Sodium erythorbate is not carcinogenic in F344 rats

EXPERIMENTAL

AND

Sodium IKUO ABE, Department

MOLECULAR

Erythorbate SACHIKO

of Oncology,

Received

41, 35-43 (1984)

PATHOLOGY

Is Not Carcinogenic

SAITO,

KATSUYOSHI HORI, HARUO SATO~

the Research Institute 4-1 Seiryomachi, September

in F344 Rats’

MAROH

for Tuberculosis and Cancer, Sendai, 980. Japan

30, 1983, and in revised

form

February

SUZUKI, Tohoku

AND University,

2, 1984

Carcinogenicity of sodium erythorbate. a widely used antioxidant food additive, was evaluated using a total of 306 eight-week-old male and female F344/DuCrj rats. Test rats were given 1.25 or 2.5% aqueous solution as drinking water for 104 weeks. Controls were given tap water. All the rats were fed commercial pellets. None of the tumors observed was attributable to sodium erythorbate in drinking water. Neither concentration of sodium erythorbate changed the pattern of spontaneous tumor development in both sexes, except for a slight reduction in aggregate tumor incidence in the 2.5% Group females. Additionally, 2.5% solution suppressed body weight gains in both males and females. These results and prior data by others together suggest that weak mutagens may be noncarcinogenic under certain conditions.

INTRODUCTION Sodium erythorbate (isoascorbate) is used widely in large quantities as a food additive for its antioxidant property (Ministry of Health and Welfare, 1974). Erythorbate as well as ascorbate (a vitamin C salt) can be either anti- or pro-oxidant in vitro for the lipids in lysosomal membranes depending on experimental conditions (Abe et al., 1979). A close relationship has been suggested between lipoperoxidation and chemical carcinogenesis (Shamberger, 1972). In a previous experiment by others no effects were demonstrated in 10 Osborn-Mendel rats given 1% sodium erythorbate for 2 years (Fitzhugh and Nelson, 1946). Sodium erythorbate was, on the other hand, positive in Ames’ mutagenicity test, but was negative in the Ret assay system and the in vitro chromosome aberration test (Kawachi, 1977). Sodium erythorbate has been shown to cause breakage of DNA strands in vitro (Omura et al., 1975). Thus, characteristic effects of sodium erythorbate in vitro are not consistent. In the present study we evaluated the carcinogenic potential of sodium erythorbate using adequate numbers of rats suitable for quantitative analyses of the tumor incidence. A particular strain of rats, F344/DuCj, was used so that possible acceleration of spontaneous tumor development could be ascertained. MATERIALS

AND METHODS

A total of 306 six-week-old Fisher rats (F344jDuCr-j) of both sexes were purchased from Charles River Japan, Inc., Tokyo. They were divided at random into three groups, each group consisting of 52 males and 50 females. Groups of 4 males and 5 females were housed in plastic cages (40 x 25 x 20 cm) with wood chip bedding and a filter cap. The room was maintained under the standard cont This work was supported by a grant-in-aid from the Ministry of Health and Welfare. 2 To whom all correspondence should be addressed. 35 0014-4800/84 $3.00 Copyright All rights

0 1984 by Academic Press. Inc. of reproduction in any form reserved

36

ABE

ET AL.

