The APM used in this study was produced by Ajinomoto (Gravelines, France) and supplied by Giusto Faravelli S.p.A. (Milan, Italy). The purity of the APM, as determined by an infrared absorption spectrophotometer assay, was > 98.7%: diketopiperazine was < 0.3% and l-phenylalanine was < 0.5%. APM was added to the standard diet, which has been used for > 30 years at the Cesare Maltoni Cancer Research Center (CMCRC)/ERF, at APM concentrations of 2,000, 400, or 0 ppm to simulate an assumed daily APM intake of 100, 20, or 0 mg/kg bw. The feed was supplied by the producer on a monthly basis. The stability of the aspartame in feed was analyzed before the start of the study and periodically confirmed throughout the course of the biophase. The daily APM consumption (milligrams per kilogram body weight) was calculated estimating the average body weight for both males and females as 400 g for the duration of the experiment and the daily consumption of feed as 20 g/day.
The feed was supplied ad libitum
to groups of 70–95 male and female Sprague-Dawley rats from the colony of the CMCRC/ERF. The basic tumorigram of this strain of rats is well known. Treatment began during fetal life, with administration of APM in feed to female breeders from the 12th day of pregnancy, when organogenesis is completed and before which time many tissues and organs are refractory to the effects of carcinogenic agents [International Agency for Research on Cancer (IARC) 1973
]. The breeders were sacrificed after weaning, and treatment of the offspring lasted until natural death. Control animals received the same feed without APM.
At 4–5 weeks of age (after weaning), the experimental animals were identified by ear punch, separated by sex, and assigned to a respective dose group, depending on the APM concentration administered to the breeder. They were then housed five per cage in poly-carbonate cages (41 × 25 × 15 cm) with stainless-steel wire tops and a shallow layer of white wood shavings as bedding, and kept in a room used only for this experiment. The room was maintained at a temperature of 23 ± 2°C and relative humidity of 50–60%.
All animals were kept under observation until natural death. The experiment was conducted according to Italian law regulating the use and humane treatment of animals for scientific purposes (Decreto Legislativo N. 116 1992
Mean daily drinking water and feed consumption were measured per cage, and body weight was measured individually, beginning at 6 weeks of age and continuing once each week for the first 13 weeks, then every 2 weeks until animals reached 110 weeks of age. Measurement of body weight continued every 2 weeks until the end of the experiment. To detect and register all gross lesions, the animals were clinically examined every 2 weeks for the duration of the experiment. To evaluate the status and behavior of the animals and to limit the postmortem modifications, a patrol was performed three times daily Monday–Friday and twice on Saturdays, Sundays, and holidays. Deceased animals were registered and kept refrigerated for a maximum of 16–19 hr at 4°C until necropsy.
The biophase ended at 147 weeks with the death of the last animal at the age of 144 weeks. Upon death, all animals underwent complete necropsy. Histopathology was routinely performed on the following organs and tissues of each animal from each group: skin and subcutaneous tissue, mammary gland, the brain (three sagittal sections), pituitary gland, Zymbal glands, salivary glands, Harderian glands, cranium (five sections, with oral and nasal cavities and external and internal ear ducts), tongue, thyroid, parathyroid, pharynx, larynx, thymus and mediastinal lymph nodes, trachea, lung and mainstem bronchi, heart, diaphragm, liver, spleen, pancreas, kidneys, adrenal glands, esophagus, stomach (fore and glandular), intestine (four levels), urinary bladder, prostate (male only), vagina (female only), gonads, interscapular brown fat pad, subcutaneous and mesenteric lymph nodes, and other organs or tissues with pathologic lesions. All organs and tissues were preserved in 70% ethyl alcohol except for bones, which were fixed in 10% formalin and then decalcified with 10% formaldehyde and 20% formic acid in water solution. The normal specimens were trimmed following CMCRC/ERF laboratory standard operating procedures. The pathologic tissue was trimmed to allow for the largest surface, including normal adjacent tissue. Trimmed specimens were processed in paraffin, and 3- to 5-μm sections of every specimen were obtained.
Sections were routinely stained with hematoxylin and eosin. All slides were examined microscopically by the same group of pathologists following the same criteria of histopathologic evaluation and classification. A senior pathologist reviewed all tumors and all other lesions of oncologic interest.
We performed statistical evaluations of the incidence and dose–response relationship of neoplastic lesions using the Cox regression model (Cox 1972
-Values are reported in the tables.