Food and Nutrition Sciences, 2013, 4, 106-112
http://dx.doi.org/10.4236/fns.2013.47A013 Published Online July 2013 (http://www.scirp.org/journal/fns)
Acute Toxicity and Genotoxic Evaluation of Metlin® and
Metlos® (Organic Agave Fructans)
M. I. Gracia1, M. M. Tinoco1, H. M. Rivera1, B. F. Sanchez1, P. G. Tapia2, L. M. Altamirano3,
R. L. Romero2, O. L. García2
1Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico; 2Facultad de Medicina Veterinaria y Zo-
otecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico; 3Facultad de Estudios Superiores-Zaragoza, Universidad
Nacional Autónoma de México, Mexico City, Mexico.
Received March 27th, 2013; revised April 27th, 2013; accepted May 7th, 2013
Copyright © 2013 M. I. Gracia et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The aim of the study was to contribute to the information on agave soluble fibers since research has been focused on
chicory fiber but not in agave products; thus we assess the acute toxicity and genotoxicity of two organic and high pu-
rity dietary soluble fibers from agave, Metlin® and Metlos®. We performed an acute toxicity assay in Hsd:ICR mice and
Hsd:Wistar rats and an in vivo genotoxic test. Results showed that there are no deaths at any doses or genotoxicity, so it
can be concluded that these products are non-toxic, at the administrated doses and none showed a cytotoxic, clastogenic
or aneuploidic effect.
Keywords: Agave Fructans; Inulin; Fructooligosacharides
1. Introduction
Chronic diseases prevention has become of great impor-
tance for Public Health, research and technology fields,
thus one way to do it is through the regular consumption
of functional foods.
Food usually refers to any product, supplying to an or-
ganism, the energy and chemical substances required for a
good health. Nutrients are chemical substances in food
that an organism uses and transforms in its own tissues to
perform basic functions. Some food contains a certain
type of chemical substances capable of causing positive
effects to promote and restore health. Functional food
implies that the food has some kind of identified value that
benefits health, or diminishes the potentiality of disease
[1].
In October, 1994, the American Dietetic Association
(ADA) adopted a position towards the functional food.
The position outlines: “Functional food, including whole
food (not modified), and fortified, enriched, improved
food have a potentially benefic effect over health when
they are consumed as part of a diet in a regular basis, in
effective levels. The Association supports a deeper re-
search on this field to define the benefits and risks for
health of the individual functional foods and their physio-
logical active components. The professional dieticians
will continue working with the food industry, the gov-
ernment, the scientific community, and the media to as-
sure that the public will be able to obtain exact informa-
tion on this emerging food science field [2].”
When functional food is evaluated in the context of a
healthy diet, it must be considered the safe intake levels.
The animal research has supplied a close look into this
ideal intake; nevertheless this information is difficult to
extrapolate to human dietetic requirements. For most of
the functional food, the daily intake levels will be estab-
lished according to clinical assays that have been pub-
lished in scientific literature.
Most of the functional food will require continued re-
search in vivo e in vitro, as well as pharmacokinetic stud-
ies, before the specific levels can be determined by
clinical assays. Once these clinical assays are completed,
the recommendations will be more specific [3].
There exist different types of functional food, but for
this matter we will focus on dietary fiber which has some
components that can be considered as prebiotics. This
type of food must have a synergic relationship with pro-
biotics, and this combination makes possible the benefic
effects in the gut [4].
Probiotic refers to a living microorganism that can be

Acute Toxicity and Genotoxic Evaluation of Metlin® and Metlos® (Organic Agave Fructans) 107
introduced in diet and has a positive effect in health,
further more than traditional nutritional effects [5].
Prebiotic refers to the non digestible food ingredients
that produce benefic effects on the host, selectively stimu-
lating the growth and/or activity of bacteria in the colon [6].
Dietary fiber refers to different carbohydrates that resist
human digestive enzyme hydrolysis, but can be ferment-
ed by the colon flora and partially excreted by the feces.
This definition would include fibers such as non starch
polysaccharides, inulin, fructiooligosaccharides (FOS),
galactooligosaccharides (GOS) [7,8].
