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VPS, a hot water extract of the Coriolus versicolor mushroom, was given at a 2% dose level in the diet of female Swiss Webster CFW outbred mice in a serial sacrifice experiment. The mice were also administered either 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) as ten weekly subcutaneous (s.c) injections of 20 microg/g body weight or physiological saline (PS) as ten weekly (s.c) injections of 0.01 ml/g body weight. The animals were sacrificed at 26 weeks or 35 weeks after the first injection of 1,2-DMH or PS. The number of mice with large intestinal tumors and the total number of these tumors were: Group I (1,2-DMH), 29 and 438; Group 2 (VPS + 1,2-DMH), 29 and 344; Group 3 (VPS + PS), 0 and 0; and Group 4 (PS), I and 1, in the mice sacrificed at 26 weeks. The corresponding tumor incidences in mice sacrificed at 35 weeks were: Group 1 (1,2-DMH), 30 and 323; Group 2 (VPS + 1,2-DMH), 29 and 521; Group 3 (VPS + PS), 1 and 2; and Group 4 (PS), 0 and 0. Histopathologically, the tumors were diagnosed as polypoid adenomas and adenocarcinomas of the cecum, colon and rectum. Contrary to expectations, the VPS treatment enhanced the development of large intestinal tumors induced by 1,2-DMH in animals sacrificed at 35 weeks after the first injection of the carcinogen.

The objective of the present evaluation was to assess the possible roles of the sex hormones (endocrine status), nutritional influences and other factors in regard to species susceptibilities to chemical carcinogens. By reviewing the experimental and epidemiological data, it was concluded that there are compelling indications, particularly in the fields of physiology and metabolism, to conclude the limited usefulness of the various animal species in sex hormone research. The findings allow only restricted inferences for the human species. On the other hand, as far as nutritional influences on cancer development are concerned, similar mechanisms operate with regard to high or low calorie, fat, protein and carbohydrate intakes in animals and humans. Consequently, the findings obtained in laboratory animals can be transmitted with some modifications to the human species. This assessment is the continuation of an earlier work, which appraised the roles of genetic and viral agents, concerning species susceptibilities to chemical carcinogens.

The carcinogenesis literature concerning the susceptibilities of the various animal species and humans to chemical carcinogens is reviewed. The fundamental issue is whether the different laboratory animal species used in the past and at the present are the appropriate systems to detect the carcinogenic properties of chemicals. It was concluded that the main problem is the presence of high to moderate incidences of spontaneously-appearing cancers in laboratory animals. These tumor incidences are, to a significant extent, either caused by genetic factors and/or viruses which render the systems excessively-susceptible to the carcinogenic action of a large proportion of chemicals. The same types of genetic factors and/or viruses do not exist in the human species. Consequently, the data obtained in the various animal species are exaggerated to an unrealistic proportion due to the inherent flawed designs, therefore, it has little bearing in the human species. It is further concluded that a new animal breeding program should be initiated with the specific aim of developing several tumor-free or near tumor-free species and strains of laboratory animals. Until then, a healthy state of mind is required to separate the reality from the fiction.

This writing details the natural occurrence, synthetic production and use of the carcinogenic hydrazines and related chemicals. Twenty-three such chemicals were found in nature in mushrooms, tobacco, bay leaves, antibiotics, soil and other sources. The synthetically-produced hydrazines and related chemicals include 61 compounds which were or are in use as pharmaceutical drugs, agricultural substances, industrial chemicals and fuels for military and space vehicles and rockets. Because there is an overlap between the naturally-occurring and synthetically-produced compounds, their total number is 76. Altogether, 98 hydrazines and related chemicals were studied for carcinogenic action thus far, of which 84 were found to be carcinogenic and the remaining 14 were inactive. This means that the human population is exposed in various degrees to a substantial number of these agents. Therefore, the hydrazine class, unlike the polycyclic aromatic hydrocarbons, N-nitroso compounds, and aromatic amines, poses a greater environmental risk to the human population. In view of these considerations, it appears reasonable to proceed with additional experimentation in this field of interest.

