- "The occurrence of brain malignancies is shown in
Table 3. Sparse malignant brain tumors were observed among males and females
in the treated groups and none in the controls."
-
- Mission Possible International is taking case histories
on aspartame brain tumors in New York, New Jersey, Mississippi, and Madison
County, Illinois. Contact Dr. Betty Martini: Bettym19@mindspring.com and
web sites: http://www.wnho.net click
on aspartame, http://www.dorway.com
-
-
- Original studies - General topic
- Eur. J. Oncol., vol. 10, n. 2, pp. 00-00, 2005 IN PRESS
-
- Aspartame induces lymphomas and leukaemias in rats a
-
- Morando Soffritti, Fiorella Belpoggi, Davide Degli Esposti,
Luca Lambertini
-
- Cancer Research Centre, European Ramazzini Foundation
of
- Oncology and Environmental Sciences, Bologna, Italy
- Received 15.3.2005 - Accepted 11.4.2005
-
- Research supported by European Ramazzini Foundation of
- Oncology and Environmental Sciences, Bologna, Italy
-
- Introduction
-
- Aspartame (APM) is a widely used artificial sweetener
consumed by hundreds of millions of people around the world1, 2. It is
found in more than 6,000 products, including soft drinks, chewing gum,
candy, yoghurt, tabletop sweeteners and some pharmaceuticals such as vitamins
and sugar-free cough drops2.
-
- Dietary surveys, performed among APM consumers, have
shown that the average APM daily intake in the general population ranged
from 2 to 3 mg/kg b.w. and was even more in children and pregnant women1.
The Accept-
-
- Summary
-
- Aspartame, a widely used artificial sweetener, was administered
with feed to male and female Sprague-Dawley rats (100-150/sex/group), 8
weeks-old at the start of the experiment, at concentrations of 100,000;
50,000; 10,000; 2,000; 400; 80 and 0 ppm. Treatment lasted until spontaneous
death of the animals. In this report we present the first results showing
that aspartame, in our experimental conditions, causes a statistically
significant, dose-related increase in lymphomas and leukaemias in females.
No statistically significant increase in malignant brain tumours was observed
among animals from the treated groups as compared to controls. Eur. J.
Oncol., 10 (2), 00-00, 2005 Key words: aspartame, artificial sweetener,
carcinogenesis, rats, lymphoma, leukaemia
-
- Daily Intake (ADI) both in the US and in Europe is 50
and 40 mg/kg b.w., respectively1. In rodents and humans, APM is metabolised
in the gastrointestinal tract into three constituents: aspartic acid, phenylalanine
and methanol3.
-
- Three long-term feeding carcinogenicity bioassays on
APM were performed on rats, and one on mice, during the 1970s. Overall,
the carcinogenicity studies were considered negative4, but it must be noted
that these studies did not comply with the basic requirements which must
nowadays be met when testing the carcinogenicity potential of a chemical
or physical agent. Because of these limitations, we decided to perform
a mega-experiment following the currently accepted Good Laboratory Practices.
In the present paper we are reporting our first results on the incidence
of haemolymphoreticular malignancies (lymphomas and leukaemias) and malignant
brain tumours.
-
- Materials and methods
-
- The APM used was produced by Nutrasweet and supplied
by Giusto Faravelli S.p.A., Milan, Italy. As an active ingredient, its
purity was more than 98%. To simulate an assumed daily intake by humans
of 5,000; 2,500; 500; 100; 20; 4; or 0 mg/kg b.w., APM was added to the
standard Corticella diet, used for 30 years at the laboratory of the Cancer
Research Centre (CRC) of the European Ramazzini Foundation (ERF), at concentrations
of 100,000; 50,000; 10,000; 2,000; 400; 80; or 0 ppm. APM-treated feed
was administered ad libitum to Sprague-Dawley rats (100-150/sex/group),
8 weeks old at the start of the experiment, and the treatment lasted until
spontaneous death. Control animals received the same feed without APM.
The plan of the experiment is shown in Table 1. Male (M) and female (F)
rats from the colony of the CRC were used. This colony of rats has been
employed2 M. Soffritti, F. Belpoggi, D. Degli Esposti, et al.
-
- IN PRESS
-
- Table 1 - Long-term carcinogenicity bioassay on aspartame
administered with feed supplied ad libitum to male (M) and female (F) Sprague-Dawley
rats from 8 weeks of age until spontaneous death. Plan of the experiment.
