex...

Liver, focal hepatocellular alteration Liver, focal biliary proliferation Stomach erosion Testicular degeneration Epididymal fibrosis Sciatic nerve: Degeneration Fibrosis

2

3

4

M

F

M

F

M

F

M

F

2 15 2 4 2

1 10 4 ---

1 17 6 8 8

3 16 4 ---

4 16 7 6 7

2 20 9 ---

0 16 6 24 22

12 22 13 ---

2 0

1 0

5 I

5 0

3 5

2 1

6 7

I1 4

*Sixty male and 60 female rats were examined in each group.

Table 4. Summary of non-neoplastic changes observed in the mammary gland of Sprague-Dawley rats fed Macrodantin in the chronic toxicity and oncogenicity studies No. of rats with lesion in Group: I Lesion

Sex...

M

2 F

M

3

4

F

M

F

M

F

No. examined... Ductal ectasia Hyperplasia Galactocoel

Chronic toxicity study 60 60 60 0 20 0 0 I 0 0 4 0

60 18 3 2

60 0 0 0

60 15 4 1

60 0 0 0

60 28 3 2

No. examined... Ductal ectasia Hyperplasia

Oncogenicity study 50 50 50 1 16 0 0 3 0

50 13 7

50 1 0

50 12 3

50 3 0

50 11 3

Table 5. Summary of mammary tumours observed in Sprague-Dawley rats fed Macrodantin in the chronic toxicity study Values for Group*: 1 Parameter

Sex...

No. of rats with mammary neoplasia Total no. of mammary neoplasms No. of malignant mammary neoplasms

2

3

4

M

F

M

F

M

F

M

F

2 2 0

21 41 11

2 2 0

17 35 14

1 1 0

23 32 11

0 0 0

17 20 3

*Sixty male and 60 female rats were examined in each Group.

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Fig. 3. Oncogenicity study: body weights of (a) male and (b) female rats fed nitrofurantoin in the diet to provide target doses of 0 (O, Group 1), 12 (O, Group 2), 24 (A, Group 3) or 48 (ll, Group 4) mg/kg body weight/day. rats showed an increase in focal biliary proliferation. This lesion is common in senile rats. However, the increased severity of the effect may be related to the route of excretion of the nitrofurantoin through the biliary system (Conklin, 1972). Both male and female rats in the highest dose group of the chronic toxicity study showed an increase in sciatic nerve demyelination and fibrosis (Table 3). Again this lesion is observed in senile rats but the increase confirms previous findings from studies in the rat (Bagar et al., 1977).

Following the observations of Wang et al. (1989) of an increased incidence of mammary fibroadenomas in rats fed nitrofurantoin, particular attention was paid to such lesions in the present studies. During the chronic toxicity study prolactin levels were assayed and thyroid function studies were conducted. In none of these investigations was there evidence of any endocrine perturbation. A detailed examination of the mammary tumours observed in both the chronic toxicity and the oncogenicity study failed to show any effect upon mammary neoplasms. These findings

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Fig. 4. Oncogenicity study: survival curves for (a) male and (b) female rats fed nitrofurantoin in the diet to provide target doses of 0 (--, Group I), 12 (0, Group 2), 24 (11, Group 3) or 48 (O, Group 4) mg/kg body weight/day. confirm the earlier results of Morris et al. (1969) and Cohen et al. (1973) which indicated that nitrofurantoin does not increase the incidence of such neoplasms. More recent results, in a study that

conformed to current guidelines (NTP, 1987), failed to demonstrate an increase in mammary plasia in Fischer F344 rats fed nitrofurantoin at levels of up to l l0mg/kg body weight/day.

also neodose The

276

W . H . Btrrl.~.a et al. Table 6. Summary of histologically diagnosed neoplasms in the oncogenicity study of Macrodantin in Sprague-Dawley rats Values for rats in Group: 1

Parameter

Sex...

2

3

4

M

F

M

F

M

F

M

F

No. of rats examined histologically No. of rats with neoplasms Percentage of rats with neoplasms

50 39 78.0

50 48 96.0

50 38 76.0

50 45 90.0

50 32 64.0

50 47 94.0

50 28 56.0

50 44 88.0

Total no. of neoplasms No. of malignant neoplasms No. of malignant with metastases Percentage of malignant neoplasms

54 9 3 16.7

79 5 2 6.3

52 9 l 17.3

82 7 0 8.5

51 3 2 5.9

78 lO 1 12.8

47 9 3 19.1

71 4 l 5.6

0 0 0 0 0 0

17 34.0 27 2 0 7.4

0 0 0 0 0 0

17 34.0 28 3 0 10.7

2 4.0 4 0 0 0

17 34.0 24 7 0 29.2

1 2.0 1 0 0 0

14 28.0 22 1 0 4.5

7 14.0

5 10.0

8 16.0

7 14.0

3 6.0

8 16.0

9 18.0

4 8.0

17.9

10.4

21.1

15.6

9.4

17.0

32.1

9.1

No. of rats with mammary neoplasms Percentage of rats with mammary neoplasms Total no. of mammary neoplasms No. of malignant mammary neoplasms No. of malignant turnouts with metastases Percentage of malignant mammary neoplasms No. of rats with malignant neoplasms Percentage of total number rats with malignant neoplasms Percentage of rats with malignant neoplasms v. number of rats with neoplasms

Table 7. Summary of histological type of mammary tumour in rats fed Macrodantin in the oncogenicity study No. of rats affected in Group*: 1

Type of mammary tumour

Sex...