Benzenediazonium sulfate (BD) was administered as 10 weekly subcutaneous injections at 25 micrograms/g b.w. and as 52 weekly oral gavages at 100 micrograms/g b.w. to Swiss mice, starting at 6 weeks of age. The subcutaneous administration induced tumors in the subcutis with an incidence of 8% in females. The oral treatment gave rise to lung tumors with incidences of 52% in females and 62% in males. In the untreated control female mice, no subcutaneous tissue tumor was observed, but the incidences of lung tumors were 28% in females and 38% in males. Histopathologically, the neoplasms were classified as fibrosarcomas of the subcutis and adenomas and adenocarcinomas of the lungs. In an earlier experiment, BD induced high incidences of subcutaneous tissue tumors in the same species when it was administered as 26 weekly subcutaneous injections at 10 micrograms/g. This indicates the length of treatment is paramount to the dose of carcinogen. The oral route, even though it was carcinogenic in the lungs, failed to elicit the development of cancer in the glandular stomach.

Benzenediazonium sulfate (BD) was given to Swiss mice by 26 subcutaneous injections of 10 micrograms/g body weight at weekly intervals. The treatment gave rise to tumors of the subcutis. The tumor incidences in the treated groups were 42% in females and 26% in males. The corresponding tumor incidences in the untreated controls were 0% in females and 2% in males. Histopathologically, the neoplasms were classified as fibrosarcomas, rhabdomyosarcomas, and osteosarcomas of the subcutaneous tissue. BD is formed during the cytochrome P-450 catalyzed metabolism of the carcinogenic 1-(phenylazo)-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14), which was used as a coloring agent for food and other materials in several countries. Further, BD is a metabolic breakdown product of different classes of nitrogen-nitrogen bond- containing chemicals. BD is the fourth benzenediazonium salt found to be carcinogenic in this laboratory.

Continuous administration of 10, 5, and 2.5% lyophilized Agaricus bisporus (AB) mushroom in the diet of six-week-old, randomly bred Swiss mice for life induced tumors in the lungs, forestomach, glandular stomach, and ovaries in certain groups. Some of the tumor incidences were found to be statistically significant, although no dose-response relationship was established. Histopathologically, the neoplasms were classified as adenomas and adenocarcinomas of lungs, glandular stomach, and ovaries and squamous cell papillomas and carcinomas of the forestomach. AB given in both raw and baked forms induced tumors in the same species in earlier experiments. Since this fungus is consumed in lyophilized form to a certain degree in the United States, the results may carry practical significance.

Agaricus bisporus, the cultivated mushroom of the western hemisphere, was baked at 220-230 degrees C for 10 minutes and subsequently fed to mice for 12 hours each day, five days each week throughout their life. After each feeding cycle, the animals received a well-balanced semisynthetic diet for 12 hours each day for five days plus the remaining two full days each week. The estimated average daily mushroom consumption per animal was 4.8 g for a female and 4.2 g for a male. Randomly bred Swiss mice, six weeks old at the start of the experiment, were used. In the baked mushroom-fed group, the incidences of tumors in the lungs, blood vessels, cecum, and colon increased when compared to the untreated controls. These increases were not, however, statistically significant. In another previous experiment, both the raw and the baked mushrooms, when used in different feeding regimens, induced statistically significant incidences of cancers in several organs of the mice. It is possible that the negative finding in the current study was due to insufficient mushroom consumption.

A comparative assessment is performed on one of the false morel mushrooms, Gyromitra esculenta, including the amounts of some hydrazines present in this fungus, the cancer-inducing doses of these chemicals or the mushroom used in animal experiments, the total amounts of the hydrazines or mushroom needed to induce neoplasms in mice and the estimated total amounts of hydrazines or mushroom needed to induce cancer in humans. When one compares the estimated amounts of hydrazines required to induce cancer with the amount of raw Gyromitra esculenta needed to yield a similar effect, it becomes clear that to date 37 percent of the carcinogenic ingredients of this fungus have been identified.

Hexanal methylformylhydrazone (HMFH) was administered to Swiss mice by intragastric instillations at weekly intervals of 52 times at 50 or 100 micrograms/g body weight. The treatment induced tumors of the lungs, liver and preputial glands. The tumor incidences in the group that received the lower dose were 74, 4 and 0% in females and 78, 8 and 6% in the males. In the group with the higher dose, the incidences were 90, 28 and 0% in females and 70, 6 and 86% in males. The corresponding tumor incidences in the untreated animals were 26, 0 and 0% in females and 26, 0 and 0% in males. The histopathological examination revealed adenomas and adenocarcinomas of lungs, benign hepatomas and liver cell carcinomas, and squamous cell papillomas and carcinomas and adenomas of preputial glands. HMFH is the sixth carcinogenic ingredient found in one of the false morel mushrooms, Gyromitra esculenta. Because this fungus is considered edible, the health hazard implications cannot be discounted.

