Comet assay tests groups of 4 mice to show sucralose genotoxicity in
stomach, colon, lung, Yu F Sasaki et al, Mutation Research 2002, full
plain text: more re aspartame and stevia: Murray 2007.11.25
http://groups.yahoo.com/group/aspartameNM/message/1494

"We tested groups of four male ddY mice once orally with each additive
at up to 0.5 LD50 or the limit dose (2000 mg/kg)
and performed the comet assay
on the glandular stomach, colon, liver, kidney, urinary bladder,
lung, brain, and bone marrow, 3 and 24 h after treatment."

This data shows that for 2000 mg/kg body weight sucralose oral dose,
24 hours later, there are statistically significant results for
genotoxicity in Stomach, Colon, Lung,
which had the longest "comet tails" from DNA damage.

However, values also over twice for  Kidney and Brain,
and rose over 50 % for Bladder and Bone marrow.
Liver was low or unchanged,
compared to the four control mice liver tissue.

"3.6. Sweeteners

Among the five tested sweeteners, sodium cyclamate
induced a statistically significant DNA damage
increase in the glandular stomach, colon, kidney, and urinary bladder,

saccharin in the colon,

sodium saccharin in the glandular stomach and colon,

and sucralose in the glandular stomach, colon, and lung.

The lowest dose that induced DNA damage was 1000 mg/kg
for saccharin and its sodium salt and

2000 mg/kg for sodium cyclamate and sucralose.

Acesulfame K, aspartame, glycyrrhizin, and stevia
did not increase DNA damage in any of the organs studied."

Sucralose has 3 significant values and 13 high values, for Stomach,
Colon, Kidney, Bladder, Lung, Brain.

However, Aspartame has high values for 2000 mg/kg body weight at 3 hr
for  Stomach, Colon, Liver, Bladder, Lung.

Sucralose fed to 4 mice----------Sampling---Migration of  "comet tail"
one dose (mg/kg body weight)---time (h)----(micro-meters, mean
--------------------------------------------- +- S.E.M. of four mice)

------------- Stomach ------- Colon --------- Liver --------- Kidney

-- 0 -------- 5.91, 0.73 ---- 5.60, 0.88----- 3.07, 0.51 ---- 1.16,
0.44

2000 --- 3 -- 7.69, 0.63 ---- 9.02, 1.09----- 1.34, 0.56 ---- 1.21,
0.74

100 -- 24 -- 6.83, 1.47 ---- 7.32, 1.61----- 1.83, 0.71 ---- 2.71,
0.75

1000 -- 24 -- 7.64, 1.31 ---- 7.95, 1.11----- 1.37, 0.17 ---- 1.32,
0.70

2000 -- 24 - 18.4 , 4.20* -- 22.6 , 0.88*---- 2.89, 0.78 ---- 4.54,
0.40
2000 -- 24 -- 3.1X ---------- 4.0X ---------- 0.94X --------- 3.9X

------- Bladder -------- Lung ---------- Brain ----------- Bone marrow

------- 6.01, 0.81 ----- 2.20, 0.44 ---- 1.06, 0.67 ------ 0.90, 0.57

------- 5.84, 0.83 ----- 3.33, 0.78 ---- 1.83, 0.63 ------ 0.88, 0.51

------- 6.30, 1.02 ----- 3.90, 0.26 ---- 1.37, 0.93 ------ 1.40, 1.23

------- 4.96, 0.62 ----- 3.05, 0.70 ---- 0.98, 0.62 ------ 0.67, 0.67

------ 10.2 , 2.87 ----- 6.56, 1.17* --- 2.76, 1.02 ------ 1.42, 0.67
------- 1.7X ----------- 3.0X ---------- 2.6X ------------ 1.6X
* significant difference: P < 0.05 (Dunnett test)

This data shows that for 2000 mg/kg body weight sucralose oral dose,
24 hours later,  there are statistically significant results for
genotoxicity in Stomach, Colon, and Lung,
which had the longest "comet tails" from DNA damage.

However, values also over twice for Kidney and Brain,
and rose over 50 % for Bladder and Bone marrow.
Liver was low or unchanged,
compared to the four control mice liver tissue.

Here we are comparing each test group of four mice with a specific
tissue average of a single group of four control mice.  With such
small numbers, there are likely to be big fluctuations -- for
instance,
for the Bone marrow in the sucralose control group, the mean value
was 0.90 plus or minus 0.57 mean Standard Error of Measurement
for the four mice.

It is reasonable to surmise that if larger numbers of mice were tested
for somewhat lower exposure levels for longer times, such as daily for
a month or longer, that statistically significant increases would be
found for almost all tissues with sucralose.

So, obviously, thorough follow-up studies are urgently needed, but
these results, to my knowledge, have never been widely discussed in
scientific or general media, nor have any more similar studies on
sucralose appeared.

Each test condition had just 4 mice, and, according to the text,
each additive had its own control group of 4 mice.
However, there are only 21 unique sets of control groups,
with 8 sets used once, 10 sets used twice, 2 sets used 3 times, and 1
set used 4 times, a total of 38 food additives listed
[Sodium erythorbic acid was left out of Table 2, while
mentioned in the report 3 times, "...erythorbic acid and its sodium
salt
did not increase DNA damage in any of the organs studied."].

Aspartame was assigned the control group that had the highest levels
of
Migration of damaged nuclear DNA for Liver and  Bladder,
and the second highest for Brain.
The same control group was used for the xanthene
dye, erythrosinc, which had Migration as high as 42.4+-2.17 um
[micro-meter], measured on 50 nuclei from stomach cells,
3 hours after ingestion.
So, the high control groups values had no effect on
the statistical analysis for erythrosinc.

The available range of the 21 control groups ranged for the Liver from
1.1 to 3.6 um.  So, the sucralose control group value of  3.07 +-
0.51 um is 3 times more than the lowest control group value of 1.1 um.

