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aspartame causes cancer in rats at levels approved for humans, Morando
Soffritti et al, Ramazzini Foundation, Italy & National Toxicology Program
of National Institute of Environmental Health Sciences 2005.11.17
Env. Health Pers. 35 pages: Murray

http://www.newswise.com/articles/view/516214/

Environmental Health Perspectives (NIEHS)
National Institute of Environmental Health Sciences
Released: Thu 17-Nov-2005, 09:00 ET

Artificial Sweetener Causes Cancer in Rats
at Levels Approved for Humans

Medical News Keywords:
ASPARTAME, CANCER, LYMPHOMA, LEUKEMIA,
ENVIRONMENTAL HEALTH PERSPECTIVES

Description

A statistically significant increase in the incidence of malignant tumors,
lymphomas and leukemias in rats exposed to varying doses of aspartame
appears to link the artificial sweetener to a high carcinogenicity rate,
according to a study.

The article appeared in the peer-reviewed journal
Environmental Health Perspectives.
http://ehp.niehs.nih.gov/ ehponline@niehs.nih.gov

Newswise -

A statistically significant increase in the incidence of malignant tumors,
lymphomas and leukemias in rats exposed to varying doses of aspartame
appears to link the artificial sweetener to a high carcinogenicity rate,
according to a study accepted for publication today by the peer-reviewed
journal Environmental Health Perspectives (EHP).
-- published by the National Institute of Environmental Health Sciences
http://www.niehs.nih.gov/
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Phone number:
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The authors of the study, the first to demonstrate
multipotential carcinogenic effects of aspartame
administered to rats in feed,
called for an "urgent reevaluation" of the current guidelines
for the use and consumption of this compound.

"Our study has shown that aspartame is a multipotential carcinogenic
compound whose carcinogenic effects are also evident at a daily dose of
20 milligrams per kilogram of body weight (mg/kg),
notably less than the current acceptable daily intake for humans,"
the authors write.

[ For a 70 kg average human, 20 mg/kg body weight is 1,400 mg,
the amount in 7 12-oz diet sodas, a little over a half-gallon daily,
the same amount as average adult daily drinking water. ]

Currently, the acceptable daily intake for humans is set
at 50 mg/kg in the United States and 40 mg/kg in Europe.

Aspartame is the second most widely used artificial sweetener in the world.

It is found in more than 6,000 products
including carbonated and powdered soft drinks, hot chocolate,
chewing gum, candy, desserts, yogurt, and tabletop sweeteners,
as well as some pharmaceutical products
like vitamins and sugar-free cough drops.

More than 200 million people worldwide consume it.

The sweetener has been used for more than 30 years,
having first been approved by the FDA in 1974.

Studies of the carcinogenicity of aspartame
performed by its producers have been negative.

Researchers administered aspartame to Sprague-Dawley rats
by adding it to a standard diet.
They began studying the rats at 8 weeks of age
and continued until the spontaneous death of each rat.
Treatment groups received feed
that contained concentrations of aspartame at dosages
simulating human daily intakes
of 5,000, 2,500, 500, 100, 20, and 4 mg/kg body weight.

Groups consisted of 100 males and 100 females
at each of the three highest dosages
and 150 males and 150 females at all lower dosages and controls.

The experiment ended after the death of the last animal at 159 weeks.

At spontaneous death, each animal underwent examination
for microscopic changes in all organs and tissues,
a process different from the aspartame studies conducted 30 years ago
and one that was designed to allow aspartame
to fully express any carcinogenic potential.

The treated animals showed extensive evidence of malignant cancers
including lymphomas, leukemias, and tumors
at multiple organ sites in both males and females.

The authors speculate the increase in lymphomas and leukemias
may be related to one of the metabolites in aspartame, namely methanol,
which is metabolized in both rats and humans to formaldehyde.

Both methanol and formaldehyde
have shown links to lymphomas and leukemias
in other long-term experiments by the same authors.

The current study included more animals
over a longer period than earlier studies.

"In our opinion, previous 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 rodents
for each experimental group
(40-86, compared to 100-150 in the current study)
and the termination of previous studies
at only 110 weeks of age of the animals," the study authors wrote.

The authors of the study were Morando Soffritti,
Fiorella Belpoggi,
Davide Degli Esposti,
Luca Lambertini,
Eva Tibaldi,
and Anna Rigano
of the Cesare Maltoni Cancer Research Center,
European Ramazzini Foundation of Oncology and Environmental Sciences,
Bologna, Italy.
Funding for the research was provided by the
European Ramazzini Foundation of Oncology and Environmental Sciences,
Bologna, Italy.
The article is available free of charge at
http://ehp.niehs.nih.gov/docs/2005/8711/abstract.html .

EHP is published by the
National Institute of Environmental Health Sciences,
part of the U.S. Department of Health and Human Services.
EHP is an Open Access journal.
More information is available online at http://www.ehponline.org/ .