The carcinogenic, mutagenic and teratogenic activity of 11 hydrazine analogues and diazonium ions are described. These chemicals are ingredients of 22 mushroom species. Of these, one is cultivated, while the remaining 21 are wild fungi. To date, many of these mushrooms are considered to be edible. The appropriate chemistry and biochemistry investigations of the mushroom chemicals are summarized briefly. Human consumption estimates also are provided in as much as possible.

3-Methylbutanal methylformylhydrazone (3-MBMFH) was given to Swiss mice by intragastric instillations at weekly intervals 53 or 2 times at 50 micrograms/g body weight. The treatments induced tumors of the lungs, liver, gallbladder, preputial glands and thyroid. The tumor incidences in the group treated 53 times were 84, 32, 10, 0 and 0% in females, and 76, 38, 6, 48 and 0% in the males, respectively. In the group treated twice the incidences were 38, 0, 4, 0 and 10% in females, and 30, 0, 2, 0 and 6% in males, respectively. The corresponding tumor incidences in the untreated controls were 26, 0, 0, 0 and 0% in females, and 26, 0, 0, 0 and 0% in males, respectively. The light microscopic examination of these neoplasms showed adenomas and adenocarcinomas of lungs, hepatomas and liver cell carcinomas, adenomas and adenocarcinomas of gallbladder, squamous cell papillomas and carcinomas of preputial glands; and, adenomas and adenocarcinomas of thyroid. Gyromitra esculenta mushroom contains 3-MBMFH along with the other hydrazine derivatives. Because at least five ingredients of this fungus exhibit carcinogenic properties, the health hazard implications should be considered.

Cyclopenteno[cd]pyrene (CPEP) is a widespread environmental pollutant. This hydrocarbon and its 3,4-dihydro derivative, cyclopentano[cd]pyrene (CPAP), were tested on skin in a two-stage initiation-promotion experiment in CD-1 mice and by repeated application in Swiss mice. The biological effect of CPEP and CPAP was compared to that of benzo[a]-pyrene (BP). Nine-week-old female CD-1 mice in groups of 30 were treated every other day over a 20-day period at mini-dose levels of 0.18, 0.06 and 0.02 mumol of CPEP or CPAP in acetone. One group was treated with BP at the low mini-dose level. Initiation was followed by twice weekly application of tetradecanoyl phorbol acetate for 40 weeks. In the second experiments, nine-week-old female Swiss mice in groups of 30 were treated at dose levels of 1.8, 0.6 and 0.2 mumol CPEP or CPAP in acetone twice weekly for 30 weeks. One group was treated with BP at the low dose. CPAP was virtually inactive in both studies. In the initiation-promotion experiment CPEP was inactive at the low dose level, whereas BP exhibited significant tumorigenicity. At the medium and high doses CPEP showed weak, but statistically insignificant, tumorigenic activity. Repeated application of CPEP at the high, medium and low doses resulted in tumor incidences of 23, 37 and 57%, respectively. This reverse dose-response may be due to the relatively high cytotoxicity of CPEP, BP, which was compared to CPEP at the low dose, elicited tumors in 100% of the mice. Most of the CPEP-induced neoplasms were malignant and some metastasized to lungs and lymph nodes. The inactivity of CPAP suggests the carcinogenicity of CPEP is probably due to formation of the ultimate metabolite CPEP 3,4-oxide. In view of the abundance of CPEP in environmental and occupational pollutants, its moderately potent carcinogenicity may represent a potential health hazard.

Agaritine (A), an ingredient of the cultivated mushroom of commerce Agaricus bisporus, was administered by subcutaneous injection to two groups of randomly bred Swiss mice. In the first group the animals of both sexes were treated at a 100 micrograms/g body weight basis five times at weekly intervals, while in the second group the mice received a single A treatment of 100 micrograms/g body weight for females and 50 micrograms/g body weight for males. The administration of the compound resulted in no detectable carcinogenic effect in the animals. Since some of the breakdown products of A were shown to be carcinogenic in mice and the mushroom itself was found to be mutagenic, the field is discussed in the light of the obtained results.

