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Lack of tumorigenicity of sodium benzoate in mice
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
4,494-496
( 1984)
SHORT COMMUNICATIONS Lack of Tumorigenicity
of Sodium Benzoate
in Mice
Lack OfTumorigenicity of Sodium Benzoate in Mice. TOTH, B. (1984). Fundam. Appl. Toxicol. 4, 494-496. Sodium benzoate was administered as a 2% solution in drinking water for life to randomly bred Swiss mice. Consumption of the chemical caused no detectable tumorigenic effect under the current experimental conditions.
Sodium benzoate (SB) is a widely used preservative throughout the world, especially in food products (National Academy of Sciences, 1965; The Merck Index, 1976). Its preservative qualities are most effective under slightly acidic conditions (Sollman, 1957), and the compound is generally considered safe for human consumption (Select Committee on GRAS Substances, 1977). The effect of this food preservative was studied in earlier experiments. SB, administered orally in food at a 5% level to rats, was without carcinogenic action (White, 194 1). Also, no cancer-inducing effect was observed when benzoic acid was administered subcutaneously or intravenously to rabbits (Hosino, 1940a,b). The present undertaking is apparently the first study in this area in which the compound was administered at a high dose level for the entire lifespan of the animals. It is also part of our long-term objective to reveal the possible carcinogenicity and mode of action of food additives and contaminants (Toth, 1975, 1980). METHODS Albino Swiss mice from a colony randomly bred by us since 195 1 were used. They were housed in plastic cages with granular cellulose bedding, separated according to sex in groups of 10, and given Wayne Lab Blox diet in regular pellets (Allied Mills, Inc., Chicago, Ill.) and tap water ad Iibitum with the exception described below. The chemical used was sodium benzoate (SB): MW, 144.11, MP, >3OO”C, purity, 99%, obtained from Fisher Scientific Co., Fair Lawn, N.J. Toxicity studies were performed with SB prior to the chronic experiment. Five dose levels of SB including 8, 4,2, 1, and 0.5% were administered in the drinking water daily for 35 days. Each group consisted of eight animals: four females and four males. By taking into account four 0272-0590184
$3.00
Copyright 8 1984 by the Society of Toxicology. All rights of reproduction in any form rew-vcd.
494
parameters, survival rate, body weight, chemical consumption, and histological changes, the 2% dose level was found to be suitable for the lifelong treatment. The 8% dose level was too toxic; ah the mice died within 3 weeks of SB administration, while at the 4% dose level 3 P and 3 d died during the 35day observation period. In addition, the body weight of the surviving mice at the 4% dose level was substantially reduced. This toxicity technique was developed in this laboratory (Toth, 1972). The solution was prepared three times weekly and the total consumption of water containing SB was measured at the same intervals during the treatment period. The solution was placed in brown bottles because of the possible light sensitivity of the chemical. The chronic experimental groups and the controls were the following. Group 1: SB was dissolved in the drinking water as a 2% solution and was given for the lifespans of 50 female and 50 male mice that were 5 weeks (39 days) old at the beginning of the experiment. The average daily water consumption with the chemical in it per animal was 5.9 ml for the females and 6.2 ml for the males. Therefore, the average daily intake of SB was 119.2 mg for a female and 124.0 mg for a male. Group 2: These were the untreated controls. Mice, 100 female and 100 male, were kept and observed from weaning time (5 weeks of age). The experimental and control animals were carefully checked and weighed at weekly intervals, and the gross pathological changes were recorded. The animals wem either allowed to die or were killed with ether when moribund. Complete necropsies were performed on all animals. All organs were examined macroscopically and fixed in 10% buffered Formahn. Histological studies were done of the liver, spleen, kidneys, bladder, thyroid, heart, pancreas, testes, ovaries, brain, nasal turbinates, at least four lobes of the lungs of each mouse and on those organs with gross pathological changes. Sections from these tissues were stained routinely with hematoxylin and eosin and examined by light microscopy.