Brogan & Partners Convergence Marketing
handles marketing and public relations for EHP,
and is responsible for the creation and distribution of this press release.

http://www.newswise.com/articles/view/516214/

http://ehp.niehs.nih.gov/docs/2005/8711/abstract.html

Environmental Health Perspectives (EHP)
is a monthly journal of peer-reviewed research
and news on the impact of the environment on human health.
EHP is published by the
National Institute of Environmental Health Sciences
and its content is free online.
Print issues are available by paid subscription.

Staff contacts for Environmental Health Perspectives:

Thomas J. Goehl, Ph.D. Editor-in-Chief goehl@niehs.nih.gov
phone 1 919 541 7961 fax 1 919 541 0273

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booker@niehs.nih.gov phone 1 919 541 1587

Research

EHP-in-Press

First Experimental Demonstration of the
Multipotential Carcinogenic Effects of Aspartame
Administered in the Feed to Sprague-Dawley Rats.
Morando Soffritti, Fiorella Belpoggi, Davide Degli Esposti,
Luca Lambertini, Eva Tibaldi, and Anna Rigano.

This EHP-in-Press article has been peer-reviewed,
revised, and accepted for publication.
The EHP-in-Press articles are completely citable
using the assigned DOI code for the article.
This document will be replaced with the copyedited
and formatted version as soon as it is available.
Through the DOI number used in the citation,
you will be able to access this document
at each stage of the publication process.
Environ Health Perspect doi:10.1289/ehp.8711
available via
http://dx.doi.org/ [Online 10 November 2005]

http://ehp.niehs.nih.gov/docs/admin/newest.html

EHP-in-Press articles are published in manuscript form
within 24 hours of acceptance.
Each EHP-in-Press article has gone through the normal process
of peer review, revision, and acceptance.
The date the article is posted on ehponline is considered
the publication date of record, and thus establishes publication priority.
Once copyedited and formatted,
the final article will replace the manuscript online
and be published in a print issue of EHP.

Environmental Health Perspectives Volume 113, Number 11
November 2005 Current print issue

The full version of this article is available for free in PDF format.

http://ehp.niehs.nih.gov/members/2005/8711/8711.pdf 35 pages

First Experimental Demonstration of the
Multipotential Carcinogenic Effects of Aspartame
Administered in the Feed to Sprague-Dawley Rats.
Morando Soffritti, Fiorella Belpoggi, Davide Degli Esposti,
Luca Lambertini, Eva Tibaldi, and Anna Rigano.

doi:10.1289/ehp.8711 -- available at http://dx.doi.org/ --
Online 17 November 2005

The National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Department of Health and Human Services
http://www.ehponline.org/

First Experimental Demonstration of the
Multipotential Carcinogenic Effects of Aspartame
Administered in the Feed to Sprague-Dawley Rats.
Morando Soffritti 1, Fiorella Belpoggi 1, Davide Degli Esposti 1, Luca
Lambertini 1, Eva Tibaldi 1,
and Anna Rigano 1
1 Cesare Maltoni Cancer Research Center,
European Ramazzini Foundation of Oncology and
Environmental Sciences, Bologna, Italy
Address of the institution:
Cesare Maltoni Cancer Research Center,
European Ramazzini Foundation of Oncology and Environmental Sciences.
Castello di Bentivoglio, Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy
Tel. +39/051/6640460 Fax +39/051/6640223 e-mail: crcfr@ramazzini.it

Address correspondence to
Dr. M. Soffritti, M.D., Scientific Director of the Cesare Maltoni
Cancer Research Center,
European Ramazzini Foundation of Oncology and Environmental
Sciences.
Castello di Bentivoglio, Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy
Tel. +39/051/6640460 Fax +39/051/6640223 e-mail: crcfr@ramazzini.it

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Running title: Aspartame carcinogenicity
Article descriptor: Carcinogenesis
Key words: aspartame, artificial sweetener, carcinogenicity, rats,
lymphomas, renal pelvis carcinomas, malignant schwannomas

Acknowledgements: A special thanks to the
US National Toxicology Program (NTP)
for convening a group of pathologists at NIEHS
in order to provide a second opinion
for a set of lesions of malignancies and their precursors
related to the APM treatment, and for the help in statistical analysis.

A special thanks also to all the staff who were involved in the project.

This research was entirely supported by European Ramazzini Foundation of
Oncology and Environmental Sciences, Bologna, Italy.

Authors do not have competing financial interests
in relation to the submitted article.

