First experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feed to Sprague-Dawley rats.
The Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation has conducted a long-term bioassay Bioassay
A method for the quantitation of the effects on a biological system by its exposure to a substance, as well as the quantitation of the concentration of a substance by some observable effect on a biological system. on aspartame aspartame: see sweetener, artificial.
Synthetic organic compound (a dipeptide) of phenylalanine and aspartic acid. It is 150–200 times as sweet as cane sugar and is used as a nonnutritive tabletop sweetener and in low-calorie (APM (Advanced Power Management) A programming interface (API) from Intel and Microsoft for battery-powered computers that lets programs communicate power requirements to slow down and speed up components. See ACPI.
APM - Advanced Power Management ), a widely used artificial sweetener. APM was administered with feed to 8-week-old Sprague-Dawley rats (100-150/sex/group), at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. The treatment lasted until natural death, at which time all deceased animals underwent complete necropsy necropsy /nec·rop·sy/ (nek´rop-se) examination of a body after death; autopsy.
examination of a body after death. See also autopsy. . Histopathologic evaluation of all pathologic lesions and of all organs and tissues collected was routinely performed on each animal of all experimental groups. The results of the study show for the first time that APM, in our experimental conditions, causes a) an increased incidence of malignant-tumor--bearing animals with a positive significant trend in males (p [less than or equal to] 0.05) and in females (p [less than or equal to] 0.01), in particular those females treated at 50,000 ppm (p [less than or equal to] 0.01); b) an increase in lymphomas and leukemias with a positive significant trend in both males (p [less than or equal to] 0.05) and females (p [less than or equal to] 0.01), in particular in females treated at doses of 100,000 (p [less than or equal to] 0.01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.05), 2,000 (p [less than or equal to] 0.05), or 400 ppm (p [less than or equal to] 0.01); a statistically significant increased incidence, with a positive significant trend (p [less than or equal to] 0.01), of transitional cell carcinomas of the renal pelvis and ureter ureter (yrē`tər), thick-walled tube that conveys urine from the kidney to the urinary bladder. It is approximately 10 in. (25. and their precursors (dysplasias) in females treated at 100,000 (p [less than or equal to] 0,01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.01), 2,000 (p [less than or equal to] 0.05), or 400 ppm (p [less than or equal to] 0.05); and d) an increased incidence of malignant schwannomas of peripheral nerves Peripheral nerves
Nerves throughout the body that carry information to and from the spinal cord.
Mentioned in: Amyloidosis, Charcot Marie Tooth Disease with a positive trend (p [less than or equal to] 0.05) in males. The results of this mega-experiment indicate that APM is a multipotential carcinogenic carcinogenic
having a capacity for carcinogenesis. agent, even at a daily dose of 20 mg/kg body weight, much less than the current acceptable daily intake acceptable daily intake
the amount of a drug or chemical residue to which an animal can be exposed daily for a lifetime without suffering a deleterious or injurious effect, on the basis of all of the facts known at the time. . On the basis of these results, a reevaluation of the present guidelines on the use and consumption of APM is urgent and cannot be delayed, Key words: artificial sweetener, aspartame, carcinogenicity carcinogenicity /car·ci·no·ge·nic·i·ty/ (kahr?si-no-je-nis´i-te) the ability or tendency to produce cancer.
the ability or tendency to produce cancer. , lymphomas, malignant schwannomas, rats, renal pelvis carcinomas. Environ Health Perspect 114:379-385 (2006). doi:10.1289/ehp.8711 available via http://dx.doi.org/ [Online 17 November 2005]
Consumers are increasingly concerned about the quality and safety of many products present in the diet of industrialized in·dus·tri·al·ize
v. in·dus·tri·al·ized, in·dus·tri·al·iz·ing, in·dus·tri·al·iz·es
1. To develop industry in (a country or society, for example).
2. countries, in particular, the use of artificial sweeteners, flavorings, colorings, preservatives, and dietary supplements. General apprehension also exists regarding the possible long-term health effects of the raw materials and technologies used for the packaging, sterilization, and distribution of foods. Of particular concern are the potential carcinogenic effects of these products and processes.
The experimental and epidemiologic data currently available to evaluate the above carcinogenic risks are insufficient and often unreliable because of the inadequate planning and conduct of previous experiments. This inadequacy, combined with the general limited knowledge about the safety and potential carcinogenic effects of substances widely present in the industrialized diet, motivated the design of an integrated project of mega-experiments in 1985 at the Cesare Maltoni Cancer Research Center (CMCRC) of the European Ramazzini Foundation (ERF n. 1. A garden plot, usually about half an acre. ). The products studied are reported in Table 1. The products and agents we selected for this project were those for which committee debate and opinions had often acted as surrogates for good laboratory work. At present, over the course of the project, 32 long-term bioassays have been performed using > 25,000 rodents. Studies have evaluated the carcinogenicity of 12 different products, including the artificial sweetener aspartame (APM).
In this article we present the results of the mega-experiment on the carcinogenicity of APM in which the sweetener Sweetener
A special feature added to a debt obligation or preferred stock to promote marketability.
Warrants and convertibles are two popular sweeteners.
See also: Convertible Bond, Kicker, Warrant
Sweetener was administered in feed to Sprague-Dawley rats for the life span.
APM, the methyl ester of the dipeptide di·pep·tide
A peptide that, on hydrolysis, yields two amino acid molecules. L-[alpha]-aspartyl-L-phenylalanine ([C.sub.14][H.sub.18][N.sub.2][O.sub.5]), is a widely used artificial sweetener with a molecular weight of 294.3. Under particular conditions (extreme pH, high temperature, lengthy storage times), APM may be contaminated by the diketopiperazine (DKP DKP Deutsche Kommunistische Partei (German Communist Party)
DKP Diketopiperazine (aspartame by-product)
DKP Dragon Kill Points (massively multiplayer online games) ) cycloaspartylphenylalanine (Butchko et al. 2002a).
For more than 30 years, APM has been widely used as a food additive because of its very strong, sweet taste. The sweetening power of APM is estimated to be 200 times that of sucrose, whereas saccharin saccharin (săk`ərĭn), C7H5NSO3, white, crystalline, aromatic compound. It was discovered accidentally by I. Remsen and C. Fahlberg in 1879. Pure saccharin tastes several hundred times as sweet as sugar. and cyclamate cyclamate (sī'kləmāt', –mət), any member of a group of salts of cyclamic acid (cyclohexanesulfamic acid). The sodium and calcium salts were commonly used as artificial sweeteners until 1969, when their use was banned by the U.S. are 300 and 30 times sweeter, respectively (Mazur 1984).
Initial commercial approval of APM in the United States was granted by the Food and Drug Administration (FDA FDA
Food and Drug Administration
n.pr See Food and Drug Administration.
n.pr the abbreviation for the Food and Drug Administration. 1974). The FDA later approved the limited use of APM in solid foods in 1981 and extended this authorization to soft drinks in 1983. APM was eventually approved as a general sweetener in 1996 (FDA 1981, 1983, 1996). In the European Union European Union (EU), name given since the ratification (Nov., 1993) of the Treaty of European Union, or Maastricht Treaty, to the
European Community , the safe use of APM was authorized in 1994 (EC Directive 1994).