Lifetime administration of 0.03125% 1,1-di-n-butylhydrazine in drinking water to Swiss mice, from 6 weeks of age, induced tumors of the lungs, forestomach and liver. The tumor incidences in these tissues in untreated controls were 25, 2 and 0.5%, whereas in the treated groups the corresponding tumor incidences increased to 68, 39 and 5%, respectively. Histopathologically these lesions were classified as adenomas and adenocarcinomas of the lungs, squamous cell papillomas and carcinomas of the forestomach, and benign hepatomas and liver cell carcinomas. The work is part of a structure activity inquiry and its specific aim is to disclose whether the dialkyl derivatives of hydrazine are more active carcinogens than the monoalkyl analogues.

Solutions of 0.002 and 0.001% N-methyl-N-formylhydrazine (MFH) were given separately daily in drinking water to 6-week-old randomly bred Swiss mice for life. The treatments resulted in induction of tumors of the lungs, blood vessels, liver and gall bladder. The 0.002% solution gave rise to tumors of the above tissues in incidences of 76, 27, 28 and 11%, respectively, while the 0.001% solution induced tumors in incidences of 75, 23, 7 and 6%. Histopathologically, the neoplasms were adenomas and adenocarcinomas of lungs, angiomas and angiosarcomas of blood vessells, benign hepatomas, liver cell carcinomas and adenomas and adenocarcinomas of the gall bladder. MFH is an ingredient of the edible mushroom, the false morel Gyromitra esculenta. The present study proves the carcinogenicity of MFH administered at low dose levels. The possible environmental significance of the findings to the human population is discussed.

N-ethyl-N-formylhydrazine (EFH) was administered as a 0.02% solution in drinking water continuously for life to randomly bred Swiss mice, from 6 weeks of age. The treatment induced tumors of the lungs, blood vessels, liver, gall bladder and preputial glands. The tumor incidences in treated females for these five tissues were 98, 94, 0, 2 and 0%, whereas in the treated males they were 78, 64, 26, 8 and 10%, respectively. Histopathologically the lesions were adenomas and adenocarcinomas of the lungs, angiomas and angiosarcomas of blood vessels, benign hepatomas, liver cell carcinomas, adenomas and adenocarcinomas of the gall bladder, and squamous cell papillomas and carcinomas of preputial glands. The study is part of a structure activity relationship inquiry and proves the carcinogenicity of EFH, a structural homologue of N-methyl-N-formylhydrazine, an ingredient of the edible false morel mushroom.

4-Methylphenylhydrazine hydrochloride was administered as 10 weekly subcutaneous injections of 140 microgram/g body weight and as 7 weekly intragastric instillations of 250 microgram/g body weight in physiological saline to randomly bred Swiss mice. Treatments given subcutaneously resulted in induction of lung tumors in incidences of 36% in females and 44% in males, while intragastric treatment caused a 40% incidence in females. In addition, it gave rise to blood vessel tumors by intragastric route in incidences of 32% in females and 18% in males. In the two physiological saline-treated control groups, the lung tumor incidence (combined) was 20% in females and 21% in males, while the blood vessel tumor incidence (combined) was 7% in females and 6% in males. Histopathologically, the lesions were classified as adenomas and adenocarcinomas of the lungs, and angiomas and angiosarcomas of blood vessels. 4-Methylphenylhydrazine was postulated to be a metabolite of 4-hydroxymethylphenylhydrazine, an ingredient of the commonly eaten mushroom Agaricus bisporus. The implications are discussed with respect to the tumorigenesis data.

4-Hydroxybenzenediazonium sulfate (HBD) was administered to Swiss mice by subcutaneous injection at weekly intervals of two or 36 times at 10 or 2 micrograms per gram body weight, respectively. The HBD given 36 times induced tumors of the subcutis in 22% of females and in 22% of males. The corresponding tumor incidences in the untreated controls were 2% in females and 8% in males. Histopathologically, the neoplasms were classified as fibromas, fibrosarcomas, myxosarcomas and rhabdomyosarcomas. The HBD given two times was, however, without tumor-inducing effect. HBD is an ingredient of the Agaricus xanthodermus, a non-cultivable unedible mushroom, which is closely related to the mushroom of commerce Agaricus bisporus. HBD is now the third compound of the diazonium class to exhibit carcinogenic activity.