Inulin is a polysaccharide that can be extracted from
plants of different families: Liliaceae, Amaryllidaceae,
Agavacae, Gramineaey compositae; although the main
source is chicory (Cichoriumintybus).
Dietary fiber is an essential part of diet, contributing to
the proper functioning of the gut; the recommended fiber
intake for adults, according to different countries, are in
the range of 25 to 35 grams per day [7,9-13]. Fructans are
dietary fibers with prebiotic activity. They are fructose
polymers with one or more fructosylfructose linkage [14].
Health Effects of Fructans
The most studied not digestible oligosaccharides (NDO)
are FOS and GOS. According to different studies, these
compounds have different benefic effects in health:
 Bifidogenic activity. There is a selective stimulation of
a limited group of beneficial bacteria in the human
colon.
 Short chain fatty acids production and associated ef-
fects. NDO fermentation in the colon produces short
chain fatty acids, such as acetic, propionic, butyric and
also gases (hydrogen and carbon dioxide). These fer-
mentation should give a caloric value of 1.5 kcal/g
[15].
 Effect over the number, frequency and weight of fecal
stools. It has been observed in animal and human as-
says that FOS increases the weight of fecal stools
[16,17].
 Colon cancer prevention.
 Elimination of diarrhea related to intestinal infections.
 Risk reduction of obesity and type 2 diabetes.
 Effect over Glucemia and Insulinemia.
 Effect over lipids metabolism. A drop in low density
lipoproteins (LDL) levels and cholesterol in patients
with non insulin dependent diabetes using oligofruc-
tose has been described [18,19] and of triglycerides
and cholesterol with inuline [20-22].
 Effect over uremia and nitrogen/ammoniac elimina-
tion.
 Effect over mineral absorption (Risk reduction of os-
teoporosis). It is known that in humans, minerals such
as Ca, P and Mg are absorbed in the small intestine, but
also are effectively absorbed in the colon [23,24].
Several studies on rats conclude that the administration
of FOS and GOS stimulate the absorption of Ca, Mg
and Fe [25-28].
Agave (Agavaceae), called the “tree of wonders”, is a
plant species of great use for indigenous groups since
prehispanic times. There exist more than 250 agave spe-
cies. From the plant it is possible to extract food, drinks,
animal fodder, construction materials, fabrics and ropes.
Since old times, agave usage has been a constant in the
cultural history of Central America; being one of the first
crops in the area.
The fermented maguey juice (aguamiel) was called
“octli” in nahuatl dialect and “seí” in otomi dialect.
Mexican mythology has two versions on its origin: one
says that goddess of earth and fertility, Mayahuel, dis-
covered aguamiel and Pachtécatl, her husband, discov-
ered the fermentation process. The second version says
that Papantzin discovered the way to extract aguamiel.
Old Mexican cultures appreciated the multiple uses
and virtues of aguamiel, even the therapeutic ones. José
Zorrilla, a Spanish poet and playwright noted: “Aguamiel
is a much appreciated drink, to which Mexicans attribute
nutritious and medicinal properties; even they make it
drink to weak nursing women because of its capacity to
increase milk production [29,30].”
Most of the attributes assigned to agave are due to the
presence of fructans on its chemical structure.
Metlin® and Metlos® are highly purified organic fruc-
tans extracted from Blue Agave (Agave tequilana Weber).
The main difference between Agave fructans and other
types of fructans (mainly inulins) is the linkages β (2-1) y
β (2-6) which make them branched molecules. These
products are not absorbed in the digestive system and are
highly soluble. These characteristics give them nutri-
tional and functional properties providing health benefits.
The solubility gives them high competitive advantages,
being extensively used as a texturizer and increasing
shelf life due to its capacity to retain water.
As pointed before, agave fructans are different from
the rest, which makes it a different molecule; also most
studies are focused in chicory fructans. Because of this, it
is necessary to realize safety and efficacy assessments.
