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Acute and Subacute Toxicity Study on Dietary Supplementation with Soy

Isoflavones in Wistar Rats

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Current Nutrition & Food Science, 2018, 14, 68-78

RESEARCH ARTICLE
ISSN: 1573-4013
eISSN: 2212-3881

Acute and Subacute Toxicity Study on Dietary Supplementation with Soy

Isoflavones in Wistar Rats
BENTHAM
SCIENCE

WCS Faria4,*, RC Martinelli1, AS Arcas1, IF Da Silva Junior2, EM Colodel3, DFLC Cavenaghi1,

AP Oliveira1 and WM Barros1

1
Faculty of Food Engineering, Federal Institute of Education, Science and Technology of Mato Grosso, IFMT. Juliano
Costa Marques Street, Bela Vista, Cuiabá, MT, Brazil, CEP 78050-560; 2Laboratory of Pharmacology, Department of
Basic Sciences of health, Faculty of Medicine, Federal University of Mato Grosso, UFMT, Fernando Corrêa da Costa
Street, nº 2367, Boa Esperança, Cuiabá, MT, Brazil, CEP 78060-900; 3Laboratory of Veterinary Pathology, Department
of Medical Clinic Veterinary, Faculty of Veterinary Medicine, Federal University of Mato Grosso, UFMT, Fernando
Corrêa da Costa Street, nº 2367, Boa Esperança, Cuiabá, MT, Brazil, CEP 78060-900; 4Faculty of Food Engineering,
Department of Food Science, State University of Campinas, UNICAMP, University City Zeferino Vaz, Barão Geraldo,
Campinas, SP, CEP 13083-970, Brazil

Current Nutrition & Food Science

ARTICLE HISTORY
Received: May 24, 2017
Revised: August 14, 2017
Accepted: August 21, 2017
DOI:
10.2174/1573401313666170821154016
Abstract: Objective: The aim of this study was to investigate the dose-dependent toxic effect of SIF
supplementation.
Methods: Single doses of 0, 100, 500, and 1000 mg/kg bw of SIF were administered by gastrogavage
to male and female Wistar rats (6/sex/group). Same dosages were gastrogavaged daily over a period of
30 days, the animals were fed with a standard ration added of 20% soybean-based protein bar
(weight/weight) ad libitum. Food consumption, Body weight, organ weights, hematological and bio-
chemical parameters were measured, and then the organs were necropsied and examined histologi-
cally.
Results: A single dose of 1000 mg/kg bw did not cause any behavioral alteration, toxicity symptoms,
or death. The SIF at 1000 mg/kg bw/day for 30 days exhibited increasing a relative liver weight in the
female. In this same dosage, a decrease in relative liver, lungs, kidneys, spleen and heart weight was
observed among males. A minimal alteration in enzymes or biochemical markers of liver function was
observed in female and male rats, but this was not considered adverse since there were no supporting
clinical or histopathologic findings.
Conclusion: The acute toxicity test indicates that SIF has a high margin of safety as shown by the
LD50 and according to subacute test the No-observed-adverse-effect level (NOAEL) of SIF was esti-
mated to be 100 mg/kg/day. However, the chronic toxicity cannot be estimated.

Keywords: Acute toxicity, glycine max, phyto-oestrogen, rodents, subacute toxicity, supplementation.

1. INTRODUCTION hormone replacement therapy, targeting the relief of meno-

Soybean products and their dominant isoflavones, that is,
genistein and daidzein [1] have become common ingredients
in many dietary supplements and functional foods [2]. Be-
sides, soyfoods have long been recognized as a source of
high-quality protein [3]. Scientific studies have shown their
involvement in preventing the onset of chronic diseases such
as various cancers [4], osteoporosis [5], type 2 diabetes [3, 6]
heart disease [7, 8] and dyslipidemia [9]. In addition, Soy
Isoflavones (SIF) have been proposed as an alternative to
*Address correspondence to this author at the Faculty of Food Engineering,
Department of Food Science, State University of Campinas, UNICAMP,
University City Zeferino Vaz, Barão Geraldo, Campinas, SP, CEP 13083-
970, Brazil; Tel: (65)99985-5106; E-mail: nessacsf@yahoo.com.br
pausal symptoms [10].
In Brazil, the Health Surveillance Agency (ANVISA) ap-
proves the use of SIF for the treatment of hot flashes and the
soy protein as an adjuvant in the reducing serum cholesterol
[11]. However, the maximum permitted amount of SIF for
food supplementation is 25 mg/day [12].
The incorporation of dietary SIF aiming to prevent and/or
controlling chronic diseases is the subject of extensive re-
search [13]. While the clinical impact of the chronic use of
isoflavones presents conflicting results, most commonly re-
ported adverse effects are related to reproduction. Isofla-
vones are identified as endocrine active compounds, since
they have a similar chemical structure to oestradiol and affin-
ity for - and -oestrogen receptors [14-16]. By contrast, a