RESULTS The survival rates of mice after weaning in weeks of age were recorded. The data clearly
999
996
Untreated
Controls
87 (51-134)
Age at death“
Lungs
23
21
92 (53-125)
95 (60-122)
14 85 (58-113)
20
%
’ Average and range in weeks. b Age at death in weeks given in parentheses.
23
21
7
506
2
10
SB, 2% in drinking water daily for life
1
509
Treatment
Effective No. of mice No.
Group
1
12
24
3
10
No.
89 (31-130)
81 (61-104)
88 (57-119)
Age at death *
12 90(40-126)
24
6
20
W
Mal. lymphomas
6
5
4
2
No.
6
5
8
4
9%
Other tissue@
88 (65-105)
112 (97-130)
2 Adenocarcinomas of breast (73, 93), 1 leiomyosarcoma of uterus (8 I), 2 adenocarcinomas of ovary (104, 106), 1 adenoma of glandular stomach (115), 1 adenoma of thyroid (116), 1 papilloma of esophagus (106), 1 papilloma of skin (109), 1 fibrosarcoma, subcutaneous (44), 1 adenoma of ovary (103), 1 malignant histiocytoma (98) 1 Fibrosarcoma, subcutaneous (82), 1 papilloma of forestomach (63), 1 adenocarcinoma of duodenum (113), 1 malignant histiocytoma (lOl), 1 carcinoma of skin (122), 1 adrenal cortical adenoma ( 130), 1 papilloma of esophagus (63)
84 (55-l 13) 4 Adenocarcinomas of breast (44, 59, 78, 100) 1 carcinoma of skin (7 1) 1 adenoma of thyroid (134) 8 1 (61-109) I Polypoid adenoma of cecum (61) 1 hepatoma (95)
Age at death”
Blood vessels
Animals with tumors of
TUMOR DISTRIBUTION IN SODIUM BENZOATE(SB)-TREATEDANDCONTROLSWISSMICE
TABLE
!E2
SHORT COMMUNICATIONS
496
showed that the treatment had no effect on the survivals when compared with that in untreated controls. The number (percentages of animals with tumors) and the age of animals with tumors at death (latent periods) are summarized in Table 1. Since the treatment had no carcinogenic action, the tumor incidences are not detailed here. DISCUSSION The present study demonstrates that SB, when given at a 2% concentration in drinking water daily for life to Swiss mice, had no ap parent carcinogenic effect. Therefore, it is concluded that the compound is not carcinogenic under present experimental conditions, where a high dose level of SB was used. The effect of SB was investigated previously by other workers, who determined that oral SB administration for 25 weeks to rats had no carcinogenic action (White, 1941). Furthermore, benzoic acid, when administered subcutaneously or intravenously for 40 days to rabbits, was also without carcinogenic effect (Hosino, 1940a,b). Even though both of these experiments yielded negative results, the present study is the first in which SB was given at a high dose level for the entire lifespan of animals. Benzoic acid is a naturally occurring compound and is also found in the urine of herbivores. Benzoic acid combines in the body with glycine to form the inactive hippuric acid. Detailed clinical investigations have recommended that healthy individuals may safely consume reasonable quantities of benzoatecontaining foods. The systemic toxicity is low and large doses produce mainly digestive derangement. However, the benzoates are probably the least harmful of food additives, since they exhibit low toxicity and do not have odor or a marked taste. This has led to their use as food preservatives, particularly for fruit and vegetable products. SB is used for such purposes at concentration of 0.1%. In acid foods, most of the benzoic acid is liberated, and such
a concentration considerably restrains bacterial growth, although it may not prevent it entirely (Goodman and Gilman, 1980; Sollman, 1957). To date sodium benzoate has been generally considered safe (GRAS) as a food additive (Select Committee on GRAS Substances, 1977), although its lifelong action has not been investigated. The present experiment is ap parently the first to fill the gap and supply needed proof that the compound is harmless in so far as its long-term tumorigenic effect is concerned. REFERENCES GOODMAN, L. S., AND GILMAN, A. (1980). The pharmacological basis of therapeutics, 8th ed. Macmillan Co., New York. HOSINO,I. (194Oa). Behavior of liver in the long-continued administration of aromatic compounds. Z&ken. Syokk. Ryogk.