ditions of 23 + 2”C, 60 r 10% relative humidity, and constant ventilation with uv-treated air. A commercial diet, CRF-1 (Charles River Japan, Inc.), was given freely to all the rats. The sodium erythorbate used was the grade specified in the Japanese Standards of Food Additives (Organ0 Co. Ltd., Toda). Sodium erythorbate was dissolved in tap water and given as drinking water. Solutions were stored in brown bottles to control decay due to photosensitivity. Ninety-four percent of the initial content of sodium erythorbate in 1.25% solution remained unchanged for 4 days. The two dose levels, 2.5 and 1.25%, were chosen based on the data from a prior subacute toxicity test. In this prior experiment, five groups of 10 males and 10 females were given the 10, 5, 2.5, 1.25, or 0.625% aqueous sodium erythorbate as drinking water for 13 weeks. All the rats given the 10% solution refused to drink and died in 2 to 5 weeks. Three males and one female out of the 10 given the 5% solution died during the first 4 days. All the rats given the 2.5% and lower concentrations survived to the end of 13 weeks. The 2.5% solution suppressed body weight gains by 12% in males and by 6% in females as compared with nontreated controls. For the current study starting at the age of 8 weeks the 2.5 and 1.25% solutions were given for the first 104 weeks and plain tap water thereafter. Controls were given plain tap water at all times. Drinking water was changed twice a week after measuring consumption. The general conditions of the rats were observed daily, and the body weights were recorded weekly up to termination at age 120 weeks. Tumors were detected first in males dying at 60 weeks and in females dying at 74 weeks with 153 males and 146 females surviving beyond this period of first detection. The causes of early mortality included pneumonia (3), pericarditis (l), accidental trauma (I), and unknown (2). Autopsy was done on all rats. Major organs and lesions were fixed with 10% Formalin. Paraffin sections were stained with hematoxylin and eosin. RESULTS Sodium erythorbate as 2.5% drinking water significantly inhibited body weight gains in both sexes (P < 0.01, Student’s t test) during the 40 through 90 experimental weeks. The greatest suppression of the body weight in the group given the 2.5% solution (2.5% Group) was 8.5% in males at 88 weeks and 15.5% in females at 85 weeks, as compared with respective nontreated controls. The group given the 1.25% solution (1.25% Group) showed no suppression of body weight gains (Fig. 1). Cummulative consumption of sodium erythorbate was 217 g per rat for the 1.25% Group males and 430 g for the 2.5% Group males. The values for the females were 206 and 583 g per rat, respectively. The high value of 583 g corresponded to the large amounts of water intake which returned to the normal level after the switch to tap water at the 104th week. More than 60% of the rats of all the groups (60 to 82%) survived this treatment period, or to 112 weeks of age (Fig. 2). Mean life span of the tumor-bearing rats was similar among the three groups: 117, 114, and 111 weeks for control, 1.25%, and 2.5% Group males, and 114, 113, and 113 weeks for control, 1.25%, and 2.5% Group females. All the male treated and control rats except two in 2.5% Group, showed testicular interstitial-cell tumor (Table I). Various other types of tumors occurred in 80% of control males, 69% of the 1.25% Group, and 78% of the 2.5% Group. Leukemia, pheochromocytoma, mammary fibroadenoma, and mesothelioma

ERYTHORBATE

0 A 0

IS NOT

37

CARCINOGENIC

Control 1.25% Sodium erythorbate I 2.5%

t 0'

' 0

I

I

,

I

I

20

40

60

80

100

ExPerImental

I 112

weeks

FIG. 1. Growth curves of the male and female rats given tap water, or 1.25 or 2.5% aqueous sodium erythorbate for 104 weeks. From the 40 through 90 treatment weeks the males and females of the 2.5% Group showed significant suppression in body weights.

were relatively frequent with 6 to 18% incidence. Aggregate tumor incidences in control females were 94%; and 88 and 78% for the 1.25% and 2.5% Groups. The value for the 2.5% Group females was significantly lower than control females by chi-square test (p < 0.05). Leukemia, mammary tibroadenoma, endometrial stromal polyp, and pituitary adenoma were observed singly or together in 20 to 43% of the female rats of the three groups. The pattern of occurrence of various types of tumors was similar among the three groups.

t A

1.25%

Sadium

erythorbate

20

80

t

0 0

20

40

60 Experfmentol

80

100

100

112

weeks

FIG. 2. Mortality rates of rats with and without tumors. Solid lines, rats with tumors; broken lines, rats without tumors. Presence or absence of tumors was ascertained histologically.

38

ABE ET AL.