2212-3881/18 $58.00+.00 © 2018 Bentham Science Publishers

Safety of Soy Isoflavones as Dietary Supplement
recent study demonstrated compelling results about the secu-
rity of this compounds to the reproductive system due to
weak estrogenic potency of the serum isoflavones in compe-
tition with endogenous oestrogens [17], suggesting that SIF
can act as a selective oestrogen receptor modulator and as anti-
estrogenic in the presence of endogenous oestrogen [18].
During metabolism, genistein and daidzein are conju-
gated with glucuronic acid uridine diphosphate-glucuro-
nosyltransferase (UDPGT) becoming inactive, in this sense
Seppen (2012) [19] demonstrated that SIF, specifically ge-
nistein, could cause infertility in individuals with partial de-
ficiency in the enzyme UDPGT. Furthermore, Guan et al.
(2008) [20] showed that soybean isoflavones extract gastro-
gavaged daily at a range of 30 to 600 mg/kg body weight,
affecting the development of reproductive system in growing
rats; these dosages are about 50 to 1000 times of human in-
take level. Nevertheless, study performed by Nakai et al.,
(2005) [21] demonstrated that consumption of SIF does not
affect the reproductive tract of healthy animals. In addition,
other findings suggest that consumption of SIF does not ex-
ert a negative effect on reproductive system or circulant hor-
mone concentration in postmenopausal healthy women [22]
since studies have shown the role of SIF and soy foods not
only in preventing breast cancer but also in benefiting
women who have breast cancer [23]. Besides, the SIF also
helps in the chemotherapy treatment as shown in an animal
model for human breast cancer [24].
The Senate Commission on Food Safety of the German
Research Foundation opines that the consumption of SIF
isolated form or associated with food matrices may be re-
lated to the development of subclinical hypothyroidism [25].
On the other hand, studies on human ingesting up to 132
mg/day isoflavones reported small effects on thyroid hor-
mone levels, which are considered clinically insignificant
[26]. Furthermore, the majority of human studies have found
no significant effect of soy isoflavones on adult thyroid func-
tion [27]. In a recent study carried out with ovariectomized
Macaca fascicularis, the feeding of an amount of soy protein
that provides 9.3 mg isoflavones/kg body weight, demon-
strated no effect on thyroid function [28]. A recent study also
did not find correlations with soy isoflavones consumption
(80 and 120 mg/day) and thyroid function in postmenopausal
healthy women [22].
Despite conflicting results on the safety of isoflavones
and soy-based foods, increasing numbers of food products
containing different ratios of soy ingredients are now avail-
able on the market. In this way, other than endocrine effects,
further evaluation of the impact of dietary supplementation
with soy isoflavones on the clinical, haematological, bio-
chemical, histopathology, and growth parameters is required.
In this sense, this study was proposed to estimate the safe
level of soy isoflavones to be applied in the supplementation
of a protein bar soy-based in an acute and subacute toxicity
study in rats.
2. MATERIAL AND METHODS
2.1. Ingredients and Reagents
The SIF used in this study was provided by Pharma Nos-
tra insumos, Rio de Janeiro, Brazil. The purity of SIF was
Current Nutrition & Food Science, 2018, Vol. 14, No. 1 69
44.55% (1.23% of genistin, 0.14% of genistein, 9.55% of
daidzin, 32.57% of daidzein, 0.54% of glycitin, and 0.52% of
glycitein, and others) according to the manufacturer's speci-
fications.
The following ingredients were used to produce the soy-
based protein bars: isolated soy protein 90% (w/w) (free of
genetically modified organisms; Bremil Food Industries,
Passo Fundo, Brazil), whey protein concentrated 30% (w/w)
(Sooro Ingredientes, Paraná, Brazil), hydrolysed collagen
(Peptiplus® SB, Gelita do Brasil, Maringá, Brazil), sucralose
sweetener, citric acid anhydrous, soy isoflavones, malic acid
(all PharmaNostra Insumos, Rio de Janeiro, Brazil), soy leci-
thin emulsion (Grings Alimentos Saudáveis, São João da
Boa Vista, Brazil), 70% sorbitol solution (v/v) (PharmaNos-
tra Insumos), vitamin E acetate oil (Via Farma, São Paulo,
Brazil), PalmFat-370B (Agropalma, Belém, Brazil), and
toasted soy (Só Soja do Brasil Ltda., Caldas Novas, Brazil).
Other ingredients, such as NaCl, orange flavouring, glycer-
ine, oat bran and diet milk chocolate, were obtained from
local markets.
The reagents used for biochemical analysis were: alanine
transaminase (ALT) aspartate transaminase (AST) UV Wie-
ner, alkaline phosphatase (ALP) UV Wiener, albumin (Alb)
AA Wiener, total/direct bilirubin (BLT/BLD) Wiener, ki-
netic AA Wiener creatinine, Total Protein (TP) AA Wiener,
urea UV kinetic Wiener, high-density lipoprotein (HDL)
cholesterol monofase AA Wiener, -glutamyltransferase
(GGT) AA kinetics, Low Density Lipoprotein (LDL) choles-
terol Wiener, enzymatic cholesterol AA Wiener, enzymatic
uricostat AA Wiener, and colour triglyceride (TG) GPO-
PAP triglyceride Wiener; all supplied by diagnostic MS
LTDA, Cuiabá-MT, Brazil.
2.2. Animals
The acute and subacute toxicity assays were performed in
Wistar rats, 12- to 16-week-old obtained from the central
animal house of the University Centre of Lowland Large
(UNIVAG), Mato Grosso, Brazil. The animals were acclima-
tised for seven days before the acute and subacute assays.
They were kept in polypropylene cages at the animal facility
of the pharmacology laboratory under controlled temperature
(20 ± 5°C), with relative humidity (50 ± 5%), 12 h light/dark
cycle, and provided with water and feed (Nuvilab® autoclav-
able CR1 Sogorb, São Paulo, Brazil) ad libitum.
This study was approved by the Committee of the Insti-
tuto Federal de Mato Grosso animal Ethics-Campus Campo
Novo do Parecis, according to Law 11.794 dated 8 October
2008, which regulates scientific research involving animals
[29].
2.3. Preparation of SIF Solution
Prior to administering SIF to the animals, it was neces-
sary to adjust the concentration to 100% by using the correc-
tion factor (fc) of 2.24. After applying the fc, SIF was
weighed on an analytical balance, solubilised in distilled
water, and administered by gavage at a dose of 1 mL/100 g
