The large consumption of biodegradable films from cassava starch acetate (FCSA) as ingredients in food and pharmaceutical products requires the assessment of the possible toxicity of these products. The aim of this study was to investigate the toxicity of biodegradable film from cassava starch acetate after oral exposure of Wistar rats for 90 days. The amount of food consumed and the body weight were weekly monitored. Blood and urine samples were obtained for the assessment of serum parameters and renal function. Histopathological analyses in target organs were also performed. No evidence of clinical toxicity in hematological, biochemical, or renal parameters in the FCSA-treated animals was found. In addition, relative organ weight and histopathological evaluations did not differ between groups treated with FCSA and control. Data obtained suggest that the subchronic exposure to FCSA does not cause obvious signs of toxicity in Wistar rats, indicating possible safety of this biofilm.
In recent decades, a new generation of biomaterials was introduced as alternative to reduce the degradation of perishable foods. In addition, pharmaceutical industries have applied edible films as adjuvants in controlled drug release systems [
Cassava (
Although these edible films are actually very promising, the risks of prolonged use of these chemically modified natural compounds remain unknown. Few toxicological studies have been conducted with modified starches, and some data suggest higher prevalence of structural changes in the kidneys and intestines of mice after prolonged use [
Given the importance of these biopolymers and their possible use on a large scale, subchronic toxicity studies are essential steps in obtaining information on the safe use of these compounds. Thus, the aim of this study was to investigate the subchronic toxicity of biodegradable film obtained from cassava starch acetate after oral exposure of 60-day-old Wistar rats.
High-amylose cassava starch (AVEBE starches, Brazil) was used. Acetic anhydride (≥99%) and D-sorbitol (≥99%) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) and Fluka Chemical Co.
Acetylated starch was prepared from high-amylose cassava starch according to methodology previously described by Xie et al. [
Forty male and 40 female Wistar rats of similar age (60 days) from the stock of the Federal University of Parana were kept under specific conditions. The animals were divided by sex and kept in groups of four in standard cages for rodents (Insight, Brazil) with the following dimensions: 49 × 34 × 16 cm (length, width, and height), temperature (
The doses used in this study were determined from the lowest dose (30 mg) required to pack an intermediate-size food product (30 g), and from this dose a 10 times safety factor was set (300 mg/kg), used to calculate the intermediate dose (100 mg/kg). The animals were divided into four groups of 10 rats (females and males), orally treated for 90 days with vehicle (control) or with different doses of FCSA (30, 100, and 300 mg/kg). Throughout the experimental period, the animals were daily weighed and observed for clinical signs of toxicity.
After 90 days of treatment, rats were evaluated for
Results are expressed as mean ± standard error of the mean of ten animals in each group. Statistical analysis was carried out using analysis of variance (ANOVA) followed by Bonferroni’s test. A
Signs of toxicity or deaths were not recorded throughout the 90-day study period. Weekly body weight and weekly body weight gain over the treatment period were similar among animals (both sexes) (Figures
Body weights of male (a) and female (b) rats treated with distilled water (control) and FCSA (3, 30, and 300 mg/kg) for 90 days. Data are the mean ± standard deviation of the weekly body weights for each treatment group.
Urine volume, pH, density, conductivity, and Na+ and K+ results for males and females rats are shown in Table
Effect of FCSA on urinary parameters of male and female Wistar rats exposed for 90 days.
Parameters | Control | FCSA 3 mg/kg/day | FCSA 30 mg/kg/day | FCSA 300 mg/kg/day | ||||
---|---|---|---|---|---|---|---|---|
Male | Female | Male | Female | Male | Female | Male | Female | |
Urine volume (mL/8 hours) | 9.76 ± 1.40 | 10.1 ± 1.34 | 10.5 ± 1.78 | 11.2 ± 1.74 | 12.0 ± 1.27 | 9.87 ± 1.31 | 11.2 ± 1.43 | 12.1 ± 1.64 |
pH | 6.36 ± 0.04 | 6.27 ± 0.13 | 6.48 ± 0.12 | 6.43 ± 0.25 | 6.15 ± 0.12 | 6.56 ± 0.15 | 6.18 ± 0.12 | 6.52 ± 0.16 |
Density (g/mL) | 1.02 ± 0.0 | 1.01 ± 0.0 | 1.01 ± 0.0 | 1.01 ± 0.0 | 1.02 ± 0.0 | 1.01 ± 0.0 | 1.02 ± 0.0 | 1.01 ± 0.0 |
Conductivity (mS/cm) | 17.6 ± 0.16 | 13.1 ± 1.91 | 17.5 ± 0.62 | 13.9 ± 1.31 | 18.0 ± 0.17 | 14.1 ± 1.15 | 18.0 ± 0.30 | 14.1 ± 1.47 |
Na+ (mmol/L) | 113 ± 1.12 | 120 ± 1.47 | 115 ± 0.93 | 121 ± 1.88 | 116 ± 1.27 | 118 ± 1.86 | 115 ± 2.04 | 119 ± 1.82 |
K+ (mmol/L) | 35 ± 3.08 | 38.7 ± 2.07 | 34 ± 2.25 | 39.8 ± 1.66 | 37 ± 2.06 | 40.9 ± 1.59 | 35 ± 0.89 | 39.4 ± 1.26 |
Values are expressed as mean ± S. E. M. of ten rats in each group in comparison to the control using one-way ANOVA followed by Bonferroni’s test.