Somach ---- Colon ---- Liver ------ Kidney ---- Bladder --- Lung

mean of 21 control groups

6.31 ------ 5.81 -----  2.15 ------ 2.25 ------ 5.40m ----- 2.61

range of values for 21 control groups

4.3-8.6 --- 4.0-8.1 --- 1.1-3.6 --- 1.2-2.9 --- 3.6-7.1 --- 1.6-4.7

Brain --------  Bone [marrow]

1.48 ---------  1.12       mean of 21 control groups

0.8-2.6 ------  0.6-1.9    range of values for 21 control groups

On quick inspection, using these means of the 21 control groups would
not have changed the statistical significance of the sucralose tissue
means.

Wouldn't the average of all the 21 control groups be the best control
values to use?
What would then be the appropriate statistical test?
How many mice would it take to reach significance with aspartame
for the 5 tissues with ratios over 1.4: Stomach, Colon, Liver,
Bladder, Lung?

Aspartame at 24 hours had levels too low to reach significance with
any
of the 21 control groups.

However, people who are heavy users of aspartame for years are bound
to accumulate toxic metabolites of the three components of aspartame:
methanol 11%, phenylalanine 50%, aspartic acid 29%, all genotoxic
[Trocho (1998), Karakis (1998)].

Comparing the mean control values to the values for the other 7
sweeteners:
Best is acesulfame K, with no significant or high values.

Good is glycyrrhizin (derived from licorice), two 1.4 ratios for
Stomach
and Brain.

Next is stevia, with one high value [above ratio 1.4],
9.48+-1.99 for Bladder, 2000 mg 3 hr,  ratio 1.8 .

Aspartame has high values for 2000 mg 3 hr for Stomach, Colon, Liver,
Bladder, Lung.

Sucralose has 3 significant values and 13 high values, for Stomach,
Colon, Kidney, Bladder, Lung, Brain.

Sodium cyclamate has 4 significant values and 10 high values for
Stomach, Colon, Liver, Kidney, Bladder, Lung, Brain, Bone.

Saccharin has 3 highly significant values for Colon, and 13 high
values
for Stomach, Colon, Kidney, Lung, Brain, Bone.

Sodium saccharin has 5 highly significant values for Stomach and
Colon,
and 14 high values for Stomach, Liver, Kidney, Bladder, Lung, Brain,
Bone.

We should keep in mind that toxicity in humans involves
many vulnerable groups,
years of daily use,
often evolution of allergies and hypersensitivity,
and complex interactions with a multitude of  diseases,
additives, other toxins, and foods.

[ Sucralose had Positive organs -- glandular stomach, colon,
with a Lowest Positive Dose of 2,000 mg/kg body weight,
and had  no references for genotoxicity data: Hayashi et al. [6];
CA, in vitro cytogenetics test,
MN, in vivo micronucleus test,
and Acceptible Daily Intake (ADI) of 15 mg/kg body weight.

[6] M. Hayashi, M. Matsui, K. Ishii, M. Kawasaki,
Genotoxicity evaluation datasheet of food additives by the MHW
(1980-1998), Environ. Mutagen Res. 22 (2000) 27-44. ]
////////////////////////////////////////////////////////////

"The no-observable adverse-effect level
in the lifetime toxicity/carcinogenicity studies of Allura Red is
2829 and 901 mg/kg per day for male and female rats, respectively,
[25],
and 7300 and 8300 mg/kg per day for male and female mice,
respectively, [26]."

This shows that in rats females
are more vulnerable to a genotoxin than males,
the same as in the Ramazzini studies on cancers from aspartame,
alcohol and methanol, acetaldehyde and formaldehyde:

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
////////////////////////////////////////////////////////////

Mutation Research 519 (2002) 103-119
The comet assay with 8 mouse organs: results with
39 currently used food additives.
Yu F. Sasaki a,*,
Satomi Kawaguchi a,
Asako Kamaya a,
Miyuki Ohshita a,
Kazumi Kabasawa a,
Kayoko Iwama a,
Kazuyuki Taniguchi b,
Shuji Tsuda c    s.tsuda@iwate-u.ac.jp

* Corresponding author. Tel.: +81-178-27-7296;
fax: +81-178-27-7296.
E-mail address: yfsasaki-c@hachinohe-ct.ac.jp (Y.F. Sasaki).

a Laboratory of Genotoxicity, Faculty of Chemical and Biological
Engineering, Hachinohe National College of Technology,
Tamonoki Uwanotai 16-1, Hachinohe, Aomori 039-1192, Japan

b Laboratory of Veterinary Anatomy, Department of Veterinary Medicine,
Faculty of Agriculture, Iwate University,
Ueda 3-18-8, Morioka, Iwate 020-8550, Japan

c Laboratory of Veterinary Public Health,
Department of Veterinary Medicine, Faculty of Agriculture,
Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan

Received 7 November 2001; accepted in revised form 17 May 2002

Abstract

We determined the genotoxicity of 39 chemicals
currently in use as food additives.

They fell into six categories -- dyes,
color fixatives and preservatives, preservatives, antioxidants,
fungicides, and sweeteners.

We tested groups of four male ddY mice once orally with each additive
at up to 0.5 LD50 or the limit dose (2000 mg/kg)
and performed the comet assay
on the glandular stomach, colon, liver, kidney, urinary bladder,
lung, brain, and bone marrow,  3 and 24 h after treatment.

Of all the additives, dyes were the most genotoxic.
Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine,
and Rose Bengal induced dose-related DNA damage
in the glandular stomach, colon, and/or urinary bladder.

All seven dyes induced DNA damage in the gastrointestinal organs
at a low dose (10 or 100 mg/kg).