body weight of the animals. The SIF solutions were prepared
daily, and administered doses were based on the body weight
of the animals.
70 Current Nutrition & Food Science, 2018, Vol. 14, No. 1
2.4. Acute Toxicity Assay
The acute toxicity test was conducted in male and female
rats (6/sex/group), which were divided in four groups, of
each sex. Animals were subdivided into two cages (3
rats/cage) and were fasted for 12 h prior to receiving a single
oral dose of SIF. The control group received only the vehicle
(water), while the test groups received 100, 500, or 1000
mg/kg of a SIF-solution. The animals were monitored indi-
vidually for clinical signs of toxicity or mortality by using
the Hippocratic screening according to Malone & Robichaud
(1962) [30] in the open 5, 10, 15, 30, 60, 120, and 240 min
after administration of SIF, and daily for a period of 15 days.
The animals were then euthanized with an overdose of keta-
mine and xylazine followed by removal of the liver, heart,
lungs, kidneys, stomach, and spleen. The relative weight of
the organs was determined [(organ weight / body weight) x
100], and the organs were not examined histologically.
2.5. Ration Formulation
The standard ration Nuvilab® CR1 was crushed and
mixed with soy-based protein bar (80:20, w/w) formulated
according to Faria et al (2017) [31]. Briefly, isolated soy
protein and whey protein concentrated (1:1, w/w) were ho-
mogenized with others dry ingredients in a powder mixer.
Batter was made by adding dry ingredients to the wet ingre-
dients (liquid and semi-solid) and then the mixture contain-
ing 80% of standard ration powder was shaped manually and
dried in a circulation oven at 35°C over 24 hours. Due to the
presence of soybean derivatives ingredients, the ration may
contain traces of isoflavones.
2.6. Subacute Toxicity Assay
The 30-days safety study was conducted in Wistar adult
rats (6/sex/group) feeding with standard ration incorporated
with 20% of soy-based protein bar ad libitum and different
dose levels of SIF of 0 (control), 100, 500 and 1000 mg/kg
body weight (bw) gastrogavaged daily.
Body weight and feed intake were monitored weekly.
Signs and symptoms of behavioural alterations were re-
corded, including, skin, eyes, gastrointestinal, respiratory,
Central Nervous System (CNS) and Peripheral Nervous Sys-
tem (PNS). At the end of the study, the animals were anes-
thetised with xylazine (10 mg/kg) and ketamine (80 mg/kg)
administered intramuscularly. Blood samples were then col-
lected by the vena cava. The animals were finally euthanized
by an overdose of ketamine and xylazine, and the sternum
bone was cut to remove the major internal organs.
The NOAEL was estimated according to Document 1A
(1993) of the United States Environmental Protection
Agency (EPA) [32], in which NOAEL is described as no-
observed-adverse-effect level, determined by no statistical
significance compared to controls with respect to decrease in
body weight gain, increased ratio of liver weight to body
weight, histopathology indistinguishable from controls and
elevated liver enzymes level.
2.7. Necropsy and Histological Analysis
After 15 days in the acute safety study and after 30 days
of treatment in the subacute safety study, all Wistar rats were
Faria et al.
weighed and necropsied. Description of all macroscopic ab-
normalities was recorded. The organs were weighed, and the
absolute and relative weights were determined. The organs
were then placed in 10% formalin prior to performing the
histopathological analysis.
Fragments of the lungs, heart, liver, spleen, kidneys, and
stomach were collected, fixed in 10% formalin, and proc-
essed by a conventional histopathological method. This was
followed by staining with haematoxylin and eosin (H&E)
and visualisation by optical microscopy. The animal tissues
were examined blindly, and the criteria for assessing the oc-
currence of lesions in organs were according to the method-
ology proposed by Jesus et al. (2012) [33].
2.8. Haematology and Serum/Plasma Chemistry
For biochemical analysis, blood samples were collected
in anticoagulant-free Vacutainer® tubes and centrifuged at
3000 g, at 4°C for 10 min. Serum was separated and stored at
-20°C until determination of glucose (GLU), urea (Ur),
creatinine (Cre), total cholesterol (CHO), HDL, LDL, and
very-low-density lipoprotein (VLDL) cholesterol, TG, uric
acid (UA), BLT, BLD, indirect bilirubin (BLI), TP, Alb,
ALT, AST, amylase (AMI), -glutamyltransferase (GGT),
and alkaline phosphatase (FS) in an automatic biochemical
analyser 3000 BT/CB 350i (Wiener lab group, Argentina).
For analysis of haematological parameters, blood samples
were collected in ethylenediaminetetraacetic acid (EDTA)
Vacutainer® tubes. The contents of red blood cells (haemo-
globin and haematocrit), platelets and leukocytes (eosino-
phils, basophils, lymphocytes, monocytes; rod and seg-
mented myelocytes, and metamyelocytes), were determined
using a veterinary haematology analyser, Poch-100iV Diff
(Sysmex of Brazil Ind. and Com. Ltd, Brazil).
2.9. Data Analysis
The results of parametric tests were expressed as mean ±
standard error of the mean (SEM). In experiments comparing
more than two means, one-way analysis of variance
(ANOVA), the significance by F-test at 5% probability, and
contrast between the means by Tukey-Kramer test were used
to identify differences between the treatments. All statistical
analyses were performed using GraphPad Prism 5.00® pro-
gram (GraphPad Software, Inc., San Diego, CA).
3. RESULTS
3.1. Acute Toxicity Assay
In the acute safety study, all animals survived the 2-
weeks observation period. Besides, neither the females nor
the males Wistar rats showed abnormal signs and behavioral
changes as evaluated by the Hippocratic test for doses up to
1000 mg/kg.
In both sexes, food consumption was unaffected by the
treatment (data not shown). Thus, body weight gain and final
body weight were similar to control values (Fig. 1) in both
the sex groups.
No significant difference was observed in the relative
weight of organs (p>0.01) of male rats in the different SIF-
treated groups as compared with the control group. However,
Safety of Soy Isoflavones as Dietary Supplement Current Nutrition & Food Science, 2018, Vol. 14, No. 1 71