-
- Groups Animals Treatment
-
- Number Age at start Sex No. Dose Duration
- (weeks) ppm mg/kg b.w. a Human ADI
- equivalent b
- I 8 M 100
- F 100 100,000 5,000 100X Life span
- M+F 200
- II 8 M 100
- F 100 50,000 2,500 50X Life span
- M+F 200
- III 8 M 100
- F 100 10,000 500 10X Life span
- M+F 200
- IV 8 M 150
- F 150 2,000 100 2X Life span
- M+F 300
- V 8 M 150
- F 150 400 20 0.4X Life span
- M+F 300
- VI 8 M 150
- F 150 80 4 0.08X Life span
- M+F 300
- VII 8 M 150
- F 150 0 - - Life span
- M+F 300
-
- a The daily assumption in mg/kg b.w. 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 per day, both for males and
females b Considering the Acceptable Daily Intake (ADI) of 50 mg/kg b.w.
for humans for various experiments in the CRC Laboratory for nearly 30
years. Data are available on the tumour incidence among untreated Sprague-Dawley
rats. These animals were monitored for feed, water consumption, and body
weight, for their life span and, at death, underwent complete necropsy
and histopathological evaluation (historical controls).
-
- The experiment was conducted according to the Italian
law regulating use of animals for scientific purposes 5. After weaning,
at 4-5 weeks of age, the experimental animals were identified by ear punch,
randomised in order to have no more than one male and one female from each
litter in the same group, and housed in groups of 5 in makrolon cages (41x25x15
cm), with stainless-steel wire tops and a shallow layer of white wood shavings
as bedding. The animals were kept in one single room, at 23 ± 2°C
and 50-60% relative humidity.
-
- Once a week for the first 13 weeks, then every two weeks
until 110 weeks of age, the mean daily drinking water and feed consumption
were measured per cage, and body weight individually. Body weight continued
to be measured every 8 weeks until the end of the experiment. Status and
behaviour of the animals were examined 3 times daily, and they were clinically
examined for gross changes every 2 weeks. All animals were kept under observation
until spontaneous death.3
-
- Aspartame, a leukaemogenic compound
-
- IN PRESS
-
- Fig. 1. Mean daily water consumption in male Sprague-Dawley
rats
-
- Fig. 2. Mean daily water consumption in female Sprague-
Dawley rats
-
- The biophase of the experiment terminated after 151 weeks,
with the death of the last animal at the age of 159 weeks.
-
- Upon death, the animals underwent complete necropsy.
Histopathology was routinely performed on the following organs and tissues
of all animals from each group: skin and subcutaneous tissue, mammary gland,
the brain (3 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,
oesophagus, stomach (fore and glandular), intestine (four levels), urinary
bladder, prostate, gonads, interscapular brown fat pad, subcutaneous and
mesenteric lymph nodes and other organs or tissues with pathological 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 the Standard Operating Procedures at
the CRC Laboratory: i.e. parenchymal organs were dissected through the
hilus to expose the widest surface, and hollow organs were sectioned across
the greatest diameter.4 M. Soffritti, F. Belpoggi, D. Degli Esposti, et
al.
-
- IN PRESS
-
- Fig. 3. Mean daily feed consumption in male Sprague-Dawley
rats
-
- Fig. 4. Mean daily feed consumption in female Sprague-Dawley
rats
-
- Any pathological tissue was trimmed through the largest
surface, including normal adjacent tissue. Trimmed specimens were processed
as paraffin blocks, and 3-5 micron sections of every specimen were obtained.
Sections were routinely stained with haematoxylin-eosin. Statistical analyses
were performed using the poly-k test (k = 3). This test is a survival-adjusted
quantal-response procedure that modifies the Cochran-Armitage linear trend
test to take survival differences into account 6-8.
-
- Results
-
- During the experiment no differences were observed among
the various groups in mean daily water consumption (figs. 1 and 2). A dose-related
difference in feed consumption was observed between the various treated
groups and the control group in both males and females (figs. 3 and 4).
No differences in mean body weight were observed among treated and control
groups in either males or females (figs. 5 and 6). No substantial difference
in survival was observed among treated and control groups, males or females
(figs. 7 and 8). Yellowing of the coat was observed in animals exposed
to APM, mainly at the highest concentrations. This change was previously
observed in our laboratory in rats exposed to formaldehyde administered
with drinking water9. The occurrence of lymphomas and leukaemias among
male and female rats in treated and control groups is5 Aspartame, a leukaemogenic
compound
-
- IN PRESS
-
- Fig. 5. Mean body weights in male Sprague-Dawley rats
-
- Fig. 6. Mean body weights in female Sprague-Dawley rats
shown in Table 2. The data indicate that APM causes a statistically significant
increase in the incidence of lymphomas and leukaemias in females, at concentrations
of 100,000 (p= 0.01); 50,000 (p= 0.01); 10,000 (p= 0.05); 2,000 (p= 0.01)
and 400 (p= 0.01) ppm as compared to untreated controls. This increase
is dose-related (p= 0.05).