Abbreviations:
ADI: Acceptable Daily Intake
APM: Aspartame
CMCRC/ERF: Cesare Maltoni Cancer Research Center/European Ramazzini
Foundation
DKP: Diketopiperazine
FDA: Food and Drug Administration
HE: Hematoxylin -Eosin
IARC International Agency for Research on Cancer
MSA: Monosodium Aspartate
MTBE Methyl-tert-butyl ether
pmm Post mortem modifications

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Outline of section headers

Abstract 286 words pg. 4
Introduction 1521 words pg. 5
Material and methods 889 words pg. 10
Results 1359 words pg. 13
Discussion 923 words pg. 18
Conclusions 158 words pg. 21
References 1158 words pg. 23
Table 1-4 1000 words pg. 29
Figure legends 158 words pg. 33
Figures 1-2 500 words pg. 34
Total paper word count 7666 words

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ABSTRACT

The Cesare Maltoni Cancer Research Center of the European Ramazzini
Foundation has conducted a long-term bioassay on aspartame
(APM), 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.
Histopathological evaluation of all pathological lesions and of all organs
and tissues collected was routinely performed on each animal
of all experimental groups. [ ~30,000 slides ]
The results of the study show for the first time that APM,
in our experimental conditions,
causes:

1) an increased incidence of malignant tumor-bearing animals with
a positive significant trend in males (p 0.05) and in females (p 0.01),
in particular those females treated at 50,000 ppm (p 0.01);

2) an increase in lymphomas and leukemias with
a positive significant trend in both males (p 0.05) and females (p 0.01),
in particular in females treated at doses of
100,000 (p 0.01), 50,000 (p 0.01),
10,000 (p 0.05), 2,000 (p 0.05), 400 (p 0.01) ppm;

3) a statistically significant increased incidence, with
a positive significant trend (p 0.01) of transitional cell carcinomas
of the renal pelvis and ureter and their precursors (dysplasias) in females
treated at 100,000 (p 0.01), 50,000 (p 0.01),
10,000 (p 0.01), 2,000 (p 0.05) and 400 ppm (p 0.05);

and 4) an increased incidence of malignant schwannomas
of peripheral nerves with a positive trend (p 0.05) in males.

The results of this mega-experiment indicate that APM is a multipotential
carcinogenic agent, even at a daily dose of 20 mg/kg b.w.,
much less than the current acceptable daily intake (ADI).

On the basis of these results,
a re-evaluation of the present guidelines on the
use and consumption of APM is urgent and cannot be delayed.

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INTRODUCTION

Nowadays consumers are increasingly concerned about the quality
and safety of many products present in the diet of industrialized 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 epidemiological data currently available to evaluate
the above carcinogenic risks are insufficient and often unreliable,
due to the inadequate planning and conduct of previous experiments.
This inadequacy, combined with the general limited knowledge
about the safety/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).

The products studied are reported in Table 1.
Products and agents selected for the project were those
for which committee debate and opinions often acted as
surrogates for good laboratory work.

Over the course of the project, up to now, 32 long term
bioassays have been performed using over 25,000 rodents.

Studies have evaluated the carcinogenicity of 12 different products,
including the artificial sweetener Aspartame (APM).

The following report presents the results of the mega-experiment on the
carcinogenicity of APM in which the sweetener was administered in feed
to Sprague-Dawley rats for the life span.

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APM, the methyl ester of the dipeptide L--aspartyl-L-phenylalanine
(C14H18N2O5), 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
cycloaspartylphenylalanine (DKP) (Butchko et al. 2002a).

For more than 30 years,
APM has been widely used as a food additive due to its very
strong, sweet taste.
The sweetening power of APM is estimated to be
200 times that of sucrose,
whereas saccharin and cyclamate are 300 and 30 times sweeter,
respectively (Mazur 1984).

Initial commercial approval of APM in the United States was granted
by the FDA in 1974.
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, the safe use of APM was authorized in 1994.

After saccharin,
APM is the second most used artificial sweetener in the world.

It is estimated that more than 8,000 tons of APM are consumed
each year in the USA (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 more than 6,000 products,
including carbonated and powdered soft drinks,
hot chocolate, chewing gum, candy, desserts, yoghurt,
table-top sweeteners and in some pharmaceutical products,
such as vitamins and sugar-free cough drops,
and is estimated by the Aspartame Information Center (2005)
to be consumed by over 200 million people worldwide.

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The average APM daily intake in the general population
has been shown, by dietary surveys performed in the United States
among APM consumers during the period 1984-1992,
to range from 2 to 3 mg/kg of body weight.

Consumption by 2 to 5 year-old children
and by females of childbearing age in these surveys
ranged from about 2.5 to 5 mg/kg b.w./day (Butchko et al. 2002b).

APM intake was also monitored in several other regions,
including 7 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 b.w.) and in Europe (40 mg/kg b.w.)
(Butchko et al. 2002b).

Investigations into the metabolism of APM have shown that,
in rodents, non-human primates and humans,
it is metabolized in the gastrointestinal tract into three constituents:
aspartic acid, phenylalanine and methanol,
which are absorbed into the systemic circulation
(Ranney et al. 1976).

For each molecule of APM, one molecule of each constituent is
produced.

After absorption, they are then utilized, metabolized and/or excreted by the
body following the same metabolic pathways
as when consumed through the ordinary diet, namely:

aspartate is transformed into alanine plus oxaloacetate (Stegink 1984);

phenylalanine is transformed mainly into tyrosine and, to a smaller extent,
phenylethylamine and phenylpyruvate (Harper 1984);

while methanol is transformed into formaldehyde and then to formic acid
(Opperman 1984).

It has been reported that APM is not genotoxic in the following tests:
dominant lethal mutation assay in rats,
host-mediated assay in rats and mice,
in vivo cytogenetic assay in rats,
the Ames test (Kotsonis and Hjelle 1996).