Among them, Amaranth, Allura Red, New Coccine, and Tartrazine
induced DNA damage in the colon
at close to the acceptable daily intakes (ADIs).

Two antioxidants (butylated hydroxyanisole
(BHA) and butylated hydroxytoluene (BHT)),
three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole),
and
four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and
sucralose) also induced DNA damage in gastrointestinal organs.

Based on these results, we believe that more extensive assessment of
food additives in current use is warranted.
PMID: 12160896  (c) 2002 Elsevier Science B.V. All rights reserved.

Keywords: Food additives; Mouse; Colon; Glandular stomach;
Gastrointestinal tract; Comet assay; Genotoxicity

1. Introduction

Food additives are used widely for various purposes, including
preservation, coloring, and sweetening.
Some food additives, however, have been prohibited
from use because of their toxicity.

AF-2, for example, was used as a food preservative in Japan
from before 1965 until it was banned because of carcinogenicity
in experimental animals [1].

Butter Yellow (p-dimethylaminoazobenzene), an azo compound,
was listed for food use in the USA and de-listed in the same year;
legislation prohibiting its use in Europe followed soon after,
because it was implicated as a carcinogen in several animal species
[2].

Many azo compounds, including Butter Yellow,
are genotoxic in short-term tests and

1383-5718/02/$ - see front matter (c) 2002 Elsevier Science B.V.
All rights reserved.
PII: S1383-5718(02)00128-6
104 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

carcinogenic in laboratory animals [3],
yet some of them are still being used.
Saccharin and its salt are widely used sweeteners
primarily because of their value to diabetes patients,
and although sodium saccharin is carcinogenic in experimental animals,
there is no clear evidence that it is in humans [4].

Although epidemiological studies of food additives
are important in the assessment of toxicological risk to humans,
they are difficult because exposure cannot be accurately assessed.

Thus, risk assessment largely depends on laboratory toxicity studies.

Currently, 337 synthesized and 488 naturally occurring food additives
are permitted for use in Japan.

Based on laboratory toxicity studies, some of them present a
risk to our health.

The usual way we evaluate chemical genotoxicity
is by a battery of in vitro and in vivo genotoxicity tests [5];
the in vivo tests reflect the compound's
absorption, distribution, excretion, and metabolism.

According to the data on food additive genotoxicity recently compiled
by the Ministry of Health and Welfare (MHW) of Japan,
some food additives currently used in Japan
are positive in at least one in vitro genotoxicity study,
and many have never been tested in vivo [6]
in spite of the importance of in vivo genotoxicity data.

The alkaline (pH >13) comet assay introduced by Singh et al. [7]
is a rapid and sensitive procedure for quantitating DNA lesions
in mammalian cells.

It detects not only single strand breaks
but also alkali-labile sites, DNA cross-linking,
and incomplete excision repair sites [7,8].

We have modified the assay by using nuclei obtained by homogenization
instead of cells obtained by enzyme treatment [9],
and we have shown that the modified assay
detects in vivo genotoxicity of various classes of chemicals [10-12].

The purpose of this study was to examine whether representative,
widely used food additives induce DNA damage in mice.

2. Materials and methods

2.1. Chemicals and animals

Table 1 lists tested food additives, their CAS numbers, and relevant
data.
Their chemical structures are shown in Fig. 1.

Regular (GP-42) and low melting point (LGT) agarose
were obtained from Nacalai Tesque (Kyoto)
and diluted, respectively, to 1 and 2 % in physiological saline.

Male ddY mice were obtained from Japan SLC Co., Shizuoka, Japan,
at 7 weeks of age and used after 1 week of acclimatization.

They were fed commercial pellets MF (Oriental Yeast Industries
Co., Tokyo, Japan) and tap water ad libitum
throughout the acclimatization period and the experiment.

The animal room was at 20-24 deg C with a 12 h light-dark cycle.

2.2. The comet assay

All food additives were administered orally at up
to 0.5 X LD50 or the limit dose of 2000 mg/kg.

In order to set the appropriate dose of each chemical for the comet
assay, we determined the approximate LD50 (Table 1)
by simple acute toxicity experiments on four-five animals.

In the comet assay, groups of four mice were treated once orally.

After 3 and 24 h,
slides were prepared at each set time as described below.

In our previous studies [11,14], we observed
no significant difference in mean migration between
DNA from vehicle control groups and the corresponding
untreated groups at any sampling time for any organ.

Therefore, we used untreated animals as controls
rather than concurrent vehicle controls.

This experiment followed the design of our previous
comet assay studies with multiple mouse organs [9-15].

The protocol followed the recently published
recommendations for comet assay genotoxicity studies [15].

From shortly after they were treated until just before they were
killed,
the animals were carefully observed for pharmacotoxic signs.

They were sacrificed 3 or 24 h after treatment, and eight organs --
glandular stomach,
colon,
liver,
kidney,
urinary bladder,
lung,
brain,
and bone marrow --
were removed.

The liver, kidney, lung, and brain were minced,
suspended in 4 ml chilled homogenizing solution (pH 7.5) containing
0.075 M NaCl and 0.024 M Na2EDTA,
and then homogenized gently using a Potter-Elvehjem type homogenizer
at 500-800 rpm, in ice [9].

The glandular stomach, colon, and urinary bladder were opened
and rinsed with physiological saline;
the mucosa was scraped into 4 ml chilled homogenizing buffer and
homogenized gently using a Potter-Elvehjem type homogenizer
at 500-800 rpm, in ice.

To obtain nuclei,
the homogenate was centrifuged at 700g for 10 min
at 0 deg C, and the precipitate was re-suspended in chilled
homogenizing
buffer at 1 g organ weight/ml [9].

