Fd Chem. Toxic. Vol. 30, No. I0, pp. 837-842, 1992 Printed in Great Britain.All rights reserved
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
LACK OF CARCINOGENICITY OF FERRIC CHLORIDE IN F344 RATS M. SATO, F. FURUKAWA,K. TOYODA, K. MITSUMORI, A. NISHIKAWAand M. TAKAHASm Division of Pathology, National Institute of Hygienic Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158, Japan
(Accepted 12 June 1992) Al~ract--The carcinogenicityof ferric chloride, a compound that is used as a food additive, a haemostatic or treatment for hypochromic anaemia, was examined in F344 rats of both sexes. It was dissolved in distilled water at levels of 0, 0.25 or 0.5%, and groups of 50 male and 50 female rats were given one of these solutions ad I/b. as their drinking water for up to 2 yr. The mean body weights of the treated rats were lower than control group values for both males and females. A variety of tumours developed in all groups, including the control group, but all these neoplasms were histologically similar to those known to occur spontaneously in this strain of rats, and no statistically significant increase in the incidence of any tumour was found in the treated groups of either sex. Thus it is concluded that under the conditions of this experiment, ferric chloride exerts no carcinogenic potential in F344 rats.
INTRODUCrION
MATERIALSAND METHODS
Ferric chloride (FeCI3' 6H20) is used in Japan and throughout the world as a food additive to modified milk powder, as well as a haemostatic or treatment for hypochromic anaemia. Ferric chloride crystals are yellowish-brown, readily soluble in water and deliquesce in ambient air. In an earlier acute toxicity study, the LDs0 values for ferric chloride were reported to be 1872 mg/kg (oral) in rats and 895 mg/kg (oral) and 58 mg/kg (iv) in mice (National Institute for Occupational Safety and Health, 1987). In a series of mutagenicity tests including Ames tests with TAI00, TA98, TA2637 or TA94 and a chromosomal aberration test using a Chinese hamster fibroblast cell line, negative results were obtained (Ishidate et aL, 1983). In recent years it has been suggested from human epidemiological studies that increased iron stores in the body are associated with an increased risk of cancer (Stevens et al., 1988; Weinberg, 1984). Furthermore, co-carcinogenic effects of iron have been reported with regard to the colon of mice initiated with dimethylhydrazine, the respiratory tract of hamsters treated with benzo[a]pyrene and the liver of mice that received a single dose of iron--dextran complex and were then fed hexachlorobenzene in the diet (Siegers et al., 1988; Smith et al., 1989; Stenb,~ck and Rowland, 1978). However, as yet, no adequate long-term carcinogenicity study of this chemical has been carried out under the international standard guidelines. The study described here was therefore performed in order to clarify whether ferric chloride has any carcinogenic effects in rats.
Subchronic toxicity study In order to determine appropriate dose levels for the long-term carcinogenicity study of ferric chloride, a subehronic toxicity study was carried out. A total of 120 specific-pathogen-free Fischer (F344) rats (5 wk old) of both sexes were purchased from Charles River Japan Inc. (Kanagawa, Japan). Ferric chloride (purity at least 98.5%) was purchased from Yoneyama Chemical Co. Ltd (Osaka, Japan) and dissolved in distilled water at concentrations of 0 (control), 0.12, 0.25, 0.5, 1.0 or 2.0% (w/v). Before animal testing, it was confirmed that a 2.0% solution of ferric chloride in distilled water was stable for 1 wk at room temperature. When the rats were 6 wk old, they were divided into six groups, each consisting of 10 males and 10 females. Each group was given one of these solutions as drinking water ad lib. for 13 wk. Ferric chloride solutions were freshly prepared twice a week. Throughout the experimental period, all rats were given a basal diet (CRF-I, Oriental Yeast Inc., Tokyo, Japan) ad lib. All rats were observed daily and clinical signs and deaths were recorded. Body weights were measured once a week. At the end of the study, all survivors were killed for haematological, blood biochemical and pathological examinations, and all major organs and tissues were taken for microscopic examination.
2-year carcinogenicity study F344 rats (5 wk old) of both sexes, purchased from Charles River Japan Inc., were maintained on basal diet (CRF-I) and tap water until they were 6 wk old, when the study was started. The ferric chloride used
837
M. SATOet al.
838
was the same lot as that used in the subchronic toxicity study. Rats were housed three to four males or five females to a plastic cage and maintained in an air-conditioned barrier-system animal room (temperature 24 + I°C and relative humidity 55 + 5%). The rats were randomly divided into three groups, each consisting of 50 males and 50 females. Ferric chloride was dissolved in distilled water at levels of 0 (control), 0.25 or 0.5% (w/v). These dose levels were selected on the basis of the results from the subehronic toxicity study mentioned above. The pH values of the test solutions containing 0, 0.25 and 0.5% ferric chloride were determined to be 6.03, 2.38 and 2.19, respectively. The rats were given the solution ad lib. as their drinking water for 2 yr. Ferric chloride solutions were freshly prepared twice a week, and the amount of solution consumed was measured for the calculation of ferric chloride intake. Control animals were given distilled water without ferric chloride in the same way. Administration of ferric chloride was stopped after 104 wk and the rats were then given distilled water for a recovery period of 8 wk. At wk 112, all surviving rats were killed and examined. Throughout the experiment, rats in all groups were given basal diet ad lib. They were observed daily and clinical signs and deaths were recorded. Body weights were measured once a week for the first 13 wk of the study and then once every 4wk. All rats that died or were killed in extremis during the study and those killed at termination were subjected to a full post-mortem examination and investigated macroand microscopically for the occurrence of neoplastic and non-neoplastic lesions. All organs, tissues and gross lesions were routinely fixed with 10% buffered formalin, sectioned, and stained with haematoxylin and eosin or Berlin blue. Body weight and the daily water intake data were analysed statistically using Student's t-test. The survival times were analysed using the generalized Wilcoxon test. The incidences of tumours were analysed statistically by Fisher's exact probability test. RESULTS
Subchronic toxicity study None of the rats in any of the groups died during the 13-wk period of the study. Figure 1 shows the growth curves for rats in each group. In both sexes given the 2.0 and 1.0% doses, a depression of body weight gain of at least 10% compared with the control group was observed at termination. A significant suppression in the intake of drinking water was observed in the groups given doses of 0.5% or more. The haematology and serum chemistry data demonstrated a higher level of serum iron in male treated groups. The level ~ug/dl) was 102 + 5 in the control group, 107 + 11 in the 0.12% group, 109 + 8 in the 0.25*/, group, 129 + 22 in the 0.5% group, 139 + 10 in the 1.0% group and 156 in the 2.0%
30O
A
0.5% 1.0%
• •
1
2
3
4
5
6
7
S
9
2.0%
10 II 12 13 Weeks
2111)
.~150
• • 0
I
2
3
4
5
6
7
8
9
1.0% 2.0%
10 I1
12 13 Weeks
Fig. 1. Mean body weights of male and female F344 rats given ferric chloride at 0 (A), 0.12 (I--I),0.25 (O), 0.5 (A), !.0 ( I ) or 2.0% (O) in the drinking water for 13 wk.