After saccharin, APM is the second most used artificial sweetener in the world. It is estimated that > 8,000 tons of APM are consumed each year in the United States (Hazardous Substances Data Bank 2005). In terms of world consumption, APM represents 62% of the value of the intense sweetener market (Fry 1999).
APM is found in > 6,000 products, including carbonated and powdered soft drinks, hot chocolate, chewing gum, candy, desserts, yogurt, tabletop sweeteners, and some pharmaceutical products, such as vitamins and sugar-free cough drops, and is estimated by the Aspartame Information Center (2005) to be consumed by > 200 million people worldwide.
Through dietary surveys performed in the United States among APM consumers during the period 1984-1992, the average APM daily intake in the general population has been shown to range from 2 to 3 mg/kg body weight (bw). Consumption by children 2-5 years of age and by females of childbearing age in these surveys ranged from about 2.5 to 5 mg/kg bw/day (Butchko et al. 2002b). APM intake was also monitored in several other regions, including seven European countries. Although survey methodologies may have differed, the APM intake was remarkably consistent across studies and was well below the acceptable daily intake (ADI) both in the United States (50 mg/kg bw) and in Europe (40 mg/kg bw) (Butchko et al. 2002b).
Investigations into the metabolism of APM have shown that, in rodents, nonhuman primates, and humans, it is metabolized in the gastrointestinal tract gastrointestinal tract
The part of the digestive system consisting of the stomach, small intestine, and large intestine.
Gastrointestinal tract into three constituents--aspartic acid, phenylalanine phenylalanine (fĕn'əlăl`ənēn'), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. , and methanol--which are absorbed into the systemic circulation systemic circulation
Circulation of blood throughout the body through the arteries, capillaries, and veins, which carry oxygenated blood from the left ventricle to various tissues and return venous blood to the right atrium. (Ranney et al. 1976). For each molecule of APM, one molecule of each constituent is produced. After absorption, they are then used, metabolized, and/or excreted by the body following the same metabolic pathways as when consumed through the ordinary diet: aspartate aspartate /as·par·tate/ (ah-spahr´tat) a salt of aspartic acid, or aspartic acid in dissociated form.
1. A salt of aspartic acid.
2. is transformed into alanine alanine (ăl`ənēn'), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of proteins (see stereochemistry). plus oxaloacetate oxaloacetate /ox·a·lo·ac·e·tate/ (ok?sal-o-as´e-tat) a salt or ester of oxaloacetic acid.
a salt or ester of oxaloacetic acid. (Stegink 1984); phenylalanine is transformed mainly into tyrosine and, to a smaller extent, phenylethylamine phen·yl·eth·yl·a·mine
An amine, C8H11N, that has pharmacological properties similar to those of amphetamine, occurs naturally as a neurotransmitter in the brain, and is present in chocolate and oil of bitter almonds. and phenylpyruvate (Harper 1984); and methanol is transformed into formaldehyde and then to formic for·mic
1. Of or relating to ants.
2. Of, derived from, or containing formic acid.
[From Latin form add (Opperman 1984).
APM was not genotoxic genotoxic /ge·no·tox·ic/ (je´no-tok?sik) damaging to DNA: pertaining to agents known to damage DNA, thereby causing mutations, which can result in cancer.
adj. in the following tests: dominant lethal mutation lethal mutation
A mutant trait that leads to a phenotype incapable of effective reproduction. assay in rats, host-mediated assay in rats and mice, in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body.
Within a living organism.
in vivo adv. cytogenetic cytogenetic /cy·to·ge·net·ic/ (-je-net´ik)
1. pertaining to chromosomes.
2. pertaining to cytogenetics.
pertaining to or originating from the origin and development of the cell. assay in rats, and the Ames test Ames test
A test in which strains of Salmonella that are unable to synthesize histidine are introduced into a test substance lacking in histidine. (Kotsonis and Hjelle 1996). Results of an assay to measure induction of unscheduled DNA synthesis in rat hepatocytes treated with APM in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.
In an artificial environment outside a living organism. were negative, indicating the absence of APM-induced DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. damage (Jeffrey and Williams 2000). In a test for the induction of chromosomal aberration in bone marrow cells of male Swiss mice, Mukhopadhyay et al. (2000) reported that a mixture of APM (up to 350 mg/kg) and a second sweetener, acesulfame potassium (up to 150 mg/kg) administered by gavage gavage /ga·vage/ (gah-vahzh´) [Fr.]
1. forced feeding, especially through a tube passed into the stomach.
1. was negative. However, a dose-related increase in the percentage of cells with chromosomal aberrations was noted with increasing doses of the two sweeteners, even though the increase was not statistically significant (Mukhopadhyay et al. 2000).
Two long-term feeding carcinogenicity bioassays on APM were performed on rats and one on mice in the early 1970s by the producer Searle & Co. Results were reviewed by the FDA and then summarized in the Federal Register (FDA 1981). To date, the details of the experiments have not been published.
In the first study, groups of 40 male and 40 female Sprague-Dawley rats were treated with 1, 2, 4, or 6--8 g/kg bw/day of APM in the diet. The treatment started at 4 weeks of age and lasted for a period of 104 weeks. A control group of 60 rats per sex was fed the same diet without APM. At the end of the treatment, all surviving animals were sacrificed and their brains, as well as other organs (not specified in the report), were examined histologically. Brain tumors were observed in 7 of 155 (4.5%) exposed males versus 1 of 59 (1.7%) controls, and in 5 of 158 (3.2%) exposed females versus 0 of 59 (0%) controls. Overall, the FDA considered the study to be negative with regard to the carcinogenicity of APM (FDA 1981).
In the second study, groups of 40 male and 40 female Sprague-Dawley rats were exposed to APM, at doses of 2 and 4 g/kg bw/day, through their mothers' diet both in utero in utero (in u´ter-o) [L.] within the uterus.
In the uterus.
in utero adv. and during lactation lactation
Production of milk by female mammals after giving birth. The milk is discharged by the mammary glands in the breasts. Hormones triggered by delivery of the placenta and by nursing stimulate milk production. , and then for 104 weeks with APM in their own diets. A control group of 60 rats per sex was fed the same diet without APM. The animals were necropsied at the time of death or at 104 weeks after weaning weaning,
n the period of transition from breast feeding to eating solid foods.
the act of separating the young from the dam that it has been sucking, or receiving a milk diet provided by the dam or from artificial sources. . Three brain tumors were observed among control males and one among control females. Brain tumors were also observed in two males and one female in the 2 g/kg bw group, and in one male and one female in the 4 g/kg bw group. Again, the FDA considered the study to be negative with regard to the carcinogenicity of APM (FDA 1981).
Regarding the third chronic APM study, in this case performed on mice, the FDA reported that the results did not show any treatment-related carcinogenic effect. In this experiment, as reported by Molinary (1984), groups of 36 male and 36 female mice were fed 1, 2, or 4 g/kg bw/day until 110 weeks of age. A group of 72 males and 72 females served as the control. There were no treatment-related effects on survival and behavior, nor were any lesions recorded during macroscopic macroscopic /mac·ro·scop·ic/ (mak?ro-skop´ik) gross (2).
mac·ro·scop·ic or mac·ro·scop·i·cal
1. Large enough to be perceived or examined by the unaided eye.
2. or microscopic analysis.