Microscopic lesions in 10 non-tumor-bearing rats out of 45 in the 2.5% Group included bronchopneumonia in 5 rats, and myocardial fibrosis, rupture of the spleen with large multiple cysts, telangiectasis and hemorrhage in the adrenal medulla, and nephropathy in 1 rat each. Two rats showed no abnormality. Certain of the rare spontaneous tumors (0.1% or less) known to occur in F344 rats were observed in the current study (Goodman et al., 1979; Sher et al., 1982; Maekawa et al., 1983). They were uterine leiomyoma, subcutaneous myxoid liposarcoma (Fig. 3) and subcutaneous hemangioma (Fig. 4), osteosarcoma of the upper arm (Fig. 5) and retroperitoneum, hemangiopericytoma in the spleen and retroperitoneum (Fig. 6), pleomorphic adenoma of the salivary gland (Fig. 7), pulmonary adenocarcinoma (Fig. 8), renal-cell carcinoma (Fig. 9), transitionalcell papilloma of the urinary bladder, and lipoma of the retroperitoneum. A subcutaneous fibroma with hyperplasia of sebaceous glands was also observed (Fig. 10). No enhancement by sodium erythorbate was observed in the development of these rare tumors. Sodium erythorbate did not appear to transform benign tumors, such as solid adenoma of the thyroid, to carcinomas (Table I). Table II shows the relationship between the age and occurrence of the tumors with the incidence greater than 10%. Interstitial-cell tumors of the testes in the groups given sodium erythorbate showed no dose-dependent acceleration according to chi-square test. Leukemia seemed to occur earlier in the males of the 2.5% Group than in controls, but the difference was not significant by Fisher’s exact probability test. DISCUSSION Spontaneous testicular interstitial-cell tumors, endometrial stromal polyps, mammary fibroadenomas, adrenal pheochromocytomas, and other endocrine tumors in control rats showed a pattern of incidence similar to that of earlier reports by others (Goodman et al., 1979; Tarone et al., 1981; Maekawa et al., 1983). The incidence of leukemias in female controls, however, was higher at 37.8% than in prior studies by others, 9.9% (Maekawa et al., 1983), 11.0% (Tarone et al., 1981), Distribution

TABLE 1 and Type of Tumors in Control F344 Rats and Those Given Two Concentrations of Sodium Erythorbate Male 0%

Number of rats surviving beyond 60 (males) or 74 (females) experimental weeks Number of rats with tumors Genitalia and mammary gland Testis, interstitial-cell tumor Uterus Endometrial stromal polyp Myoma Adenocarcinoma Endometrial sarcoma Mammary gland Fibroadenoma Fibroma Adenocarcinoma

Female

1.25%

2.5%

0%

1.25%

2.5%

49 49

48 48

50 50

48 45

49 43

45 35*

49

48

48

-

-

-

11 0 0 1

11 1 0 0

10 0 2 I

13 1 4

20 2 0

12 2 2

-

-

-

-

-

-

4 1 0

6 2 0

3 3 0

ERYTHORBATE TABLE

IS NOT CARCINOGENIC

39

I-Continued Male 0%

Endocrine system Pituitary adenoma Thyroid Solid adenoma Solid carcinoma Adrenal Pheochromocytoma Cortical adenoma Pancreas, insuloma Integument and musculoskeletal system Skin Papilloma Basal-cell carcinoma Squamous-cell carcinoma Keratoacanthoma Adenoacanthoma Subcutis Fibroma Lipoma Liposarcoma, myxoid Hemangioma Fibrosarcoma Sarcoma Bone osteosarcoma Preputial or clitoral gland, adenoacanthoma Hematopoietic system Hematopoietic organs, leukemia Spleen, hemangiopericytoma Digestive system Tongue, papilloma Salivary gland, pleomorphic adenoma Colon and rectum, adenoma Liver, hepatocellular adenoma Pancreas, acinar-cell adenoma Respiratory system Lung Adenocarcinoma Adenoacanthoma Urinary system Kidney, renal-cell carcinoma Bladder, papilloma Abdominal cavity Peritoneum, mesothelioma Retroperitoneum Fribroma Lipoma Osteosarcoma Hemangiopericytoma

Female

1.25%

2.5%

0%

1.25%

2.5%~

4

1

1

11

10

6

2 3

4 I

3 2

1 0

2 1

1 0

6 1 4

3 0 0

9 0 1

0 2 0

2 0 2

2 1 0

0 0 0 1 0

0 0 0 0 1

0 0 0 0 0

0 0 0 0 0

5 0 1 0 1 0 0

1 0 0 0 0 0 lb

0 0 0 0 0 0 0

0 0 0 0 0 1 0

a With sebaceous gland hyperplasia. b With distant metastases to the lungs, kidneys, and ribs. * Statistically significant at P < 0.05 (chi-square test).