group. The counts of red blood cells in male treated groups were also significantly increased, compared with the control value. In histological examinations on the sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the ,esophageal mucosa in the groups given doses of 0.25% or more and in the laminae propriae of the large intestine in the 2.0% group. In sections stained with Berlin blue, increased numbers of positive pigments were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney, and the adipose tissues of the groups given doses of 0.25% or more, in addition to the organs described above. The intensity of staining was marked in the intestine and liver. From the results of this subchronic toxicity study, it was concluded that the probable maximum tolerated dose of ferric chloride in the drinking water would be 0.5% in both sexes. Therefore, 0.5 and 0.25% were selected as appropriate dose levels for the long-term carcinogenicity study.
2-year carcinogenicity study Figure 2 shows the growth curves in each group. The mean body weights of the treated males and females were significantly lower than those of the
Carcinogenicity tests of ferric chloride
839
500 MALE 400
.~ 3oo '~
FEMALE
] 0
=
o. 5%
;
0.5%
t 0
20
40
60 WEEKS
80
100
120
Fig. 2. Mean body weights of male and female F344 rats given ferric chloride at 0 (©), 0.25 (11) or 0.5% ( 0 ) in the drinking water for 2 yr.
corresponding control groups. Data for final body weight, drinking water intake, ferric chloride intake, final survival rate and mean survival time are shown in Table 1. The final body weight and the mean daily water intake of the treated groups were significantly lower than those of the control groups. The mean daily ferric chloride intakes in the 0.25 and 0.5% groups calculated from the water intake were, respectively, 169.7 and 319.7 mg/kg body weight/day in males, and 187.9 and 336.0 mg/kg body weight/day in females. The cumulative mortality at termination in males of the 0.5% group was significantly decreased compared with the control value. The sites, histological types and incidence of tumours found in each group of both sexes are summarized in Table 2. The first autopsy was performed at wk 49, when a female rat in the 0.5% group was killed in extremis because of a malignant fibrous histiocytoma. This rat and all of the animals that survived beyond this week were included in the effective numbers, with the exception of rats for
which histopathological examinations could not be performed owing to advanced autolysis. There were no statistically significant differences in the overall tumour incidence between control and treated groups of either sex. Tumours were found in many organs or tissues in all groups, including the control group. In males of all groups, tumours of the testis were most frequent, followed by neoplasms of the haematopoietic organs, adrenal gland, mammary gland, thyroid, pituitary and pancreas. In females, tumours of the uterus, pituitary, haematopoietic organs, mammary gland, adrenal gland and thyroid were common. Tumours were also detected in the other organs or tissues in all groups of both sexes, but the incidence was very low. None of the treated groups demonstrated a significant increase in the incidence of any specific tumour over that in the corresponding control group. All tumours observed in this study were similar to those that are known to occur spontaneously in this strain of rats (Goodman et al., 1979; Haseman et al., 1990; Solleveld et aL, 1984).
Table I. Final body weight, intake of drinking water and chemical, and survival time in F344 rats given ferric chloride in the drinking water for 2 yr Average daily intake
Group Male Control 0.25% 0.5% Female Control 0.25% 0.5%
Final body weight (mean :t: SD)
Drinking water (g/kg body weight)
Chemical (mg/kg body weight)
Final survival rate (%)
Mean survival time and range (wk)
458.4 _+40 431.0 _.+35* 430.6 + 24*
95.8 67.4* 63.2*
0 169.7 319.7
62 54 82*
107.0 (69-112) 104.5 (60-I 12) 109.3 (64-112)
317.6 _4-33 287.3 + 29* 271.1 + 24*
105.0 73.6* 67.2*
0 187.9 336.0
72 56 62
106.5 (50- I 12) 106.9 (73- I 12) 105.6 (49-112)
*Significantly different from control value at P < 0.05.
840
M. S^ro et al. Table 2. Incidence, sites and types of turnouts in F344 rats given ferric chloride in the drinking water for 2 yr No. of rats with tumours Dose (%) ...
