Accepted Manuscript Title: Subacute and subchronic toxicity of Avalon® mixture (bentazone+dicamba) to rats Author: Dragica Brki´c Ilona Szakonyne-Pasics Slavica Gaˇsi´c Ivana Teodorovi´c Boˇzidar Raˇskovi´c Nenad Brki´c Neˇsko Neˇskovi´c PII: DOI: Reference:
S1382-6689(15)00068-X http://dx.doi.org/doi:10.1016/j.etap.2015.03.004 ENVTOX 2221
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
12-9-2014 28-2-2015 3-3-2015
Please cite this article as: Brki´c, D., Szakonyne-Pasics, I., Gaˇsi´c, S., Teodorovi´c, I., Raˇskovi´c, B., Brki´c, N., Neˇskovi´c, N.,Subacute and subchronic toxicity of Avalonregd mixture (bentazone+dicamba) to rats, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Subacute and subchronic toxicity of Avalon mixture (bentazone+dicamba) to rats Dragica Brkić1a, Ilona Szakonyne-Pasics1b, Slavica Gašića, Ivana Teodorovićc, Božidar Raškovićd, Nenad Brkiće and Neško Neškovića 1
Corresponding author:
Ac ce p
te
d
Dragica Brkić Banatska 31-b 11080 Belgrade-Zemun Serbia Telephone: +381113076136 Mobile: +38163571500 Fax: +381113076133 E-mail: [email protected]
M
an
us
cr
ip t
LAB International Hungary Ltd., Szabadságpuszta, H-8200 Veszprém, Hungary (now CiToxLAB Hungary Ltd., Szabadságpuszta, H-8200 Veszprém, Hungary) a Institute of Pesticides and Environmental Protection, Banatska 31-b, 11080 BelgradeZemun, Serbia, [email protected], [email protected], [email protected] b Toxi-Coop, Toxicological Research Center Zrt., Pálya u. 2, H-2120 Dunakeszi, Hungary, [email protected] c University of Novi Sad, Faculty of Sciences, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia, [email protected] d University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-Zemun, Serbia [email protected] e Ministry of Agriculture and Environmental Protection, Directorate for Water, Bulevar umetnosti 2a, 11070 Belgrade, Serbia, [email protected]
Page 1 of 29
ip t
Subacute and subchronic toxicity of Avalon mixture (bentazone+dicamba) to rats
ABSTRACT
cr
Subacute and subchronic toxicity of the herbicide Avalon®, a mixture of bentazone and dicamba, were tested on rats. Avalon® was administered at dose levels of 250, 500 and 1000
us
mg/kg body weight/day for 28 and 90 days. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were monitored together
an
with biochemistry parameters. The results showed that the mixture caused increases in the activities of ALT, AST and ALP, elevated concentrations of sodium, albumin and albumin/globulin ratio in males. In females, ALT activity, cholesterol and phosphate levels
M
were increased. The changes generally were dose related and, in most cases, females exhibited lower susceptibility than males. The effects of a mixture are, in the most cases, different from the effects of the individual substances. The effects of bentazone were not
te
d
prevalent which would be expected taking the composition of the mixture into account.
Ac ce p
Keywords: Mixture; Bentazone; Dicamba; Rat; Subacute Toxicity; Subchronic Toxicity
Page 2 of 29
1. INTRODUCTION Pesticides are biologically active substances designed to be toxic, which causes growing concerns about their health impacts on many non-target species. Unlike other pollutants, pesticides are deliberately spread into the living and working environments. Because of their
ip t
ubiquitous presence people are constantly exposed to low levels of pesticides (Corsini et al., 2013).
cr
Pesticide active substances are commonly formulated before application. In the process of
developing formulations, manufacturers may use one or more active substances in order to
us
produce effective and long lasting products (Knowles, 2005). The combined effects of two or more active substances are very difficult to predict, and individual effect thresholds do not
an
necessarily protect against combination effects (Kortenkamp, 2014). Depending on similarity / dissimilarity of mode of action (MoA) of substances in mixture, two distinct concepts embedded in simple mathematical models are still being used: dose/concentration addition
M
(CA) and independent action (IA) (Kortenkamp et al., 2009). For compounds that act either similarly or differently, the null-models dose/concentration addition and IA are used respectively (Webster, 2013). Interactions such as potentiation, antagonism, synergies are
d
continually in question, as well as differences in the manifestation of joint toxic effects
te
compared to individual effects (Lydy et al., 2004; Kortenkamp et al., 2009). Among hot toxicological issues, toxicity of mixtures has attracted growing awareness and attention due to
Ac ce p
rising health problems without an obvious explanation (Demur et al., 2013; Løkke et al, 2013; Taxvig, 2013).