2. Material and Methods
2.1. Metlin® an d Metlo s®
Metlin® and Metlos® are highly purified organic fructans
extracted from Blue Agave (Agave tequilana Weber);
these fructans are reported to be of the branched group
with both (β 2→1) and (β 2→6) fructosyl-fructose link-
ages; their main difference from current commercial
fructans is their molecular structure; the structure of sev-
eral fructan molecules present in Metlin® and Metlos®
which are representative of the highly branched nature of

Acute Toxicity and Genotoxic Evaluation of Metlin® and Metlos® (Organic Agave Fructans) 108
agave fructans were elucidated by Praznik et al. [10].
The main difference between Metlin® and Metlos® is
their degree of polymerization (DP) distribution. Metlin®
contains mostly fructans over DP 10 and a very low
mono and disaccharide content. Metlos® contains pre-
dominantly fructans under DP 10, with slightly higher
monosaccharide content. Since both products consist of
ramified molecules, they are extremely soluble in cold
water. A major advantage is that these products are the
only organic-certified long chain fructans available on
the market today.
2.2. Animals
Experiments were performed using certified animals as
Specific Pathogen Free “SPF” obtained from Harlan
Laboratories-México. Genotoxicitywas performed using
Hsd:ICR mice 4 - 5 weeks old (Average weight 28 g) and
for acute toxicity we used Hsd:ICR mice 4 - 5 weeks old
(Average weight 28 g) and Hsd:WI rats 8 - 9 weeks old
(Average weight 195 g) [11]. The experiments were con-
ducted after getting prior permission from the Institutional
Committee for Care and Use of Laboratory Animals. They
were housed in the Animal Experimentation Unit, in the
Chemistry School at the National Autonomous University
of Mexico, in well-ventilated sterile propylene cages; each
cage contained 5 animal of the same sex. There were
maintained at a controlled temperature of 22 ± 2˚C and
relative humidity 60% ± 10% and provided 12 h light/dark
cycles. Experiments were started after acclimatizing the
animals for one week. They were fed with normal pelleted
rodent diet (Teklad 2018S) and water ad libitum. The
composition of the feed is 18.6% crude protein, 3.5%
crude fiber and 6.2% fat.
2.3. Chemicals
Metlin® and Metlos® were purchased from Nekutli S.A.
de C.V., México. Mitomycin-C (MMC), Giemsa, Hema-
toxilin-Eosine solution and Acridine Orange (AO) were
obtained from Sigma-Aldrich.
2.4. Preparation of Metlin® and Metlos®
Metlin® and Metlos® were dissolved in bidistilled, deion-
ized and filtered water with a 0.22 μm membrane and each
one was administered to the animals at different doses in
no more than 2 ml/100g of body weight for acute oral
toxicity assay.
We used a control group administered with the same
quality of water mentioned above. For the genotoxic
study the solution was immediately administered intrap-
eritoneally (ip) in a proper volume as showed using Mi-
tomycine-C (powerful clastogen agent) as positive control
and PBS as negative control. All treatments aredescribed
in Table 1.
Table 1. Treatments for acute toxicity and genotoxic assays.
Product Animal
model
Doses
mg/kg Sex Animals/dose (total animals) Assay
17.5
55
175
550
1750
Male 5 (30)
Metlin®Hsd:ICR
mice
5000 Female 5 (30)
Acute oral
toxicity
17.5
55
175
550
Male 5 (30)
1750
Metlos®Hsd:WI
rats
5000 Female 5 (30)
143
357.5
Metlin®Hsd:ICR
mice
715
Male 5 (15)
143
357.5
Metlos®Hsd:ICR
mice
715
Male 5 (15)
Genoto-xicity
assay
2.5. Acute Toxicity Studies
Each product (Metlin® or Metlos®) was administered as a
single dose through oral gavage, and the animals were
monitored for 14 days. The body weight, mortality, clini-
cal and behavioral symptoms were noted; observations
included assessment of fur, eyes, mucosal membranes,
respiratory system, activity, with particular attention on
possible tremors, convulsions, salivation, and diarrhea.