0
Males 0.25
Adenoma C-cell adenoma C-cell carcinoma Follicular cell adenoma Follicular cell carcinoma Phaeochromocytoma Malignant phaeochromocytoma Ganglioneuroma Schwannoma Cortical adenoma Islet cell adenoma Islet cell carcinoma Acinar cell adenoma Interstitial cell tumour Adenoma Endometrial stromal polyp Carcinoma Endometrial stromal sarcoma Granulosa/theca cell tumour Fibroma Fibroadenoma Adenoma Carcinoma Large granular lymphocytic leukaemia Malignant lymphoma Malignant lymphoma Histiocytic sarcoma Adenoma Adenocarcinoma Adenosquamous cell carcinoma Adenocarcinoma Leiomyosarcoma Adenocarcinoma Neoplastic nodule Histiocytic sarcoma Nephroblastoma Granular cell tumour Glioma Sehwannoma Squamous cell papilloma Squamous cell carcinoma Trichoepithelioma Sebaceous adenoma Sebaceous carcinoma Preputial/clitoral adenoma Preputial/clitoral carcinoma Rhabdomyosarcoma Malignant fibrous histiocytoma Mesothelioma Osteosarcoma Squamous cell carcinoma Rhabdomyosarcoma
7 6 0 I 0 I0 1 1 0 0 5 2 2 48 I ---
6 7 1 0 0 7 0 0 0 0 5 I 1 46 I ---
Site and type of tumour Pituitary Thyroid
Adrenal
Pancreas Testis Prostate Uterus Ovary Mammary gland
Spleen Lymph node Bone marrow Lung Small i n t e s t i n e Large i n t e s t i n e Liver Kidney Brain Peripheral n e r v e Skin
Subcutis Abdominal c a v i t y Undetermined origin
Effective no. of rats No. of rats with tumours
In a d d i t i o n to these t u m o u r s , v a r i o u s types o f n o n - n e o p l a s t i c lesions were o b s e r v e d in e a c h group. Age-related chronic n e p h r o p a t h y was present in all g r o u p s . T e s t i c u l a r a t r o p h y , a c c o m p a n i e d by o l i g o s p e r m i a o r a s p e r m i a , w a s seen in a l m o s t all m a l e r a t s o f all g r o u p s . D u c t u l a r p r o l i f e r a t i o n a n d altered cell foci, w h i c h are k n o w n to be c o m m o n in a g e i n g F344 rats ( B u r e k , 1978; Eustis et al., 1990), were o b s e r v e d in the livers o f b o t h c o n t r o l a n d t r e a t e d rats. I n sections stained w i t h Berlin blue, a l t h o u g h slight s t a i n i n g w a s seen in v a r i o u s o r g a n s / t i s s u e s f r o m t r e a t e d a n d c o n t r o l rats, n o m a r k e d increase in p i g m e n t d e p o s i t i o n w a s o b s e r v e d in a n y o r g a n o r tissue o f the 0 . 5 % g r o u p . T h e r e w e r e n o specific
--
--
Females 0.5
0
0.25
0.5
5 5 2 1 0 I0 3 0 I 0 3 3 0 48 1 ---
12 4
16 I
15 I
-7 1 0 1 I0 I I 2 6 0 1 I 2 0 2 I 0 0 0 1 0 0 0 0 0 3 I 0 0 3 I 0 0
-6 0 1 0 10 0 0 0 2 0 0 2 0 0 3 0 0 I I 0 0 0 I 0 0 0 0 0 I 2 0 0 0
50 50
48 47
49 49
0
0
0
0
I
0
0
4 0 0 0
I 0 0 0
6 0 0 0
I
0
0
0 0 0
0 0 0
2 0 0
14 4
11 4
15 5
I I
I 0
1 0
0 4 2 0 8 0 0 0
2 2 0 0 I1 0 0 0
I 2 0 2 4 0 0 0
--
-9 4 2 0 I0 0 0 0 1 1 0 1 0 I 2 0 0 0 1 1 I l 0 0 0 4 0 0 l 2 0 0 0
I
0
I
I
2
0 0 0 0 0 2 0
0 0 I 0 0 0 0
0 0 0 0 0 I 0
1
0
0
0 0 0 0 0 0 0 0
0 1 2 0 0 0 1 I
0 0 1 0 0 0 0 0
1
0
0
0 0 2 0 0 0 0
1 0 I 0 0 0 1
0 I 1 0 0 I 0
50
49 39
40
48 42
lesions c o n s i d e r e d to be a t t r i b u t a b l e to ferric c h l o r i d e treatment. DISCUSSION I n the c a r c i n o g e n i c i t y s t u d y described here, a l t h o u g h a variety o f t u m o u r s were detected in all g r o u p s , including the c o n t r o l g r o u p , their o r g a n d i s t r i b u t i o n a n d histological types were essentially similar to t h o s e o f the s p o n t a n e o u s t u m o u r s described p r e v i o u s l y ( G o o d m a n e t a / . , 1979; H a s e m a n et al., 1990; Solleveld et al., 1984). T h e r e were n o significant increases in the incidence o f lesions in a n y o r g a n o r tissue t h a t c o u l d be a t t r i b u t e d to the t r e a t m e n t w i t h ferric chloride.