Results of an assay to measure induction
of unscheduled DNA synthesis in rat hepatocytes
treated with APM in vitro were negative,

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indicating the absence of APM-induced DNA damage
(Jeffrey and Williams 2000).

In vivo, a mixture of aspartame (up to 350 mg/kg) and a second sweetener,
acesulfame potassium (up to 150 mg/kg), was reported to be negative
in a test for the induction of chromosomal aberration
in bone marrow cells of male Swiss mice,
when administered by gavage.
However a dose-related increase
in the percentage of cells with chromosomal aberrations
was noted with increasing doses of the two sweeteners,
but 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 of 1981 (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 to 8 g/kg b.w./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 7/155 (4.5%) exposed males vs 1/59 (1.7%) controls,
and in 5/158 (3.2%) exposed females vs 0/59 (0%) in controls.
Overall, the FDA considered the study to be negative with regard to the
carcinogenicity of APM.

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 b.w./day,
through their mothers' diet both in utero and during lactation,
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

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at the time of death or at 104 weeks after weaning.
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 b.w. group,
and in one male and one female in the 4 g/kg b.w. group.
Again, the FDA considered the study to be negative with regard to the
carcinogenicity of APM.

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, 4 g/kg b.w./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 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 b.w./day
from 6 to 110 weeks of age.
No increase in the incidence of brain tumors
was observed in the treated groups as compared to the controls.
Exhaustive experimental details of this study were not published.

Epidemiological 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.

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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
(Soffritti et al. 1999; Soffritti et al. 2002c).

MATERIALS AND METHODS

The APM, as a food grade material, was produced
by Nutrasweet and supplied by Giusto Faravelli S.p.A. in Milan, Italy.
Its purity was more than 98%:
DKP was less than 1.5%
and L-phenylalanine was less than 0.5%.
The method used to determine APM purity
was an infrared absorption spectrophotometer assay.

An assumed daily intake by humans of
5,000; 2,500; 500; 100; 20; 4 or 0 mg/kg b.w. was simulated
by adding APM to the standard Corticella diet,
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 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.

APM was administered with feed ad libitum to Sprague-Dawley rats
(100-150/sex/group).
The experiment started when the animals were 8 weeks old.
The treatment lasted until natural death.
Control animals received the same feed without APM.
The experiment was conducted according to the Italian law regulating the

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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 (M) and female (F) 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 histopathological evaluation.
The health status of the animals was regularly checked by the veterinarians
of the Local and National Health Services.
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 randomised 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 5, in makrolon cages (41x25x15 cm),
with stainless-steel wire tops and a shallow layer of white wood-shavings
as bedding, and kept in rooms destined only to this experiment, at
a temperature of 23 ± 2°C and relative humidity of 50-60%, respectively.
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 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 entire duration of the experiment.
In order to evaluate the status and behaviour of

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the animals and to limit the post mortem modifications (pmm),
a patrol was performed three times daily from Monday to Friday and twice
on Saturdays and Sundays and festivities.
Dead animals were registered and kept refrigerated at 4°C until necropsy.
Based on this procedure (part of our longstanding Standard Operating
Procedures, SOP) very few animals were affected by pmm
and, on very rare occasions, this interfered 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 was routinely performed on the following organs and tissues
of each animal 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 CMCRC/ERF
Laboratory SOP.
Trimmed specimens were processed as paraffin blocks,
and 3-5 micron sections of every specimen were obtained.
Sections were routinely stained with Hematoxylin-Eosin (HE).
Immunohistochemical staining for S100 was performed
to characterize malignant Schwannoma, while

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chromogranin A staining was used to characterize olfactory neuroblastoma.
For S100 staining, a polyclonal rabbit Anti-S100 (Dakocytomation code no.
Z0311) was used as primary antibody while, for chromogranin A staining,
a polyclonal rabbit anti-human chromogranin A (Dakocytomation code no.
N1535) was used (Information Centre for Immunohistochemistry 2005).

Two statistical tests were used to analyze neoplasm and non neoplastic
lesion incidence data.
The Cochran-Armitage trend test (Armitage 1971; Gart et al. 1979)
was used to test for linear trends in tumor incidence.

Also used was the poly K-test (Bailer and Portier 1988; Portier and Bailer
1989; Piegorisch and Bailer 1997),
a survival-adjusted quantal response modification of the Cochran-Armitage
test that takes survival into account.
The test used and the p-values are reported in the tables.

RESULTS

The study proceeded smoothly without unexpected occurrences.
No differences were observed 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 (Figure 1C).
No substantial difference in survival was observed among the groups
(Figure 1D, E).
No evident behavioral changes were observed among treated animals
compared to the controls.
In animals exposed to the highest dose of APM,
yellowing of the coat was

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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 type and site,
of different type in the same site,
of the same type in bilateral organs,
of the same type in the skin,
in the subcutaneous tissue,
and in 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 malignant tumor-bearing animals occurred
with a significant positive trend in males (p 0.05) and in females (p 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 0.01)
compared to the control group (Table 3).