An APM carcinogenicity study was also conducted in Japan during this period (Ishii 1981; Ishii et al. 1981). Groups of 86 male and 86 female Wistar rats were treated with APM in feed at doses of 0, 1, 2, or 4 g/kg bw/day from 6 to 110 weeks of age. No increase in the incidence of brain tumors was observed in the treated groups compared with the controls. Exhaustive experimental details of this study were not published.
Epidemiologic studies to evaluate the relationship between APM intake and cancer development in humans are not currently available.
Although all of the aforementioned studies were considered negative with respect to the carcinogenicity of APM, in our opinion, these studies did not comply with today's basic requirements for testing the carcinogenic potential of a physical or chemical agent, in particular concerning the number of animals for each experimental group and the duration of the experiment until 110 weeks of age of the animals.
For these reasons, and in light of the ever-increasing diffusion of APM in the diet of industrialized countries (particularly in products consumed by young children and pregnant women), we considered it important to perform a mega-experiment following today's internationally recognized good laboratory practices for carcinogenicity bioassays and, more specifically, the life-span carcinogenicity bioassay design followed for many years at the CMCRC and described in previous publications (Soffritri et al. 1999, 2002c).
Materials and Methods
APM, as a food-grade material, was produced by Nutrasweet and supplied by Giusto Faravelli S.p.A. (Milan, Italy). Its purity was > 98%: DKP was < 1.5% and L-phenylalanine was < 0.5%. An infrared absorption spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. assay was used to determine APM purity. An assumed daily intake by humans of 5,000, 2,500, 500, 100, 20, 4, or 0 mg/kg bw was simulated by adding APM to the standard Corticella diet (Laboratori Dottori Piccioni, Milan Italy), used for 30 years at the CMCRC/ERF laboratory, at concentrations of 100,000, 50,000, 10,000, 2,000, 400, 80, or 0 ppm. The APM daily assumption in milligrams per kilogram body weight was calculated considering the average weight of a rat for the duration of the experiment as 400 g, and the average consumption of feed as 20 g/day, both for males and females. APM was administered with feed ad libitum ad libitum
ad libitum feeding
food available at all times with the quantity and frequency of consumption being the free choice of the animal. to Sprague-Dawley rats (100-150/sex/group). The experiment started when the animals were 8 weeks of age, and the treatment lasted until natural death. Control animals received the same feed without APM. The experiment was conducted according to Italian law regulating the use of animals for scientific purposes (Decreto Legislativo 116 1992), which provides the guidelines on how to treat animals humanely and without suffering.
Rodents used for the experiment were male and female Sprague-Dawley rats from the colony of the CMCRC/ERF. This colony of rats has been employed for various experiments in the laboratory for nearly 30 years, and extensive historical data are available on the tumor incidence among untreated rats. All control animals were monitored for feed and water consumption and body weight for their life span and, upon death, underwent complete necropsy and histopathologic evaluation.
The health status of the animals was regularly checked by the veterinarians of the local and national health services health services Managed care The benefits covered under a health contract . Before matching, the breeders were clinically observed for their health status, in order to exclude any diseased animals, and the experimental animals were clinically examined monthly until the end of the experiment.
At 4-5 weeks of age, after weaning, the experimental animals were randomized ran·dom·ize
tr.v. ran·dom·ized, ran·dom·iz·ing, ran·dom·iz·es
To make random in arrangement, especially in order to control the variables in an experiment. in order to have no more than one male and one female from each litter in the same group. They were then housed, in groups of five, in Makrolon cages (41 cm x 25 cm x 15 cm), with stainless-steel wire tops and a shallow layer of white wood shavings as bedding, and kept in rooms used only for this experiment, at a temperature of 23 [+ or -] 2[degrees]C and relative humidity relative humidity
The ratio of the amount of water vapor in the air at a specific temperature to the maximum amount that the air could hold at that temperature, expressed as a percentage. of 50-60%.
Once a week for the first 13 weeks, then every 2 weeks until the rats were 110 weeks of age, the mean daily drinking water drinking water
supply of water available to animals for drinking supplied via nipples, in troughs, dams, ponds and larger natural water sources; an insufficient supply leads to dehydration; it can be the source of infection, e.g. leptospirosis, salmonellosis, or of poisoning, e.g. and feed consumption was measured per cage, and body weight was measured individually. Measurement of body weight continued every 8 weeks until the end of the experiment. The animals were clinically examined for gross changes every 2 weeks for the duration of the experiment. To evaluate the status and behavior of the animals and to limit the postmortem postmortem /post·mor·tem/ (post-mort´im) performed or occurring after death.
Relating to or occurring during the period after death.
See autopsy. modifications (pmm), a patrol was performed three times daily from Monday through Friday and twice on Saturdays and Sundays and holidays. Dead animals were registered and kept refrigerated at 4[degrees]C until necropsy. Based on this procedure [part of our longstanding standard operating procedures (SOP)], very few animals were affected by pmm, and only on very rare occasions did this interfere with the ability to histologically diagnose and interpret some lesions.
The biophase ended at 151 weeks, with the death of the last animal at the age of 159 weeks. Upon death, the animals underwent complete necropsy. Histopathology his·to·pa·thol·o·gy
The science concerned with the cytologic and histologic structure of abnormal or diseased tissue.
The study of diseased tissues at a minute (microscopic) level. was routinely performed on the following organs and tissues of each animal from each group: skin and subcutaneous tissue subcutaneous tissue
A layer of loose, irregular connective tissue immediately beneath the skin; it contains fat cells except in the auricles, eyelids, penis, and scrotum. , mammary gland mammary gland, organ of the female mammal that produces and secretes milk for the nourishment of the young. A mammal may have from 1 to 11 pairs of mammary glands, depending on the species. Generally, those mammals that bear larger litters have more glands. , the brain (three sagittal sagittal /sag·it·tal/ (saj´i-t'l)
1. shaped like an arrow.
2. situated in the direction of the sagittal suture; said of an anteroposterior plane or section parallel to the median plane of the body. sections), pituitary gland pituitary gland, small oval endocrine gland that lies at the base of the brain. It is sometimes called the master gland of the body because all the other endocrine glands depend on its secretions for stimulation (see endocrine system). , Zymbal glands, salivary glands salivary glands (săl`əvâr'ē), in humans, three pairs of glands that secrete the alkaline digestive fluid, saliva, into the mouth. , Harderian glands, cranium cranium: see skull. (five sections, with oral and nasal cavities and external and internal ear ducts), tongue, thyroid, parathyroid parathyroid /par·a·thy·roid/ (-thi´roid)
1. situated beside the thyroid gland.
2. see under gland.
1. , pharynx pharynx (fâr`ĭngks), area of the gastrointestinal and respiratory tracts which lies between the mouth and the esophagus. In humans, the pharynx is a cone-shaped tube about 4 1-2 in. (11.43 cm) long. , larynx, thymus thymus
Pyramid-shaped lymphoid organ (see lymphoid tissue) between the breastbone and the heart. Starting at puberty, it shrinks slowly. It has no lymphatic vessels draining into it and does not filter lymph; instead, stem cells in its outer cortex develop into and mediastinal mediastinal /me·di·as·ti·nal/ (-as-ti´n'l) of or pertaining to the mediastinum.
of or pertaining to the mediastinum. lymph nodes Lymph nodes
Small, bean-shaped masses of tissue scattered along the lymphatic system that act as filters and immune monitors, removing fluids, bacteria, or cancer cells that travel through the lymph system. , trachea trachea (trā`kēə) or windpipe, principal tube that carries air to and from the lungs. It is about 4 1-2 in. (11.4 cm) long and about 3-4 in. (1.9 cm) in diameter in the adult. , lung and mainstem bronchi bronchi /bron·chi/ (brong´ki) plural of bronchus.