Carcinogenicity tests of ferric chloride In humans, epidemiological studies have indicated that there is a relationship between an increase in organ or tissue iron concentration and an increased risk of cancer (Stevens et al., 1988; Weinberg, 1984). Iron is thought to catalyse the formation, from superoxide anions and hydrogen peroxide, of highly reactive species (e.g. hydroxyl radicals or reactive iron-oxygen complexes) that can subsequently react with chromosomes, damage DNA and result in carcinogenesis (Aust et al., 1985; Cerutti, 1985; Halliwell and Gutteridge, 1988). In addition, iron is known to have a major role in the initiation and propagation of the enzymatic lipid peroxidation reaction (Minotti and Aust, 1987), and dietary iron overloading increases lipid peroxidation in vivo ( W u et al., 1990; Younes et al., 1990). It has also been reported that orally administered ferric chloride induces nuclear aberrations in the stomach and colon of mice (Bianchini et al., 1988), and co-carcinogenic effects of iron were observed in some studies involving mice and hamsters, using two-stage initiation/ promotion carcinogenesis models (Siegers et al., 1988; Stenb/ick and Rowland, 1978). From the positive results of Berlin blue staining in many organs and the increased serum iron levels in the treated groups in the study of the subchronic toxicity of ferric chloride, it had been predicted initially that the condition of iron overload would be maintained by administration of 0.5% ferric chloride for 2 yr. However, iron deposition was not increased in any organ/tissue of the high dose group compared with the control group at the termination of the present carcinogenicity study. Park et al. (1987) reported that the chronic iron overload produced by dietary supplementation with 2.5% carbonyl iron for 1 yr resulted in an accumulation of large amounts of iron in many tissues, associated with hepatocellular damage and fibrosis leading to cirrhosis. It is considered, therefore, that the level of iron overload was too low to increase lipid peroxidation in the study described here, although no quantitative assessment of lipid peroxidation was performed. The lack of any toxic lesions in the high dose group, despite lowered body weights, suggests that the dose levels selected may possibly have been too low for the carcinogenicity study. However, the design and conduct of the present ferric chloride bioassay was in accordance with recent guidelines for carcinogenicity tests and the evaluation of resultant data (National Cancer Institute, 1976; National Toxicology Program, 1984; Odashima, 1980). The highest dose currently recommended is that which, when given for the duration of the chronic study, is just high enough to elicit signs of minimal toxicity without significantly altering the normal lifespan of the animal through toxic effects other than carcinogenicity. In this study, the maximal tolerated dose was based on the weight-gain decrement observed in the subchronic study: since the highest dose in carcinogenicity studies should not cause more than a 10%
841
reduction in weight gain, a dose level of 0.5% was selected as the maximal tolerated dose. From the above results, it was concluded that ferric chloride exerts no carcinogenic activity in F344 rats when administered as a 0.25 or 0.5% solution continuously in the drinking water for up to 2 yr. REFERENCES
Aust S. D., Morehouse L. A. and Thomas C. E. (1985) Role of metals in oxygen radical reactions. Journal of Free Radicals in Biology and Medicine 1, 3-25. Bianchini F., Caderni G., Dolara P. and Tanganelli E. (1988) Nuclear aberrations and micronuclei induction in the digestive tract of mice treated with different iron salts. Journal of Applied Toxicology 8, 179-183. Burek J. D. (1978) Pathology of Aging Rats. Edited by J. D. Burek. pp. 58-70. CRC Press, Boca Raton, FL. Cerutti P. A. (1985) Prooxidant states and tumor promotion. Science 227, 375-381. Eustis S. L., Boorman G. A., Harada T. and Popp J. A. (1990) Liver. In Pathology of the Fischer Rat. Edited by G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, Jr and W. F. MacKenzie. pp. 71-94. Academic Press, San Diego, CA. Goodman D. G., Ward J. M., Squire R. A., Chu K. C. and Linhart M. S. (1979) Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicology and Applied Pharmacology 48, 237-248. Halliwell B. and Gutteridge J. M. C. (1988) Free radicals and antioxidant protection: mechanisms and significancein toxicology and disease. Human Toxicology 7, 7-13. Haseman J. K., Arnold J. and Eustis S. L. (1990) Tumor incidences in Fischer 344 Rats: NTP historical data. In Pathology of the FLwher Rat. Edited by G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, Jr and W. F. MacKenzie. pp. 555-564. Academic Press, San Diego, CA. Ishidate M., Jr, Yoshikawa K. and Sofuni T. (1983) Mutagenicity tests of food additives. Toxicology Forum 6, 671~178. (In Japanese). Minotti G. and Aust S. D. (1987) The role of iron in the initiation of lipid peroxidation. Chemistry and Physics of Lipids 44, 191-208. National Cancer Institute (1976) Guidelines for Carcinogen Bioassay in Small Rodents (Carcinogenesis Technical Report Series 1). National Institute of Health, Betbesda, MD. National Institute for Occupational Safety and Health (NIOSH) (1987) Registry of Toxic Effects of Chemical Substances (RTECS) Edited by D. V. Sweet. 3. pp. 2397. US Government Printing Office, Washington, DC. National Toxicology Program (1984) Report of the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation. Board of Scientific Counselors, National Toxicology Program, DHHS. Odashima S. (1980) Cooperative program on long-term assays for carcinogenicity in Japan. In Molecular and Cellular Aspects of Carcinogen Screening Tests. IARC Scientific Publication no. 27. Edited by R. Montesano, H. Bartsch and L. Tomatis. p. 315. International Agency for Research on Cancer, Lyon. Park C. H., Bacon B. R., Brittenham G. M. and Tavill A. S. (1987) Pathology of dietary carbonyl iron overload in rats. Laboratory Investigation 57, 555-563. Siegers C. P., Bumann D., Baretton C. and Younes M. (1988) Dietary iron enhances the tumor rate in dimethylhydrazine-induced colon carcinogenesis in mice. Cancer Letters 41, 251-256.
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Smith A. G., Cabral J. R. P., Carthew P., Francis J. E. and Manson M. M. (1989) Carcinogenicity of iron in conjunction with a chlorinated environmental chemical, hexachlorobenzene, in C57BL/10ScSn mice. International Journal of Cancer 43, 492--496. Solleveld H. A., Haseman J. K. and McConnel E. E. (1984) Natural history of body weight gain, survival, and neoplasia in the F344 rat. Journal of the National Cancer Institute 72, 929-940. Stenb~ck F. and Rowland J. (1978) Carcinogenic activation of benzo(a)pyrene by iodine and ferric chloride in the respiratory tract of Syrian golden hamsters. Experientia 34, 1065-1066. Stevens R. G., Jones Y., Micozzi M. S. and Taylor P. R.
(1988) Body iron stores and the risk of cancer. New England Journal of Medicine 319, 1047-1052. Weinberg E. D. (1984) Iron withholding: a defense against infection and neoplasia. Physiological Reviews 64, 65-102. Wu W., Meydani M., Meydani S. N., Burklund P. M., Blumberg J. B. and Munro H. N. (1990) Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats. Journal of Nutrition 120, 280-289. Younes M., Trepkau H. D. and Siegers C. P. (1990) Enhancement by dietary iron of lipid peroxidation in mouse colon. Research Communications in Chemical Pathology and Pharmacology 70, 349-354.