15, 5-49.
HOSINO,I. (194Ob). Behavior of liver in the long-continued administration of aromatic compounds. Continued. Zikken. Syokk. Ryogk. 15, 117-15 1. National Academy of Sciences,National Research Council ( 1965). Chemicals Used in Food Processing. Publication 1274, Washington, D.C. Select Committee on GRAS Substances (1977). Evaluation of health aspects of GRAS food ingredients: Lessons learned and questions unanswered. Fed. Proc. X$25 192582. SOLLMANN, T. (1957). A Manual of Pharmacology and Its Application
to Therapeutics
and Toxicology,
8th ed.
Saunders, Philadelphia. The Merck Index (1976). 9th ed. Merck, Rahway, N.J. TOTH, B. (1972). A toxicity method with calcium cyclamate for chronic carcinogenesis experiments. Tumori 58, 137-142. TOTH, B. (1975). Synthetic and naturally occurring hydrazines as possible cancer causative agents. Cancer Res. 35, 3693-3697.
TOTH, B. (1980). Actual new cancer-causing hydrazines, hydrazides and hydrazones. J. Cancer Res. C/in. Oncol. 97,97-108. WHITE, A. (1941). Growth-inhibition produced in rats by the oral administration of sodium benzoate. Effects of various dietary supplements. Yale J. Biol. Med. 13, 759-768. BELA TOTH The Eppley Institute for Research in Cancer Diseases University of Nebraska Medical Center Omaha, Nebraska 68105
and AIlied
AND
APPLIED
TOXICOLOGY
4,494-496
( 1984)
SHORT COMMUNICATIONS Lack of Tumorigenicity
of Sodium Benzoate
in Mice
Lack OfTumorigenicity of Sodium Benzoate in Mice. TOTH, B. (1984). Fundam. Appl. Toxicol. 4, 494-496. Sodium benzoate was administered as a 2% solution in drinking water for life to randomly bred Swiss mice. Consumption of the chemical caused no detectable tumorigenic effect under the current experimental conditions.
Sodium benzoate (SB) is a widely used preservative throughout the world, especially in food products (National Academy of Sciences, 1965; The Merck Index, 1976). Its preservative qualities are most effective under slightly acidic conditions (Sollman, 1957), and the compound is generally considered safe for human consumption (Select Committee on GRAS Substances, 1977). The effect of this food preservative was studied in earlier experiments. SB, administered orally in food at a 5% level to rats, was without carcinogenic action (White, 194 1). Also, no cancer-inducing effect was observed when benzoic acid was administered subcutaneously or intravenously to rabbits (Hosino, 1940a,b). The present undertaking is apparently the first study in this area in which the compound was administered at a high dose level for the entire lifespan of the animals. It is also part of our long-term objective to reveal the possible carcinogenicity and mode of action of food additives and contaminants (Toth, 1975, 1980). METHODS Albino Swiss mice from a colony randomly bred by us since 195 1 were used. They were housed in plastic cages with granular cellulose bedding, separated according to sex in groups of 10, and given Wayne Lab Blox diet in regular pellets (Allied Mills, Inc., Chicago, Ill.) and tap water ad Iibitum with the exception described below. The chemical used was sodium benzoate (SB): MW, 144.11, MP, >3OO”C, purity, 99%, obtained from Fisher Scientific Co., Fair Lawn, N.J. Toxicity studies were performed with SB prior to the chronic experiment. Five dose levels of SB including 8, 4,2, 1, and 0.5% were administered in the drinking water daily for 35 days. Each group consisted of eight animals: four females and four males. By taking into account four 0272-0590184
$3.00
Copyright 8 1984 by the Society of Toxicology. All rights of reproduction in any form rew-vcd.