Two main branches of the trachea that go into the lungs. This then further divides into the bronchioles and alveoli. , heart, diaphragm, liver, spleen, pancreas, kidneys, adrenal glands Adrenal glands
The two glands that are located on top of the kidneys. These glands secrete several hormones, including the glucocorticoids which, among other things, influence the way the immune system works, and the mineralocorticoids, which affect retention of , esophagus, stomach (fore and glandular glandular /glan·du·lar/ (glan´du-ler)
1. pertaining to or of the nature of a gland.
1. ), intestine (four levels), urinary bladder urinary bladder
A musculomembranous elastic receptacle in the anterior part of the pelvic cavity serving as the temporary storage place for urine. , prostate, gonads, interscapular brown fat pad fat pad
An accumulation of encapsulated adipose tissue. , subcutaneous and mesenteric mesenteric /mes·en·ter·ic/ (-ter´ik) pertaining to the mesentery.
pertaining to or emanating from the mesentery. lymph nodes, and other organs or tissues with pathologic lesions. All organs and tissues were preserved in 70% ethyl alcohol ethyl alcohol: see ethanol. , except for bones, which were fixed in 10% formalin formalin /for·ma·lin/ (for´mah-lin) formaldehyde solution.
An aqueous solution of formaldehyde that is 37 percent by weight. and then decalcified with 10% formaldehyde and 20% formic acid formic acid or methanoic acid (mĕth'ənō`ĭk), HCO2H, a colorless, corrosive liquid with a sharp odor; it boils at 100.7°C; and solidifies at 8.4°C;. in water solution. The normal specimens were trimmed following the CMCRC/ERF laboratory SOP. Trimmed specimens were processed as paraffin blocks, and 3-5 [micro]m sections of every specimen were obtained.
Sections were routinely stained with hematoxylin hematoxylin /he·ma·tox·y·lin/ (he?mah-tok´si-lin) an acid coloring matter from the heartwood of Haematoxylon campechianum; used as a histologic stain and also as an indicator. and eosin eosin /eo·sin/ (e´o-sin) any of a class of rose-colored stains or dyes, all being bromine derivatives of fluorescein; eosin Y, the sodium salt of tetrabromofluorescein, is much used in histologic and laboratory procedures. (H&E). Immunohistochemical staining for $100 was performed to characterize malignant schwannoma, whereas chromogranin A chromogranin A
see parathyroid secretory protein. staining was used to characterize olfactory olfactory /ol·fac·to·ry/ (ol-fak´ter-e) pertaining to the sense of smell.
Of, relating to, or contributing to the sense of smell. neuroblastomas. For $100 staining, we used a polyclonal polyclonal /poly·clo·nal/ (-klon´'l)
1. derived from different cells.
2. pertaining to several clones.
derived from different cells; pertaining to several clones. rabbit anti-S100 (Z0311; Dakocytomation, Carpinteria, CA, USA) as primary antibody, whereas for chromogranin A staining, we used a polyclonal rabbit anti-human chromogranin A (N1535, Dakocytomation) (Information Center for Immunohistochemistry 2005).
Two statistical tests were used to analyze neoplastic neoplastic /neo·plas·tic/ (ne?o-plas´tik)
1. pertaining to a neoplasm.
2. pertaining to neoplasia.
pertaining to neoplasia or a neoplasm. and nonneoplastic lesion incidence data. We used the Cochran-Armitage trend test (Armitage 1971; Gartet al. 1979) in test for linear trends in tumor incidence. We also used the poly-k test (Bailer and Portier 1988; Piegorsch and Bailer 1997; Portier and Bailer 1989), a survival-adjusted quantal quantal
pertaining to specific quantities; used usually in reference to drugs and their dose rates.
quantal drug-receptor relationship
the variation in effect observed with increasing doses of a drug. response modification of the Cochran-Armitage test that takes survival into account. The tests used and the resulting p-values are reported in the tables.
The study proceeded smoothly without unexpected occurrences. We observed no differences in water consumption between the treated and the untreated groups, whereas a dose-related difference in feed consumption was observed between the various treated groups and the control group in both males and females (Figure 1A, B). No substantial differences in mean body weight were observed between the treated and control groups, apart from a slight decrease in females treated at 100,000 ppm APM (Figure 1C). No substantial difference in survival was observed among the groups (Figure 1D,E).
[FIGURE 1 OMITTED]
No evident behavioral changes were observed among treated animals compared with controls. In animals exposed to the highest dose of APM, yellowing of the coat was observed; this change had previously been observed in our laboratory in rats exposed to formaldehyde administered with drinking water (Soffritti et al. 2002b).
The carcinogenic effects of APM are reported in Table 2 for males and Table 3 for females. Multiple tumors of different types and sites; of different types in the same site; of the same types in bilateral organ; of the same types in the skin, subcutaneous tissue, or mammary glands; or at distant sites of diffuse tissue (i.e., bones and skeletal muscle) were plotted as single/independent tumors. Multiple tumors of the same type in the same tissue and organ, apart those above mentioned, were plotted only once.
Total malignant tumors. The incidence of animals bearing malignant tumors occurred with a significant positive trend in males (p [less than or equal to] 0.05) and in females (p [less than or equal to] 0.01), as reported in Tables 2 and 3. A statistically significant increase of the incidence of malignant tumors was observed in females treated at 50,000 ppm (p [less than or equal to] 0.01) compared with the control group (Table 3). Tumor types that contributed most are presented below.
Lymphomas/leukemias. The data on the occurrence of lymphomas/leukemias, reported in Tables 2 and 3, indicate that APM causes a significant positive trend in males (p [less than or equal to] 0.05) and in females (p [less than or equal to] 0.01). Compared with untreated control groups, the increased incidence of lymphomas/leukemias in treated females was statistically significant at doses of 100,000 (p [less than or equal to] 0.01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.05), 2,000 (p [less than or equal to] 0.05), or 400 ppm (p [less than or equal to] 0.01). The most frequent histocytotypes observed in the experiment were lymphoimmunoblastic lymphomas, mainly involving lung and mediastinal/peripheral nodes, and histiocytic histiocytic
pertaining to histiocytes.
see malignant histiocytosis.
prolymphocyte. sarcomas Sarcomas Definition
A sarcoma is a bone tumor that contains cancer (malignant) cells. A benign bone tumor is an abnormal growth of noncancerous cells.
A primary bone tumor originates in or near a bone. , involving mainly lung, liver, spleen, and nodes. The distribution of lymphomas/leukemias by histocytotypes is presented in Table 4. The differential diagnoses were based on the morphologic criteria followed in our laboratory for several decades and are in line with the guidelines of the International Classification of Rodent Tumors [International Agency for Research on Cancer The International Agency for Research on Cancer (IARC, or CIRC in its French acronym) is an intergovernmental agency forming part of the World Health Organisation of the United Nations.