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
LACK OF CARCINOGENICITY OF FERRIC CHLORIDE IN F344 RATS M. SATO, F. FURUKAWA,K. TOYODA, K. MITSUMORI, A. NISHIKAWAand M. TAKAHASm Division of Pathology, National Institute of Hygienic Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158, Japan
(Accepted 12 June 1992) Al~ract--The carcinogenicityof ferric chloride, a compound that is used as a food additive, a haemostatic or treatment for hypochromic anaemia, was examined in F344 rats of both sexes. It was dissolved in distilled water at levels of 0, 0.25 or 0.5%, and groups of 50 male and 50 female rats were given one of these solutions ad I/b. as their drinking water for up to 2 yr. The mean body weights of the treated rats were lower than control group values for both males and females. A variety of tumours developed in all groups, including the control group, but all these neoplasms were histologically similar to those known to occur spontaneously in this strain of rats, and no statistically significant increase in the incidence of any tumour was found in the treated groups of either sex. Thus it is concluded that under the conditions of this experiment, ferric chloride exerts no carcinogenic potential in F344 rats.
INTRODUCrION
MATERIALSAND METHODS
Ferric chloride (FeCI3' 6H20) is used in Japan and throughout the world as a food additive to modified milk powder, as well as a haemostatic or treatment for hypochromic anaemia. Ferric chloride crystals are yellowish-brown, readily soluble in water and deliquesce in ambient air. In an earlier acute toxicity study, the LDs0 values for ferric chloride were reported to be 1872 mg/kg (oral) in rats and 895 mg/kg (oral) and 58 mg/kg (iv) in mice (National Institute for Occupational Safety and Health, 1987). In a series of mutagenicity tests including Ames tests with TAI00, TA98, TA2637 or TA94 and a chromosomal aberration test using a Chinese hamster fibroblast cell line, negative results were obtained (Ishidate et aL, 1983). In recent years it has been suggested from human epidemiological studies that increased iron stores in the body are associated with an increased risk of cancer (Stevens et al., 1988; Weinberg, 1984). Furthermore, co-carcinogenic effects of iron have been reported with regard to the colon of mice initiated with dimethylhydrazine, the respiratory tract of hamsters treated with benzo[a]pyrene and the liver of mice that received a single dose of iron--dextran complex and were then fed hexachlorobenzene in the diet (Siegers et al., 1988; Smith et al., 1989; Stenb,~ck and Rowland, 1978). However, as yet, no adequate long-term carcinogenicity study of this chemical has been carried out under the international standard guidelines. The study described here was therefore performed in order to clarify whether ferric chloride has any carcinogenic effects in rats.
Subchronic toxicity study In order to determine appropriate dose levels for the long-term carcinogenicity study of ferric chloride, a subehronic toxicity study was carried out. A total of 120 specific-pathogen-free Fischer (F344) rats (5 wk old) of both sexes were purchased from Charles River Japan Inc. (Kanagawa, Japan). Ferric chloride (purity at least 98.5%) was purchased from Yoneyama Chemical Co. Ltd (Osaka, Japan) and dissolved in distilled water at concentrations of 0 (control), 0.12, 0.25, 0.5, 1.0 or 2.0% (w/v). Before animal testing, it was confirmed that a 2.0% solution of ferric chloride in distilled water was stable for 1 wk at room temperature. When the rats were 6 wk old, they were divided into six groups, each consisting of 10 males and 10 females. Each group was given one of these solutions as drinking water ad lib. for 13 wk. Ferric chloride solutions were freshly prepared twice a week. Throughout the experimental period, all rats were given a basal diet (CRF-I, Oriental Yeast Inc., Tokyo, Japan) ad lib. All rats were observed daily and clinical signs and deaths were recorded. Body weights were measured once a week. At the end of the study, all survivors were killed for haematological, blood biochemical and pathological examinations, and all major organs and tissues were taken for microscopic examination.
2-year carcinogenicity study F344 rats (5 wk old) of both sexes, purchased from Charles River Japan Inc., were maintained on basal diet (CRF-I) and tap water until they were 6 wk old, when the study was started. The ferric chloride used
837
M. SATOet al.
838
was the same lot as that used in the subchronic toxicity study. Rats were housed three to four males or five females to a plastic cage and maintained in an air-conditioned barrier-system animal room (temperature 24 + I°C and relative humidity 55 + 5%). The rats were randomly divided into three groups, each consisting of 50 males and 50 females. Ferric chloride was dissolved in distilled water at levels of 0 (control), 0.25 or 0.5% (w/v). These dose levels were selected on the basis of the results from the subehronic toxicity study mentioned above. The pH values of the test solutions containing 0, 0.25 and 0.5% ferric chloride were determined to be 6.03, 2.38 and 2.19, respectively. The rats were given the solution ad lib. as their drinking water for 2 yr. Ferric chloride solutions were freshly prepared twice a week, and the amount of solution consumed was measured for the calculation of ferric chloride intake. Control animals were given distilled water without ferric chloride in the same way. Administration of ferric chloride was stopped after 104 wk and the rats were then given distilled water for a recovery period of 8 wk. At wk 112, all surviving rats were killed and examined. Throughout the experiment, rats in all groups were given basal diet ad lib. They were observed daily and clinical signs and deaths were recorded. Body weights were measured once a week for the first 13 wk of the study and then once every 4wk. All rats that died or were killed in extremis during the study and those killed at termination were subjected to a full post-mortem examination and investigated macroand microscopically for the occurrence of neoplastic and non-neoplastic lesions. All organs, tissues and gross lesions were routinely fixed with 10% buffered formalin, sectioned, and stained with haematoxylin and eosin or Berlin blue. Body weight and the daily water intake data were analysed statistically using Student's t-test. The survival times were analysed using the generalized Wilcoxon test. The incidences of tumours were analysed statistically by Fisher's exact probability test. RESULTS
Subchronic toxicity study None of the rats in any of the groups died during the 13-wk period of the study. Figure 1 shows the growth curves for rats in each group. In both sexes given the 2.0 and 1.0% doses, a depression of body weight gain of at least 10% compared with the control group was observed at termination. A significant suppression in the intake of drinking water was observed in the groups given doses of 0.5% or more. The haematology and serum chemistry data demonstrated a higher level of serum iron in male treated groups. The level ~ug/dl) was 102 + 5 in the control group, 107 + 11 in the 0.12% group, 109 + 8 in the 0.25*/, group, 129 + 22 in the 0.5% group, 139 + 10 in the 1.0% group and 156 in the 2.0%
30O
A
0.5% 1.0%
• •
1
2
3
4
5
6
7
S
9
2.0%
10 II 12 13 Weeks
2111)
.~150
• • 0
I
2
3
4
5
6
7
8
9
1.0% 2.0%
10 I1
12 13 Weeks
Fig. 1. Mean body weights of male and female F344 rats given ferric chloride at 0 (A), 0.12 (I--I),0.25 (O), 0.5 (A), !.0 ( I ) or 2.0% (O) in the drinking water for 13 wk.