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 105

Table 1

Tested food additives

Food additive CAS no. Color index Purity (%) Source a Vehicle b
LD50 c (mg/kg)

Food dyes

Azo dyes

Amaranth 915-67-3 CI-16185 T S >2000
Allura Red 25956-17-6 CI-16035 T S >2000
New Coccine 2611-82-7 CI-16255 T S >2000
Tartrazine 1934-21-0 CI-19140 T S >2000
Sunset Yellow FCF 2783-94-0 CI-15985 T S >2000

Xanthene dyes

Erythrosine 16423-68-0 CI-45430 T S >2000
Phloxine 18472-87-2 CI-45410 T S >2000
Rose Bengal 632-69-9 CI-45440 T S >2000
Acid Red 3520-42-1 CI-45100 T S >2000

Triphenylmethane and other dyes

Fast Green FCF 2353-45-9 CI-42053 T S >2000
Brilliant Blue FCF 3844-45-9 CI-42090 T S >2000
Indigocarmine 860-22-0 CI-73015 T S >2000

Color fixative and preservative

Sodium nitrite 7632-00-0 K S 200

Preservatives

Benzoic acid 65-85-0 99.5 K O 2000
Sodium benzoate 532-32-1 >98.0 K S 2000
p-Hydroxybenzoic acid n-butyl ester 94-26-8 >98.0 K O >2000
p-Hydroxybenzoic acid iso-butyl ester 4247-02-3 T O >2000
p-Hydroxybenzoic acid ethyl ester 120-47-8 >99.0 W O >2000
p-Hydroxybenzoic acid n-propyl ester 94-13-3 >95.0 W O >2000
p-Hydroxybenzoic acid iso-propyl ester 4191-73-5 >99.0 T O >2000
Sodium dehydroacetic acid 4418-26-2 K S 2000
Sorbic acid 110-44-1 K O >2000
Potassium sorbate 24634-61-5 K S >2000

Antioxidants

BHA 25013-16-5 >98.0 T O 2000
BHT 128-37-0 >98.0 T O 2000
Erythorbic acid 89-65-6 K O >2000
Sodium erythorbic acid 7378-23-6 K S >2000
Gallic acid n-propyl ester 121-79-7 K O >2000

Fungicides

Biphenyl 92-52-4 >99.5 T O >2000
Sodium o-phenylphenol 132-27-4 >98 T O >2000
Thiabendazole 148-79-8 >98.0 T O 400

Sweeteners

Acesulfame K 55589-62-3 >98.0 W S >2000

Aspartame 22839-47-0 T S >2000

Sodium cyclamate 100-8-9 Z S >2000

Glycyrrhizin 1405-86-3 T S >2000

Saccharin 81-07-2 98+ K O >2000

Sodium saccharin 128-44-9 99+ K S >2000

Stevia M S >2000

Sucralose S S >2000

a  K, Kanto Chemical Co. Inc., Tokyo, Japan;
M, Maruzen Pharmaceuticals Inc., Kyoto, Japan;
S, San-Ei Gen F.F.I. Inc., Osaka, Japan;
T, Tokyo Kasei Kogyo Industry Ltd., Tokyo, Japan;
W, Wako Pure Chemical Industry Ltd., Osaka, Japan;
Z, Zhong Hua Fang Da Ltd., Hong Kong, PR China.

b  O, olive oil; S, saline.

c  In order to set appropriate doses for the assay,
we determined approximate LD50 by simple acute toxicity experiments
on four-five animals.
When no death was observed at 2000 mg/kg,
the LD50 was defined as  >2000 mg/kg.

106 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

Fig. 1. Structures of studied food additives.

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 107

Fig. 1. (Continued ).

Seventy-five microliters agarose GP-42 was quickly layered on a slide
(Matsunami Glass Ind. Ltd., Osaka, Japan)
coated with agarose GP-42 and covered with another slide.

The slide sandwiches were placed horizontally
to allow the agarose to solidify.

The nucleus suspension was mixed 1:1 (v/v) with 2 %, 45 deg C,
agarose LGT, and 75 micro-l of the nucleus mixture was quickly layered
in the same manner after removal

108 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

Fig. 1. (Continued ).

of the covering slide.

Finally, 75 micro-l of agarose GP-42 was quickly layered on again.

Slides prepared from nuclei isolated by homogenization were placed in
a chilled lysing solution (2.5 M NaCl, 100 mM
Na2EDTA, 10 mM Trizma, 1 % sarkosyl, 10 %  DMSO,
and 1 % Triton X-100, pH 10) [9] and kept at 0 deg C
in the dark for about 1 night, then in chilled alkaline solution
(300 mM NaOH and 1 mM Na2EDTA, pH 13)
for 10 min in the dark at 0 deg C [9].

Electrophoresis was conducted at 0 deg C in the dark for 15 min at
25 V (0.96 V/cm) and approximately 250 mA.

The slides were neutralized and then stained with 50 micro-l
of 20 micro-g/ml ethidium bromide
(Wako Pure Chemical Industries Ltd.) [9].

We examined and photographed 50 nuclei per slide
at 200X magnification with the aid of a fluorescence microscope.

The length of the whole comet ("length") and the diameter of the head
("diameter") were measured for 50 nuclei per organ per animal.

We calculated migration as the difference
between length and diameter for each of 50 nuclei.

Mean migration of 50 nuclei from each organ was
calculated for each individual animal.

The differences

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 109

between the averages of four treated animals and the
untreated control animals were compared
with the Dunnett test after one-way ANOVA.

A P value less than 0.05 was considered statistically significant.

2.3. Histopathological examination

Necropsies were performed, and the organs were examined
for changes in size, color, and texture.

A small portion of each organ was fixed in 10 % formaldehyde,
dehydrated, and embedded in paraffin.

When positive results were obtained in the comet assay,
tissue sections stained by the hematoxylin-eosin and TUNEL
methods were observed histopathologically.

In the TUNEL method, tissue sections were incubated with
20 mg/ml proteinase K (Sigma) for 20 min at 37 deg C after
deparaffinization and hydration.