Avalon® is an herbicide mixture, containing the active substances bentazone and dicamba (in the form of sodium salts), intended to eliminate annual and some perennial broad-leaved weeds. It has been registered for use and been on the market in Serbia since 2006. Similar or near identical products have been registered in Europe under various trade names. Avalon® is formulated as a soluble liquid concentrate (SL) with 349 g/L of bentazone (Na-salt) and 98 g/L of dicamba (Na-salt). Bentazone 3-(isopropyl)-1H-2,1,3-benzothiadiazin-4(3H)-one2,2dioxide is a post-emergence, selective, contact herbicide classified in the group of benzothiazinones. Bentazone is a photosynthetic electron transport inhibitor at the photosystem II receptor site (MacBean, 2012). The acute oral toxicity of bentazone is moderate. The oral median lethal dose (LD50) is between 1400-1800 mg/kg body weight (bw), dermal and inhalation toxicity is low; its LD50 exceeds 5000 mg/kg bw, and the inhalation median lethal concentration (LC50)
Page 3 of 29
is over 5.1 mg/L (EC, 2000). Tennekes et al. (1987, cit. DAR, 2013) reported the following effects of bentazone in subchronic studies on rats: decrease in body weight, increases in prothrombin times (PT) and partial thromboplastin times (PTT), elevated serum albumin level and albumin/globulin ratio (A/G), as well as elevated cholesterol, sodium and chloride levels. Experimental data for bentazone exposure suggested that females were less affected than
ip t
males. Bentazone was not found to be carcinogenic, genotoxic or teratogenic (DAR, 2013). Dicamba (3,6-dichloro-o-anisic or anisole acid; 3,6-dichloro-2-methoxybenzoic acid) is a
cr
selective and systemic hormone herbicide, a derivative of benzoic acid. The MoA of dicamba in mammals is not known, while it is an auxin-like growth regulator in plants (MacBean,
us
2012).
The oral toxicity of dicamba is moderate and the lowest LD50 is 1600 mg/kg bw. Dermal and
an
inhalation toxicity is low; dermal LD50 exceeds 2000 mg/kg bw, and the inhalation LC50 is over 9.6 mg/L. Decreased glucose, total protein and cholesterol levels, as well as increased creatinine, phosphate and urea levels, and alkaline phosphatase (ALP), aspartate
M
aminotransferase (AST), alanine aminotransferase (ALT) activities have been recorded in subchronic rat feeding studies. Dicamba has not been found to be carcinogenic or teratogenic (DAR, 2007). Literature data on the genotoxicity of dicamba is inconclusive, and both
d
negative and positive data have been reported (González et al., 2007).
te
The present study focused on investigating the combined effects of bentazone and dicamba formulated as a SL, which is considered to be an environmentally friendly composition based
Ac ce p
on water. The results of previous studies (Brkić et al., 2009) showed that the mixture of these two active substances is of moderate acute oral toxicity; its LD50 according to the acute toxic class method is between 300 and 2000 mg/kg bw. It was not found to be genotoxic either in a bacterial reverse mutation test (Brkić et al., 2006) or in an in vivo mammalian erythrocyte micronucleus test (Brkić et al., 2007). Haematological effects of the mixture of bentazone and dicamba included dose-related decreases in haemoglobin, haematocrit and erythrocyte indexes in male and female rats. Weak anaemia was more pronounced in males than in females (Brkić et al., 2011).
It is well known that subacute and subchronic toxicities of mixtures may be different from the toxicities of their active substances. However, subacute and subchronic toxicity tests of formulated pesticide products are not required for the current registration process in the EU. The need to consider the combined exposure of chemicals in mixtures, in future risk assessments, was stressed by the EU Council and furthermore that research in this area should be supported by additional development of legislation and methodology (EC, 2009).