Continuous administration of 0.04% N-n-butyl-N-formylhydrazine (BFH) in drinking water to 6-week-old randomly bred Swiss mice for life produced tumors of the lungs, preputial and clitoral glands. The tumor incidences in these three tissues of the treated animals were 87, 66, and 10%, whereas in the untreated controls they were 25, 0, and 0%, respectively. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of the lungs, squamous cell papillomas and carcinomas, angio-, fibro-, and myxo- sarcomas of preputial glands and squamous cell papillomas and carcinomas of clitoral glands. N-n-Butyl-N-formylhydrazine is a structural homologue of the carcinogenic N-methyl-N-formylhydrazine and N-ethyl-N-formylhydrazine. These studies are integral parts of structure activity relationship inquiries.

Continuous administration of 0.0625% 1,2-diallylhydrazine dihydrochloride in drinking water for life to 6-week-old randomly bred to Swiss mice induced lung tumors. In comparison with the untreated controls, the lung tumor incidence rose from 25 to 80% in the females and from 26 to 80% in the males. The treatment had no apparent effect on the development of other tumor types. Histopathologically, the lesions were classified as adenomas and adenocarcinomas of the lungs. The work is a continuation of our structure activity inquiry concerning the relative carcinogenic potencies of the dialkyl versus the monoalkyl series of hydrazine analogues.

N-Methyl-N-formylhydrazine (MFH) was administered in drinking water as a 0.0078% solution to randomly bred Syrian golden hamsters for life beginning at 6 weeks of age. The treatment gave rise to benign and malignant liver cell tumors, malignant histiocytomas and tumors of the gall bladder and bile ducts. The tumor incidence in these four treated tissues was 43, 34, 11,8%, while in untreated controls it was 0, 0, 0, 0%, respectively. Histopathologically, tumors were classified as benign hepatomas, liver cell carcinomas, malignant histiocytomas, adenomas and adenocarcinomas of the gall bladder, cholangiomas, and cholangiocarcinomas.

The sulfate form of 4-(hydroxymethyl) benzenediazonium ion (HMBD) of Agaricus bisporus was given by 26 weekly subcutaneous injections at 50 micrograms/g body weight to Swiss mice. As a result of treatment, tumors of subcutis and skin developed in 32% and 14% of the females and 40% and 4% of the males, respectively. The corresponding tumor incidences in the sodium sulfate-injected solvent control groups were 0% and 2% in females and 4% and 0% in males, respectively. Histopathologically, the tumors were fibrosarcomas, rhabdomyosarcomas and angiosarcomas of the subcutis, squamous cell papillomas and carcinomas of skin. The present finding discounts an earlier allegation that the tetrafluoroborate portion of the diazonium ion acted as a cocarcinogen. HMBD was found at a 0.6 ppm level in the mushroom of commerce Agaricus bisporus.

A solution of 2% succinic acid 2,2-dimethylhydrazide was given continuously in the drinking water of 6-week-old randomly bred albino mice for the remainder of their lives. The treatment gave rise to tumors of blood vessels, lungs, and kidneys. The tumor incidences in these tissues in the controls were 6, 18, and 0%, whereas in the treated groups the corresponding tumor incidences were 73, 73, and 5%. Light microscopic examination revealed typical angiomas and angiosarcomas of blood vessels, adenomas and adenocarcinomas of lungs, and adenomas of kidneys. The study thus demonstrates the tumorigenicity of the herbicide, succinic acid 2,2-dimethylhydrazide. Since the residues of this chemical occur in fruit, the human population is exposed to it. The environmental implication of this finding and the fact that the hydrazines as a class have tumorigenic properties are discussed.

4-(Hydroxymethyl-benzenediazonium tetrafluoroborate was administered as a single intragastric instillation at 400 micrograms/g to Swiss albino mice. The treatment gave rise to glandular stomach tumours in incidences of 30% in females and 32% in males. Histopathologically, the tumours were classified as polypoid adenomas and adenocarcinomas. This diazonium ion is an ingredient of the cultivated mushroom of commerce, Agaricus bisporus. The implications are self-evident.

1,2-Dimethylhydrazine dihydrochloride (1,2-DMH) was given to Swiss mice in 1 or 10 weekly intracolonic instillations of 60 and 40 micrograms/g body weight, respectively. In mice that received a single treatment, the tumor incidences in the lungs, blood vessels and subcutis were 34% (p less than 0.006), 8 and 25 in females and 2, 20 and 0% in males, respectively. In mice treated repeatedly with the carcinogen, the corresponding tumor incidences were 34% (p less than 0.006), 38% (p less than 0.000001) and 12% (p less than 0.03) in females and 34, 10 and 0% in males, respectively. In control mice the tumor incidences in lungs, blood vessels and subcutis were 15, 8 and 2% in females and 22, 5 and 2% in males, respectively. The induction of large intestinal cancer, which was the main objective of this investigation, however, failed to materialize.