Sections were immersed in TdT buffer after inactivation of endogenous
peroxidase and were reacted with TdT (Gibco-BRL, MD)
and biotinylated dUTP (Boehringer Mannheim, Mannheim, Germany)
in TdT buffer at 37 deg C for 90 min.

The reaction was terminated with a TB buffer
(300 mM NaCl, 30 mM Na citrate) for 30 min, and
the sections were covered with 10% rabbit serum for 10 min
and avidin-biotin-peroxidase complex for 30 min at room temperature.

Finally, the sections were reacted
in 3,3-diaminobenzidine tetrahydrochloride solution.
After each step, sections were washed with
double distilled water or phosphate buffered saline.

3. Results

An increase in DNA damage was indicated by an
increase in DNA migration (Table 2).

No death, morbidity, or clinical signs were observed after any
treatment.

Necropsy and histopathological examination of
tissue sections stained by the hematoxylin-eosin and
TUNEL methods revealed no treatment effect on any organ examined.

Thus, any DNA damage observed
was not likely to be due to general cytotoxicity (necrosis)
and apoptosis.

3.1. Food dyes

Food dyes included four groups --
azo, xanthene, triphenylmethane, and other.

Among the five azo dyes,
Amaranth, Allura Red, New Coccine, and Tartrazine,
starting at 10 mg/kg, induced dose-related
DNA damage in the colon that was most prominent
3 h after administration.

Other organs that showed DNA damage at 3 h were:
(1) glandular stomach at >10 mg/kg Tartrazine,

1000 mg/kg Amaranth;

(2) urinary bladder at >100 mg/kg New Coccine.
At 24 h, Amaranth,
Allura Red, and
Tartrazine had induced DNA damage in a gastrointestinal organ,
and New Coccine in the liver, kidney, urinary bladder, and lung.

Sunset Yellow FCF did not yield a statistically significant
increase in DNA damage in any of the organs studied.

Among the four xanthene dyes,
Erythrosine, Phloxine, and Rose Bengal had induced dose-related DNA
damage in the glandular stomach, colon, and urinary bladder at 3 h;
no DNA damage was evident at 24 h.

The lowest dose that induced statistically significant
DNA damage in the glandular stomach and colon was
10 mg/kg for Rose Bengal and
100 mg/kg for Erythrosine and Phloxine.

In the urinary bladder,
Phloxine and Rose Bengal induced DNA damage at >10 mg/kg
and Erythrosine at >100 mg/kg.

Acid Red did not yield a statistically significant increase in DNA
damage in any of the organs studied.

Two triphenylmethane dyes --
Fast Green FCF and Brilliant Blue FCF,
and Indigocarmine
did not yield a statistically significant
increase in DNA damage in any of the organs studied.

3.2. Color fixative and preservative

Sodium nitrite did not yield a statistically significant
increase in DNA damage in any of the organs studied.

3.3. Preservatives

Benzoic acid and its sodium salt,
five p-hydroxybenzoic acid esters,
sodium dehydroacetic acid, and
sorbic acid and its potassium salts
did not yield a statistically significant increase in DNA damage
in any of the organs studied.

3.4. Antioxidants

Among the five antioxidants,
butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT)
had induced DNA damage in the glandular stomach
and colon 3 h after their administration,
and BHT was also positive in the urinary bladder and brain.

110 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 111

112 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 113

114 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

The lowest doses that induced a statistically significant increase in
DNA damage within 3 h in the colon
were 500 mg/kg for BHA and 100 mg/kg for BHT.

Gallic acid n-propyl ester and
erythorbic acid and its sodium salt
did not increase DNA damage in any of the organs studied.

3.5. Fungicides

Biphenyl,
sodium o-phenylphenol, and
thiabendazole
all induced dose-related DNA damage in the colon.

Sodium o-phenylphenol was positive at 3 h
and biphenyl at 24 h.

The lowest dose that induced
DNA damage in the colon was 100 mg/kg for all three fungicides.

Some other organs also showed DNA damage:
sodium o-phenylphenol induced DNA
damage in the glandular stomach, urinary bladder,
and lung at >1000 mg/kg and
in the liver and kidney at 2000 mg/kg.

Biphenyl and thiabendazole
induced DNA damage in all the organs studied at >1000 and
200 mg/kg, respectively.

3.6. Sweeteners

Among the five tested sweeteners, sodium cyclamate
induced a statistically significant DNA damage increase
in the glandular stomach, colon, kidney, and urinary bladder,

saccharin in the colon,

sodium saccharin in the glandular stomach and colon,

and sucralose in the glandular stomach, colon, and lung.

The lowest dose that induced DNA damage was 1000 mg/kg
for saccharin and its sodium salt and
2000 mg/kg for sodium cyclamate and sucralose.

Acesulfame K, aspartame, glycyrrhizin, and stevia
did not increase DNA damage in any of the organs studied.

4. Discussion

In vitro and in vivo genotoxicity tests detect compounds
that induce genetic damage directly or indirectly by various
mechanisms.

Since, no single test is capable of detecting all genotoxic agents,
the usual approach is to carry out a battery of tests [5].

A standard test battery includes the following:
(1) a bacterial reverse mutation test;
(2) a mammalian cell genotoxicity test to detect damage not detected
in bacteria; and
(3) an in vivo test so that additional factors
(absorption, distribution metabolism, excretion)
that influence the genotoxicity of a compound are included [16].

According to the recent review by
the MHW of Japan of food additive genotoxicity [6],
however, only 8 out of the 39 food additives studied
here have been assessed for in vivo genotoxicity (Table 3).

So further evaluation is needed.

If a compound shows negative results in vitro,
it is usually sufficient to carry out a single in vivo cytogenetics
assay [5].