body weight of female rates (grams)

500
400

body weightmale rates (grams)

initial weight lnitial weight
450 final weight
Final weight
300
400

350 200

300
100
250

200 0

kg
kg
kg

kg
l

kg
kg
ro
ro

g/
g/
g/

g/

g/
nt

g/
nt

m
m
m

m
m
Co
Co

00
0
0

00
0
50
10

10

50
10

10
Isoflavones dosage Isoflavones dosage

Fig. (1). Change in body weight of male and female Wistar rats during acute toxicity study.

Table 1. Final body weights and selected absolute and relative organ weights from the acute study with Wistar rats treated with
singles doses of soy isoflavones.

Males Females

Control 100 mg/kg 500 mg/kg 1000 mg/kg Control 100 mg/kg 500 mg/kg 1000 mg/kg

Final body weight (g) 415.66±16.20 455.5±24.54 453.00±24.24 418.66±15.66 279.5±13.30 262.05±13.01 263.66±8.86 260.16±11.53

Spleen weight (g) 0.96±0.05 1.08±0.05 1.08±0.06 1.05±0.1 0.90±0.07 0.77±0.04 0.87±0.04 0.93±0.06

Relative spleen weight

0.23±0.009 0.23±0.007 0.23±0.008 0.24±0.01 0.33±0.03 0.29±0.01 0.34±0.02 0.36±0,03
((g/g body weight) x 100)

Kidneys weight (g) 2.76±0.22 3.17±0.16 3.04±0.17 3.02±0.16 2.25±0.14 2.22±0.11 2.31±0.11 2.16±0.10

Relative kidneys weight

0.66±0.03 0.70±0.03 0.66±0.01 0.72±0.01 0.81±0.03 0.85±0.03 0.91±0.05 0.84±0.07
((g/g body weight) x 100)

Liver weight (g) 11.93±0.73 14.58±0.90 13.83±0.77 13.52±0.40 9.47±0.37 8.68±0.49 8.91±0.26 10.48±0.28

Relative liver weight

2.86±0.09 3.22±0.15 3.04±0.1 2.99±0.04 3.34±0.11 3.32±0.12 3.50±0.09 4.08±0.25*
((g/g body weight) x 100)

Heart weight (g) 1.17±0.08 1.23±0.08 1.24±0.04 1.21±0.07 0.90±0.06 0.93±0.07 0.89±0.04 0.89±0.06

Relative heart weight

0.28±0.01 0.27±0.006 0.27±0.003 0.29±0.008 0.32±0.01 0.35±0.01 0.35±0.02 0.35±0.03
((g/g body weight) x 100)

Lungs weight (g) 1.77±0.14 2.13±0.08 2.01±0.18 1.64±0.07 1.57±0.16 1.54±0.13 1.42±0.04 1.52±0.11

Relative lungs weight

0.42±0.02 0.47±0.02 0.44±0.03 0.39±0.009 0.56 ± 0.04 0.58 ± 0.03 0.56±0.01 0.59±0.06
((g/g body weight) x 100)

Stomach weight (g) 2.14±0.10 2.55±0.18 2.43±0.21 2.23±0.15 1.97±0.14 1.90±0.17 1.85±0.07 1.78±0.12

Relative stomach weight

0.52±0.02 0.55±0.0.1 0.53±0.03 0.52±0.01 0.68±0.04 0.72±0.03 0.73±0.04 0.69±0.07
((g/g body weight) x 100)
The results were expressed as mean ± standard error of mean (SEM). Analysis by ANOVA followed by Tukey-Kramer test. *p  0.05 and **p  001 represent significant differences
from the control group.

in female rats, there was an increase in the relative liver 3.2. Subacute Toxicity Assay
weight in the group treated with SIF at 1000 mg/kg (10.66
3.2.1. Body Weight and Feed Intake
%, p<0.05) (Table 1), but no macroscopic findings were
noted at necropsy. It has been concluded that SIF has a low As can be seen in Fig. (2) over the study period, males
order of acute toxicity and that the oral lethal dose for male treated with 100, 500, and 1000 mg/kg bw of SIF showed
and female rats is more than 1000 mg/kg bw. 3.68, 4.07, and 3, 41% decrease in body weights, respec-
72 Current Nutrition & Food Science, 2018, Vol. 14, No. 1 Faria et al.

Control

body weight of male rates (grams)

body weight of female rates (grams)

Control
400 100 mg/kg b.w.
240 100 mg/kg b.w.
500 mg/kg b.w.
500 mg/kg b.w.
100 mg/kg b.w. 100 mg/kg b.w.

360 200

320 160
0 10 20 30 0 10 20 30

Treatment time Treatment time

Fig. (2). Effects of diet and isoflavones (SIF) on body weight at 30-days subacute toxicity assay.

120

F e m a l e f e e d i n ta k e ( g r a m s )
Control Control
M a l e f e e d i n ta k e ( g r a m s )

100 mg/kg b.w. 100 mg/kg b.w.

90
500 mg/kg b.w. 500 mg/kg b.w.

1000 mg/kg b.w. 1000 mg/kg b.w.

60

30

2 3 4 5 2 3 4 5
1

W
1

W W W W W W W
W
W

Feed intake from each six days Feed intake from each six days

Fig (3). W1-W5: Average feed intake every 6 days. Results are expressed as mean ± standard error of mean (SEM) and analysed by ANOVA
followed by Tukey-Kramer test. *p  0.05 and **p  0.01 represent significant differences compared to the control group.

tively, while the control group showed a weight gain of 3, (23.46%, p<0.01), and 1000 mg/kg bw/ week (20.44%,
73% (no statistically significant), but the decrease in weight p<0.01) as compared to that of the control. Among females,
gain of group treated with 500 mg/kg bw was significant as decrease in food consumption was approximately 17% in
compared to control (p<0.05). On the other hand, in treated overall, but the significant decrease was observed in the first
females, body weight gains and final body weights were week by groups treated with 500 (34, 20%, p<0.01) and
similar to the control values, showing that the group that 1000 mg/kg bw/week (31,04%, p<0.01) as compared to con-
received 1000mg/kg showed a gain of 9.8% compared to trol group. Despite the reduced food consumption observed
control (p>0.05). Throughout the study, only control and in the first week, all female treated groups showed an in-
female treated with 1000 mg/kg bw gained weight, with crease in the last four weeks of treatment (W2 - W5).
mean female body weight gain (± standard error) being
16.1±3.1g and control being 22.9±4.8 g to 1000 mg/kg bw; 3.2.2. Necropsy and Histological Analysis
while SIF-treated lowest and mid-dose showed a decrease of The male Wistar rats treated with 1000 mg/kg of SIF,
1.38±0.6 g and 2.34±0.9g (mean± standard error, p>0.05). showed significant (p>0.01) decreases in relative and abso-
The overall food consumption was decreased by ap- lute weight of all the major organs analysed compared to the
proximately 14% in males at 500mg/kg bw/week and ap- control group, except for the stomach (Table 2). However,
proximately 23% in the last 2-weeks (W4-W5); these values values for other groups did not differ significantly from the
were statistically significant based on the first (W1, p<0.01), control. On the other hand, female that received 1000mg/kg
fourth (W4, p<0.01) and fifth (W5, p<0.01) week of con- showed an increase in relative and absolute liver weight, but
trolled feed intake compared to that of the control (Fig. 3). no significant changes were observed in relative or absolute
weight of other organs.
A significant decrease in feed intake was observed during
the last 12 days of treatment (W4-W5, 19-30 days) in the At necropsy, there were no macroscopic findings consid-
male groups treated with 100 (19.13%, p  0.05), 500 ered to be related to treatment.
Safety of Soy Isoflavones as Dietary Supplement Current Nutrition & Food Science, 2018, Vol. 14, No. 1 73