Its main offices are in Lyon, France. (IARC) 1993]. Lymphomas/leukemias (this term includes all types of hemolymphosarcomas and leukemias) are neoplasias arising from hemolymphoreticular tissues, and their aggregation is widely used in experimental carcinogenesis car·ci·no·gen·e·sis
The production of cancer.
production of cancer.
viruses and some parasites are capable of initiating neoplasia. because both solid and circulating phases are present in many lymphoid lymphoid /lym·phoid/ (lim´foid) resembling or pertaining to lymph or tissue of the lymphoid system.
Of or relating to lymph or the lymphatic tissue where lymphocytes are formed. neoplasms, and distinction between them is artificial (Harris et al. 2001).
Preneoplastic and neoplastic lesions of the renal pelvis and ureter. The incidences of preneoplastic and neoplastic lesions of the transitional cell transitional cell
cells which make up an epithelium, e.g. in the urinary bladder, consisting of several layers of soft cuboidal cells which flatten out when stretched.
transitional cell tumors
1. epithelium of the renal pelvis and ureter are reported in Tables 2 and 3. A dose-related increase in the incidence of dysplastic dysplastic
emanating from or pertaining to abnormality of development. hyperplasias and dysplastic papillomas of the renal pelvis and ureter was observed in females. Carcinomas in females occurred with a positive trend (p [less than or equal to] 0.05), and the incidence in females exposed at 100,000 ppm was significantly higher (p [less than or equal to] 0.05) compared with the controls. Carcinomas were also observed among males treated at 100,000, 50,000, 10,000, or 2,000 ppm. In females, dysplastic lesions and carcinomas combined show a significant positive trend (p [less than or equal to] 0.01) and a statistically significant increase in those treated at 100,000 (p [less than or equal to] 0.01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.01), 2,000 (p [less than or equal to]0.05), or 400 ppm (p [less than or equal to] 0.05). A 3-fold increase is also observed in the group treated with 80 ppm. We did not observe substantial differences in the incidence of inflammation between males and females treated at the different doses, compared with controls. Increased incidence of calcification calcification /cal·ci·fi·ca·tion/ (kal?si-fi-ka´shun) the deposit of calcium salts in a tissue.
dystrophic calcification was observed in females, particularly in those treated at 100,000 ppm (39%), 50,000 ppm (25%), or 10,000 ppm (19%), compared with controls (8%); this effect was not observed in males. Although transitional cell carcinomas of the renal pelvis and ureter are extremely rare in male and female untreated rats, the APM male and female groups had a total of 21 transitional cell carcinomas of the renal pelvis, whereas the controls had none. Microscopically, the carcinomas were invading, with various levels of extension, the papilla papilla /pa·pil·la/ (pah-pil´ah) pl. papil´lae [L.] a small nipple-shaped projection or elevation.
circumvallate papillae vallate papillae. and the kidney parenchyma Parenchyma
A ground tissue of plants chiefly concerned with the manufacture and storage of food. The primary functions of plants, such as photosynthesis, assimilation, respiration, storage, secretion, and excretion—those associated with living ; the cells were of transitional type, and several mitotic figures were present (Figure 2A, B).
[FIGURE 2 OMITTED]
Malignant schwannomas of peripheral nerves. As shown in Table 2, the incidence of malignant schwannomas of the peripheral nerves occurred with a positive trend (p [less than or equal to]0.05) in males. In females, nine malignancies were observed among treated animals of the different dosage groups, and none among the controls (Table 3). All lesions, in males and females, diagnosed as malignant schwannomas were positive for $100 staining. The most frequent site of origin of the malignant schwannomas was in the cranial nerves Cranial nerves
The set of twelve nerves found on each side of the head and neck that control the sensory and muscle functions of a number of organs such as the eyes, nose, tongue face and throat. (72%); the other cases arose at the spinal nerve roots Spinal nerve roots can refer to:
A tumor growth or deposit that has spread via lymph or blood to an area of the body remote from the primary tumor.
Mentioned in: Malignant Melanoma of cranial nerve cranial nerve
Any of 12 pairs of nerves that emerge from or enter the brain, comprising the olfactory (I), optic (II), oculomotor (III), trochlear (IV), trigeminal (V), abducent (VI), facial (VII), vestibulocochlear (VIII), glossopharyngeal (IX), malignant schwannomas were observed in three males treated at the highest dose. The metastases were found in submandibular lymph nodes The submandibular lymph nodes (submaxillary glands in older texts), three to six in number, are placed beneath the body of the mandible in the submaxillary triangle, and rest on the superficial surface of the submaxillary salivary gland. in two cases, and the tumor metastatized to the lung and liver in one case. Histologically the feature of malignant schwannomas was Antoni B type (Figure 2C,D).
Preneoplastic and neoplastic lesions of the olfactory epithelium olfactory epithelium
Pseudostratified epithelium that contains olfactory, receptor, and nerve cells whose axons connect with the olfactory bulb of the brain. . Incidence of hyperplasia of the olfactory epithelium increased with a significant positive trend in males and females. The observed incidences were, respectively, 14.0% and 18.0% in males and females exposed at 100,000 ppm, 12.0% and 21.0% at 50,000 ppm, 7.0% and 17.0% at 10,000 ppm, 2.7% and 8.7% at 2,000 ppm, 6.0% and 7.3% at 400 ppm, 2.0% and 3.3% at 80 ppm, and 0.7% and 4.0% at 0 ppm. The differences were statistically significant (p [less than or equal to] 0.01) at 100,000, 50,000, or 10,000 ppm in both males and females and at 400 ppm in males. Among females treated at the highest dose, one case of dysplastic hyperplasia, one adenoma adenoma: see neoplasm. , and one olfactory neuroblastoma Neuroblastoma Definition
Neuroblastoma is a type of cancer that usually originates either in the tissues of the adrenal gland or in the ganglia of the abdomen or in the ganglia of the nervous system. were observed. The neuroblastoma invaded the cranium, compressing the forebrain forebrain: see brain. , and was positive for chromogranin A immunohistochemical staining.
Malignant brain tumors. Concerning the incidence of malignant tumors in the brain, it should be noted that, as previously reported (Soffritti et al. 2005), 12 malignant tumors (10 gliomas, 1 medulloblastoma and 1 meningioma meningioma /me·nin·gi·o·ma/ (me-nin?je-o´mah) a benign, slow-growing tumor of the meninges, usually next to the dura mater, which may invade the skull or cause hyperostosis, and often causes increased intracranial pressure; it is usually ) were observed, without dose relationship, in male and female APM-treated groups, whereas none were observed in controls.
Other malignant tumors. The other malignant tumors were among those commonly observed in Sprague-Dawley rats, apart from two transitional cell carcinomas of the bladder observed in males exposed to 10,000 ppm, one in females exposed to 2,000 ppm, and none among the controls. Because this type of tumor is extremely rare among the historical controls of our colony of Sprague-Dawley rats, this occurrence cannot be disregarded.
Historical controls. Over the last 20 years in our laboratory, when we consider only groups of > 100 animals/sex, the numbers of the untreated males and females total 1,934 and 1,945 respectively. Concerning the renal pelvis and ureter transitional cell carcinomas, no carcinomas were observed in either males or females. The overall incidence of malignant schwannomas was 0.5% (range, 0-2.0%) in males and 0.1% (range, 0-1.0%) in females. The overall incidence of lymphomas/ leukemias was 20.7% (range, 8.0-30.9%) in males and 12.4% (range, 7.0-18.4%) in females. The overall incidence of olfactory neuroblastoma was 0.1% (0-1.8%) in both males and females.