Page 4 of 29
With the aim of clarifying some issues regarding the possible toxic effects of the mixture, this study focused on the subacute and subchronic toxic effects of the mixture Avalon® after oral administration to rats. Transferase and alkaline phosphatase activities were monitored together with biochemical parameters (glucose, total protein, albumin, cholesterol, urea, creatinine, total bilirubin, calcium, phosphate, potassium, sodium and chloride) in 28 and 90-
ip t
day studies.
cr
2. MATERIAL AND METHODS
us
2.1. Chemicals
an
The mixture Avalon®, kindly provided by “Galenika-Fitofarmacija” (Belgrade, Serbia) is formulated as a SL with the active substances dicamba (98 g/L as Na-salt) and bentazone (349 g/L as Na-salt). Among inert substances, the mixture contains ethylenediaminetetraacetic acid
M
(EDTA) as a stabiliser (in the form of Na-salts) and water, as the solvent. All other reagents, purchased from different producers, were standard, laboratory grade,
d
commercial products of the highest purity.
te
2.2. Animals and experimental design
Ac ce p
Albino rats strain: CRL: (WI) BR Wistar of both sexes, 9 weeks old, with body weight between 150-180 g (females) and 220-240 g (males), were used in the experiment. The animals were obtained from the Charles River (Europe) Laboratory, Toxi Coop Ltd., Budapest, Hungary. After the acclimatisation period of 12 days, ten groups of 10 animals/sex/group (plus 10 animals/sex randomisation reserve) were compiled for randomisation. During the experiment the rats were kept in polypropylene-polycarbonate cages, at 22 ± 3 °C, under a 12 hour-light cycle, fed with standard, prepared food for laboratory animals (Ssniff SM R/M-Z+H, Germany), and had ad libitum access to food and water. The mixture was administered daily for 28 or 90 days by oral gavage at three dose levels: 250, 500 and 1000 mg/kg bw/day. The following parameters were monitored: liver enzyme activity (ALP, AST, ALT) and biochemical parameters (glucose, albumin, total protein, total bilirubin, urea, cholesterol, creatinine, phosphate, calcium, chloride, sodium and potassium concentrations). Animals from the recovery (satellite) groups (5 animals/sex) were treated
Page 5 of 29
with the high dose level (1000 mg/kg bw/day) for 90 days and afterwards observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of 28 days. A control group of 10 animals (5 animals/sex) were added to the recovery group, and treated with the vehicle. The study was performed in full accordance with the requirements described by the following internationally accepted guidelines: OECD Guidelines for Testing No. 408 - Repeated Dose 90-day Oral Toxicity Study in Rodents (1998).
ip t
of Chemicals, No. 407 - Repeated Dose 28-day Oral Toxicity Study in Rodents (2003), and
cr
The conduct of the study was permitted by the Animal Ethics Committee of LAB
International Research Centre Hungary Ltd. Also, the study was completely in compliance
us
with the principles of the Hungarian Acts LXVII 2002 and CIX 2006 (modification of
an
Hungarian Act 1998 XXVIII) regulating animal protection.
2.3. Enzyme activity
M
The activities of AST, ALT and ALP were measured by Vitros 250 Chemistry System
te
2.4. Biochemical parameters
d
(Ortho-Clinical Diagnostics, Inc., Johnson & Johnson Company).
Serum biochemical parameters including glucose, albumin, A/G ratio, total proteins, urea,
Ac ce p
total bilirubin, cholesterol, creatinine as well as serum electrolytes (phosphorus, calcium, chloride, sodium and potassium) were determined by Vitros 250 Chemistry System (OrthoClinical Diagnostics, Inc., Johnson & Johnson Company).