On Day 14 of the experiment, we proceeded to eutha-
nasia in a CO2 chamber followed by necropsy for the
determination of any possible macroscopically finding
and to obtain the next organs: stomach, small intestine
(duodenum, jejunum, and ileum), large intestine (cecum,
colon, and rectum), and liver. The organs were washed
with a 7.4 pH PBS solution and fixed in 10% buffered
formaldehyde for the histopathology study. Even the in-
formation obtained from acute experiments is unsubstan-
tial; we decided to perform the histopatologic analysis to
get more information.
2.6. Genotoxic Studies
2.6.1. Evaluation of Chromosomal Aberrations
After 24 hours of started the treatment, we proceeded to
euthanasia in a CO2 chamber and the femur’s bone mar-
row was extracted from mice. The preparations with the
bone marrow cells were stained for 10 minutes with a 5%
Giemsa solution (Sigma). The chromosomal aberration

Acute Toxicity and Genotoxic Evaluation of Metlin® and Metlos® (Organic Agave Fructans) 109
analysis (CA) was performed in a double-blind study in
100 cells in metaphase for each animal. The structural
alterations in the chromosomes were scored, both chro-
mosomic and chromatid type following the OECD [31]
and EPA [32] procedures.
2.6.2. Micronu clei Assay
Five micro liters of peripheral blood from tail vein of
each animal was collected before the sacrifice. Collected
blood was placed in the center of an AO-coated glass
slide and covered with a 24 mm × 40 mm cover slip.
These slides were kept at 4˚C for no more than 5 days
until analysis. AO supravitally stained erythrocytes and
the frequencies of micro nucleated polychromatic eryth-
rocytes (MN-PCE) were examined in a fluorescence mi-
croscope [33,34].
2.7. Statistical Analysis
Data from the acute toxicity were analyzed using the
statistical SPSS® 19 IBM package. Metlin® and Metlos®
were analyzed separately.
A Generalized Linear Model was used in the modality
Multinomial logistic regression [35] using, as predictor
variables: Treatments, species (Rats, mice) and sex for all
kind of lesions in the liver and small intestine (dependent
variables). Each lesion has three categories (+slight, ++
moderate, +++sever) so the ordinary logistic link was
selected. The Odd Ratios and their Wald Confidence In-
tervals were obtained for all treatments against the control
group.
For the liver, the analyzed lesions were: degeneration
and inflammation. For the small intestine the analyzed
lesions were: Atrophy and villus fusion and inflammation.
The significance level considered in this study was 0.05. It
was also obtained the powers of all tests, using the soft-
ware G*Power 3.1.2, with the following parameters: no
centrality parameter λ = 10, significance obtained with
SPSS software, degrees of freedom obtained with the
SPSS software.
The weights were analyzed with a polynomial model of
repeated measurements, with day as intra-subjects factor
and dose and sex as inter-subjects factor, with Green-
house-Geisser correction for sphericity. In case of dif-
ferences between groups (doses), a Dunnett measure-
ments comparison test was applied. A significance level
of 0.05 was considered for all the analysis, also with sta-
tistical SPSS® 19 IBM package.
For the genotoxic study, the obtained data was analyzed
by comparing the control group against the treated group.
A Chi Squaretest [36,37] was used for the analysis of the
Chromosome Aberrations frequencies (CA) induced. For
Mitotic Index (MI), significant differences were detected
with the statistical proportion Z test.
3. Results
3.1. Acute Toxicity
3.1.1. General Status of Treated Groups
All treatments had no important variations observed in
body weights during the study and no changes were ob-
served in their physical appearance and behavior.
There were no significant differences (P > 0.05, 1 − β =
0.69) attributable to the intake of Metlos® or Metlin® (P =
0.10, 1 − β = 0.69) regarding to weight. All had a signifi-
cant weight increase (P = 0.001); although males were heav-
ier than females in all weight measurements (P = 0.001) and
these increase is normal due to the growth of the animals.
3.1.2. Hi stopathology
1) Macroscopic and microscopic description
Gastrointestinal tract of 3 animals per dose per species
per product were examined included: stomach, liver,
duodenum, jejunum, ileum, cecum, and colon. No sig-
nificant macroscopic changes were observed. Micro-
scopic changes were subjected to statistical.