Tumor types which contributed most are presented as follows.
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 0.05) and in females (p 0.01).

When compared to untreated control groups,
the increased incidence of lymphomas-leukemias in treated females was
statistically significant at doses of
100,000 (p 0.01), 50,000 (p 0.01), 10,000 (p 0.05),
2,000 (p 0.05) and 400 (p 0.01) ppm.

The most frequent histocytotypes observed in the experiment were
lymphoimmunoblastic lymphomas,
mainly involving lung and mediastinal/peripheral nodes,
and histiocytic sarcomas, 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 morphological criteria followed in our laboratory

- 15 -

for several decades and are in line with the guidelines of the International
Classification of Rodent Tumors (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.
The reason is, as has been stated, that both solid and circulating phases
are present in many lymphoid 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 epithelium of the renal pelvis and ureter are reported in Tables 2 and 3.

A dose-related increase in the incidence of dysplastic hyperplasias and
dysplastic papillomas of the renal pelvis and ureter
were observed in females.

Carcinomas in females occurred with a positive trend (p 0.05)
and the incidence in females exposed at 100,000 ppm
was significantly higher (p 0.05)
compared to the controls.

Carcinomas were also observed among males treated at 100,000, 50,000,
10,000, and 2,000 ppm.

In females, dysplastic lesions and carcinomas combined show a
significant positive trend (p 0.01)
and a statistically significant increase in those treated at
100,000 (p 0.01), 50,000 (p 0.01), 10,000 (p 0.01),
2,000 (p 0.05) and 400 ppm (p 0.05).

A threefold increase is also observed in the 80 ppm treated group.

We did not observe substantial differences in the incidence of inflammation
between males and females treated at the different doses,
as compared to controls.

Increased incidence of calcification was observed in females,
particularly in those treated at
100,000 ppm (39%), 50,000 ppm (25%) and 10,000 ppm (19%),
when compared to controls (8%);
this effect was not observed in males.

It should be pointed out that,
while transitional cell carcinomas of the renal pelvis and ureter
are extremely rare in male and female untreated rats,
the APM male and female

- 16 -

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 and the kidney parenchyma;
the cells were of transitional type and several mitotic figures were present
(Figure 2A, B). [ color microscopic images ]

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 0.05) in males.
In females, 9 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 S100 staining.
The most frequent site of origin of the malignant schwannomas
was in the cranial nerves (72%);
the other cases arose at the spinal nerve roots.

Microscopically, malignant schwannomas invaded the soft tissues locally.

Metastases of cranial nerve malignant schwannomas
were observed in 3 males treated at the highest dose.

The metastases were found in submandibular lymph nodes in 2 cases,
and in 1 case, the tumor metastatized to the lung and to the liver.

Histologically the feature of malignant schwannomas was Antoni B type
(Figure 2C, D). [ color microscopic photo ]

Preneoplastic and neoplastic lesions of the olfactory epithelium.
Incidence of hyperplasia of the olfactory epithelium increased
with a significant positive trend in males and females.
The observed incidences were respectively:
4.0% 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 0.01) at 100,000, 50,000 and 10,000 ppm
in both males and females and also, in males, at 400 ppm.

It is noteworthy that among females treated at the highest dose, one case of

- 17 -

dysplastic hyperplasia, one adenoma
and one olfactory neuroblastoma were observed.
The neuroblastoma invaded the cranium,
compressing the forebrain and was positive
for chromogranin A immunohistochemical staining.

Brain malignant tumors.
Concerning the incidence of brain malignant tumors,
it should be noted that, as previously reported (Soffritti et al. 2005),
12 malignant tumors (10 gliomas, 1 medulloblastoma and 1 meningioma)
were observed, without dose relationship, in male and female APM-treated
groups, while none were observed in controls.

Other malignant tumors.
The other malignant tumors were among those commonly observed
in Sprague-Dawley rats,
apart from 2 transitional cell carcinomas of the bladder
observed in males exposed to 10,000 ppm
and 1 in females exposed to 2,000 ppm
and none among the controls.

Since 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 or more animals per sex,
the numbers of the untreated males and females
total 1934 and 1945 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% (0-2.0%) in males and 0.1% (0-1.0%) in females.

The overall incidence of lymphomas-leukemias
was 20.7% (8.0-30.9%) in males and 12.4% (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 less than 100 animals per sex,
the numbers of untreated males and females
total 2265 and 2274 respectively.

The overall incidence of the renal pelvis
and ureter transitional cell carcinomas
was 0.04% (0-1.0%) in females, while

- 18 -

no carcinomas were observed in males.

The overall incidence of malignant schwannomas
was 0.4% (0-2.0%) in males and 0.1% (0-2.0%) in females.

The overall incidence of lymphomas-leukemias
was 20.6% (8.0-30.9%) in males and 13.3% (4.0-25.0%) in females.

The overall incidence of olfactory neuroblastomas
was 0.1% (0-1.8%) in both males and females.