Heart Liver Kidney

A1. Control B1. Control C1. Control

A2. 100 mg/kg B2. 100 mg/kg C2. 100 mg/kg

A3. 500 mg/kg B3. 500 mg/kg C3. 500 mg/kg

A4. 1000 mg/kg B4. 1000 mg/kg C4. 1000 mg/kg

Fig (4). Photomicrographs of the major organs (200 μ) analysed histologically.

Table 2. Final body weights and selected absolute and relative organ weights from the subacute study with Wistar rats treated with
singles doses of soy isoflavones.

Males Females

Control 100 mg/kg 500 mg/kg 1000 mg/kg Control 100 mg/kg 500 mg/kg 1000 mg/kg

Final body weight (g) 383.89±7.50 359.98±15.41 369.89±4.33 370.92±10.52 209.06±10.80 201.91±11.00 194.79±9.88 229.56±8.25

Spleen weight (g) 1.03±0.03 1.07±0.06 1.09±0.02 0.74±0.02** 0.66±0.02 0.74±0.04 0.67±0.03 0.85±0.04

Relative spleen weight

0.27±0.008 0.29±0.01 0.28±0.007 0.20±0.009** 0.31±0.01 0.36±0.007 0.34±0.01 0.037±0.007
((g/g body weight) x 100)

Kidneys weight (g) 2.73±0.07 2.56±0.12 2.86±0.12 1.87±0.05** 1.74±0.09 1.62±0.08 1.49±0.06 2.05±0.1

Relative kidneys weight

0.71±0.01 0.71±0.04 0.74±0.02 0.51±0.03** 0.83±0.03 0.80±0.02 0.76±0.02 0.84±0.04
((g/g body weight) x 100)

Liver weight (g) 10.37±0.27 8.63±0.36* 9.95±0.31 6.77±0.29** 6.44±0.27 6.20±0.34 6.12±0.11 8.73±0.17**

Relative liver weight ((g/g

2.70±0.07 2.42±0.15 2.60±0.06 1.83±0.09** 3.09±0.11 3.07±0.06 3.18±0.13 3.83±0.13**
body weight) x 100)

Heart weight (g) 1.21±0.06 1.05±0.05 1.24±0.03 0.77±0.03** 0.79±0.04 0.77±0.05 0.72±0.03 0.83±0.02

Relative heart weight

0.31±0.01 0.29±0.02 0.32±0.008 0.20±0.01** 0.38±0.01 0.38±0.01 0.037±0.01 0.036±0.01
((g/g body weight) x 100)

Lungs weight (g) 1.55±0.09 1.50±0.09 1.64±0.03 1.13±0.03** 1.14±0.04 1.15±0.06 1.09±0.05 1.36±0.03

Relative lungs weight

0.40±0.02 0.41±0.01 0.43±0.01 0.30±0.01* 0.55±0.02 0.57±0.01 0.56±0.01 0.59±0.01
((g/g body weight) x 100)

Stomach weight (g) 1.94±0.10 1.83±0.15 1.93±0.10 1.59±0.11 1.42±0.13 1.34±0.08 1.40±0.12 1.74±0.06

Relative stomach weight

0.50±0.02 0.50±0.03 0.50±0.02 0.43±0.04 0.66±0.03 0.67±0.05 0.71±0.04 0.76±0.03
((g/g body weight) x 100)