2

2

2

3

0

10 1

18 0

17 0

21 0

20 0

0 0 1 0 0

0 0 1 1 0

1 0 0 1 0

0 0 2 1 0

0 0 0 0 1

1 0

0 0

0 0

1 0

1 0

0 0

1 0

0 0

0 0

0 1

1

7

0

0

0

0 0 0 0

0 0 1 0

0 0 0 0

1 1 0 0

0 0 0 0

40

ABE ET AL.

FIG. 3. Myxoid liposarcoma in the subcutis (male, 1.25% Group). A soft tumor consisted of sparse stellate cells with pleomorphic nuclei and a dense vascular stroma. Mitotic figures were frequent. x 200. FIG. 4. Hemangioma in the subcutis (male, 2.5% Group). Blood-filled spaces are lined by flat endothelium and are separated by fibrous septa. x 100. FIG. 5. Osteosarcoma in the humerus (control female). Calcified osseous matrix forms characteristic trabeculae in both the primary and metastatic lesions, the latter being seen in the lungs, kidneys, and ribs. x 100. FIG. 6. Hemangiopericytoma in the retroperitoneum (control male). “Staghorn-shaped” vascular spaces are lined by endothelial cells. Reticulin stain showed the pattern typical of vessels. x 100.

ERYTHORBATE

IS NOT CARCINOGENIC

41

FIG. 7. Pleomorphic adenoma in the salivary gland (control male). The matrix is myxomatous and contains scattered tubules. Some of the tubules contain eosinophilic secretion. x 100. FIG. 8. Pulmonary adenocarcinoma(male, 1.25% Group). Papillary, tubular, and sheet-like structures consist of columnar cells. x ZOO. FIG. 9. Renal-cell carcinoma (male. 2.5% Group). Large clear cells having pale nuclei with distinct nucleoli (upper field) and relatively dark cells with vesicular nuclei (lower field) form nests and sheets supported by sparse fibrovascular tissue. A tubular structure is in the right lower corner. x 200. FIG. 10. Subcutaneous fibroma and hyperplastic sebaceous gland (control male). The tumor is dense fibrotic. Sparse spindle cells surround hyperplastic sebaceous glands which are connected to blind distended ducts. x 100.

42

ABE ET AL. TABLE II Age-Related Occurrence of Tumors with More Than 10% Incidence Concentration of sodium erythorbate (%) Male Interstitialcell tumor of testis Leukemia

Pheochromocytoma Mammary fibroadenoma Mesothelioma Female Leukemia Mammary tibroadenoma Endometrial stromal polyp Pituitary adenoma

Total incidence

1.25 2.5 0 1.25 2.5 0 1.25 2.5 0 1.25 2.5 0 1.25 2.5

49 48 48 13 10 18 6 3 9 4 6 3 5 0 I

0 1.25 2.5 0 1.25 2.5 0 1.25 2.5 0 1.25 2.5

17 21 20 13 20 12 11 10 10 11 10 6

0

Age-related occurrence of tumors (weeks)
79- 104

1

40 37 31 9 6 I 6 3 5 4 6 2 4 6

I 8 8 2 2 1 4 3 4 3 2 2

10 12 12 11 18 11 7 7 6 8 8 4

2

-

1 -

1

>104

4

1 1 -

and 21.9% (Goodman et al., 1979). We have no specific explanation for this difference. Variations in the tumor incidence among and within laboratories are well known, and Tarone et al. (1981) stressed the importance as well as limitations of historical controls. The males of the nontreated and treated groups showed similar aggregate tumor incidences (Table I). The females of the 2.5% Group, however, showed less aggregate tumor incidence than that of untreated control females. Further investigations are necessary to determine whether this apparent reduction may simply be due to the shorter life span of the rats dying before the development of tumors or to specific suppression of tumorigenesis by sodium erythorbate. No effect of the lower dose of 1.23% was demonstrable in aggregate tumor incidence (Table I). The current data indicate that sodium erythorbate is not carcinogenic in F344 rats. Sodium erythorbate did not significantly enhance the incidence of any tumor including those which are known to occur rarely as spontaneous tumors. Similarly