2) Liver
No significant differences were found in any the lesions
analyzed, attributable to Metlin® intake (P > 0.05; 1 − β >
0.73) (Table 2). The same results were found to Metlos®
(Table 3).
Table 2. Liver histopathology results in rats and mice treat-
ed with Metlin®1 and the control group.
Lesion Treatment Specie Sex
Degrees of freedom 6 1 1
Wald chi-square
Degeneration 6.50 1.31 3.38
P 0.37 0.25 0.06
1 − β 0.82 0.90 0.73
Inflammation 3.90 0.22 1.27
P 0.69 0.64 0.26
1 − β 0.94 0.97 0.90
1Doses: control, 17.5, 55, 175, 550, 1750, 5000 mg/Kg; 2Test power.
Table 3. Liver histopathology results in rats and mice treat-
ed with Metlos®1 and the control group.
Lesion Treatment Specie Sex
Degrees of freedom 6 1 1
Wald chi-square
Degeneration 4.88 0.68 0.002
P 0.56 0.41 0.96
1 − β2 0.91 0.94 0.97
Inflammation 1.08 1.82 1.81
P 0.29 0.18 0.29
1 − β 0.76 0.86 0.91
1Doses: control, 17.5, 55, 175, 550, 1750, 5000 mg/Kg; 2Test power.

Acute Toxicity and Genotoxic Evaluation of Metlin® and Metlos® (Organic Agave Fructans) 110
3) Small intestine
In the intestine lesions, no significant differences were
attributable to the intake of Metlin® (P > 0.05, 0.53 ≤ 1 −
β ≤ 0.99) (Table 4) or Metlos® (P > 0.05, 0.91 ≤ 1 − β ≤
0.99 (Table 5).
4) Stomach
No relevant lesion or findings at microscope analysis.
3.2. Chromosome Aberrations
The results from the bone marrow chromosome analysis
treated with Metlin® and Metlos® are shown on Tables 6
and 7.
Data showed that in both treated groups the simple
chromosomal aberrations frequency, such as fragments
and deletions are within the normal range, when com-
pareing against the negative control group.
In the case of the positive control groups treated with
MMC, the chromosomal alteration frequency was sub-
stantially higher and statistically significant when com-
pared with the negative control group.
Finally, when the mitotic index was analyzed as a cy-
totoxicity parameter, it was observed that in none of these
treatments the index was modified, with the exception of
the group treated with MMC, which decreased signifi-
cantly.
3.3. Micronuclei Analysis
As an additional parameter for the genotoxic study, an
analysis of the micronucleus frequency present in the
peripheral blood erythrocytes from mice. The results
shown in Tables 8 and 9, indicate that neither Metlin® or
Metlos® increased the micronuclei frequency. The ani-
mals treated with MMC increase significantly when com-
pared with the negative control group.
Table 4. Small intestine histopathology in rats and mice
treated with Metlin®1 and the control group.
Lesion Treatment Specie Sex
Degrees of freedom 6 1 1
Wald chi-square
Atrophy and villus fusion2.21 0.041 0.697
P 0.89 0.84 0.40
1 − β 0.99 0.99 0.99
Inflammation 5.92 2.69 1.73
P 0.43 0.10 0.18
1 − β 0.99 0.96 0.98
1Doses: control, 17.5, 55, 175, 550, 1750, 5000 mg/Kg; 2Test power.
Table 5. Small Intestine histopathology in rats and mice
treated with Metlos®1 and the control group.
Lesion Treatment Specie Sex
Degrees of freedom 6 1 1
Wald chi-square
Atrophy and villus fusion1.09 0.30 0.11
P 0.30 0.58 0.74
1 − β 0.91 0.99 0.99
Inflammation 2.77 0.01 1.61
P 0.84 0.99 0.20
1 − β 0.99 0.99 0.96
1Doses: control, 17.5, 55, 175, 550, 1750, 5000 mg/Kg; 2Test power.
Table 6. Chromosomal alterations frequency in bone marrow cells of mice treated with Metlin®.