2.5. Statistical Analysis
The results were processed using the statistical software SPSS/PC+ (SPCC, Inc., Chicago, IL). The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorov-Smirnov test. In cases of non-normal distribution, the non-parametric Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, inter-group comparisons were performed
Page 6 of 29
using the Mann-Whitney U-test. All parameters were expressed by the mean values and standard deviations. Probabilities of 0.05 and 0.01 were used as criteria for significance of differences.
ip t
3. RESULTS
cr
3.1. Enzyme activity
The effect of the mixture Avalon® on serum enzyme activity (ALP, AST, ALT) in the 28-day
us
experiment are depicted in Figure 1. Increases in ALT activity were registered in males from the groups receiving 250 and 500 mg/kg (p
S1382-6689(15)00068-X http://dx.doi.org/doi:10.1016/j.etap.2015.03.004 ENVTOX 2221
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
12-9-2014 28-2-2015 3-3-2015
Please cite this article as: Brki´c, D., Szakonyne-Pasics, I., Gaˇsi´c, S., Teodorovi´c, I., Raˇskovi´c, B., Brki´c, N., Neˇskovi´c, N.,Subacute and subchronic toxicity of Avalonregd mixture (bentazone+dicamba) to rats, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Subacute and subchronic toxicity of Avalon mixture (bentazone+dicamba) to rats Dragica Brkić1a, Ilona Szakonyne-Pasics1b, Slavica Gašića, Ivana Teodorovićc, Božidar Raškovićd, Nenad Brkiće and Neško Neškovića 1
Corresponding author:
Ac ce p
te
d
Dragica Brkić Banatska 31-b 11080 Belgrade-Zemun Serbia Telephone: +381113076136 Mobile: +38163571500 Fax: +381113076133 E-mail: [email protected]
M
an
us
cr
ip t
LAB International Hungary Ltd., Szabadságpuszta, H-8200 Veszprém, Hungary (now CiToxLAB Hungary Ltd., Szabadságpuszta, H-8200 Veszprém, Hungary) a Institute of Pesticides and Environmental Protection, Banatska 31-b, 11080 BelgradeZemun, Serbia, [email protected], [email protected], [email protected] b Toxi-Coop, Toxicological Research Center Zrt., Pálya u. 2, H-2120 Dunakeszi, Hungary, [email protected] c University of Novi Sad, Faculty of Sciences, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia, [email protected] d University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-Zemun, Serbia [email protected] e Ministry of Agriculture and Environmental Protection, Directorate for Water, Bulevar umetnosti 2a, 11070 Belgrade, Serbia, [email protected]
Page 1 of 29
ip t
Subacute and subchronic toxicity of Avalon mixture (bentazone+dicamba) to rats
ABSTRACT
cr
Subacute and subchronic toxicity of the herbicide Avalon®, a mixture of bentazone and dicamba, were tested on rats. Avalon® was administered at dose levels of 250, 500 and 1000
us
mg/kg body weight/day for 28 and 90 days. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were monitored together
an
with biochemistry parameters. The results showed that the mixture caused increases in the activities of ALT, AST and ALP, elevated concentrations of sodium, albumin and albumin/globulin ratio in males. In females, ALT activity, cholesterol and phosphate levels
M
were increased. The changes generally were dose related and, in most cases, females exhibited lower susceptibility than males. The effects of a mixture are, in the most cases, different from the effects of the individual substances. The effects of bentazone were not
te
d
prevalent which would be expected taking the composition of the mixture into account.
Ac ce p
Keywords: Mixture; Bentazone; Dicamba; Rat; Subacute Toxicity; Subchronic Toxicity
Page 2 of 29
1. INTRODUCTION Pesticides are biologically active substances designed to be toxic, which causes growing concerns about their health impacts on many non-target species. Unlike other pollutants, pesticides are deliberately spread into the living and working environments. Because of their
ip t
ubiquitous presence people are constantly exposed to low levels of pesticides (Corsini et al., 2013).
cr
Pesticide active substances are commonly formulated before application. In the process of
developing formulations, manufacturers may use one or more active substances in order to
us
produce effective and long lasting products (Knowles, 2005). The combined effects of two or more active substances are very difficult to predict, and individual effect thresholds do not
an
necessarily protect against combination effects (Kortenkamp, 2014). Depending on similarity / dissimilarity of mode of action (MoA) of substances in mixture, two distinct concepts embedded in simple mathematical models are still being used: dose/concentration addition
M
(CA) and independent action (IA) (Kortenkamp et al., 2009). For compounds that act either similarly or differently, the null-models dose/concentration addition and IA are used respectively (Webster, 2013). Interactions such as potentiation, antagonism, synergies are
d
continually in question, as well as differences in the manifestation of joint toxic effects
te
compared to individual effects (Lydy et al., 2004; Kortenkamp et al., 2009). Among hot toxicological issues, toxicity of mixtures has attracted growing awareness and attention due to
Ac ce p
rising health problems without an obvious explanation (Demur et al., 2013; Løkke et al, 2013; Taxvig, 2013).
