III.

5 Genetic Toxicity
All food additives have to be tested on their mutagenic potential. Likewise the food
additive which is designated for direct human consumption, all metabolites have also
to be tested to exclude any risk.
Therefore, in the case of Stevia rebaudiana, Stevioside and the major metabolite
Steviol was tested.
In the past have been some controverse opinions about the genetic toxicity of
Stevioside and its degradation products after human consumption.
The controverse raised with studies on mutagenic activity of steviol. In a specific test
(Forward Mutation Assay) Steviol was treated with S-9 mix of 9000 x g liver
supernatant of Aroclor 1254 treated male Sprague-Dawley rats (Pezutto, J. et al.:
Metabolically activated steviol, the agylcone of Stevioside is mutagenic; Proc. Natl.
Acad. Sci. USA 82 (1985) p.2478-2482)
In this study, Steviol expressed a mutagenic activity after metabolic activation of the
above described S-9 mix. Without the addition of S-9 mix Steviol did not express a
mutgenic activity.
This result suggests that a metabolic product of Steviol is produced in the liver by S-9
during the liver passage. However, until now the responsible agent was never found.
All Steviol derivates have been identified and synthesized but, however, none of them
exerted any mutagenicity under the same conditions of the "Forward Mutation Assay"
(Compadre et al.: Mass Spectral Analysis of some derivates and in vitro metabolites of
Steviol, the agyclone of the natural sweeteners Stevioside, Rebaudioside A and
Rubusoside; Biomedical and Environmental Mass Spectrometry 15 (1988) p.211-222).
With a synthetically produced 15-Oxosteviol further Forward Mutation Tests have
been done and a strong mutgenicity was found (Pezutto, J. et al.: Characterization of
bacterial mutagenicity mediated by 13-hydroxy-ent-kaurenoic acid (Steviol) and
severall structurally- related derivates and evaluation of potential to induce glutathione
S-transferase in mice; Mutation Research 169 (1986) p.93-106). However, this
suggested metabolite of Steviol was never found, neither in vitro nor in vivo.
In the meanwhile it is recognized that Steviol produces a mutagenic response by using
the "Forward Mutation Assay". It was detected that Steviol is mutagenic in a dose
depend manner in five laboratory animal species (hamster, rat, guinea pig, rabit and
mouse) when testing with a dose from 0,5 mg/l to 5 mg/l (Temcharoen, Punya et al.:
Mutagenic activity of Steviol to Salmonela typhimurium TM677: Comparison of
activity of S9 liver fractions from five laboratory animal species; Bulletin of Health
Science and Technology 1 (1998) p. 38-45). Previously it was suggested by Procinska,
that the mutagenic response of Steviol is a result of its purity (Procinska, E. et al.
Interpretation of results with then 8-azaguanine resistance system in Salmonella
1

thyhimurium: No evidence for direct acting mutagenesis by 15-Oxosteviol, a possible

metabolite of steviol, Mutagenesis 6 (1991) 165-167). However, this interpretation
was rejected by others.
However, the controversy remains as never the responsible mutagenic metabolite of
Steviol was found. This urges the questions about the significance of those results. In
the past a set of genetic toxicity tests have been developed which is obligate for food
additives. The so called "Forward Mutation Test" is not in that list, therefore it makes
sense to concentrate on the official obligate testing procedure. Nevertheless, for
approval of Stevioside it will be necessary to bridge this knowledge gap and to
evaluate with exactitude the significance for human consumption of the already
obtained results for Steviol.
The official test programm is called "Gentoxicity Test Battery" and is composed by
the following test types:
Test Typ

Proposed Tests Battery

Bacterial in vitro tests
1. In vitro Tests Bacterial reverse mutation test
TA 1535, TA1537 or TA97 or TA97a,
TA98, TA100, E.coli WP2uvrA or E.coli
WP2uvrA(pKM101) or TA102
Non bacterial in vitro tests
- Mammalian cell mutation1 or
- Chromosomal aberration2
2. In vivo Tests

- Chromosomal aberration or
- Micronucleus

Proposed Dose
5 mg/plate or
5 l/plate

5 l/ml, 5mg/ml or 10
mM
5 l/ml, 5mg/ml or 10
mM
2,000 mg/kg
2,000 mg/kg

1 = the only proposed test typ is L5178Y mouse lymphoma gene and chromosomal mutation test
2 = the only proposed test types are a) Chinese hamster fibroblasts (CHO, Chinese hamster ovary, or
CHL, Chinese hamster lung) or b)human lymphocytes or c) rat lymphocytes

The above presented tables shows that the only recommended test type for "Bacterial
in vitro tests" is the so called "Ames-Test". This test type has a minimum set of tests
with the following bacteria strains TA97, TA98, TA100, TA 1535, TA1537 and
TA102. The so called "Forward Mutation Test" which caused the controversy about
Steviol is not part of the official test battery.
The further sections of this chapter describes the tests results obtained by Stevioside
and then the obtained results of Steviol according to the above given test battery
schedule.