Treatment mg/kg No. of miceNo. of cells Del. Fragm.Trans.Gaps % cells w/+ gaps % cells w/- gapsIM
Control 5 500 5 6 - 7 3.6 2.2 4.2
+Control (MMC) 4 400 35 42 4 25 26.5 20.25* 1.1**
143 5 500 7 8 - 9 4.8 3.0 5.4
357.5 5 500 6 7 - 10 4.6 2.6 3.9
715 5 500 4 9 - 8 4.2 2.6 4.0
Del = deletions; Fragm = fragmentations; Trans = translocations; IM = mitotic index; *P < 0.001 vs. negative control; **P < 0.01 vs. negative control.
Table 7. Chromosomal alterations frequency in bone marrow cells of mice treated with Metlos®.
Treatment mg/kg No. of miceNo. of cells Del. Fragm.Trans. Gaps % cells w/+ gaps % cells w/- gapsIM
Control 5 500 4 6 - 6 3.2 2.0 3.9
+Control (MMC) 5 500 51 42 6 24 24.6* 19.80* 1.3**
143 5 500 8 8 - 7 4.6 3.2 4.4
357.5 5 500 3 3 1 11 3.6 1.4 3.7
715 5 500 8 6 - 9 4.6 2.8 3.0
D
el = deletions; Fragm = fragmentations; Trans = translocations; IM = mitotic index; *P < 0.001 vs. negative control; **P < 0.01 vs. negative control.

Acute Toxicity and Genotoxic Evaluation of Metlin® and Metlos® (Organic Agave Fructans) 111
Table 8. Micronucleus frequenc y in peripheral blood eryth-
rocytes in mice treated with Metlin®.
Treatment mg/kg No. of animals Micronuleated cells/100cells
(X ± E.E.)
Control 5 1.50 ± 0.27
+Control (MMC) 4 6.94 ± 0.90
143 5 1.67 ± 0.37
357.5 5 1.89 ± 0.35
715 5 2.10 ± 0.22
P < 0.005 vs. negative control.
Table 9. Micronucleus frequenc y in peripheral blood eryth-
rocytes in mice treated with Metlos®.
Treatment mg/kg No. of animalsMicronuleated cells/100cells
(X ± E.E.)
Control 5 1.10 ± 0.031
+ Control (MMC) 5 9.19 ± 0.45
143 5 1.30 ± 0.33
357.5 5 1.10 ± 0.21
715 5 1.67 ± 0.55
P < 0.001 vs. negative control.
4. Discussion
This study shows that organic agave inulin andorganic
agave fructooligosacharides (Metlin® and Metlos®), do
not causes any toxicity up to the highest dose (5000
mg/Kg). No treatment-related effects were observed in
body weight, or microscopic pathology, in vivo genotoxic
studies have also shown the absence of mutagenic or
genotoxic potential. Lack of adverse toxicity seen with
this products at this dosage is consistent with a similar
lack of significant toxicity exhibited by inulin or fructoo-
ligosacharides from other sources like chicory [38] or a
poly- and oligomers of fructose joined by β(2-1) fructo-
syl-fructose bonds call inulin-type fructans [39].
5. Final Comments and Conclusions
There was no death at any doses, concluding that these
compounds are not toxic at the administered doses. It is
worth mentioning that maximum doses, permitted by
internationally accepted protocols (FDA and OECD)
were reached: 5 g/kg weight (OECD, 2001-Guideline for
testing on acute oral toxicity). The appearance and be-
havior of mice treated with Metlin® and Metlos® showed
no changes during the study.
The frequency analysis of the chromosome aberrations
presented in mice treated with Metlin® and Metlos®
showed that none of each compounds are genotoxic, as
well as the mitotic index which not showed a cytotoxic
effect.
The frequency of micronuclei presence in polychro-
matic erythrocytes confirmed the fact that none of the
compounds tested are clastogens (chromosome-breakers)
or induced aneuploidies.
There were no significant histopatology changes in
any of organs studied.
In conclusion, we can say that under the model and
experimental conditions used, both Metlin® and Metlos®
are non toxic, cytotoxic or genotoxic. The further valida-
tion using clinical trial is still needed.
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