494
parameters, survival rate, body weight, chemical consumption, and histological changes, the 2% dose level was found to be suitable for the lifelong treatment. The 8% dose level was too toxic; ah the mice died within 3 weeks of SB administration, while at the 4% dose level 3 P and 3 d died during the 35day observation period. In addition, the body weight of the surviving mice at the 4% dose level was substantially reduced. This toxicity technique was developed in this laboratory (Toth, 1972). The solution was prepared three times weekly and the total consumption of water containing SB was measured at the same intervals during the treatment period. The solution was placed in brown bottles because of the possible light sensitivity of the chemical. The chronic experimental groups and the controls were the following. Group 1: SB was dissolved in the drinking water as a 2% solution and was given for the lifespans of 50 female and 50 male mice that were 5 weeks (39 days) old at the beginning of the experiment. The average daily water consumption with the chemical in it per animal was 5.9 ml for the females and 6.2 ml for the males. Therefore, the average daily intake of SB was 119.2 mg for a female and 124.0 mg for a male. Group 2: These were the untreated controls. Mice, 100 female and 100 male, were kept and observed from weaning time (5 weeks of age). The experimental and control animals were carefully checked and weighed at weekly intervals, and the gross pathological changes were recorded. The animals wem either allowed to die or were killed with ether when moribund. Complete necropsies were performed on all animals. All organs were examined macroscopically and fixed in 10% buffered Formahn. Histological studies were done of the liver, spleen, kidneys, bladder, thyroid, heart, pancreas, testes, ovaries, brain, nasal turbinates, at least four lobes of the lungs of each mouse and on those organs with gross pathological changes. Sections from these tissues were stained routinely with hematoxylin and eosin and examined by light microscopy.
RESULTS The survival rates of mice after weaning in weeks of age were recorded. The data clearly
999
996
Untreated
Controls
87 (51-134)
Age at death“
Lungs
23
21
92 (53-125)
95 (60-122)
14 85 (58-113)
20
%
’ Average and range in weeks. b Age at death in weeks given in parentheses.
23
21
7
506
2
10
SB, 2% in drinking water daily for life
1
509
Treatment
Effective No. of mice No.
Group
1
12
24
3
10
No.
89 (31-130)
81 (61-104)
88 (57-119)
Age at death *
12 90(40-126)
24
6
20
W
Mal. lymphomas
6
5
4
2
No.
6
5
8
4
9%
Other tissue@
88 (65-105)
112 (97-130)
2 Adenocarcinomas of breast (73, 93), 1 leiomyosarcoma of uterus (8 I), 2 adenocarcinomas of ovary (104, 106), 1 adenoma of glandular stomach (115), 1 adenoma of thyroid (116), 1 papilloma of esophagus (106), 1 papilloma of skin (109), 1 fibrosarcoma, subcutaneous (44), 1 adenoma of ovary (103), 1 malignant histiocytoma (98) 1 Fibrosarcoma, subcutaneous (82), 1 papilloma of forestomach (63), 1 adenocarcinoma of duodenum (113), 1 malignant histiocytoma (lOl), 1 carcinoma of skin (122), 1 adrenal cortical adenoma ( 130), 1 papilloma of esophagus (63)
84 (55-l 13) 4 Adenocarcinomas of breast (44, 59, 78, 100) 1 carcinoma of skin (7 1) 1 adenoma of thyroid (134) 8 1 (61-109) I Polypoid adenoma of cecum (61) 1 hepatoma (95)
Age at death”
Blood vessels
Animals with tumors of
TUMOR DISTRIBUTION IN SODIUM BENZOATE(SB)-TREATEDANDCONTROLSWISSMICE
TABLE
!E2
SHORT COMMUNICATIONS
496