Results for hematologic and biochemical analysis are shown in Tables
Effect of FCSA on hematological parameters of male and female Wistar rats exposed for 90 days.
Parameters | Control | FCSA 3 mg/kg/day | FCSA 30 mg/kg/day | FCSA 300 mg/kg/day | ||||
---|---|---|---|---|---|---|---|---|
Male | Female | Male | Female | Male | Female | Male | Female | |
RBC (106/mL) | 7.9 ± 0.6 | 7.4 ± 0.1 | 8.6 ± 0.1 | 7.3 ± 0.1 | 8.5 ± 0.1 | 7.4 ± 0.1 | 8.5 ± 0.2 | 7.5 ± 0.1 |
Hemoglobin (g/mL) | 14.7 ± 0.4 | 14.4 ± 0.2 | 15.4 ± 0.2 | 14.3 ± 0.2 | 15.1 ± 0.1 | 14.4 ± 0.2 | 16.1 ± 0.8 | 14.7 ± 0.1 |
Hematocrit (%) | 43 ± 1.2 | 41 ± 0.6 | 45 ± 0.5 | 40 ± 0.6 | 44 ± 0.4 | 40 ± 0.7 | 44 ± 1.0 | 41 ± 0.4 |
MCV (fL) | 51.9 ± 0.5 | 56.2 ± 0.6 | 52.5 ± 0.5 | 55.7 ± 0.4 | 52.3 ± 0.5 | 55.1 ± 0.6 | 51.7 ± 0.3 | 55.8 ± 0.4 |
MCH (pg) | 17.7 ± 0.3 | 19.7 ± 0.3 | 18.0 ± 0.2 | 19.6 ± 0.2 | 17.8 ± 0.2 | 19.6 ± 0.3 | 18.8 ± 0.7 | 19.7 ± 0.2 |
MCHC (%) | 34 ± 0.3 | 35 ± 0.2 | 34 ± 0.2 | 35 ± 0.3 | 34 ± 0.2 | 36 ± 0.3 | 36 ± 1.3 | 35 ± 0.2 |
Platelets (103/mm3) | 1032 ± 79 | 951 ± 60 | 1045 ± 30 | 843 ± 120 | 1070 ± 42 | 881 ± 85 | 1098 ± 60 | 972 ± 71 |
WBC (103/mm3) | 4.4 ± 0.5 | 2.9 ± 0.3 | 5.4 ± 0.8 | 3.2 ± 0.8 | 4.4 ± 0.3 | 2.9 ± 0.5 | 4.1 ± 0.3 | 3.7 ± 1.3 |
Neutrophils (%) | 24.8 ± 4.0 | 26.3 ± 2.4 | 22.2 ± 1.4 | 21.5 ± 3.4 | 21.9 ± 2.1 | 28.1 ± 2.9 | 24.5 ± 3.8 | 23.8 ± 1.9 |
Lymphocytes (%) | 69.4 ± 3.4 | 67.3 ± 3.5 | 71.3 ± 2.9 | 71.6 ± 2.1 | 70.6 ± 1.7 | 65.5 ± 3.3 | 68.2 ± 2.3 | 69.4 ± 2.7 |
Monocytes (%) | 4.4 ± 0.7 | 5.5 ± 0.7 | 4.9 ± 1.2 | 6.1 ± 1.2 | 5.8 ± 0.9 | 5.3 ± 0.8 | 6.1 ± 1.1 | 5.8 ± 1.0 |
Eosinophils (%) | 1.4 ± 0.5 | 0.9 ± 0.3 | 1.6 ± 0.6 | 0.8 ± 0.2 | 1.7 ± 0.3 | 1.1 ± 0.5 | 1.2 ± 0.3 | 1.0 ± 0.3 |
Values are expressed as mean ± S. E. M. of ten rats in each group in comparison to the control using one-way ANOVA followed by Bonferroni’s test.