Tetramethylhydrazine hydrochloride (TEMH) was administered in drinking water as a 0.125% solution to randomly bred Swiss mice for life beginning at 6 weeks of age. As a result of treatment, the incidence of blood vessel tumors rose from 5 to 96% in females and from 6 to 88% in males, while that of lung tumors increased from 21 to 36% in females and from 23 to 28% in males, as compared with untreated controls. The increased incidence of blood vessel tumors, but not of lung neoplasms, was statistically significant. Histopathologically, the tumors exhibited the characteristics of angiomas and angiosarcomas of blood vessels and adenomas of the lung. The investigation proved for the first time the tumorigenicity of TEMH. The possible role of increased methyl substitution on hydrazine in tumorigenesis was also discussed, as well as hydrazine derivatives as a tumor-producing class.

Nicotinic acid hydrazide was administered as a 0.125% solution in drinking water continuously for life from 6 weeks of age to randomly bred Swiss albino mice. As a result of treatment, the lung tumor incidence rose from 25 to 76% in females and from 26 to 42% in males. The treatment had no statistically significant effect on the development of other types of tumors. Histopathologically the tumors were classified as adenomas and adenocarcinomas of the lungs. The study thus demonstrates the tumorigenicity of this compound which is a structural homologue of the tuberculostatic and carcinogenic isonicotinic acid hydrazide.

Continuous administration of 0.0078% N-methyl-N-formylhydrazine (MFH) in drinking water to 6-week-old outbred Swiss mice for life produced tumors of the liver, lung, gallbladder, and bile duct. The incidences of tumors in these four tissues were 33, 50, 9, and 7%, whereas in the untreated controls they were 1, 18, 0, and 0%, respectively. The higher dose (0.0156% MFH) given under identical conditions had no tumorigenic effect, since it proved too toxic for the animals. Histopathologically, the lesions were classified as benign hepatomas, liver cell carcinomas, adenomas and adenocarcinomas of the lungs, adenomas of the gallbladder, cholangiomas, and cholangiocarcinomas. Since the edible false morel Gyromitra esculenta contains a high amount of MFH, the human population should be dissuaded from consumption of this dangerous mushroom.

Capsaicin, the pungent principle of hot pepper, was administered at concentrations of 1, 0.5, 0.25, 0.125 and 0.0625% in powdered diet for 35 days to five groups of Swiss albino mice. In the capsaicin-treated groups 4 mice (10%) developed 4 adenocarcinomas of the duodenum (one tumor at each dose level, except for the highest dose), while no such tumor occurred in the untreated control mice. Capsaicin exhibited a low level of mutagenicity in the Ames assay with S. typhimurium strain TA98 in the presence of liver activating enzymes from Aroclor-induced rats.

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.

The various synthetic substituted hydrazines, which cause tumors in animals, are briefly enumerated. To date, 19 of them have proved to be tumorigenic in animals. A number of these chemicals are found today in the environment, in industry, in agriculture, and in medicine, and the human population is exposed to a certain degree to some of them. Hydrazine also occurs in nature in tobacco and tobacco smoke. The three other naturally occurring hydrazine compounds are N-methyl-N-formylhydrazine, which occurs in the wild edible mushroom, Gyromitra esculenta, and beta-N-[gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine and 4-hydroxymethylphenylhydrazine, which are found in the commonly eaten cultivated mushroom, Agaricus bisporus. Tumorigenesis studies with the naturally occurring hydrazines are in progress.