DISCUSSION

The mega-experiment performed in our laboratory on APM
(administered with feed to Sprague-Dawley rats from 8 weeks-old
until natural death) has shown for the first time the
multipotential carcinogenic effects of this compound.

In fact, the results indicate that APM causes, in our experimental
conditions:

1) an increased incidence of malignant tumor-bearing animals
with a positive significant trend
in males (p 0.05) and in females (p 0.01),
particularly in the females treated at 50,000 ppm (p 0.01);

2) a statistically significant dose-related increase of the incidence of
lymphomas-leukemias in females treated at the doses
of 100,000 (p 0.01), 50,000 (p 0.01), 10,000 (p 0.05),
2,000 (p 0.05) and 400 (p 0.01) ppm
and a positive significant trend
in both males (p 0.05) and females (p 0.01);

3) in females, dysplastic lesions and carcinomas
of the renal pelvis and ureter combined
show a significant positive trend (p 0.01)
and a statistically significant increase in those treated
at 100,000 (p 0.01), 50,000 (p 0.01), 10,000 (p 0.01),
2,000 (p 0.05) and 400 (p 0.05) ppm;

and 4) an increased incidence of malignant schwannomas of the
peripheral nerves with a positive trend (p 0.05) in males.

The increase in lymphomas-leukemias in APM-treated females
could be related to its metabolite methanol,
which is in turn metabolized to formaldehyde in both humans and rats

- 19 -

(Ranney et al. 1976).

In fact, previous experiments performed at the CMCRC Laboratory
have shown that:

1) methanol administered in drinking water, at doses ranging from 20,000
to 500 ppm, induced a statistically significant increase in the incidence of
lymphomasleukemias in female rats, (Soffritti et al. 2002a);

2) 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; Soffritti et al. 2002b);

and 3) the same effect was observed in females treated with the
gasoline oxygenated additive methyl-tert-butyl-ether (MTBE), which
metabolizes to methanol (Belpoggi et al. 1995).

The important role of formaldehyde in the induction of hematological
malignancies in rodents is further highlighted by these results.

In a recent re-evaluation of the carcinogenicity of formaldehyde by the
International Agency for Research on Cancer (IARC),
strong (although not considered sufficient) evidence
of an association between formaldehyde exposure and leukemias
in humans was found (IARC, in press).

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 of APM are responsible in
inducing these lesions,
it is of paramount importance to perform adequate life-span
carcinogenicity studies on aspartic acid or phenylalanine.

It is worthy of note that,
in a long-term carcinogenicity study on monosodium aspartate (MSA)
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

- 20 -

hyperplasia of the renal pelvis was observed
in males and in females (Kitahori et al. 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 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,
when compared to the controls.

The fact that we observed an increased incidence of kidney
calcification in females and not in males,
when compared to 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 which 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.

Secondly, in our experiment,
rodents were not killed at 110 weeks of age,
but rather were observed until natural death,
to allow APM to

- 21 -

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
(Soffritti et al. 2002c) and toluene (Soffritti 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 Spague-Dawley rats.

CONCLUSIONS

Our study has shown that APM is a multipotential carcinogenic compound whose
carcinogenic effects are evident
even at a daily dose of 20 mg/kg b.w.,
much less than the current ADI for humans in Europe (40 mg/kg b.w.)
and in the United States (50 mg/kg b.w.).

It has been shown that the results of carcinogenicity bioassays in rodents
are consistent predictors of human cancer risks
(Huff 1999; Tomatis et al. 1989; Rall 1995).

The results of our study therefore call for an urgent re-examination
of the present guidelines on the use and consumption of APM.

The decision to use experimental data to protect public health is
important as the time span of widespread aspartame use
is still too brief to have produced solid epidemiologic data.

Moreover, it is unlikely that sufficient epidemiological data will

- 22 -

be available in the near future,
given the difficulty of finding a control group that has not
been exposed to this widely diffused compound.

- 23 -

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Table 1.

Beverages and diet products studied at the CMCRC/ERF:
status of studies

No. Products No. of bioassays Animals Status of studies a
---------------------------------- Species No.

1. Water in polyvinylchloride bottles 2 Rat b 2200 P c

2. Coca-cola 4 Rat b 1999 RP

3. Pepsi-Cola 1 Rat 400 E

4. Ethyl alcohol (10% v/v) 4 Rat b, mice 1458 P d

5. Sucrose 1 Rat 400 E

6. Aspartame 6 Rat, mice b 4460 BO, PP e

7. Sucralose 1 Mice b 760 BO

8. Caffeine 1 Rat 800 E

9. Vitamin A 5 Rat 5100 PP f

10. Vitamin C 5 Rat 3680 E

11. Vitamin E 5 Rat 3680 E

12. Feed sterilized by gamma rads 1 Rat b 2000 E

TOTAL 36 26937

a P = published;
PP = partially published;
RP = ready for publication;
E = in elaboration;
BO = biophase ongoing
b Treatment started from embryonal life;
c Maltoni et al. 1997;
d Soffritti et al. 2002a;
e Soffritti et al. 2005;
f Soffritti et al. 1992

- 30 -

Table 2. Incidence of the preneoplastic and neoplastic lesions
in male Sprague-Dawley rats
in a life-span feed carcinogenicity study of Aspartame

- 31 -

Table 3. Incidence of the preneoplastic and neoplastic lesions
in female Sprague-Dawley rats
in a life-span feed carcinogenicity study of Aspartame

- 32 -

Table 4. Incidence and distribution by hystocytotype of
lymphomas-leukemias in female Sprague-Dawley rats
in a life-span feed carcinogenicity study of Aspartame

- 33 -

Figure 1.