Avalon® is an herbicide mixture, containing the active substances bentazone and dicamba (in the form of sodium salts), intended to eliminate annual and some perennial broad-leaved weeds. It has been registered for use and been on the market in Serbia since 2006. Similar or near identical products have been registered in Europe under various trade names. Avalon® is formulated as a soluble liquid concentrate (SL) with 349 g/L of bentazone (Na-salt) and 98 g/L of dicamba (Na-salt). Bentazone 3-(isopropyl)-1H-2,1,3-benzothiadiazin-4(3H)-one2,2dioxide is a post-emergence, selective, contact herbicide classified in the group of benzothiazinones. Bentazone is a photosynthetic electron transport inhibitor at the photosystem II receptor site (MacBean, 2012). The acute oral toxicity of bentazone is moderate. The oral median lethal dose (LD50) is between 1400-1800 mg/kg body weight (bw), dermal and inhalation toxicity is low; its LD50 exceeds 5000 mg/kg bw, and the inhalation median lethal concentration (LC50)
Page 3 of 29
is over 5.1 mg/L (EC, 2000). Tennekes et al. (1987, cit. DAR, 2013) reported the following effects of bentazone in subchronic studies on rats: decrease in body weight, increases in prothrombin times (PT) and partial thromboplastin times (PTT), elevated serum albumin level and albumin/globulin ratio (A/G), as well as elevated cholesterol, sodium and chloride levels. Experimental data for bentazone exposure suggested that females were less affected than
ip t
males. Bentazone was not found to be carcinogenic, genotoxic or teratogenic (DAR, 2013). Dicamba (3,6-dichloro-o-anisic or anisole acid; 3,6-dichloro-2-methoxybenzoic acid) is a
cr
selective and systemic hormone herbicide, a derivative of benzoic acid. The MoA of dicamba in mammals is not known, while it is an auxin-like growth regulator in plants (MacBean,
us
2012).
The oral toxicity of dicamba is moderate and the lowest LD50 is 1600 mg/kg bw. Dermal and
an
inhalation toxicity is low; dermal LD50 exceeds 2000 mg/kg bw, and the inhalation LC50 is over 9.6 mg/L. Decreased glucose, total protein and cholesterol levels, as well as increased creatinine, phosphate and urea levels, and alkaline phosphatase (ALP), aspartate
M
aminotransferase (AST), alanine aminotransferase (ALT) activities have been recorded in subchronic rat feeding studies. Dicamba has not been found to be carcinogenic or teratogenic (DAR, 2007). Literature data on the genotoxicity of dicamba is inconclusive, and both
d
negative and positive data have been reported (González et al., 2007).
te
The present study focused on investigating the combined effects of bentazone and dicamba formulated as a SL, which is considered to be an environmentally friendly composition based
Ac ce p
on water. The results of previous studies (Brkić et al., 2009) showed that the mixture of these two active substances is of moderate acute oral toxicity; its LD50 according to the acute toxic class method is between 300 and 2000 mg/kg bw. It was not found to be genotoxic either in a bacterial reverse mutation test (Brkić et al., 2006) or in an in vivo mammalian erythrocyte micronucleus test (Brkić et al., 2007). Haematological effects of the mixture of bentazone and dicamba included dose-related decreases in haemoglobin, haematocrit and erythrocyte indexes in male and female rats. Weak anaemia was more pronounced in males than in females (Brkić et al., 2011).
It is well known that subacute and subchronic toxicities of mixtures may be different from the toxicities of their active substances. However, subacute and subchronic toxicity tests of formulated pesticide products are not required for the current registration process in the EU. The need to consider the combined exposure of chemicals in mixtures, in future risk assessments, was stressed by the EU Council and furthermore that research in this area should be supported by additional development of legislation and methodology (EC, 2009).