ERYTHORBATE

IS NOT

CARCINOGENIC

43

negative were temporal acceleration of tumor development and malignant metamorphosis of benign tumors. These results are in accord with those obtained in B6C3F, mice (Inai et al., 1981). Thus weak mutagens may not be carcinogenic under certain conditions (Tanaka et al., 1979). Cocarcinogenicity of weak mutagens should be further investigated in view of the multiplicity of environmental chemical exposures in man. Sodium erythorbate was shown not to affect carcinogenesis by N-methyl-IV-nitro-N-nitrosoguanidine in the rat stomach (Abe et al., 1983). Ascorbic acid has been shown to enhance carcinogenesis by methylcholanthrene in guinea pigs (BaniC, 1981). The wide distribution in foodstuffs and heavy consumption of sodium erythorbate warrant further investigations to determine whether or not this food additive enhances tumorigenesis by methylcholanthrene and other potent carcinogens. ACKNOWLEDGMENTS The authors thank the late Dr. Shigeyoshi Odashima and Dr. Yuji Kurokawa, National Institute of Hygienic Sciences, Tokyo, and Dr. Hideshige Imai, Albany Medical College, Albany, New York, for valuable suggestions. Dr. Masahiro Nakadate performed the stability test of solutions of sodium erythorbate.

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Univ.

Ser. C 31, 40-55.

BANI~, S. (1981). Vitamin C acts as a cocarcinogen to methylcholanthrene Lett.

in guinea-pigs. Cancer

11, 239-242.

FITZHUGH, 0. G., and NELSON, A. A. (1946). Subacute and chronic toxicities of ascorbyl palmitates. Proc.

Sot.

Exp.

Biol.

Med.

61, 195-198.

GOODMAN, D. G., WARD, J. M., SQUIRE, R. A., CHU, K. C., and LINHART, M. S. (1979). Neoplastic and nonneoplastic lesions in aging F344 rats. Toxicol. Appl. Phnrmacol. 48, 237-248. INAI, K., KOBUKE, T., MATSUKI, K., and TOKUOKA, S. (1981). Pathological studies in mice by longterm oral administration of sodium erythorbate. In “Proc. Jpn. Cancer Assoc. 40th Annual Meeting,” p. 69. KAWACHI, T. (1977). Development of technics based on mutagenicity for screening carcinogens. In “Annual Report of the Cancer Research,” pp. 773-789. The Ministry of Health and Welfare, Tokyo. MAEKAWA, A.. KUROKAWA, Y., TAKAHASHI, M., KOKUBO, T., OGIU, T., ONODERA, H., TANIGAWA. H., OHNO, Y., FURUKAWA, F., and HAYASHI, Y. (1983). Spontaneous tumors in F-344iDuCrj rats. Gann

74, 365-372.

Ministry of Health and Welfare. (1974). “The Japanese Standards of Food Additives,” 3rd ed., pp. 424-426. The Ministry of Health and Welfare, Tokyo. OMURA, H., IIYAMA, S., TOMITA, Y., NARAZAKI, Y., SHINOHARA, K., and MURAKAMI, H. (1975). Breaking action of ascorbic acid on nucleic acids. J. Nutr. Sci. Vitaminol. 21, 237-249. SHAMBERGER,R. J. (1972). Increase of peroxidation in carcinogenesis. J. Narl. Cancer Inst. 48, 14911497. SHER, S. I?, JENSEN, R. D., and BOKELMANN, D. L. (1982). Spontaneous tumors in control F344 and Charles River-CD rats and Charles River CD-l and B6C3HFl mice. Toxicol. Let?. 11, 103-110. TANAKA, T., FUJII, M., MORI, H., and HIRONO, I. (1979). Carcinogenicity test of potassium metabisulfite in mice. Ecotoxicol. Environ. Safety 3, 451-453. TARONE, R. E., CHU, K. C., and WARD, J. M. (1981). Variability in the rates of some common naturally occurring tumors in Fisher 344 rats and (C57BL/6N x C3H/HeN)F, (B6C3F,) mice. J. Natl. Cancer Inst. 66. 1175- 1181.