It is well recognized that Stevioside itself it not mutagenic. An enormous testing was
done in the past. The most important results are given in the table below.
Genetic Toxicity Tests on Stevioside
Institute

Testsystem

National Cancer
Center (Japan)

Ames-Test with TA 98 a) Stevia Herb Extract

and TA 100 with and
without S-9
b)Crystallized Sweet
Constituents

National Institute of
Hygienic Science
(Japan)

Ames-Test with TA a) Stevia Herb Extract

98, TA 100 and TA
1537 with and without
S-9
b) Crystallized Sweet
Constituents
Ames-Test with TA a) Crude Juice
98, TA 100, TA 1535, b) Stevia Herb Extract
TA 1537 TA 1538 and c) Crystallized Sweet
E.coli WP 2 with and Constituents
without S-9
Micronucleus test in c) Crystallized Sweet
hamster, mice and rats Constituents

Omiya Research
Research Laboratory
of Nikken Chemicals
(Japan)
Department of
Biochemistry, Chiang
Mai University,
(Thailand)
Omiya Research
Research of Nikken
Chemicals (Japan)

Testsample

Mutant
Frequency
Test with Salmonella
typhimurium G 46 in
mice
University of Illionois, Ames-Test with TM
Medical Center,
677 with and without
Chicago (USA)
S-9
National Institute of
Hygienic Science
(Japan)

National Institute of
Hygienic Science
(Japan)
School of Medicine,
Kobe University
(Japan)
Department of
Biochemistry, Chiang
Mai University,
(Thailand)
Department of
Biochemistry, Chiang
Mai University,
(Thailand)

a) Crude Juice
b) Stevia Herb Extract
c) Crystallized Sweet
Constituents
a) Pure Chemical
Substance
b) Derivates of Pure
Chemical Substance
Chromosomal Aber- a) Stevia Herb Extract
ration Test in vitro
with
celline
of
hamster lung
b) Crystallized Sweet
Constituents
Chromosomal Aber- Crystallized Sweet
ration Test in vitro Constituents
with
celline
of
hamsterfibroblasts
Chromosomal Aber- a) Stevia Herb Extract
ration in vivo in bone b) Crystallized Sweet
marrow cells of rats
Constituents
Amest-Test with TA Pure Chemical
98 and TA 100.
Substance
99% Stevioside
Chromosomal Aber- Pure Chemical
ration
in
human Substance
lympocytes with and 99% Stevioside
without
metabolic
activation

Results
Negative with TA 98 with and without metabolic activation.
Negative with TA 100 without metabolic activation; with
metabolic activation slightly postive.
Negative with TA 98 with and without metabolic activation.
Negative with TA 100 without metabolic activation; with
metabolic activation slightly postive.
Negative with TA 98, TA 100 and TA 1537 with and without
metabolic activation.
Negative with TA 98, TA 100 and TA 1537 with and without
metabolic activation.

Results are presented in Table 1

Results are presented in Table 2

Results are presented in Table 3
All results negative

a) Stevia Herb Extract without metabolic activation was

positive.
b) Crystallized Sweet Constituents have been negative in all
tests with and without metabolic activation.
The highest dose of 12mg/ml induced after 48 hours of
incubation 1% of Chromosomal Aberrations. The controll
showed a Chromosomal Aberration of <3%. All test results are
negative.
The test sample was administered to the rats via i.p. The
bonemarrow cells were screened for Chromosomal Aberration
after killing of the rats. All test results have been negative.
a) Stevioside was found to exhibit no mutagenicity in both
strains up to 25 mg/plate with and without metabolic
activation. At 50 mg/plate stevioside did show in TA 98 a
mutagenic activity which was not shown in TA 100.
Stevioside did not cause any significant aberrations of
metapasic chromosomes between 1-10 mg/ml in all samples.

III.5. Genetic Toxicity - Table 1

Ames-Test
Conducted by Omiya Research Laboratory, Nikken Chemicals, 1978

Test sample

A) Crude Juice
B) Stevia Herb Extract
C) Chrystallized Sweet Constituents

Testsystem

Ames-Test with E. coli WP2 and S.

typhimurium TA 1535, TA 100, TA 1537,
TA 1538 and TA 98 with and without
addition of S-9.

Test Dose

10 g - 0,010 g/plate

Observations

See Figure 1

Conclusion from the authors

Crude Juice (in text "preparations"), Stevia

Herb Extract and Chrystallized Sweet
Constituents underwent an Ames-Test. All
test samples produced no mutagenicity with
or without metabolic activation.