A. Mean daily feed consumption in males.
B. Mean daily feed consumption in females.
C. Mean body weights in males (M) and females (F).
D. Survival in males.
E. Survival in female.

- 34 -

Figure 2. [ color microscopic photos ]

A. Carcinoma of the renal pelvis in a female rat administered
100,000 ppm aspartame in feed. Hematoxylin-Eosin (HE)
X25. Scale bar 500 mm.

B. A detail of the carcinoma shown in A. HE X400. Scale bar 20 mm.

C. Malignant schwannoma of cranial nerves resembling
Antoni B type pattern in a male rat administered
100,000 ppm aspartame in feed. HE X200. Scale bar 50 mm.

D. Immunohistochemical characterization with S-100 protein of the
schwannoma shown in C. X1000. Scale bar 10 mm.
************************************************** ********

http://groups.yahoo.com/group/aspartameNM/message/1016
President Bush & formaldehyde (aspartame) toxicity: Ramazzini Foundation
carcinogenicity results Dec 2002: Soffritti: Murray 2003.08.03 rmforall

p. 88 "The sweetening agent aspartame hydrolyzes in the gastrointestinal
tract to become free methyl alcohol, which is metabolized in the liver
to formaldehyde, formic acid, and CO2. (11)"
Medinsky MA & Dorman DC. 1994; Assessing risks of low-level
methanol exposure. CIIT Act. 14: 1-7.

Ann N Y Acad Sci. 2002 Dec; 982: 87-105.
Results of long-term experimental studies on the carcinogenicity of
formaldehyde and acetaldehyde in rats.
Soffritti M, Belpoggi F, Lambertin L, Lauriola M, Padovani M, Maltoni C.
Cancer Research Center, European Ramazzini Foundation for Oncology
and Environmental Sciences, Bologna, Italy. crcfr@tin.it

Formaldehyde was administered for 104 weeks in drinking water supplied
ad libitum at concentrations of 1500, 1000, 500, 100, 50, 10, or 0 mg/L
to groups of 50 male and 50 female Sprague-Dawley rats beginning at
seven weeks of age.
Control animals (100 males and 100 females) received tap water only.
Acetaldehyde was administered to 50 male and 50 female Sprague-Dawley
rats beginning at six weeks of age at concentrations of 2,500, 1,500,
500, 250, 50, or 0 mg/L.
Animals were kept under observation until spontaneous death.
Formaldehyde and acetaldehyde were found to produce an increase in total
malignant tumors in the treated groups and showed specific carcinogenic
effects on various organs and tissues. PMID: 12562630

Ann N Y Acad Sci. 2002 Dec; 982: 46-69.

Results of long-term experimental studies on the carcinogenicity of
methyl alcohol and ethyl alcohol in rats.
Soffritti M, Belpoggi F, Cevolani D, Guarino M, Padovani M, Maltoni C.
Cancer Research Center, European Ramazzini Foundation for Oncology
and Environmental Sciences, Bologna, Italy. crcfr@tin.it

Methyl alcohol was administered in drinking water supplied ad libitum at
doses of 20,000, 5,000, 500, or 0 ppm to groups of male and female
Sprague-Dawley rats 8 weeks old at the start of the experiment.
Animals were kept under observation until spontaneous death.
Ethyl alcohol was administered by ingestion in drinking water at a
concentration of 10% or 0% supplied ad libitum to groups of male and
female Sprague-Dawley rats; breeders and offspring were included in the
experiment.
Treatment started at 39 weeks of age (breeders), 7 days before mating,
or from embryo life (offspring) and lasted until their spontaneous death.
Under tested experimental conditions, methyl alcohol and ethyl alcohol
were demonstrated to be carcinogenic for various organs and tissues.
They must also be considered multipotential carcinogenic agents.
In addition to causing other tumors, ethyl alcohol induced malignant
tumors of the oral cavity, tongue, and lips.
These sites have been shown to be target organs in man by epidemiologic
studies. Publication Types: Review Review, Tutorial PMID: 12562628

Surely the authors deliberately emphasized that aspartame is well-known
to be a source of formaldehyde, which is an extremely potent, cumulative
toxin, with complex, multiple effects on all tissues and organs.