p-Hydrazinobenzoic acid (HBA), an ingredient of the cultivated mushroom Agaricus bisporus, was given in hydrochloride form at a dosage of 0.125% in drinking water for life to randomly bred Swiss mice. Previous studies had demonstrated that either synthetic or naturally occurring hydrazines are carcinogenic in mice with the main tumors so-induced being peripheral angiomas and angiosarcomas. As a result of HBA treatment in the present experiments, smooth muscle cell tumors of the aorta and large arteries were induced in 14% of females and 42% of males, whereas the corresponding frequency of tumors in untreated female and male controls was 0 and 4%, respectively. Tumors were observed as early as at 17 weeks of age. Numerous experimental animals (32% of females and 50% of males) died of aortic rupture. Histopathologically, two major changes were observed to explain both the ruptures and tumors. First, the intimal and inner medial aspect of the aortic walls had undergone effacement with widespread fibrinoid necrosis, accompanied by medial elastinolysis. Second, a proliferation of cells arising in the media, benign in some aortae and frankly malignant in others, was strikingly positive by immunohistochemistry for cytoplasmic actin and myosin, moderately positive for desmin, weakly positive for vimentin, and negative for factor VIII-related antigen. When malignant, the tumors extended into the periaortic adventitial connective tissue. The tumors were classifiable as leiomyomas and leiomyosarcomas. Thus, HBA is an additional carcinogenic ingredient of the widely consumed mushroom, A. bisporus. A continuum from toxic tissue injury to cellular hyperplasia, dysplasia, and ultimate neoplasia is well-illustrated by HBA.

beta-Phenylisopropylhydrazine hydrochloride and thiocarbamylhydrazine were separately administered as 0.0312 and 0.0156% solutions in drinking water for life to randomly bred Swiss mice. The consumption of the chemicals resulted in no detectable tumorigenic effects in the treated animals, and it is therefore concluded that these compounds are apparently noncarcinogenic under the present experimental conditions.

The experimental techniques, methods of evaluation, and environmental implications of the findings of chemical carcinogenesis studies and related subject areas are herein critically reviewed. It is concluded that firstly, certain presently used procedures provide the best possibilities for the study of the hazardous nature of chemicals. Secondly, other matters, including the dosage of chemicals, animal species, meaning of the term "tumor induction", values of short-term tests, evaluation of tumor incidences, relation of the responses of humans and animals to carcinogenic stimuli, require new attitudes, innovative approaches, and meaningful changes. Thirdly, some of the issues are and will remain controversial until our basic knowledge substantially improves. Finally, it appears advisable to abandon some of the dogmatic attitudes for a more flexible approach.

N'-Acetyl-4-(hydroxymethyl)phenylhydrazine was administered as a 0.0625% solution in drinking water continuously for the life span of Swiss mice, from 6 weeks of age. Compared to that in untreated controls, in treated animals the lung tumor incidence rose from 15 to 34% in females and 22 to 48% in males, whereas the incidence of blood vessel tumors increased from 8 to 32% in females and from 5 to 30% in males. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of the lungs and angiomas and angiosarcomas of the blood vessels. The commonly eaten mushroom Agaricus bisporus contains beta-N-[gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine, which under certain conditions yields 4-hydroxymethylphenylhydrazine and L-glutamic acid. Since 4-hydroxymethylphenylhydrazine is relatively unstable, its acetyl derivative was synthesized for this study. The possible environmental significance of the findings is discussed.

4-(Methyl)benzenediazonium sulfate (MBD) was administered to Swiss mice by subcutaneous injection at weekly intervals 19 or 16 times at 50 micrograms/g body weight. The treatment gave rise to tumors of the subcutaneous tissue and skin. The tumor incidences in the groups with 19 injections were 12 and 6% in females and 12 and 4% in males, respectively, while in the group with 16 injections were 24 and 14% in females and 50 and 4% in males, respectively. The corresponding tumor incidences in the untreated controls were 2 and 2% in females and 8 and 2% in males, respectively. Histopathologically, the tumors were classified as fibromas, fibrosarcomas, myxosarcomas, fibromyxosarcomas, rhabdomyosarcomas and angiosarcomas in subcutis and squamous cell papillomas and carcinomas of skin. MBD is closely related to 4-(hydroxymethyl)benzenediazonium ion (HMBD), an ingredient of the cultivated mushroom Agaricus bisporus. The conversion of HMBD to MBD appears to be a distinct possibility.

A dose response study in carcinogenesis by 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) in Swiss mice was performed. The compound was administered continuously in drinking water for life from 6 weeks of age at dose levels of 0.002, 0.001, 0.0005, 0.00025, 0.000125, 0.0000625, 0.00003125 and 0.000015625%. A positive correlation was established between the dose levels of 1,2-DMH and the yield of blood vessel tumors. In addition, the latency periods for these tumors diminished when the dose levels of the carcinogen decreased. Although some of the treatments induced lung tumors, no association was observed between the dose levels of the carcinogen and the lung tumor incidence. The study thus provides an example in which the continuous lifespan administration of a carcinogen resulted in a partial dose response effect. This method of administration closely resembles some of the human exposure situation.