group. The counts of red blood cells in male treated groups were also significantly increased, compared with the control value. In histological examinations on the sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the ,esophageal mucosa in the groups given doses of 0.25% or more and in the laminae propriae of the large intestine in the 2.0% group. In sections stained with Berlin blue, increased numbers of positive pigments were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney, and the adipose tissues of the groups given doses of 0.25% or more, in addition to the organs described above. The intensity of staining was marked in the intestine and liver. From the results of this subchronic toxicity study, it was concluded that the probable maximum tolerated dose of ferric chloride in the drinking water would be 0.5% in both sexes. Therefore, 0.5 and 0.25% were selected as appropriate dose levels for the long-term carcinogenicity study.
2-year carcinogenicity study Figure 2 shows the growth curves in each group. The mean body weights of the treated males and females were significantly lower than those of the
Carcinogenicity tests of ferric chloride
839
500 MALE 400
.~ 3oo '~
FEMALE
] 0
=
o. 5%
;
0.5%
t 0
20
40
60 WEEKS
80
100
120
Fig. 2. Mean body weights of male and female F344 rats given ferric chloride at 0 (©), 0.25 (11) or 0.5% ( 0 ) in the drinking water for 2 yr.
corresponding control groups. Data for final body weight, drinking water intake, ferric chloride intake, final survival rate and mean survival time are shown in Table 1. The final body weight and the mean daily water intake of the treated groups were significantly lower than those of the control groups. The mean daily ferric chloride intakes in the 0.25 and 0.5% groups calculated from the water intake were, respectively, 169.7 and 319.7 mg/kg body weight/day in males, and 187.9 and 336.0 mg/kg body weight/day in females. The cumulative mortality at termination in males of the 0.5% group was significantly decreased compared with the control value. The sites, histological types and incidence of tumours found in each group of both sexes are summarized in Table 2. The first autopsy was performed at wk 49, when a female rat in the 0.5% group was killed in extremis because of a malignant fibrous histiocytoma. This rat and all of the animals that survived beyond this week were included in the effective numbers, with the exception of rats for
which histopathological examinations could not be performed owing to advanced autolysis. There were no statistically significant differences in the overall tumour incidence between control and treated groups of either sex. Tumours were found in many organs or tissues in all groups, including the control group. In males of all groups, tumours of the testis were most frequent, followed by neoplasms of the haematopoietic organs, adrenal gland, mammary gland, thyroid, pituitary and pancreas. In females, tumours of the uterus, pituitary, haematopoietic organs, mammary gland, adrenal gland and thyroid were common. Tumours were also detected in the other organs or tissues in all groups of both sexes, but the incidence was very low. None of the treated groups demonstrated a significant increase in the incidence of any specific tumour over that in the corresponding control group. All tumours observed in this study were similar to those that are known to occur spontaneously in this strain of rats (Goodman et al., 1979; Haseman et al., 1990; Solleveld et aL, 1984).
Table I. Final body weight, intake of drinking water and chemical, and survival time in F344 rats given ferric chloride in the drinking water for 2 yr Average daily intake
Group Male Control 0.25% 0.5% Female Control 0.25% 0.5%
Final body weight (mean :t: SD)
Drinking water (g/kg body weight)
Chemical (mg/kg body weight)
Final survival rate (%)
Mean survival time and range (wk)
458.4 _+40 431.0 _.+35* 430.6 + 24*
95.8 67.4* 63.2*
0 169.7 319.7
62 54 82*
107.0 (69-112) 104.5 (60-I 12) 109.3 (64-112)
317.6 _4-33 287.3 + 29* 271.1 + 24*
105.0 73.6* 67.2*
0 187.9 336.0
72 56 62
106.5 (50- I 12) 106.9 (73- I 12) 105.6 (49-112)
*Significantly different from control value at P < 0.05.
840
M. S^ro et al. Table 2. Incidence, sites and types of turnouts in F344 rats given ferric chloride in the drinking water for 2 yr No. of rats with tumours Dose (%) ...