showed that the treatment had no effect on the survivals when compared with that in untreated controls. The number (percentages of animals with tumors) and the age of animals with tumors at death (latent periods) are summarized in Table 1. Since the treatment had no carcinogenic action, the tumor incidences are not detailed here. DISCUSSION The present study demonstrates that SB, when given at a 2% concentration in drinking water daily for life to Swiss mice, had no ap parent carcinogenic effect. Therefore, it is concluded that the compound is not carcinogenic under present experimental conditions, where a high dose level of SB was used. The effect of SB was investigated previously by other workers, who determined that oral SB administration for 25 weeks to rats had no carcinogenic action (White, 1941). Furthermore, benzoic acid, when administered subcutaneously or intravenously for 40 days to rabbits, was also without carcinogenic effect (Hosino, 1940a,b). Even though both of these experiments yielded negative results, the present study is the first in which SB was given at a high dose level for the entire lifespan of animals. Benzoic acid is a naturally occurring compound and is also found in the urine of herbivores. Benzoic acid combines in the body with glycine to form the inactive hippuric acid. Detailed clinical investigations have recommended that healthy individuals may safely consume reasonable quantities of benzoatecontaining foods. The systemic toxicity is low and large doses produce mainly digestive derangement. However, the benzoates are probably the least harmful of food additives, since they exhibit low toxicity and do not have odor or a marked taste. This has led to their use as food preservatives, particularly for fruit and vegetable products. SB is used for such purposes at concentration of 0.1%. In acid foods, most of the benzoic acid is liberated, and such
a concentration considerably restrains bacterial growth, although it may not prevent it entirely (Goodman and Gilman, 1980; Sollman, 1957). To date sodium benzoate has been generally considered safe (GRAS) as a food additive (Select Committee on GRAS Substances, 1977), although its lifelong action has not been investigated. The present experiment is ap parently the first to fill the gap and supply needed proof that the compound is harmless in so far as its long-term tumorigenic effect is concerned. REFERENCES GOODMAN, L. S., AND GILMAN, A. (1980). The pharmacological basis of therapeutics, 8th ed. Macmillan Co., New York. HOSINO,I. (194Oa). Behavior of liver in the long-continued administration of aromatic compounds. Z&ken. Syokk. Ryogk.
15, 5-49.
HOSINO,I. (194Ob). Behavior of liver in the long-continued administration of aromatic compounds. Continued. Zikken. Syokk. Ryogk. 15, 117-15 1. National Academy of Sciences,National Research Council ( 1965). Chemicals Used in Food Processing. Publication 1274, Washington, D.C. Select Committee on GRAS Substances (1977). Evaluation of health aspects of GRAS food ingredients: Lessons learned and questions unanswered. Fed. Proc. X$25 192582. SOLLMANN, T. (1957). A Manual of Pharmacology and Its Application
to Therapeutics
and Toxicology,
8th ed.
Saunders, Philadelphia. The Merck Index (1976). 9th ed. Merck, Rahway, N.J. TOTH, B. (1972). A toxicity method with calcium cyclamate for chronic carcinogenesis experiments. Tumori 58, 137-142. TOTH, B. (1975). Synthetic and naturally occurring hydrazines as possible cancer causative agents. Cancer Res. 35, 3693-3697.
TOTH, B. (1980). Actual new cancer-causing hydrazines, hydrazides and hydrazones. J. Cancer Res. C/in. Oncol. 97,97-108. WHITE, A. (1941). Growth-inhibition produced in rats by the oral administration of sodium benzoate. Effects of various dietary supplements. Yale J. Biol. Med. 13, 759-768. BELA TOTH The Eppley Institute for Research in Cancer Diseases University of Nebraska Medical Center Omaha, Nebraska 68105
and AIlied