Page 4 of 29
With the aim of clarifying some issues regarding the possible toxic effects of the mixture, this study focused on the subacute and subchronic toxic effects of the mixture Avalon® after oral administration to rats. Transferase and alkaline phosphatase activities were monitored together with biochemical parameters (glucose, total protein, albumin, cholesterol, urea, creatinine, total bilirubin, calcium, phosphate, potassium, sodium and chloride) in 28 and 90-
ip t
day studies.
cr
2. MATERIAL AND METHODS
us
2.1. Chemicals
an
The mixture Avalon®, kindly provided by “Galenika-Fitofarmacija” (Belgrade, Serbia) is formulated as a SL with the active substances dicamba (98 g/L as Na-salt) and bentazone (349 g/L as Na-salt). Among inert substances, the mixture contains ethylenediaminetetraacetic acid
M
(EDTA) as a stabiliser (in the form of Na-salts) and water, as the solvent. All other reagents, purchased from different producers, were standard, laboratory grade,
d
commercial products of the highest purity.
te
2.2. Animals and experimental design
Ac ce p
Albino rats strain: CRL: (WI) BR Wistar of both sexes, 9 weeks old, with body weight between 150-180 g (females) and 220-240 g (males), were used in the experiment. The animals were obtained from the Charles River (Europe) Laboratory, Toxi Coop Ltd., Budapest, Hungary. After the acclimatisation period of 12 days, ten groups of 10 animals/sex/group (plus 10 animals/sex randomisation reserve) were compiled for randomisation. During the experiment the rats were kept in polypropylene-polycarbonate cages, at 22 ± 3 °C, under a 12 hour-light cycle, fed with standard, prepared food for laboratory animals (Ssniff SM R/M-Z+H, Germany), and had ad libitum access to food and water. The mixture was administered daily for 28 or 90 days by oral gavage at three dose levels: 250, 500 and 1000 mg/kg bw/day. The following parameters were monitored: liver enzyme activity (ALP, AST, ALT) and biochemical parameters (glucose, albumin, total protein, total bilirubin, urea, cholesterol, creatinine, phosphate, calcium, chloride, sodium and potassium concentrations). Animals from the recovery (satellite) groups (5 animals/sex) were treated
Page 5 of 29
with the high dose level (1000 mg/kg bw/day) for 90 days and afterwards observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of 28 days. A control group of 10 animals (5 animals/sex) were added to the recovery group, and treated with the vehicle. The study was performed in full accordance with the requirements described by the following internationally accepted guidelines: OECD Guidelines for Testing No. 408 - Repeated Dose 90-day Oral Toxicity Study in Rodents (1998).
ip t
of Chemicals, No. 407 - Repeated Dose 28-day Oral Toxicity Study in Rodents (2003), and
cr
The conduct of the study was permitted by the Animal Ethics Committee of LAB
International Research Centre Hungary Ltd. Also, the study was completely in compliance
us
with the principles of the Hungarian Acts LXVII 2002 and CIX 2006 (modification of
an
Hungarian Act 1998 XXVIII) regulating animal protection.
2.3. Enzyme activity
M
The activities of AST, ALT and ALP were measured by Vitros 250 Chemistry System
te
2.4. Biochemical parameters
d
(Ortho-Clinical Diagnostics, Inc., Johnson & Johnson Company).
Serum biochemical parameters including glucose, albumin, A/G ratio, total proteins, urea,
Ac ce p
total bilirubin, cholesterol, creatinine as well as serum electrolytes (phosphorus, calcium, chloride, sodium and potassium) were determined by Vitros 250 Chemistry System (OrthoClinical Diagnostics, Inc., Johnson & Johnson Company).
2.5. Statistical Analysis
The results were processed using the statistical software SPSS/PC+ (SPCC, Inc., Chicago, IL). The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorov-Smirnov test. In cases of non-normal distribution, the non-parametric Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, inter-group comparisons were performed
Page 6 of 29
using the Mann-Whitney U-test. All parameters were expressed by the mean values and standard deviations. Probabilities of 0.05 and 0.01 were used as criteria for significance of differences.
ip t
3. RESULTS
cr
3.1. Enzyme activity
The effect of the mixture Avalon® on serum enzyme activity (ALP, AST, ALT) in the 28-day
us
experiment are depicted in Figure 1. Increases in ALT activity were registered in males from the groups receiving 250 and 500 mg/kg (p