0
Males 0.25
Adenoma C-cell adenoma C-cell carcinoma Follicular cell adenoma Follicular cell carcinoma Phaeochromocytoma Malignant phaeochromocytoma Ganglioneuroma Schwannoma Cortical adenoma Islet cell adenoma Islet cell carcinoma Acinar cell adenoma Interstitial cell tumour Adenoma Endometrial stromal polyp Carcinoma Endometrial stromal sarcoma Granulosa/theca cell tumour Fibroma Fibroadenoma Adenoma Carcinoma Large granular lymphocytic leukaemia Malignant lymphoma Malignant lymphoma Histiocytic sarcoma Adenoma Adenocarcinoma Adenosquamous cell carcinoma Adenocarcinoma Leiomyosarcoma Adenocarcinoma Neoplastic nodule Histiocytic sarcoma Nephroblastoma Granular cell tumour Glioma Sehwannoma Squamous cell papilloma Squamous cell carcinoma Trichoepithelioma Sebaceous adenoma Sebaceous carcinoma Preputial/clitoral adenoma Preputial/clitoral carcinoma Rhabdomyosarcoma Malignant fibrous histiocytoma Mesothelioma Osteosarcoma Squamous cell carcinoma Rhabdomyosarcoma
7 6 0 I 0 I0 1 1 0 0 5 2 2 48 I ---
6 7 1 0 0 7 0 0 0 0 5 I 1 46 I ---
Site and type of tumour Pituitary Thyroid
Adrenal
Pancreas Testis Prostate Uterus Ovary Mammary gland
Spleen Lymph node Bone marrow Lung Small i n t e s t i n e Large i n t e s t i n e Liver Kidney Brain Peripheral n e r v e Skin
Subcutis Abdominal c a v i t y Undetermined origin
Effective no. of rats No. of rats with tumours
In a d d i t i o n to these t u m o u r s , v a r i o u s types o f n o n - n e o p l a s t i c lesions were o b s e r v e d in e a c h group. Age-related chronic n e p h r o p a t h y was present in all g r o u p s . T e s t i c u l a r a t r o p h y , a c c o m p a n i e d by o l i g o s p e r m i a o r a s p e r m i a , w a s seen in a l m o s t all m a l e r a t s o f all g r o u p s . D u c t u l a r p r o l i f e r a t i o n a n d altered cell foci, w h i c h are k n o w n to be c o m m o n in a g e i n g F344 rats ( B u r e k , 1978; Eustis et al., 1990), were o b s e r v e d in the livers o f b o t h c o n t r o l a n d t r e a t e d rats. I n sections stained w i t h Berlin blue, a l t h o u g h slight s t a i n i n g w a s seen in v a r i o u s o r g a n s / t i s s u e s f r o m t r e a t e d a n d c o n t r o l rats, n o m a r k e d increase in p i g m e n t d e p o s i t i o n w a s o b s e r v e d in a n y o r g a n o r tissue o f the 0 . 5 % g r o u p . T h e r e w e r e n o specific
--
--
Females 0.5
0
0.25
0.5
5 5 2 1 0 I0 3 0 I 0 3 3 0 48 1 ---
12 4
16 I
15 I
-7 1 0 1 I0 I I 2 6 0 1 I 2 0 2 I 0 0 0 1 0 0 0 0 0 3 I 0 0 3 I 0 0
-6 0 1 0 10 0 0 0 2 0 0 2 0 0 3 0 0 I I 0 0 0 I 0 0 0 0 0 I 2 0 0 0
50 50
48 47
49 49
0
0
0
0
I
0
0
4 0 0 0
I 0 0 0
6 0 0 0
I
0
0
0 0 0
0 0 0
2 0 0
14 4
11 4
15 5
I I
I 0
1 0
0 4 2 0 8 0 0 0
2 2 0 0 I1 0 0 0
I 2 0 2 4 0 0 0
--
-9 4 2 0 I0 0 0 0 1 1 0 1 0 I 2 0 0 0 1 1 I l 0 0 0 4 0 0 l 2 0 0 0
I
0
I
I
2
0 0 0 0 0 2 0
0 0 I 0 0 0 0
0 0 0 0 0 I 0
1
0
0
0 0 0 0 0 0 0 0
0 1 2 0 0 0 1 I
0 0 1 0 0 0 0 0
1
0
0
0 0 2 0 0 0 0
1 0 I 0 0 0 1
0 I 1 0 0 I 0
50
49 39
40
48 42
lesions c o n s i d e r e d to be a t t r i b u t a b l e to ferric c h l o r i d e treatment. DISCUSSION I n the c a r c i n o g e n i c i t y s t u d y described here, a l t h o u g h a variety o f t u m o u r s were detected in all g r o u p s , including the c o n t r o l g r o u p , their o r g a n d i s t r i b u t i o n a n d histological types were essentially similar to t h o s e o f the s p o n t a n e o u s t u m o u r s described p r e v i o u s l y ( G o o d m a n e t a / . , 1979; H a s e m a n et al., 1990; Solleveld et al., 1984). T h e r e were n o significant increases in the incidence o f lesions in a n y o r g a n o r tissue t h a t c o u l d be a t t r i b u t e d to the t r e a t m e n t w i t h ferric chloride.