This is even more significant, considering that they have already tested
aspartame, but not yet released the results:

p. 29-32 Table 1: The Ramazzinni Foundation Cancer Program
Project of [200] Long-Term Carcinogenicity Bioassays: Agents Studied

No. No. of Bioassays Species No. Route of Exposure
108. "Coca-Cola" 4 Rat 1,999 Ingestion, Transplantal Route

109. "Pepsi-Cola" 1 Rat 400 Ingestion
110. Sucrose 1 Rat 400 Ingestion
111. Caffeine 1 Rat 800 Ingestion
112. Aspartame 1 Rat 1,800 Ingestion

http://members.nyas.org/events/conference/conf_02_0429.html
Soffritti said that Coca-Cola showed no carcinogenicity.
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Rich Murray, MA Room For All rmforall@comcast.net
505-501-2298 1943 Otowi Road Santa Fe, New Mexico 87505

http://groups.yahoo.com/group/aspartameNM/messages
group with 146 members, 1,250 posts in a public, searchable
archive http://RoomForAll.blogspot.com
http://AspartameNM.blogspot.com

Dark wines and liquors, as well as aspartame, provide
similar levels of methanol, above 100 mg daily, for
long-term heavy users, 2 L daily, about 6 cans.

Methanol is inevitably largely turned into formaldehyde,
and thence largely into formic acid.
It is the major cause of the dreaded symptoms of "next
morning" hangover.

Fully 11% of aspartame is methanol -- 1,120 mg aspartame
in 2 L diet soda, almost six 12-oz cans, gives 123 mg
methanol (wood alcohol). If 30% of the methanol is turned
into formaldehyde, the amount of formaldehyde, 37 mg,
is 18.5 times the USA EPA limit for daily formaldehyde in
drinking water, 2.0 mg in 2 L average daily drinking water,

185 times the New Jersey limit,
615 times the California and Maine limits,
1850 times the Maryland limit.

The 1999 July EPA 468-page formaldehyde profile admits that
four states substantially exceed the federal EPA limit:

Environmental Protection Agency 2.00 mg in 2 L daily
drinking water

California and Maine------------ 0.06 mg
Maryland---------------------- 0.02 mg
New Jersey-------------------- 0.20 mg

http://groups.yahoo.com/group/aspartameNM/message/1108
faults in 1999 July EPA 468-page formaldehyde profile:
Elzbieta Skrzydlewska PhD, Assc. Prof., Medical U. of
Bialystok, Poland, abstracts -- ethanol, methanol,
formaldehyde, formic acid, acetaldehyde, lipid peroxidation,
green tea, aging: Murray 2004.08.08 2005.07.11

http://groups.yahoo.com/group/aspartameNM/message/1106
hangover research relevant to toxicity of 11% methanol in
aspartame (formaldehyde, formic acid): Calder I (full text):
Jones AW: Murray 2004.08.05 2005.09.28

Since no adaquate data has ever been published on the exact
disposition of toxic metabolites in specific tissues in
humans of the 11% methanol component of aspartame, the many
studies on morning-after hangover from the methanol impurity
in alcohol drinks are the main available resource to date.

Jones AW (1987) found next-morning hangover from red wine
with 100 to 150 mg methanol (9.5% w/v ethanol, 100 mg/L
methanol, 0.01%, one part in ten thousand).

http://groups.yahoo.com/group/aspartameNM/message/1237
ubiquitous potent uncontrolled co-factors in nutrition research are
formaldehyde from wood and tobacco smoke and many sources,
including from methanol in dark wines and liquors, in pectins
in fruits and vegetables, and in aspartame: Murray 2005.11.14

http://groups.yahoo.com/group/aspartameNM/message/1141
Nurses Health Study can quickly reveal the extent of aspartame
(methanol, formaldehyde, formic acid) toxicity: Murray 2004.11.21
[ Any scientist can get access to this data for free by submitting a proper
research proposal. No one has admitted mining the extensive data on diet
soda use and many symptoms for decades for about 100,000 nurses. ]

http://groups.yahoo.com/group/aspartameNM/message/1052
DMDC: Dimethyl dicarbonate 200mg/L in drinks adds methanol 98 mg/L
[ becomes formaldehyde in body ]: EU Scientific Committee on Foods
2001.07.12: Murray 2004.01.22

http://groups.yahoo.com/group/aspartameNM/message/1226
USA National Institutes of Health National Toxicology
Program aids eminent Ramazzini Foundation, Bologna, Italy,
in more results on cancers in rats from lifetime low levels
of aspartame (methanol, formaldehyde), Felicity Lawrence,
www.guardian.co.uk: (http://www.guardian.co.uk:) Murray 2005.09.30

http://groups.yahoo.com/group/aspartameNM/message/1186
aspartame induces lymphomas and leukaemias in rats, full plain text,
M Soffritti, F Belpoggi, DD Esposti, L Lambertini: Ramazzini
Foundation study 2005.07.14: main results agree with their previous
methanol and formaldehyde studies: Murray 2005.09.03

http://groups.yahoo.com/group/aspartameNM/message/1189
Michael F Jacobson of CSPI now and in 1985 re aspartame
toxicity, letter to FDA Commissioner Lester Crawford;
California OEHHA aspartame critique 2004.03.12; Center for
Consumer Freedom denounces CSPI: Murray 2005.07.27
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