The results were expressed as mean ± standard error of mean (SEM). Analysis by ANOVA followed by Tukey-Kramer test. *p  0.05 and **p  001 represent
significant differences from the control group.
74 Current Nutrition & Food Science, 2018, Vol. 14, No. 1
Fig. (4) illustrates photomicrographs of heart, liver, and
kidney tissues. There were no treatment related histopa-
thologic findings observed after 30-days subacute safety
study of SIF. No apparent histological changes were found in
the comparative analysis of tissue from the organs of treated
and control groups, and there were no histologic correlation
for changes in the organ’s weight.
3.2.3. Haematology and Serum/Plasma Chemistry
After 30 days of treatment, both male and female rats had
a significant increase in platelets cell counts. The increase
among male rats was observed at dosage of 100 (4.04%,
p<0.05), 500 (9%, p<0.05), and 1000 mg/kg bw (14.15%,
p<0.05), while among females, the increase was seen at the
highest dosage (25.48%, p<0.05), as compared to the control
(Table 3).
A significant decrease was observed in the relative values
of leukocytes in male groups treated at the doses of 100
(21.47%, p<0.05), 500 (21.15%, p<0.05) and 1000 mg/kg
(33.05%, p<0.05) compared to that of control.
Biochemical findings in treated male rats showed signifi-
cant increase in the uric acid levels of 122.22% (p<0.01) and
91.53% (p<0.01) at 500 and 1000 mg/kg bw of SIF com-
pared to control, respectively. A no dose-dependent increase
in AST enzyme was seen among males receiving 100
(19.8%, p<0.05), 500 (60.59%, p<0.05), and 1000 mg/kg bw
(29.34%, p<0.05), but in female groups, the AST value
showed a dose-dependent decrease (p<0.01). However, it is
worth highlighting that AST value was within the reference
value [34].
Other biochemical alteration showed by female data was
a dose-dependent increase in ALP, in which, 500 and 1000
mg/kg bw of ISF increased in 39.48% (p<0.01) and 50.28%
(p<0.01) compared to control, nevertheless all ALP values
obtained were also within the reference value [34].
No other statistically significant difference between the
treatment and control groups was observed in haematological
and biochemical analyses.
3.2.4. The NOAEL
Considering the NOAEL as the highest experimentally
determined dose without a statistically or biologically sig-
nificant adverse effect (EPA) [32], in front of the results
achieved in this study, the SIF NOAEL was determined to
100mg/kg/day since the significant haematological or bio-
chemical alterations observed were within the reference val-
ues and no other clinical or histopathological findings were
observed.
4. DISCUSSION
The acute toxicity test with SIF did not show any clinical
signs, behavioural abnormalities, or death in treated animals.
It was, therefore, impossible to determine the LD50 of the
SIF containing a larger amount of daidzein (~33%) followed
by daidzin (~10%), genistin (~2%), and other soy isofla-
vones at doses up to 1000 mg/kg bw. Following acute ad-
ministration of a soybean extract that contains 10% of glyco-
side isoflavones (~50% of daidzin, ~40% of glycitin, and
~10% of genistein) combined to L-carnitine, a single limit
Faria et al.
dose of 2000 mg/kg bw did not cause mortality or adverse
effects were observed during a 14-day period or upon gross
pathological examination [35]. Dosage of 4500mg/kg of isofla-
vones (25.6% daidzein, 50.0% genistein, 8.4% glycetin, and
others) was assessed in subchronic study performed in male
rats by Zhang et al. (2009) [2] and no death was observed;
although they found an endocrine disruption at this dosage
that caused hyperplastic and lactational mammary glands.
They showed a decrease in blood total testosterone levels in
the 0.5, 1.5 and 4.5 g/kg without dose-response relationship.
Although no adverse effects were observed, in the acute
assay, there were a significant difference in the relative or-
gan weight of females’ liver between the highest dosage SIF-
treated and control groups, but no alteration was observed
upon gross pathological examination. One of the major ob-
jectives of any preclinical toxicity study is to identify target
organs which help the clinicians to monitor related adverse
effects during clinical development [36] and organ weight is
one of the most sensitive drug toxicity indicators, and
changes in it often precede morphological changes [37]. In
the subacute study, the female rats that received the highest
dosage of SIF also showed an increase in relative and abso-
lute weight of the liver. According to Nirog et al. (2014)
[36], a minimal increase in the liver weight without any mi-
croscopic lesions can be correlated with enzyme induction.
Though the serum AST and ALT values in treated groups
were lower than in the control group, ALP, frequently corre-
lated to bone or hepatobiliary diseases, was seen to signifi-
cantly increase (p<0.01) in 500 and 1000 mg/kg bw; how-
ever, both were within the reference values.
The results of the analysis of the biochemical parameters
showed a significant change in the serum AST enzyme on
male treated with 500 mg/kg SIF as compared to control.
However, this increase is within the limit stipulated in the
table forwarded by Giknis and Clifford (2008) [34]. The re-
sults of the determination of serum AST show that this de-
viation was not dose-dependent since the group that received
1000 mg/kg of SIF showed lower serum levels of this en-
zyme as compared to the group treated with 500 mg/kg. In-
crease in serum AST and ALT enzymes can be indicative of
some hepatic damage or possible damage to the heart muscle
[38]. The enzyme ALT has increased specificity for diagnos-
ing liver injury. However, another nonhepatic abnormality as
rhabdomyolysis can also increase these enzymes according
findings reported by Wheibrecht et al. (2010) [39].
A dose-dependent decrease in the content of AST was
observed when the soybean extract with purity of 94% (w/w)
saponins and 6% of isoflavones (3.7% daidzein, 1.6% gly-
citein and 0.6% genistein) was administered at dosages of
2.5 and 5% [40]. Recent studies have demonstrated the hepa-
toprotective role of soy isoflavones. Liu et al. (2016) [41]
found decreased plasma ALT and AST activity in rats with
the liver injury induced by acetaminophen treated with 120
mg/kg of SIF. Sarhan et al. (2012) [42], also showed a lower
plasma AST and ALT activity in rats with liver damage in-
duced by CCl4 treated with isoflavones-enriched soy protein
(167.3 mg/100 g soy protein) containing a higher amount of
daidzin (~90 mg/100 g), followed by daidzein (~50 mg/100
g), genistin (~20 mg/100 g), and genistein (~10 mg/100 g).
Besides, findings about the protective performance of soy
Safety of Soy Isoflavones as Dietary Supplement Current Nutrition & Food Science, 2018, Vol. 14, No. 1 75

Table 3. Effects of diet and soy isoflavone supplementation on haematological and biochemical parameters of female and male rats after 30 days.

Haematological Males Females

parameters
Control 100 mg/kg 500 mg/kg 1000 mg/kg Reference Control 100 mg/kg 500 mg/kg 1000 mg/kg Reference

Red blood cells

8.2±0.06 8.3±0.11 8.05±0.16 8.14±0.17 7.62 -9.99 8.0±0.10 6.9±0.34 6.9±0.42 7.57±0.14 7.16-9.94
(106/L)

Haemoglobin
15.6±0.16 15.6±0.25 15.04±0.34 15.3±0.33 13.6-17.4 15.63±0.15 13.7±0.73 13.46±0.78 15±0.24 13.7-17.2
(g/dL)

Haematocrit (%) 46.1±0.64 46.5±0.69 47.4±1.34 46±1.13 38.5-52 53.33±0.98 47±2.75 43.66±2.51 45±0.78 38.5-49.2

Platelets (103/ L) 544±9.64 566±8.36* 593.2±9.52* 621.3±9.31* 574-1253 416±10 376±11.3 457±28.86 522±11.86* 599-1114

Leukocytes
4033±19.24 3167±30.42* 3180±17.88* 2700 ± 23.57* 1980-11060 3967±98.13 3180±71.5 4067±153 3117±160 960-7880
(103/mm3)

Segmented (%) 23.5±1.07 19.8±1.49 25.2±3.27 26.3±2.56 9-49.3 16.6±1.78 16.6±0.45 18.3±1.61 24±2.12 8.8-43.8

Eosinophils (%) 2.0±0.47 2.5±0.84 2.8±0.91 1.5±1.19 0.4-4 3±0.27 3±0.43 4±0.20 2±0.15 0.3-4.7