Carcinogenicity tests of ferric chloride In humans, epidemiological studies have indicated that there is a relationship between an increase in organ or tissue iron concentration and an increased risk of cancer (Stevens et al., 1988; Weinberg, 1984). Iron is thought to catalyse the formation, from superoxide anions and hydrogen peroxide, of highly reactive species (e.g. hydroxyl radicals or reactive iron-oxygen complexes) that can subsequently react with chromosomes, damage DNA and result in carcinogenesis (Aust et al., 1985; Cerutti, 1985; Halliwell and Gutteridge, 1988). In addition, iron is known to have a major role in the initiation and propagation of the enzymatic lipid peroxidation reaction (Minotti and Aust, 1987), and dietary iron overloading increases lipid peroxidation in vivo ( W u et al., 1990; Younes et al., 1990). It has also been reported that orally administered ferric chloride induces nuclear aberrations in the stomach and colon of mice (Bianchini et al., 1988), and co-carcinogenic effects of iron were observed in some studies involving mice and hamsters, using two-stage initiation/ promotion carcinogenesis models (Siegers et al., 1988; Stenb/ick and Rowland, 1978). From the positive results of Berlin blue staining in many organs and the increased serum iron levels in the treated groups in the study of the subchronic toxicity of ferric chloride, it had been predicted initially that the condition of iron overload would be maintained by administration of 0.5% ferric chloride for 2 yr. However, iron deposition was not increased in any organ/tissue of the high dose group compared with the control group at the termination of the present carcinogenicity study. Park et al. (1987) reported that the chronic iron overload produced by dietary supplementation with 2.5% carbonyl iron for 1 yr resulted in an accumulation of large amounts of iron in many tissues, associated with hepatocellular damage and fibrosis leading to cirrhosis. It is considered, therefore, that the level of iron overload was too low to increase lipid peroxidation in the study described here, although no quantitative assessment of lipid peroxidation was performed. The lack of any toxic lesions in the high dose group, despite lowered body weights, suggests that the dose levels selected may possibly have been too low for the carcinogenicity study. However, the design and conduct of the present ferric chloride bioassay was in accordance with recent guidelines for carcinogenicity tests and the evaluation of resultant data (National Cancer Institute, 1976; National Toxicology Program, 1984; Odashima, 1980). The highest dose currently recommended is that which, when given for the duration of the chronic study, is just high enough to elicit signs of minimal toxicity without significantly altering the normal lifespan of the animal through toxic effects other than carcinogenicity. In this study, the maximal tolerated dose was based on the weight-gain decrement observed in the subchronic study: since the highest dose in carcinogenicity studies should not cause more than a 10%
841
reduction in weight gain, a dose level of 0.5% was selected as the maximal tolerated dose. From the above results, it was concluded that ferric chloride exerts no carcinogenic activity in F344 rats when administered as a 0.25 or 0.5% solution continuously in the drinking water for up to 2 yr. REFERENCES
Aust S. D., Morehouse L. A. and Thomas C. E. (1985) Role of metals in oxygen radical reactions. Journal of Free Radicals in Biology and Medicine 1, 3-25. Bianchini F., Caderni G., Dolara P. and Tanganelli E. (1988) Nuclear aberrations and micronuclei induction in the digestive tract of mice treated with different iron salts. Journal of Applied Toxicology 8, 179-183. Burek J. D. (1978) Pathology of Aging Rats. Edited by J. D. Burek. pp. 58-70. CRC Press, Boca Raton, FL. Cerutti P. A. (1985) Prooxidant states and tumor promotion. Science 227, 375-381. Eustis S. L., Boorman G. A., Harada T. and Popp J. A. (1990) Liver. In Pathology of the Fischer Rat. Edited by G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, Jr and W. F. MacKenzie. pp. 71-94. Academic Press, San Diego, CA. Goodman D. G., Ward J. M., Squire R. A., Chu K. C. and Linhart M. S. (1979) Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicology and Applied Pharmacology 48, 237-248. Halliwell B. and Gutteridge J. M. C. (1988) Free radicals and antioxidant protection: mechanisms and significancein toxicology and disease. Human Toxicology 7, 7-13. Haseman J. K., Arnold J. and Eustis S. L. (1990) Tumor incidences in Fischer 344 Rats: NTP historical data. In Pathology of the FLwher Rat. Edited by G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, Jr and W. F. MacKenzie. pp. 555-564. Academic Press, San Diego, CA. Ishidate M., Jr, Yoshikawa K. and Sofuni T. (1983) Mutagenicity tests of food additives. Toxicology Forum 6, 671~178. (In Japanese). Minotti G. and Aust S. D. (1987) The role of iron in the initiation of lipid peroxidation. Chemistry and Physics of Lipids 44, 191-208. National Cancer Institute (1976) Guidelines for Carcinogen Bioassay in Small Rodents (Carcinogenesis Technical Report Series 1). National Institute of Health, Betbesda, MD. National Institute for Occupational Safety and Health (NIOSH) (1987) Registry of Toxic Effects of Chemical Substances (RTECS) Edited by D. V. Sweet. 3. pp. 2397. US Government Printing Office, Washington, DC. National Toxicology Program (1984) Report of the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation. Board of Scientific Counselors, National Toxicology Program, DHHS. Odashima S. (1980) Cooperative program on long-term assays for carcinogenicity in Japan. In Molecular and Cellular Aspects of Carcinogen Screening Tests. IARC Scientific Publication no. 27. Edited by R. Montesano, H. Bartsch and L. Tomatis. p. 315. International Agency for Research on Cancer, Lyon. Park C. H., Bacon B. R., Brittenham G. M. and Tavill A. S. (1987) Pathology of dietary carbonyl iron overload in rats. Laboratory Investigation 57, 555-563. Siegers C. P., Bumann D., Baretton C. and Younes M. (1988) Dietary iron enhances the tumor rate in dimethylhydrazine-induced colon carcinogenesis in mice. Cancer Letters 41, 251-256.
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Smith A. G., Cabral J. R. P., Carthew P., Francis J. E. and Manson M. M. (1989) Carcinogenicity of iron in conjunction with a chlorinated environmental chemical, hexachlorobenzene, in C57BL/10ScSn mice. International Journal of Cancer 43, 492--496. Solleveld H. A., Haseman J. K. and McConnel E. E. (1984) Natural history of body weight gain, survival, and neoplasia in the F344 rat. Journal of the National Cancer Institute 72, 929-940. Stenb~ck F. and Rowland J. (1978) Carcinogenic activation of benzo(a)pyrene by iodine and ferric chloride in the respiratory tract of Syrian golden hamsters. Experientia 34, 1065-1066. Stevens R. G., Jones Y., Micozzi M. S. and Taylor P. R.
(1988) Body iron stores and the risk of cancer. New England Journal of Medicine 319, 1047-1052. Weinberg E. D. (1984) Iron withholding: a defense against infection and neoplasia. Physiological Reviews 64, 65-102. Wu W., Meydani M., Meydani S. N., Burklund P. M., Blumberg J. B. and Munro H. N. (1990) Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats. Journal of Nutrition 120, 280-289. Younes M., Trepkau H. D. and Siegers C. P. (1990) Enhancement by dietary iron of lipid peroxidation in mouse colon. Research Communications in Chemical Pathology and Pharmacology 70, 349-354.