Effect of FCSA on biochemical parameters of male and female Wistar rats exposed for 90 days.
Parameters | Control | FCSA 3 mg/kg/day | FCSA 30 mg/kg/day | FCSA 300 mg/kg/day | ||||
---|---|---|---|---|---|---|---|---|
Male | Female | Male | Female | Male | Female | Male | Female | |
Glucose (mg/dL) | 139 ± 7.4 | 122 ± 5.1 | 139 ± 6.0 | 144 ± 7.1 | 130 ± 6.0 | 131 ± 5.1 | 137 ± 4.9 | 138 ± 6.2 |
Total cholesterol (mg/dL) | 59 ± 1.4 | 60 ± 3.4 | 56 ± 2.1 | 57 ± 2.5 | 59 ± 1.7 | 63 ± 3.4 | 60 ± 2.7 | 63 ± 3.0 |
HDL cholesterol (mg/dL) | 35 ± 1.1 | 36 ± 2.4 | 33 ± 1.4 | 33 ± 1.8 | 36 ± 1.1 | 38 ± 1.8 | 37 ± 1.4 | 39 ± 2.1 |
Triglycerides (mg/dL) | 54 ± 6.7 | 56 ± 3.9 | 51 ± 6.7 | 64 ± 5.8 | 48 ± 5.1 | 54 ± 4.1 | 53 ± 6.5 | 55 ± 3.3 |
Urea (mg/dL) | 36 ± 2.5 | 33 ± 3.4 | 38 ± 2.0 | 34 ± 3.4 | 41 ± 3.1 | 34 ± 2.9 | 44 ± 2.4 | 34 ± 2.8 |
Creatinine (mg/dL) | 0.47 ± 0.02 | 0.48 ± 0.04 | 0.49 ± 0.01 | 0.49 ± 0.04 | 0.49 ± 0.01 | 0.50 ± 0.03 | 0.47 ± 0.01 | 0.48 ± 0.04 |
Sodium (mEq/L) | 141 ± 1.0 | 138 ± 1.4 | 142 ± 1.3 | 139 ± 1.1 | 139 ± 0.6 | 137 ± 1.0 | 141 ± 1.3 | 137 ± 0.6 |
Potassium (mEq/L) | 5.6 ± 0.1 | 6.0 ± 0.2 | 5.5 ± 0.2 | 6.0 ± 0.3 | 5.5 ± 0.1 | 5.8 ± 0.3 | 5.7 ± 0.3 | 5.6 ± 0.3 |
Uric acid (mg/dL) | 1.0 ± 0.1 | 1.2 ± 0.1 | 1.2 ± 0.2 | 1.5 ± 0.2 | 1.0 ± 0.1 | 1.4 ± 0.1 | 1.1 ± 0.2 | 1.5 ± 0.1 |
Total protein (g/dL) | 5.4 ± 0.1 | 5.4 ± 0.1 | 5.3 ± 0.1 | 5.4 ± 0.1 | 5.3 ± 0.1 | 5.5 ± 0.1 | 5.2 ± 0.1 | 5.5 ± 0.2 |
Albumin (g/dL) | 3.5 ± 0.1 | 3.7 ± 0.1 | 3.5 ± 0.1 | 3.7 ± 0.1 | 3.5 ± 0.1 | 3.8 ± 0.1 | 3.4 ± 0.1 | 3.8 ± 0.1 |
Globulin (g/dL) | 1.9 ± 0.1 | 1.7 ± 0.1 | 1.8 ± 0.1 | 1.7 ± 0.1 | 1.8 ± 0.1 | 1.7 ± 0.1 | 1.8 ± 0.1 | 1.7 ± 0.1 |
Amylase (U/L) | 732 ± 24 | 722 ± 76 | 699 ± 41 | 667 ± 58 | 703 ± 37 | 664 ± 67 | 756 ± 41 | 596 ± 59 |
Alkaline phosphatase (U/L) | 154 ± 11 | 106 ± 9 | 154 ± 11 | 107 ± 15 | 139 ± 9 | 106 ± 4 | 153 ± 11 | 100 ± 7 |
AST (U/L) | 99 ± 9 | 124 ± 6 | 120 ± 9 | 117 ± 7 | 98 ± 8 | 117 ± 6 | 99 ± 10 | 134 ± 12 |
ALT (U/L) | 49 ± 2 | 42 ± 2 | 58 ± 6 | 40 ± 2 | 50 ± 1 | 42 ± 2 | 53 ± 5 | 41 ± 2 |
Total bilirubin (mg/dL) | 0.15 ± 0.01 | 0.12 ± 0.01 | 0.17 ± 0.02 | 0.13 ± 0.01 | 0.16 ± 0.01 | 0.19 ± 0.04 | 0.15 ± 0.01 | 0.15 ± 0.01 |
Direct bilirubin (mg/dL) | 0.10 ± 0.01 | 0.07 ± 0.01 | 0.11 ± 0.02 | 0.08 ± 0.01 | 0.11 ± 0.01 | 0.10 ± 0.02 | 0.09 ± 0.02 | 0.10 ± 0.01 |
Indirect bilirubin (mg/dL) | 0.05 ± 0.01 | 0.05 ± 0.01 | 0.06 ± 0.01 | 0.05 ± 0.01 | 0.05 ± 0.01 | 0.09 ± 0.01 | 0.06 ± 0.02 | 0.05 ± 0.01 |
Values are expressed as mean ± S. E. M. of ten rats in each group in comparison to the control using one-way ANOVA followed by Bonferroni’s test.
There were no FCSA-related changes in the relative weight of organs of all experimental groups. Furthermore, no significant change was observed by autopsy or histopathological analysis in all samples. Due to alterations reported in literature, intestine and kidney samples from both male and female rats from control and FCSA groups (300 mg/kg) are shown in Figure