-
- Although not statistically significant, an increase was
also observed in females treated with 80 ppm and in males treated with
the highest dose.
-
- The haemolymphoreticular neoplasias observed in the experiment
include: lymphoblastic lymphoma and leukaemia, lymphocytic lymphoma, lymphoimmunoblastic
lymphoma, histiocytic sarcoma and monocytic leukaemia, myeloid leukaemia.
The most frequent type of neoplasia was the lymphoimmunoblastic lymphoma
(figs. 9 and 10). Lymphomas and leukaemias are considered together, since
both solid and circulating phases are present in many lymphoid neoplasms,
and distinction between them is artificial10.
-
- The occurrence of brain malignancies is shown in Table
3. Sparse malignant brain tumours were observed among males and females
in the treated groups and none in the controls. 6 M. Soffritti, F. Belpoggi,
D. Degli Esposti, et al.
-
- IN PRESS
-
- Fig. 7. Survival in male Sprague-Dawley rats
-
- Fig. 8. Survival in female Sprague-Dawley rats7
-
- Aspartame, a leukaemogenic compound
-
- IN PRESS
-
- Fig. 9. Lymphoimmunoblastic lymphoma in a female rat
administered
- 80 ppm aspartame in feed (lung). HE X 25
-
- Fig. 10. A detail of the lymphoimmunoblastic lymphoma
shown
- in fig. 9. HE X 400
-
- In our historical controls over the last 20 years, when
we consider groups of 100 or more animals per sex (1934 males and 1957
females), the overall incidence of lymphomas and leukaemias in males is
21.8% (8.0-30.9) and in females 13.4% (7.0-18.4). The overall incidence
of malignant brain tumours is 1.7% (0-5.0) in males and 0.7% (0-2.0) in
females respectively.
-
- Conclusions
-
- In our experimental conditions, it has been demonstrated,
for the first time, that APM causes a dose-related statistically significant
increase in lymphomas and leukaemias in females at dose levels very near
those to which humans can be exposed. Moreover, it can hardly be overlooked
that at the lowest exposure of 80 ppm, there was a 62% increase in lymphomas
and leukaemias compared to controls, even though this was not statistically
significant. When compared to the concurrent control group, an increase
in the incidence of these neoplasias was also observed in males exposed
to the highest dose; even though not statistically significant, this observation
confirms and extends the result in females.
-
- The significance of the increase in haemolymphoreticular
neoplasias is further reinforced by the following considerations, based
on the results of experiments performed in the CRC laboratory.
-
- These experiments demonstrate that the increase in lymphomas
and leukaemias, observed in the APM study, could be related to methanol,
a metabolite of APM, which is metabolised to formaldehyde and then to formic
acid, both in humans and rats3. In fact we have shown that: 1) methanol
administered in drinking water increased the in-8 M. Soffritti, F. Belpoggi,
D. Degli Esposti, et al.
-
- IN PRESS
-
- Table 2 - Long-term carcinogenicity bioassay on aspartame
administered with feed supplied ad libitum to male (M) and female (F) Sprague-Dawley
rats from 8 weeks of age until spontaneous death.
-
- Incidence of lymphomas and leukaemias
-
- Group Animals Treatment Animals with No. lymphomas and
leukaemias
- Age at start Sex No. Dose Duration Number %
- (weeks) ppm mg/kg b.w.a Human ADI
- equivalent b
-
- I 8 M 100 29 29.0
- F 100 100,000 5,000 100X Life span 25 25.0**
- M+F 200 54 27.0
- II 8 M 100 20 20.0
- F 100 50,000 2,500 50X Life span 25 25.0**
- M+F 200 45 22.5
- III 8 M 100 15 15.0
- F 100 10,000 500 10X Life span 19 19.0*
- M+F 200 34 17.0
- IV 8 M 150 33 22.0
- F 150 2,000 100 2X Life span 28 18.7*
- M+F 300 61 20.3
- V 8 M 150 25 16.7
- F 150 400 20 0.4X Life span 30 20.0**
- M+F 300 55 18.3
- VI 8 M 150 23 15.3
- F 150 80 4 0.08X Life span 22 14.7
- M+F 300 45 15.0
- VII 8 M 150 31 20.7
- F 150 0 - - Life span 13 8.7
- M+F 300 44 14.7
-
- a Considering the life-span average weight of a rat (male
and female) as 400 g and the average consumption of food as 20 g per day
-
- b Considering the Acceptable Daily Intake (ADI) of 50
mg/kg b.w. for humans
-
- * Statistically significant p= 0.05; ** Statistically
significant p= 0.01 using poly-k test (k = 3) cidence of lymphomas and
leukaemias in female rats11; 2) the same effect was induced in females
treated with the gasoline oxygenated additive methyl-tert-butyl-ether (MTBE),
which is also metabolised to methanol12 ; and finally
-
- 3) an increase in the incidence of lymphomas and leukaemias
was also observed in females treated with formaldehyde9, 13.