The few in vivo assays that have been
validated involve either an analysis of chromosome aberrations
in bone marrow cells
or of micronuclei in bone marrow or peripheral blood erythrocytes [5].

Although this micronucleus test is simple,
its performance is related to whether a chemical reaches
the hematopoietic system.
Its sensitivity for carcinogens
targeting the liver is only approximately 40 %,
and some rodent carcinogens that are dialkyl-type
N-nitrosoamines show poor micronucleus induction [17].

Thus, if a compound shows positive results in vitro,
it is recommended that an in vivo test using
non-hematopoietic tissue be conducted [16].

Although most of the 39 food additives are negative in the Ames test,
14 are clastogenic to cultured cells (Table 3).

More noteworthy is that reliable in vivo data are not
available for nine food additives
(Amaranth, Sunset Yellow FCF, Brilliant Blue FCF, sodium benzoate,
p-hydroxybenzoic acid ethyl ester, potassium sorbate,
gallic acid n-propyl ester, sodium saccharin, and stevia)
that induce a positive result in an in vitro test [6] (Table 3).

In this study, 16 food additives were positive in vivo.

Interestingly, those included
only 4 of the 14 food additives that are clastogenic to cultured cells
but that 7 of 15 that are not.

That discordance, which may be due to the fact that
in vivo genotoxicity tests
reflect the absorption, distribution, metabolism, and excretion
of the test agent,
supports the significance of such tests.

One important objective of genotoxicity tests
is to predict chemical carcinogenicity.

Some carcinogenicity data for some of the food additives studied here
have already been published (Table 3).

Among them,
although BHA, BHT, sodium o-phenylphenol, and sodium saccharin
are carcinogenic to rodents,
they are permitted for use because of their various positive
effects on health.

BHA, which is used as an antioxidant
in fat-containing foods and in edible fats and oils
so that they do not to become rancid and develop

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 115

Table 3

Comet assay of food additives vs. results of other studies

Food additives---Positive organs---LPD a
------------------------------------(mg/kg)
-----------------Genotoxicity b ------------------Carcinogenicity
---------------------------------------------------in rodents c
-------------------------------------------------------------ADI d (mg/
kg
-------------------------------------------------------------------per
day)
------------------Ames---CA---MN---------Mouse--Rat
Food dyes

Azo dyes

Amaranth C 10 - + I [28] I [28] 0.50
S, Ub 1000
Allura Red C 10 - -[26] - [25] 7.00
S 100
Lu 1000
New Coccine C 10- -[29] 4.00
S, Ub 100
L, K, Lu 2000
Tartrazine S, C 10 - + - -[30] - [31,32] 7.50
Sunset Yellow FCF - - + 2.50

Xanthene dyes

Erythrosine S, C, Ub 100 - + - -[33] - [34] 0.10
Phloxine Ub 10 - - S, C 100
Rose Bengal S, C, Ub 10 - - L 100
K, Lu, Br 2000
Acid Red - - +-

Triphenylmethane and other dyes

Fast Green FCF - - ---[35] - [35] 25.0
Brilliant Blue FCF - - + 12.5
Indigocarmine - - - 5.00

Color fixative and preservative

Sodium nitrite - +,- + - -[36] 0.2

Preservatives

Benzoic acid - - - -[37] 5.0
Sodium benzoate - - + -[37] 5.0e
p-Hydroxybenzoic acid n-butyl ester- - - -[38] 10e
p-Hydroxybenzoic acid iso-butyl ester- - - -[38] 10e
p-Hydroxybenzoic acid ethyl ester - - + 10e
p-Hydroxybenzoic acid n-propyl ester- - 10e
p-Hydroxybenzoic acid iso-propyl ester- - - 10e
Sodium dehydroacetic acid - Sorbic acid - - -[39] 25
Potassium sorbate - - + -[39] 25e

Antioxydants

BHA C 500 - - - S [18] 0.5
S 1000
BHT C 100 - - Lu [40] Ptg [40]
S, Ub 500
Br 1000
Erythorbic acid - +,- - -

116 Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119

Table 3 (Continued )

Food additives Positive organs LPD a (mg/kg)
Genotoxicity b
Carcinogenicity in rodents c
ADI d (mg/kg per day)

Ames CA MN Mouse Rat

Sodium erythorbic acid- -- -[41]
Gallic acid n-propyl ester- -,+ + 1.4

Fungicides

Biphenyl C 100 - - S, L, K, Ub, Lu, Br, BM 1000
Sodium o-phenylphenol C 100 - + -[42] Ub [42] 0.2
S, Ub, Lu 1000
L, K 2000
Thiabendazole C 100 - - -[43] - [43]
S, L, K, Ub, Lu, Br, BM 200

Sweeteners

Acesulfame K - - [44] 15

Aspartame - - -[44] 40

Sodium cyclamate S, C, K, Ub 2000 - [45] I [45]

Glycyrrhizin - -+--[46] 0.2 [46]

Saccharin C 1000 - - -[4] - [4] 5

Sodium saccharin S, C 1000 - + -[4] Ub [4] 5e

Stevia - +,- -[47]

Sucralose S, C 2000 15

[ Sucralose had Positive organs -- glandular stomach, colon, lung,
with a Lowest Positive Dose of 2,000 mg/kg body weight,
and had no references for genotoxicity data: Hayashi et al. [6];
CA, in vitro cytogenetics test,
MN, in vivo micronucleus test,
and Acceptible Daily Intake (ADI) of 15 mg/kg body weight.