Basophils (%) 0 0 0 0 0-0.6 0 0 0 0 0-0.7

Lymphocytes (%) 73±1.13 76.3±3.24 70.6±3.24 70.5±1.5 44.7-87.1 78.66±3.14 78.6±1.73 76.66±2.24 56.16±10.77 48.9-88.1

Monocytes (%) 1.5±0.20 1.3±0.21 1.4±0.21 1.6±0.19 1-3.6 2±0.0 2±0.21 2±0.20 2±0.19 1-3.6

Serum/Plasma
Control 100 mg/kg 500 mg/kg 1000 mg/kg Reference Control 100 mg/kg 500 mg/kg 1000 mg/kg Reference
Chemistry

Glu (mg/dL) 158.167±14.54 139±10.67 153±7.88 161±21.75 106-184 160.6±15.17 154±10.63 151.2±16.50 149.8±13.42 89-163

AMI (U/L) 551.83±9.33 487±24.64 506.83±29.96 512.167±19.93 1223-2109 364±30.57 316±15.71 379±20.70 406±37.78 866-1642

Ur (mg/dL) 44.83±1.57 49.5±2.27 40±2.62 45.5±1.83 12.3-48.6 43.2±1.71 38.8±2.24 35.6±2.18 45.3±1.89 11.7 - 45

Cre (mg/dL) 0.56167±0.01 0.5083±0.09 0.6367±0.04 0.5967±0.01 0.2-0.5 0.45±0.01 0.36±0.04 0.40±0.02 0.54±0.02 0.3-0.6

CHO (mg/dL) 40.67±1.42 43.83±1.47 43.6±1.57 39.83±2.85 37-95 35.4±2.63 30.2±2.50 28±2.68 32.8±2.32 23-97

LDL (mg/dL) 8.67±1.33 8.83±1.62 7.5±0.6 7.167±1.01 3.6±0.8 3.8±0.4 2.8±0.5 3.6±0.3

VLDL (mg/dL) 5.167±0.30 6±0.51 7.1±1.67 6.67±1.40 9,6±1.28 6.53±1.02 7.8±0.73 10.1±1.37

HDL (mg/dL) 27.3±0.71 28.83±0.94 29.3±1.62 26±1.50 21.6±2.2 19.83±1.85 17.6±2.15 19±1.15

TG (mg/dL) 30.66±1.33 29.167±2.65 34.83±1.42 32.33±1.05 27-170 46.2±2.8 45.5±2.6 46.4±3.26 44.16±3.07 16-175

UA (mg/dL) 0.567±0.03 0.75±0.03 1.26±0.06** 1.083±0.09** 1.58±0.07 1.58±0.1 1.38±0.1 1.11±0.1

AST (U/L) 96±2.92 115±4.75* 154.16±1.93** 124.16± 5.66** 63-175 146±10.12 100±4.63** 85.4±2.67** 85.1±3.71** 64-222

ALT (U/L) 44±1.50 41.83±3.33 49±2.14 43.83±2.006 19-48 44.4±3.15 33.5±1.52* 39.2±1.68 36.8±2.65 14-64

GGT (U/L) 3.167±0.47 2.83±0.4 3.16±0.30 3±0.25 2.60±0.2 2.16±0.3 2.2±0.2 3±0.2

ALP (U/L) 150.83±1.9 156.83±1.19 152.5±3.82 155.67±2.33 62-230 70.4±2.78 75±0.8 98.2±3.82** 105.8±0.9** 62-230

BLT (mg/dL) 0.08167±0.01 0.1067±0.01 0.08167±0.024 0.14±0.01 0.04-0.2 0.07±0.001 0.07±0.009 0.07±0.006 0.07±0.004 0.07-0.21

BLD (mg/dL) 0.02±0.003 0.005±0.003 0.07±0.025* 0.1167±0.016* 0.03-0.06 0.014±0.004 0.006±0.003 0.014±0.002 0.016±0.002 0.03-0.07

-18 -18
BLI (mg/dL) 0.1±6.2010 0.1±6.2010 0.0183*±0.01* 0* 0-0.1 0.058±0.007 0.06±0.01 0.06±0.005 0.05±0.004 0.02-0.13

TP (g/dL) 6.483±0.07 6.483±0.10 6.6167±0.14 6.7167±0.11 5.6-7.6 6.16±0.09 5.40±0.13* 6.08±0.14 6.58±0.12 5.7-8.3

Alb (g/dL) 4.1±0.03 4.13±0.06 4.1167± 007 4.083±0.14 3.7-4.6 4.12±0.05 3.63±0.10* 3.96±0.02 4.28±0.06 3.7-5.8