2

3

4

M

F

M

F

M

F

M

F

0 0 0 0

12 7 3 2

0 0 0 0

11 6 4 3

2 0 0 0

10 5 0 6

1 0 0 0

10 5 0 1

Fibroadenoma Adenoma Cystadenoma Adenocarcinoma

*Fifty male and 50 female rats were examined in each Group.

two out of 50 rats in the low-dose group and one out of 50 high-dose rats. In a review of the NTP study the slides were examined (W. H. Butler) and focal proximal tubular hyperplasia was found in six out of control, six out of 50 low-dose and seven out of 50 high-close male groups. In addition to the focal hyperplasia, one tubular adenoma in the low-dose males and two in the high-dose males were confirmed. An adenoma carcinoma was also confirmed in the high-dose group. Tubular adenomas are uncommon in both the Sprague-Dawley and F344 rat and their association with chronic progressive nephropathy is uncertain. Large lesions such as the reported adenocarcinoma (NTP, 1987) do not present a problem in diagnosis, but focal hyperplasias do present problems of differential diagnosis between hyperplasia and adenoma (Hard, 1987). Such lesions possibly present a continuous spectrum of change making any division arbitary. In view of the uncertainty of the diagnosis of small adenomas, the lack of statistical significance and the low incidence within the overall historical range such a finding does not suggest a carcinogenic

studies reported here suggest that the observations of Wang et al. (1984) reflect the small numbers of rats used (group size 11-12), and do not demonstrate an oncogenic effect of nitrofurantoin. Nitrofurantoin is excreted unchanged through the urinary system (Conklin, 1972). In an oncogenicity study in mice (Butler et al., 1990) there was evidence of increased severity of the chronic progressive nephropathy that is common in ageing mice, but no evidence of a renal oncogenic effect. In contrast, neither of the studies reported here provide evidence of an increased severity of chronic progressive nephropathy in Sprague-Dawley rats at doses of up to 116 mg/kg/day. Neither was there any evidence of primary renal neoplasia. In the NTP carcinogenicity study using the Fischer F344 rat (NTP, 1987) there was an increased severity of chronic progressive nephropathy in males given 60 or 110 mg nitrofurantoin/kg body weight/day. This effect was not observed in the females. In association with the nephropathy, regeneration of the tubular epithelium was reported and hyperplasia of the epithelium was reported in two out of 50 controls,

Table 8. Summary of hepatic changes in rats fed Macrodantin in the oncogenicity study No. of rats affected in Group*: 1

Hepatic change Fatty change Biliary proliferation

Sex...

2

3

4

M

F

M

F

M

F

M

F

13 13

11 9

44 17

37 7

33 18

28 18

41 12

33 17

*Fifty male and 50 female rats were examined in each Group.

Oncogenicity study of nitrofurantoin in rats effect of nitrofurantoin on the kidney of the F344 rat. Nitrofurantoin is possibly more toxic to the kidney of the F344 rat than to that of the Sprague-Dawley rat. However at comparable doses there is no evidence of renal neoplasia in the rats used in the present studies, lending weight to the view that the lesions observed in the F344 rat are not c o m p o u n d related. F r o m the results of the present studies in the Sprague-Dawley rat and a review of studies in Fischer F344 and Holtzman (Morris et al., 1969) rats, nitrofurantoin does result in toxicity at a number of sites but, under the conditions of the study, does not have an oncogenic effect. Acknowledgements--The authors wish to thank the staff of the National Toxicology Program for their continued help and co-operation, and also the staff of the Environmental Pathology Laboratories Archives for their assistance in the course of the reviews of the studies. REFERENCES

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Grizzle J. E., Starmer C. F. and Koch G. G. (1969) Analysis of categorical data by linear models. Biometrics 25, 489-504. Hard G. C. (1987) Chemically induced epithelial tumours and carcinogenesis of the renal parenchyma. In Nephrotoxicity in the Experimental and Clinical Situation. Edited by P. H. Bach and E. A. Lock. pp. 211-250. IARC Working Group (1980) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Suppl. 2. Long-term and Short-term Screening Assays for Carcinogens: A Critical Appraisal. International Agency for Research on Cancer, Lyon. McConnell R. F. (1987) The Procter and Gamble Company Pilot feeding study in mice with NF-153: 4, 8, 12 and 16 Week Interim Sacrifice. Histopathology Report. Project no. 270.09.00-ks. Procter and Gamble Co., Cincinnati, OH, USA. Morris J. E., Price J. M., Lalich J. J. and Stein R. S. (1969) The carcinogenic activity of some 5-nitrofuran derivatives in the rat. Cancer Res. 29, 2145-2156. National Cancer Institute (1975) Guidelines for Carcinogen Bioassay in Small Rodents. Carcinogens Division of Cancer Cause and Prevention, National Cancer Institute, National Institute of Health, Bethesda, MD, USA. National Toxicology Program (1987) NTP Technical Report no. 341. Nitrofurantoin. Tarone R. E. (1975) Tests for trends in life table analysis. Biometrika 62, 679-682. Thomas D. G., Breslow N. and Gart J. J. (1977) Trend and homogeneity analysis of proportions and life table data. Comput. biomed. Res. 10, 373-381. Tracor-Jitco Inc. (1980) Subchronic Toxicity Report on Nitrofurantoin (C55 196) in Fischer 344 Rats and B6C3F1 Mice. Final Report of Study Carried Out at Southern Research Institute, Alabama, USA. Wang C. Y., Croft N. A. and Bryan G. T. (1984) Tumor production in germ-free rats fed 5-nitrofurans. Cancer Lett. 21, 303-308. WHO (1969) Principles for Testing and Evaluation of Drugs for Carcinogenicity. WHO Tech. Rep. Series no. 426. WHO, Geneva.