[6] M. Hayashi, M. Matsui, K. Ishii, M. Kawasaki, Genotoxicity
evaluation datasheet of food additives by the MHW
(1980-1998), Environ. Mutagen Res. 22 (2000) 27-44. ]

Abbreviations and marks:

+, positive;
-, negative;
I, inadequate data for evaluation;
BM, bone marrow;
Br, brain;
C, colon;
K, kidney;
L, liver;
Lu, lung;
Ptg, pituitary gland;
S, glandular stomach;
Ub, urinary bladder.
a Lowest positive dose.
b References for genotoxicity data: Hayashi et al. [6];
CA, in vitro cytogenetics test;
MN, in vivo micronucleus test.
c Affected organs are shown for some tests.
d Except for glycyrrhizin, references for acceptable daily intake
(ADI) data is MHW [26].
e ADI was calculated for the weight of the free acid or base.

objectionable odors, is negative in the Ames,
in vitro cytogenetics, and micronucleus tests [18].
Although sodium o-phenylphenol is negative in the Ames test,
its free acid, o-phenylphenol,
induces chromosome aberrations in cultured CHO cells [19].
Therefore, further in vivo genotoxicity and carcinogenicity tests
would be significant.

In this study, the four food additives carcinogenic to rodents were
genotoxic in mouse gastrointestinal organs,
suggesting that the comet assay would be
another useful further in vivo test.

Of all the food additives,
dyes were the most potent genotoxins in the gastrointestinal organs
(it may be surprising that their lowest effective dose was 10 mg/kg),
but carcinogenicity has not been confirmed for any food dyes (Table
3).

Considering the large number of persons exposed to food additives,
it is important to explore the significance of the genotoxic effects
revealed by the in vivo comet assay and
to re-evaluate those comet assay-positive chemicals
for use as food additives.

Whether the DNA damage is repaired or persists is important to the
fate of organs targeted by chemical carcinogens [20].

The development of tumors in target organs
depends not only on the initial levels
of induced DNA damage and its repair,
but also on other contributing factors
including the production of reactive metabolite(s),
their distribution, and their effect on cell proliferation.

In our comet assay studies, mice were killed 24 h after a single,
relatively high dose of the test chemical.

Under those conditions,
the proportion of mouse carcinogens that was positive
in the colon, glandular stomach, and urinary bladder
in the comet assay was high,
in spite of the fact that

Y.F. Sasaki et al. / Mutation Research 519 (2002) 103-119 117

tumor incidence was rare at those sites [11].

One possible explanation for this discrepancy
is that the assay detects DNA damage induced shortly after
administration
of a relatively high dose,
while carcinogenicity is detected after long treatment
with relatively low doses [11].

Metabolic processes may become saturated at high tissue
concentrations,
and the rates and pathways of metabolic activation and detoxification
may be different at high single doses and low long-term doses.

In our most recent study, we found that many chemicals,
including Amaranth, that were positive in mouse gastrointestinal
organs
within 24 h of a single dosing at the MTD
and daily levels used in the chronic carcinogenicity test
were negative at these sites after
triple dosing at the daily levels used in the carcinogenicity test
[21].
This finding suggests that the treatment schedule
is responsible for the discrepancy
between genotoxicity and carcinogenicity data at those sites.

An alternative explanation is that the power to
detect effects in colon might be lower for rodent carcinogenicity
studies than for the comet assay [11].

Human carcinomas of the gastrointestinal tract have
high incidence and mortality rates,
and colorectal cancer incidence is as much as 10 times higher in
industrialized countries than in developing countries,
suggesting environmental causes [22].

Therefore, it would be important to re-evaluate whether the food
additives that induce DNA damage in mouse colon
at low levels are carcinogenic at this site.

Three food dyes
(Allura Red, Tartrazine, and Erythrosine)
that induced DNA damage in the colon in this study
(ddY mice) did not do so in CD1 mice [26,30,33].

MeIQ, however, which is also potent genotoxic but not carcinogenic
in CDF1 mouse colon [23],
is carcinogenic in C57BL mouse colon (incidences, 42 % (8/19)) [24].

These results suggest strain differences in colon sensitivity to
carcinogens.

Therefore, to ensure that food additives that are positive in the
colon in the comet assay do not induce colon tumors,
their carcinogeniocities should be studied in different strains of
animals with high sensitivity to colon carcinogenesis.

In general, in vivo genotoxicity is evaluated at
high doses, such as the MTD.

In the present study,
the lowest dose that induced DNA damage in at least
one organ was 10 mg/kg for six dyes
(Amaranth, Allura Red, and New Coccine, Tartrazine, Phloxine,
and Rose Bengal),
which was even lower
than the doses generally used in carcinogenicity studies.

The no-observable adverse-effect level
in the lifetime toxicity/carcinogenicity studies of Allura Red is
2829 and 901 mg/kg per day for male and female rats, respectively,
[25],
and 7300 and 8300 mg/kg per day for male and female mice,
respectively, [26].

The acceptable daily intake (ADI) recommended by the
Joint FAO/WHO Expert Committee on Food Additives for
Amaranth, Allura Red, New Coccine, and Tartrazine
is 0.5, 7.00, 4.00, and 7.50 mg/kg, respectively, [27].

Most noteworthy is that the four food dyes we tested
induced DNA damage, mainly in colon, at close to those ADIs.

In this study, we found that some currently used
food additives induce DNA damage in mouse organs.

In vivo genotoxicity does not always reflect carcinogenicity.

The results shown in this study, however,
warrant a more extensive safety assessment of food additives.

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**********************************************************

http://groups.yahoo.com/group/aspartameNM/message/935
Comet assay finds DNA damage from sucralose, cyclamate, saccharin in
mice: Sasaki YF & Tsuda S  Aug 2002: Murray 2003.01.01 rmforall
[ Also borderline evidence, in this pilot study of 39 food additives,
using test groups of 4 mice, for DNA damage from for stomach, colon,
liver, bladder, and lung 3 hr after oral dose of 2000 mg/kg aspartame
--
a very high dose.]