When we also consider control groups of < 100 animals/sex, the numbers of untreated males and females total 2,265 and 2,274, respectively. The overall incidence of the renal pelvis and ureter transitional cell carcinomas was 0.04% (range, 0-1.0%) in females, whereas no carcinomas were observed in males. The overall incidence of malignant schwannomas was 0.4% (range, 0-2.0%) in males and 0.1% (range, 0-2.0%) in females. The overall incidence of lymphomas/leukemias was 20.6% (range, 8.0-30.9%) in males and 13.3% (range, 4.0-25.0%) in females. The overall incidence of olfactory neuroblastomas was 0.1% (range, 0-1.8%) in both males and females.
The mega-experiment performed in our laboratory on APM (administered with feed to Sprague-Dawley rats from 8 weeks of age until natural death) has shown for the first time the muhipotential carcinogenic effects of this compound. In fact, the results indicate that APM causes, in our experimental conditions, a) an increased incidence of malignant-tumor-bearing animals with a positive significant trend in males (p [less than or equal to] 0.05) and in females (p [less than or equal to]0.01), particularly in the females treated at 50,000 ppm (p [less than or equal to] 0.01); b) a statistically significant dose-related increase of the incidence of lymphomas/leukemias in females treated at the doses of 100,000 (p [less than or equal to] 0.01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.05), 2,000 (p [less than or equal to] 0.05), or 400 ppm (p [less than or equal to] 0.01) and a positive significant trend in both males (p [less than or equal to] 0.05) and females (p [less than or equal to]0.01); c) in females, dysplastic lesions and carcinomas of the renal pelvis and ureter combined show a significant positive trend (p [less than or equal to] 0.01) and a statistically significant increase in those treated at 100,000 (p [less than or equal to] 0.01), 50,000 (p [less than or equal to] 0.01), 10,000 (p [less than or equal to] 0.01), 2,000 (p [less than or equal to] 0.05), or 400 ppm (p [less than or equal to] 0.05); and d) an increased incidence of malignant schwannomas of the peripheral nerves with a positive trend (p [less than or equal to] 0.05) in males.
The increase in lymphomas/leukemias in APM-treated females could be related to its metabolite metabolite, organic compound that is a starting material in, an intermediate in, or an end product of metabolism. Starting materials are substances, usually small and of simple structure, absorbed by the organism as food. methanol, which is in turn metabolized to formaldehyde in both humans and rats (Ranney et al. 1976). In fact, previous experiments performed at the CMCRC laboratory have shown that a) methanol administered in drinking water, at doses ranging from 20,000 to 500 ppm, induced a statistically significant increase in the incidence of lymphomas/ leukemias in female rats (Soffritti et al. 2002a); b) a dose-related increase in the incidence of lymphomas/leukemias was also observed in females treated with formaldehyde, administered in drinking water at doses ranging from 1,500 to 50 ppm (Soffritti et al. 1989, 2002b); and c) the same effect was observed in females treated with the gasoline oxygenated additive methyt-tert-butyl ether (MTBE MTBE Methyl-tert-butyl-ether Surgery An aliphatic ether that rapidly dissolves cholesterol stones in vivo, introduced under local anesthesia via a percutaneous transhepatic cholecystectomy catheter, as a non-invasive method for treating gallstones; after injection, ), which metabolizes to methanol (Belpoggi et al. 1995).
The important role of formaldehyde in the induction of hematologic malignancies in rodents is further highlighted by these results. In a recent reevaluation of the carcinogenicity of formaldehyde by the IARC (in press), strong (although not considered sufficient) evidence of an association between formaldehyde exposure and leukemias in humans was found.
Moreover, carcinogenic effects for the renal pelvis and ureter, peripheral nerves and proliferative changes of the olfactory epithelium were not observed in the long-term bioassays performed in the same conditions at the CMCRC on methanol, MTBE, or formaldehyde. To investigate if the other two metabolites Metabolites
Substances produced by metabolism or by a metabolic process.
Mentioned in: Interactions of APM are responsible for inducing these lesions, it is of paramount importance to perform adequate life-span carcinogenicity studies on aspartic acid aspartic acid (əspär`tĭk), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of proteins. or phenylalanine.
In a long-term carcinogenicity study on monosodium aspartate (MSA (Metropolitan Service Area) An urban area with at least 50,000 people plus surrounding counties. There are 306 MSAs and 428 RSAs (rural service areas) in the U.S. MSAs and RSAs are used to allocate cellular licenses. ) administered with drinking water to groups of 50 male and 50 female Fischer-344 rats (beginning at 6 weeks of age for 100 weeks and then sacrificed), a dose-related hyperplasia of the renal pelvis was observed in males and in females (Kitahori et am. 1996). The same effect was found by the same group of investigators in another study in which MSA was administered in drinking water to groups of male and female Fischer-344 rats to evaluate its promoting activity of carcinogenesis of the transitional epithelium of the renal pelvis (Kitamura et al. 1996). In both studies, clear evidence was provided of a relationship between MSA treatment and transitional cell hyperplasia. The authors indicated that calcification could have an important role in inducing simple and papillary papillary /pap·il·lary/ (pap´i-lar?e) pertaining to or resembling a papilla, or nipple.
adj similar to a small, nipple-shaped elevation or projection. hyperplasia of the renal pelvis transitional cell epithelium and, consequently, in the induction of transitional cell tumors. In our study performed on 1,800 Sprague-Dawley rats, which are less susceptible to the spontaneous development of nephropathies than Fischer rats, we observed a dose-related, statistically significant increase in the incidence of dysplastic hyperplasia and carcinoma of the renal pelvis in females, but none in males, compared with the controls. The fact that we observed an increased incidence of kidney calcification in females and not in males, compared with the controls, gives added weight to the hypothesis that aspartic acid may cause preneoplastic and neoplastic lesions of the renal pelvis, and that calcification may be the mechanism responsible for this effect.
The carcinogenic effects of APM observed in our experiment are in contrast with the results obtained with long-term carcinogenicity bioassays, performed almost 30 years ago on Sprague-Dawley rats, which did not reveal APM to have any carcinogenic effects (FDA 1981). There are several reasons that can explain this difference. First of all, in our experiment the number of animals per sex per group was much greater, allowing a more thorough and reliable statistical analysis. Second, in our experiment, rodents were not killed at 110 weeks of age but rather were observed until natural death, to allow APM to fully express its carcinogenic potential. Had we stopped the experiments at 110 weeks of age, we would most likely never have demonstrated the carcinogenicity of important industrial compounds such as xylenes, mancozeb, vinyl acetate monomer (Soffritri et al. 2002c), and toluene toluene (tōl`yēn') or methylbenzene (mĕth'əlbĕn`zēn), C7H8 (Soffritri et al. 2004).
Finally, concerning the absence of carcinogenic effects observed in the experiment performed on Wistar rats (Ishii 1981; Ishii et al. 1981), it cannot be disregarded that this strain is more resistant than Sprague-Dawley rats to developing cancer, a characteristic shown in our experiments on benzene (Maltoni et al. 1989). Moreover, the aforementioned experiment on Wistar rats was terminated at the age of 110 weeks. Given these differences, the results of the Wistar rat study are not comparable with those performed on Sprague-Dawley rats.