Reference

No. 5

III.5. Genetic Toxicity - Table 2

Rec-Assay Test
Conducted by Omiya Research Laboratory, Nikken Chemicals, 1978.
Test sample

A) Crude Juice
B) Stevia Herb Extract
C) Crystallized Sweet Constituents

Test system
Test Dose

Rec-Assay Test with Bacillus subtilis H

17 and M 45
20-2000 g/disk

Observation

See Figure 1

Conclusion from the authors

Reference

No inhibition could be established.

No. 5

III.5. Genetic Toxicity - Table 3

Mutant Frequency Test
Conducted by Omiya Research Laboratory, Nikken Chemicals,1978
Test sample

A) Crude Extract
B) Stevia Herb Extract
C) Crystallized Sweet Constituents

Test system
Maximum Dose

Mutant frequency test with Salmonella

typhimurium G 46 in mice
20 g/kg body weight

Observation
Conclusion from the authers

See figure 1
Tests were negative

Reference

No. 5

III.5 Genetic Toxicity - Table 4

Micronucleous Test
Conducted by Department of Biochemistry, Chiang Mai University, (Thailand),
2002.
Test sample

Test Dose

Pure Chemical Substance Stevioside

(96% purity)
Micronucleous
Test
after
oral
administration with hamster, mouse and
rat
10 g/kg body weigth

Observation

See Figure 1

Conclusion from the authors

Stevioside at the dose of 10 g/kg body

weight had no effect on the frequencies of
micronucleous formation in the bone
marrow erythrocytes of either sex of
hamsters, mice and rats at any time point
when compared with the negative control.
Moreover, there are no apparent changes
in the PCE:NCE ratio in either sexes of
the tested species with an exception of
male
hamsters
which
exhibited
significantly lower PCE:NCE ratio at 48
h after the treatment. The results suggets
that Stevioside at the given dose had no
chromosome damaging effect on the bone
marrow cells of the tested species
No. 5

Test system

Reference

10

11

III.5. Genetic Toxicity - Table 5

Forward Mutation Assay
Conducted by University of Illinois, Medical Center, Chicago, USA.,1982.
Test sample

Pure chemical substances of Stevia

rebaudiana sweet constituents which are
Dulcosid
A,
Rebaudioside
A,
Rebaudioside C and Stevioside.

Test system

Forward
Mutation
Assaywith
S.
typhimurium TM 677 with and without
metabolic activation obtained from
Aroclor 1254 pretreated rats.

Test Dose

Not given

Observaton

No significant mutagenic or bactericidal

activity was noted when assayed either in
presence or in absence of a metabolic
System obtained from Aroclor 1254.

Conclusion from the authors

The primary sweetening constituents (see

above at Test sample) of Stevia
rebaudiana are neither toxic nor
mutagenic when evaluated as described
above.
No. 1

Reference

12

III.5. Genetic Toxicity

Genetic Toxicity Tests on Steviol

Test Typ
1. Bacterial in vitro tests
a) Reverse Mutation
TA 98 und TA 1001
TA 97, TA 98, TA 102, TA 104,
TA 1535, TA 15372
E.coli WP 2uvrA
(pKM101)2
b) Non bacterial in vitro tests
Chromosomal aberration test
with CHL cells2
Chromosomal aberration test
with human lympocytes1
Mammalian
cell
mutation:
L5178Y mouse lymphoma gene
and chromosomal mutation test3
2. In Vivo Tests
Micronucleus test with bone
marrow and hepatocytes in mice3
Micronucleus test with bone
marrow in mice2
Micronucleous test with bone
marrow of rats and mice1
Micronucleous test with bone
marrow of hamster1

Dose

Result

Proposed
Dose OECD

- S9 Mix + S9 Mix
1 - 20 mg/plate
negativ
0 - 5000 g/plate negativ

negativ
negativ

5 mg/plate
5 mg/plate

0 - 5000 g/plate negativ

negativ

5 mg/plate

0 - 1,5 mg/ml

negativ

positiv

0 mg/ml

0 - 0,2 mg/ml

negativ

negativ

340 g/ml

negativ

5 l/ml

200 mg/kg

negative

2000 mg/kg

0 - 1000 mg/kg

negativ

2000 mg/kg

8000 mg/kg

negativ

2000 mg/kg

4000 mg/kg

negativ

2000 mg/kg

1,5

1 = executed by Chiang Mai Universitt, Thailand, various publications

2 = Japanese Research group, published in Mutagenesis 11 (1996) 573 - 579
3 = Korea Food Administration. Oh, Hye-Young et al.: In vitro and in vivo evaluation
of genotoxicity of stevioside and steviol, natural sweetener; Yakhak Hoechi 43 (1999)
p. 614 - 622.
The overall conclusion: Steviol will fullfil all requirements of genetic toxicity testing
according to OECD standards. The test on "Chromosomal aberration with CHL cells
which caused a positive response when adding S-9 mix is voluntary and no obligate
requirement.
13