Results are expressed as mean ± standard error of mean (SEM). One-way ANOVA followed by Tukey-Kramer test. * p  0.05 and ** p  0.01 represent significant differences from
the control group. Glu, glucose; AMI, amylase; Ur, urea; LDL, low density lipoprotein; VLDL, very low-density lipoprotein; HDL, high density lipoprotein; TG, triglycerides; CHO,
total cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, -glutamyltransferase; Alb, albumin; TP, total protein; UA, uric
acid; Cre, creatinine; ALP, alkaline phosphatase; BLT, total bilirubin; BLD, direct bilirubin; BLI, indirect bilirubin. Giknis and Cilfford (2008)34
76 Current Nutrition & Food Science, 2018, Vol. 14, No. 1
isoflavones against fatty liver disease such as non-alcoholic
and alcoholic fatty liver diseases have also been reported
[43, 44].
A significant decrease was observed in the relative and
absolute weight of liver, lungs, kidneys, spleen, and heart in
the present subacute toxicity test at a dose of 1000 mg/kg
bw. This finding does not corroborate the results obtained by
Cho et al., (2009) [40]; the authors reported a significant
dose-dependent increase in the relative liver weight after
treatment with extracts containing 6% soy isoflavones ad-
ministered at doses of 1.25% and above groups of inbred
F344 male rats for 13 weeks. The decrease in organs’ weight
is frequently associated with organs’ dysfunction and atro-
phy [37] which can be correlated with biochemical findings.
The biochemical liver findings in males suggest a possible
biliary obstruction, due to the predominance of increased
direct bilirubin observed in the dosage of 1000 mg/kg bw.
However, other biochemical markers of liver function, such
as GGT, ALP and serum albumin did not show significant
alteration. Another parameter that can be correlated to he-
patic dysfunction is the platelets count. The reduction in the
amount of platelets in the blood is common in individuals
with liver diseases, which may be accompanied by sple-
nomegaly by the increase of sequestration and destruction of
these; but these alterations were not found on hematological
and necropsy analysis of both the sex. In addition, rather
than splenomegaly, in male treated with the highest dose of
SIF, a decrease in spleen mass was observed.
Urea, a marker of renal function, was within the refer-
ence range. However, the serum creatinine concentration was
slightly higher than the reference range values in the males
treated with 500 mg/kg bw, and 13.35% higher than the con-
trol group (p>0.05). This finding does not appear to be re-
lated to decrease in absolute and relative weight of kidneys
since the increase in serum creatinine was not dose-
dependent and was not statistically significant.
A significant increase in the serum UA in 500 and 1000
mg/kg bw SIF-treated groups compared to control was ob-
served. Its increase may be correlated with renal dysfunction
affecting the UA excretion. This increase was not dose-
dependent, as can be seen in Table 3. Moreover, the values
obtained were below the reference range values reported by
taconic, animal models (1998) by Diniz et al. apud (2006)
[45]. The reference value for this parameter is 4.3 ± 0.35
mg/dL, which indicates that this difference is not related to
renal damage or any other metabolic disorder.
Mean biweekly body weights in subacute assay de-
creased from those of controls in treated males throughout
the study, besides, male rats exhibited a consistent pattern of
reduced feed intake. This pattern appears to reflect poor pal-
atability of the modified diet or stress caused by daily gas-
trogavage. In treated females, some variability in feed con-
sumption, body weight gain, and feed efficiency (data not
shown) was seen, generally indicating feed avoidance for the
first few days, but recovery was evident by about day 7.
These effects on body weight was similar as reported by
Guan et al., (2008) [20], where the authors found a dose-
dependent suppression on body weight gain after soybean
isoflavones extract’s administration, and the effect was
greater on male than on female rats.

Faria et al.
According to Che et al., (2011) [5], body weight change
is an important parameter to be considered in the side effects
of drugs and chemicals under the subchronic toxicity study.
Nevertheless, these signs were not accompanied by macro-
scopic or microscopic changes in the organs of animals or
any symptoms of toxicity, which may indicate that these
changes are not toxicologically significant. Zhang et al.,
(2009) [2] and Cho et al., (2009) [40] showed similar results
with the suppression of appetite and loss of body weight fol-
lowing administration of 1.5 and 4.5 g/kg of soybean extract
containing6% of isoflavones (3.7% daidzein, 1.6% glycitein
and 0.6% genistein), but it is worth emphasizing that the SIF
composition used in this study is different from those as-
sessed in other studies, with daidzein being the main isofla-
vone present. Che et al. (2011) [5] assessed the subchronic
toxicity of soybean extract that contains 10% of glycoside
isoflavones composed of ~50% daidzin, 40% glycitin, and
10% genistin, combined with L-carnitine (5:3 w/w) and
found a decrease in body weight of female adult rats treated
with 2000mg/kg/day for 90 days; but no others side effects
or histopathological abnormalities were observed. Cederroth
et al. (2007) [46] correlated diets high in soy isoflavones
with lower caloric and fat intakes. These data can also be
related to the ability of SIF to suppress weight gain by in-
creasing the levels of leptin and decreasing adiponectin in
plasma [47]. In addition, increase in the levels of serotonin
[48] and decrease in the intestinal absorption of lipids may
be involved [49].
A dose-dependent decrease in serum leukocytes level
was observed in the SIF-treated male groups. However, these
values are within the reference range for male rats aged 17
weeks or greater, indicating that these results present no
clinical value [34].
The SIF and other soybean ingredients have shown bene-
ficial effects on lipid profile in mice46 and human [50].
However, this study showed no significant effects (p>0.05)
in reducing the total cholesterol, reducing LDL, increasing
HDL, or decreasing TG, contrary to the findings reported by
Zhang et al. (2009) [2] that showed a decrease in blood
triglycerides and increase in HDL. Furthermore, no decrease
in glucose rate was observed in this study, as also reported in
a study by Liu et al. (2010) [51], in which the authors as-
sessed the effect of soy protein and isoflavones on post-
menopausal Chinese woman.
CONCLUSION
In conclusion, the results obtained in the acute toxicity
test indicate that SIF has a high margin of safety as shown by
the LD50.In addition, subacute toxicity assay showed that a
snack soybean-based protein bar containing traces of isofla-
vones supplemented with SIF could be safe for daily con-
sumption, since the No-Observed-Adverse-Effect Level
(NOAEL) of SIF in male and female rats in this study was
considered to be 100 mg/kg/day. However, the chronic toxic-
ity cannot be estimated.
LIST OF ABBREVIATIONS
Alb = Albumin
ALP = Alkaline Phosphatase
Safety of Soy Isoflavones as Dietary Supplement Current Nutrition & Food Science, 2018, Vol. 14, No. 1 77

ALT = Alanine Transaminase CONSENT FOR PUBLICATION

AMI = Amylase Not applicable.
ANOVA = Analysis of Variance
CONFLICT OF INTEREST
ANVISA = Health Surveillance Agency
The authors declare no conflict of interest, financial or
AST = Aspartate Aminotransferase otherwise.
BLD = Direct Bilirubin
ACKNOWLEDGEMENTS
BLI = Indirect Bilirubin
We thank the National Council for Scientific and Tech-
BLT = Total Bilirubin
nological Development (CNPq) and the Research Support
BLT/BLD = Total/Direct Bilirubin Foundation of Mato Grosso for financial support.
CHO = Total Cholesterol We also thank the Federal Institute of Mato Grosso, Bela
Vista Campus, and the Laboratory of Instrumental Analysis
CNS = Central Nervous System
at the Federal University of Mato Grosso and University
Cre = Creatinine Centre of Várzea Grande for providing the infrastructure
CVD = Cardiovascular Disease required to carry out this study.

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