Photomicrographs of jejunum and kidney histopathology from representative male Wistar rats treated with vehicle (control group) or the highest dosage of FCSA (300 mg/kg) for 90 days. At the top, the jejunum (200x) and its respective control group are shown. In the line below, a sample of the left kidney (400x) is also represented. Hematoxylin and eosin stain.
In recent years, a large amount of biomaterials has been developed with the aim of producing edible films used for food quality preservation [
Despite the potential use of starch-derived biopolymers and the belief that their natural origin ensures low toxicity, some studies have shown that even natural products commonly used as additives or coatings in food products may have toxic effects. Nevertheless, most of these studies have only investigated the toxicity of compounds obtained from microorganisms, crustaceans, or nonstarch derivatives [
For the first time, a study on the subchronic toxicology (90 days) was conducted to provide a comprehensive assessment of the risk of possible prolonged FCSA consumption. The study aimed at exploring the main parameters that are commonly affected by prolonged exposure to toxic agents, such as hematopoietic, reproductive, and nervous systems [
A study conducted about 30 years ago showed that mice chronically treated with modified starch had increased water consumption and increased amounts of calcium and amorphous crystals in urine. Moreover, a slightly increased incidence of intratubular nephrosis was also observed [
Literature data have shown that many polysaccharides, including starch, can cause some changes in small and large intestines. Cases of changes in the cecal and colonic enlargement have been reported after use of modified starches [
Another contentious issue on starches refers to their metabolic effect on the small intestine. Diets high in carbohydrates can induce hypertriglyceridemia, hyperglycemia, and atherosclerosis as well as changes in liver function. One explanation for this fact is the inadequate evolutionary adaptation to starch and sugars in foods [
In addition to the above data, studies have shown that different types of starch, including starch acetate, may contribute to the development of microbial population groups and short-chain fatty acids (SCFA) in the cecum and feces of rats [
Treatments with FCSA did not induce toxicity signs in all clinical parameters evaluated, including gross and microscopic pathologies. Furthermore, FCSA treatment did not affect hematopoiesis, serum biochemical parameters, or physicochemical aspects of urine after prolonged administration. Additional preclinical toxicology studies (such as mutagenicity and genotoxicity studies) and clinical trials should be carried out to complement the safety evaluation in the use of FCSA in humans, mainly due to the possibility of its use for prolonged periods of time.
The authors declare that there is no conflict of interests regarding the publication of this this study.
The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) and Diretoria Executiva de Gestão da Pesquisa e Pós-Graduação (DEGPP/UNIPAR-Brazil) for financial support.