-
- These results further highlight the important rôle
that formaldehyde has on the induction of haematological malignancies in
rodents. Moreover, in a recent reevaluation of the carcinogenicity of formaldehyde
by the International Agency for Research on Cancer (IARC), strong, although
not considered sufficient, evidence of an association with leukaemias in
humans was found14.
-
- Since the results of carcinogenicity bioassays in rodents,
mainly rats and mice, have been shown to be a consistent predictor of human
cancer risk15-17, the first results of our study call for urgent re-examination
of permissible exposure levels of APM in both food and beverages, especially
to protect children.
-
- References
-
- 1. Butchko HH, Stargel WW, Comer CP, et al. Preclinical
safety
- evaluation of aspartame. Regul Toxicol Pharmacol 2002;
35:
- S7-S12.
- 2. Aspartame Information Center. Available on http://www.aspartame.
- org, 2004.
- 3. Ranney RE, Opperman JA, Maldoon E, et al. Comparative
metabolism
- of aspartame in experimental animals and humans.
- Toxicol Environ Health 1976; 2: 441-51.
- 4. Food and Drug Administration. Aspartame: Commissioner,s
Final
- Decision; 1981 Fed Regist 46, 38285-308.
- 5. Repubblica Italiana. Decreto Legislativo 116. Attuazione
della
- direttiva n. 86/609/CEE in materia di protezione degli
animali
- 9
- Aspartame, a leukaemogenic compound
-
- IN PRESS
-
- Table 3 - Long-term carcinogenicity bioassay on aspartame
administered with feed supplied ad libitum to male (M) and female (F) Sprague-Dawley
rats from 8 weeks of age until spontaneous death.
-
- Incidence of malignant brain tumours
-
- Group Animals Treatment Animals with
- Number malignant brain tumoursa
- Age at start Sex No. Dose Duration Number %
- (weeks) ppm mg/kg b.w.b Human ADI
- equivalent c
- I 8 M 100 1 1.0
- F 100 100,000 5,000 100X Life span 1 1.0
- M+F 200 2 1.0
- II 8 M 100 2 2.0
- F 100 50,000 2,500 50X Life span 1 1.0
- M+F 200 3 1.5
- III 8 M 100 0 -
- F 100 10,000 500 10X Life span 1 1.0
- M+F 200 1 0.5
- IV 8 M 150 2 1.3
- F 150 2,000 100 2X Life span 1 0.7
- M+F 300 3 1.0
- V 8 M 150 0 -
- F 150 400 20 0.4X Life span 0 -
- M+F 300 0 -
- VI 8 M 150 2 1.3
- F 150 80 4 0.08X Life span 1 0.7
- M+F 300 3 1.0
- VII 8 M 150 0 -
- F 150 0 - - Life span 0 -
- M+F 300 0 -
- a The malignancies observed were: 10 malignant gliomas
or mixed gliomas, 1 medulloblastoma, and 1 malignant meningioma
- b Considering the life-span average weight of a rat (male
and female) as 400 g and the average consumption of food as 20 g per day
- c Considering the Acceptable Daily Intake (ADI) of 50
mg/kg b.w. for humans
- utilizzati a fini sperimentali o ad altri fini scientifici.
Supplemento
- ordinario alla Gazzetta Ufficiale 1992; 40: 5-25.
- 6. Bailer AJ, Portier CJ. Effects of treatment-induced
mortality
- and tumor-induced mortality on tests for carcinogenicity
in
- small samples. Biometrics 1988; 44: 417-31.
- 7. Portier CJ, Bailer AJ. Testing for increased carcinogenicity
using
- a survival-adjusted quantal response test. Fundam Appl
- Toxicol 1989; 12: 731-7.
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