Our study shows that APM is a multi-potential carcinogenic compound whose carcinogenic effects are evident even at a daily dose of 20 mg/kg bw, much less than the current ADI for humans in Europe (40 mg/kg bw) and in the United States (50 mg/kg bw).
The results of carcinogenicity bioassays in rodents are consistent predictors of human cancer risks (Huff 1999; Rail 1995; Tomatis et al. 1989). The results of our study therefore call for an urgent reexamination re·ex·am·ine also re-ex·am·ine
tr.v. re·ex·am·ined, re·ex·am·in·ing, re·ex·am·ines
1. To examine again or anew; review.
2. Law To question (a witness) again after cross-examination. of the present guidelines on the use and consumption of APM. The decision to use experimental data to protect public health is important because the time span of widespread APM use is still too brief to have produced solid epidemiologic data. Moreover, it is unlikely that sufficient epidemiologic data will be available in the near future, given the difficulty of finding a control group that has not been exposed to this widely diffused compound.
Received 3 October 2005; accepted 16 November 2005.
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Substances in the environment that cause cancer, presumably by inducing mutations, with prolonged exposure.
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Morando Soffritti, Fiorella Belpoggi, Davide Degli Esposti, Luca Lambertini, Eva Tibaldi, and Anna Rigano
Cesare Maltoni Cancer Research Center, European Ramazzini Foundation of Oncology and Environmental Sciences, Bologna, Italy
Address correspondence to M. Soffritti, Cesare Maltoni Cancer Research Center, European Ramazzini Foundation of Oncology and Environmental Sciences, Castello di Bentivoglio, Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy. Telephone: 39-051-6640460. Fax: 39-051-6640223. E-mail: firstname.lastname@example.org
We thank the U.S. National Toxicology Program National Toxicology Program Environment A program that conducts toxicologic tests on substances frequently found at the EPA's National Priorities List sites, which have the greatest potential for human exposure for convening a group of pathologists at the National Institute of Environmental Health Sciences The National Institute of Environmental Health Sciences (NIEHS) is one of 27 Institutes and Centers of the National Institutes of Health (NIH),which is a component of the Department of Health and Human Services (DHHS). The Director of the NIEHS is Dr. David A. Schwartz. to provide a second opinion for a set of malignant lesions and their precursors related to aspartame treatment, and for their help in statistical analysis. We also thank all of the staff involved in the project.
This research was supported by the European Ramazzini Foundation of Oncology and Environmental Sciences. The authors declare they have no competing financial interests.
Address correspondence to M. Soffritti, Cesare of Protection of the Human Environment, World Health Organization, 1211 Geneva Geneva, canton and city, Switzerland
Geneva (jənē`və), Fr. Genève, canton (1990 pop. 373,019), 109 sq mi (282 sq km), SW Switzerland, surrounding the southwest tip of the Lake of Geneva. 27, Switzerland. Telephone: +41 22 791 4979. Fax: +41 22 791 1383. E-mail. email@example.com
We thank F. Gore (WHO) and D. Barnes (World Bank) for their essential contributions to compiling the most up-to-date data on solid fuel use. We also thank S. Chatterji and N. Naidoo (WHO), who provided us with preliminary World Health Survey data and assistance in analyzing them, and L. Tzala (Imperial College London History
Imperial College was founded in 1907, with the merger of the City and Guilds College, the Royal School of Mines and the Royal College of Science (all of which had been founded between 1845 and 1878) with these entities continuing to exist as "constituent colleges". ) for her analysis of the World Health Survey solid fuel use data. We are grateful to them and to C. Corvalan (WHO) for comments on the draft article.
The views expressed in this article are those of the authors and do not necessarily reflect the position of the World Health Organization or the Health Effects Institute The Health Effects Institute (HEI) is a non-partisan, non-profit corporation specializing in research on the health effects of air pollution. It is headquartered in Charlestown, Massachusetts, USA. .
The authors declare they have no competing financial interests.
Table 1. Beverages and diet products studied at the CMCRC/ERF: status of studies. Animals No. of Study No. Products bioassays Species No. status 1 Water in 2 Rat (a) 2,200 P (b) polyvinyl chloride bottles 2 Coca-Cola 4 Rat (a) 1,999 RP 3 Pepsi Cola 1 Rat 400 E 4 Ethyl alcohol 4 Rat, (a) 1,458 P (c) (10% vol/vol) mouse 5 Sucrose 1 Rat 400 E 6 APM 6 Rat, 4,460 BO, mouse (a) PP (d) 7 Sucralose 1 Mouse (a) 760 BO 8 Caffeine 1 Rat 800 E 9 Vitamin A 5 Rat 5,100 PP (e) 10 Vitamin C 5 Rat 3,680 E 11 Vitamin E 5 Rat 3,680 E 12 Feed sterilized by 1 Rat (a) 2,000 E gamma radiation Total 36 26,937 Abbreviations: BO, biophase ongoing; E, in elaboration; P, published; PP, partially published; RP, ready for publication. (a) Treatment started from embryonic life. (b) Data from Maltoni et al. (1997). (c) Data from Soffritti et al. (2002a). (d) Data from Soffritti et al. (2005). (d) Data from Soffritti et al. (1992). Table 2. Incidence of the preneoplastic and neoplastic lesions in male Sprague-Dawley rats in a life-span feed carcinogenicity study of APM. Malignant tumors (a) Tumor-bearing Total tumors Animals animals (b) Dose, ppm at Per 100 (mg/kg bw) start No. % No. animals 100,000 (5,000) 100 43 43.0 55 55.0 50,000 (2,500) 100 38 38.0 45 45.0 10,000 (500) 100 34 34.0 42 42.0 2,000 (100) 150 60 40.0 69 46.0 400 (20) 150 48 32.0 52 34.7 80 (4) 150 44 29.3 49 32.7 0 (0) 150 53 35.3 * 59 39.3 Animals bearing dysplastic lesions and carcinomas of