The Single Cell Gel Assay is able to detect single-strand and
double-strand DNA breaks in individual eukaryotic cells;
requires small numbers of cells (<20,000 per sample);
can detect DNA damage from low levels of toxic or physical insults;
and is rapid, simple and efficient.

In this assay, cells are treated with the agent of interest,
embedded in agarose on a histological slide,
the cell membranes are lysed, and the
slides are placed in an electric field.

If the DNA has single or double-strand breaks,
it will flow out of the cells and move toward the anode,
causing the cell and its DNA to resemble a comet.

The more DNA released from the cell, the greater the DNA damage.
A computerized imaging system is used to score and measure the comets.

The Comet assay is not FDA approved as a human medical test, so it is
not covered by insurance.

It is used in many human research studies.

http://cometassay.com/  Comet Assay Interest Group

http://www.geocities.com/cometassay/pioneers.htm

http://www.geocities.com/cometassay/related_links.htm

http://www.ems-us.org/   Environmental Mutagen Society

http://www.geocities.com/cometassay/related_links.htm
DNA repair Interest Group  about a thousand members

http://www.ils-inc.com/dna_damage_assessment.html
Integrated Laboratory Systems    Comet assays for $155-300

http://www.mdbiotechinc.com/  MD Biotech, Inc.
Comet assays on four 10 ml blood samples for $800

http://groups.yahoo.com/group/aspartameNM/message/961
genotoxins, Comet assay in mice: Ace-K, stevia fine; aspartame poor;
sucralose, cyclamate, saccharin bad: Y.F. Sasaki Aug 2002:
Murray 2003.01.27 [A detailed look at the data]
////////////////////////////////////////////////////////////

http://groups.yahoo.com/group/aspartameNM/message/1123
genotoxins, Comet assay in mice: Ace-K, stevia fine; aspartame poor;
sucralose, cyclamate, saccharin bad: Sasaki YF, Aug, Dec 2002:
Rencuzogullari E et al, Aug 2004: Murray 2003.01.27, 2004.10.17

J Toxicol Sci. 2002 Dec; 27 Suppl 1: 1-8.
[Genotoxicity studies of stevia extract and steviol by the comet
assay]
[Article in Japanese]
Sekihashi K, Saitoh H, Sasaki Y.   yfsasaki-c@hachinohe-ct.ac.jp
Safety Research Institute for Chemical Compounds Co., Ltd.,
363-24 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan.

The genotoxicity of steviol, a metabolite of stevia extract,
was evaluated for its genotoxic potential using the comet assay.

In an in vitro study, steviol at 62.5, 125, 250, and 500 micrograms/ml
did not damage the nuclear DNA of TK6 and WTK1 cells in the presence
and absence of S9 mix.

In vivo studies of steviol were conducted by two independent
organizations.

Mice were sacrificed 3 and 24 hr after one oral administration of
steviol at 250, 500, 1000, and 2000 mg/kg.

DNA damage in multiple mouse organs was measured by the comet assay
as modified by us.

After oral treatment, stomach, colon, liver, kidney and testis DNA
were not damaged.

The in vivo genotoxicity of stevia extract was also evaluated for its
genotoxic potential using the comet assay.

Mice were sacrificed 3 and 24 hr after oral administration of stevia
extract at 250, 500, 1000, and 2000 mg/kg.

Stomach, colon and liver DNA were not damaged.

As all studies showed negative responses, stevia extract and steviol
are
concluded to not have DNA-damaging activity in cultured cells and
mouse organs.  PMID: 12533916
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"Of course, everyone chooses, as a natural priority, to enjoy peace,
joy, and love by helping to find, quickly share, and positively act
upon evidence about healthy and safe food, drink, and environment."

Rich Murray, MA Room For All rmforall@comcast.net
505-501-2298 1943 Otowi Road, Santa Fe, New Mexico 87505

http://RMForAll.blogspot.com  new primary archive

http://groups.yahoo.com/group/aspartameNM/messages
group with 113 members, 1,494 posts in a public,
searchable archive

details on 6 epidemiological studies since 2004 on diet soda (mainly
aspartame) correlations, as well as 14 other mainstream studies on
aspartame toxicity since summer 2005: Murray 2007.11.24
http://rmforall.blogspot.com/2007_11_01_archive.htm
Wednesday, November 14, 2007
http://groups.yahoo.com/group/aspartameNM/message/1490

[ This layman review gives detailed access to the gist of six
epidemiological studies since 2004, two in 2007, that show
correlations of diet soda (largely aspartame) with health issues.

Probably studies of the correlations at the top 0.1 to 1.0 % level of
use over periods of years by people in vulnerable groups are needed.

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

The Nurses Health Study is a bonanza of information about the health
of
probably hundreds of nurses who use 6 or more cans daily of diet soft
drinks -- they have also stored blood and tissue samples from their
immense pool of subjects, over 100,000 for decades.

In total, there are 20 mainstream studies about negative effects with
aspartame since summer, 2005, listed in this review, included many
about the detailed biochemistry involved. ]

http://rmforall.blogspot.com/2007_09_01_archive.htm
Saturday, September 15, 2007
http://groups.yahoo.com/group/aspartameNM/message/1472
bias, omissions, incuriosity = opportunity, aspartame safety
evaluation, Magnuson BA, Burdock GA, Williams GM, 7 more, 2007 Sept,
Ajinomoto funded 98 pages  html [$ 32 781888262_content.pdf]: Murray
2007.09.15

http://groups.yahoo.com/group/aspartameNM/message/1491
industry scientists praise aspartame safety and benefits in Paris on
2006.05.30, Herve Nordmann, Andrew G. Renwick, Carlo La Vecchia, Tommy
Visscher, Jaap Seidell, France Bellisle, Adam Drewnowski, Margaret
Ashwell, Anne de la Hunty, Sigrid A. Gibson, Alan R. Boobis: Murray
2007.11.18
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