the renal pelvis and Total animals ureter (a) bearing lymphomas/ Dysplastic leukemias (a,b) hyperplasias Dose, ppm (mg/kg bw) No. % No. % 100,000 (5,000) 29 29.0 3 3.0 50,000 (2,500) 20 20.0 2 2.0 10,000 (500) 15 15.0 2 2.0 2,000 (100) 33 22.0 4 2.7 400 (20) 25 16.7 4 (c) 2.7 80 (4) 23 15.3 3 (c) 2.0 0 (0) 31 20.7 *,# 1 0.7 Animals bearing dysplastic lesions and carcinomas of the renal pelvis and ureter (a) Dysplastic papillomas Carcinomas Dose, ppm (mg/kg bw) No. % No. % 100,000 (5,000) 0 -- 1 1.0 50,000 (2,500) 0 -- 1 1.0 10,000 (500) 0 -- 1 1.0 2,000 (100) 0 -- 1 0.7 400 (20) 1 (c) 0.7 0 (c) -- 80 (4) 0 (c) -- 0 (c) -- 0 (0) 0 -- 0 -- Animals bearing dysplastic lesions Animals bearing and carcinomas of peripheral nerve the renal pelvis malignant and ureter (a) schwannomas (a) Total Cranial Dose, ppm (mg/kg bw) No. % No. % 100,000 (5,000) 4 4.0 3 3.0 50,000 (2,500) 3 3.0 3 3.0 10,000 (500) 3 3.0 2 2.0 2,000 (100) 5 3.3 2 1.3 400 (20) 5 (c) 3.4 1 0.7 80 (4) 3 (c) 2.0 1 0.7 0 (0) 1 0.7 1 0.7 Animals bearing peripheral nerve malignant schwannomas (a) Other sites Total (b) Dose, ppm (mg/kg bw) No. % No. % 100,000 (5,000) 1 1.0 4 4.0 50,000 (2,500) 0 -- 3 3.0 10,000 (500) 0 -- 2 2.0 2,000 (100) 0 -- 2 1.3 400 (20) 2 1.3 3 2.0 80 (4) 0 -- 1 0.7 0 (0) 0 -- 1 0.7 *,# (a) The tumor rates are based on the number of animals examined (necropsied). (b) p-Values associated with the trend test are near the control incidence. (c) Tissues from 149 animals were analyzed. * Statistically significant (p [less than or equal to] 0.05) using Cochran-Armitage test. # Statistically significant (p [less than or equal to] 0.05) using poly-k test (k = 3). Table 3. Incidence of the preneoplastic and neoplastic lesions in female Sprague-Dawley rats in a life-span feed carcinogenicity study of APM. Malignant tumors (a) Tumor-bearing Total tumors Animals animals (b,c) at Per 100 start No. % No. animals 100,000 (5,000) 100 51 51.0 64 64.0 50,000 (2,500) 100 58 58.0 # 84 84.0 10,000 (500) 100 40 40.0 62 62.0 2,000 (100) 150 67 44.7 86 57.3 400 (20) 150 70 46.7 95 63.3 80 (4) 150 64 42.7 85 56.7 0 (0) 150 55 36.7 ** 69 46.0 Animals bearing dysplastic lesions and carcinomas of the renal pelvis and Total animals ureter (a,b,c) bearing lymphomas/ Dysplastic leukemias (a,b,c) hyperplasias No. % No. % 100,000 (5,000) 25 25.0 ## 8 8.0 50,000 (2,500) 25 25.0 ## 6 (e) 6.1 10,000 (500) 19 19.0 # 6 6.0 2,000 (100) 28 18.7 # 6 4.0 400 (20) 30 20.0 ## 5 3.3 80 (4) 22 14.7 4 2.7 0 (0) 13 8.7 **,# 2 1.3 ** Animals bearing dysplastic lesions and carcinomas of the renal pelvis and ureter (a,b,c) Dysplastic papillomas Carcinomas (d) No. % No. % 100,000 (5,000) 3 3.0 4 4.0 # 50,000 (2,500) 1 (e) 1.0 3 (e) 3.0 10,000 (500) 1 1.0 3 (4) 3.0 2,000 (100) 1 0.7 3 (4) 2.0 400 (20) 1 0.7 3 2.0 80 (4) 1 0.7 1 0.7 0 (0) 0 -- * 0 -- Animals bearing dysplastic lesions Animals bearing and carcinomas of peripheral nerve the renal pelvis malignant and ureter (a,b,c) schwannomas (a) Total Cranial No. % No. % 100,000 (5,000) 15 15.0 ## 1 1.0 50,000 (2,500) 10 (e) 10.1 ## 1 1.0 10,000 (500) 10 10.0 ## 1 1.0 2,000 (100) 10 6.7 # 1 0.7 400 (20) 9 6.0 # 0 -- 80 (4) 6 4.0 1 0.7 0 (0) 2 1.3 **,## 0 -- Animals bearing peripheral nerve malignant schwannomas (a) Other sites Total (b) No. % No. % 100,000 (5,000) 1 1.0 2 2.0 50,000 (2,500) 0 -- 1 1.0 10,000 (500) 0 -- 1 1.0 2,000 (100) 2 1.3 3 2.0 400 (20) 0 -- 0 -- 80 (4) 1 0.7 2 1.3 0 (0) 0 -- 0 -- (a) The tumor rates are based on the number of animals examined (necropsied). (b) p-Values corresponding to pairwise comparisons between the controls and the dosed group are near the dosed group incidence. (c) p-Values associated with the trend test are near the control incidence. (d) Values in parentheses indicate the number of tumors (one animal can bear bilateral tumor). (e) Tissues from 99 animals were analyzed. * Statistically significant (p [less than or equal to] 0.05) using Cochran-Armitage test. ** Statistically significant (p [less than or equal to] 0.01) using Cochran-Armitage test. # Statistically significant (p [less than or equal to] 0.05) using poly-k test (k = 3). ## Statistically significant (p [less than or equal to] 0.01) using poly-k test (k = 3). Table 4. Incidence and distribution by hystocytotype of lymphomas/ leukemias in female Sprague-Dawley rats in a life-span feed carcinogenicity study of APM. Lymphomas/ leukemias (a) Total lymphomas/ Lymphoblastic Animals leukemias (b) lymphoma Dose, ppm at (mg/kg bw) start No. % No. % 100,000 (5,000) 100 25 25.0 ## 1 4.0 50,000 (2,500) 100 25 25.0 ## 2 8.0 10,000 (500) 100 19 19.0 # 2 10.5 2,000 (100) 150 28 18.7 # 5 17.8 400 (20) 150 30 (c) 20.0 ## 7 23.3 80 (4) 150 22 14.7 3 13.6 0 (0) 150 13 8.7 **, # 2 15.4 Lymphomas/leukemias (a) Lymphoblastic Lymphocytic Lymphoimmunoblastic leukemia lymphoma lymphoma Dose, ppm (mg/kg bw) No. % No. % No. % 100,000 (5,000) 0 -- 2 8.0 11 44.0 50,000 (2,500) 0 -- 0 -- 10 40.0 10,000 (500) 0 -- 2 10.5 3 15.8 2,000 (100) 1 3.6 1 3.6 8 28.6 400 (20) 0 -- 2 6.7 8 26.7 80 (4) 0 -- 5 22.7 6 27.2 0 (0) 0 -- 2 15.4 5 38.5 Lymphomas/leukemias (a) Histiocytic Monocytic Myeloie sarcoma leukemia leukemia Dose, ppm (mg/kg bw) No. % No. % No. % 100,000 (5,000) 7 28.0 2 8.0 2 8.0 50,000 (2,500) 8 32.0 4 16.0 1 4.0 10,000 (500) 10 52.6 2 10.5 0 -- 2,000 (100) 8 28.6 4 14.3 1 3.6 400 (20) 9 30.0 5 16.7 0 -- 80 (4) 6 27.3 2 9.1 0 -- 0 (0) 4 30.8 0 -- 0 -- (a) Percentage of animals bearing specific histocytotype refer to the total number of animals bearing lymphomas/leukemias. (b) Percentage of animals at start to bear lymphomas/leukemias. (c) One animal had two types of neoplasias: lymphoblastic lymphoma and histocytic sarcoma. ** Statistically significant (p [less than or equal to] 0.01) using Cochran-Armitage test. # Statistically significant (p [less than or equal to] 0.05) using poly-k test (k = 3). ## Statistically significant (p [less than or equal to] 0.01) using poly-k test (k = 3).