Acute toxicity and chromosomal aberration toxicity of insect wax and its policosanol

Jinju Ma, Kun Li, Wenwen Zhang, Liyi Ma, Juan Xu, Lanxiang Liu,Xiaoming Chen*, Hong Zhang*

Peculiar Biological Resources Technology Engineering Center, Research Institute of Resources Insects, Chinese Academy of Forestry, Kunming 650224, China

Keywords:

Insect wax

Policosanol from insect wax

Acute toxicity

Chromosomal aberration

A B S T R A C T

An acute toxicity test in Sprague Dawley (SD) rats and a chromosomal aberration toxicity test in Chinese hamster lung (CHL) fibroblasts were conducted to promote the application of insect wax and its policosanol.Results of oral acute toxicity test showed that the LD50 values of insect wax and its policosanol were higher than 5 000 mg/kg. The rats showed no obvious toxic symptoms and survived, suggesting that these substances were not toxic. The chromosomal aberration rates of the CHL cells incubated with insect wax for 3–6 h under metabolic activation conditions and for 3–6 h and 24 h under non-metabolic activation conditions were lower than 5% . No significant difference was found compared with the negative control group. Also, no significant difference in chromosomal aberration rates was found between each dose group of policosanol under metabolic or non-metabolic activation conditions and the negative control group. Therefore, the chromosomal aberration rates of CHL cells treated with insect wax and its policosanol were negative regardless of metabolic activation conditions. These results indicate the absence of acute toxicity and potential chromosomal aberration in the tested dose range of insect wax and its policosanol.

1. Introduction

Policosanol generally refers to saturated linear monohydric alcohols containing 20–36 carbon atoms. They are widely found in rice bran wax, sugarcane wax, insect wax, beeswax, plant seed husk,and so on in the form of higher fatty acid wax esters, which have important physiological activities [1-3]. Hexacosanol is a policosanol that can induce neuronal maturation, execute nerve protection and regeneration, and alleviate neuronal degeneration. Thus, it can be applied to the treatment of some neurodegenerative disorders, such as Alzheimer’s disease. Octacosanol is a new functional food additive that can reduce cholesterol and triglycerides levels, alleviate fatigue,enhance physical strength and endurance, improve metabolic rate,and treat and prevent gastrointestinal inflammation. The combination of octacosanol and hexacosanol can inhibit atherosclerosis and thrombosis. Triacontanol can regulate plant growth [1,4-8].

Insect wax secreted by the maleEricerus pela(Chavannes)parasitizing onPrivetorFraxinustrees, also called white wax, is a unique product in China. It is a traditional Chinese medicine used for nourishing the lung, strengthening the intestine and the stomach,killing parasites, and treating blood sore hemorrhage, hematochezia,hematuria, hemostasis, and analgesia. It can be applied externally or internally and used as excipients and lubricants during pill and tablet preparation [9,10]. The major components of wax are ester mixtures of fatty acid monoacids and monoalcohols, accounting for about 93% -95% of the total amount. Hexacosanoic acid esters and octacosanoic acid esters are prominent components of wax and excellent raw materials for extracting policosanol, which is mainly composed of hexacosanol and octacosanol. Policosanol can be prepared via reduction or saponification from insect wax [11-14].

Insect wax and its policosanol have great potential for developing health products or drugs. However, current studies on insect wax mainly focused on its properties, applications, and the separation and extraction of policosanol from insect wax. Zhang et al. [15]studied the thermal characteristics of insect wax by differential scanning calorimetry. Also, Lin et al. [16]reported that the crude polysaccharide extracted from the insect wax scale could cure atopic dermatitis. And the chemistry and biosynthesis of insect waxes were reported in detail by Blomquist [17]. Moreover, policosanol was prepared via saponification and reduction from insect wax, having high yield, respectively [12,13,18]. Ma et al. [9]prepared an insect wax compound ointment which could promote wound healing.However, the toxicity and safety of insect wax remain unclear.TheCompendium of Materia Medicaand some ancient books have recorded the sweetness in taste, the warm in temperament,and nontoxicity of insect wax [19]. Feng and Zeng et al. [20,21]reported no mutagenic and teratogenic effects of the egg and whole worm meal prepared with insects in a wide range of safe dose.On the other hand, many studies on policosanol also focused on its separation, extraction, and functions. Policosanol derived from insect wax was emulsified to improve its poor water solubility by Ma et al. [22].Kim et al. [23]reported that policosanol extracted from sugarcane wax was homogeneously dispersed in aqueous medium containing a dextrin produced after a mild hydrolysis of amylomaize starch. Also,Venturelli et al. [4]proposed a new procedure for the extraction and purification of policosanol from yellow beeswax by using microwaveassisted technology. Moreover, policosanol plus fenofibrate combination therapy on 102 patients significantly improved lipid parameters,arterial stiffness, and quality of life, with good tolerability [24].However, few studies evaluated the toxicity of policosanol derived from rice bran wax and sugarcane wax, and they were mainly concerned with the oral toxicity of policosanol. Aleman et al. [25]used policosanol derived from sugarcane wax to feed mice at a dose of 0.5–500 mg/kg for 12 months and observed no oral toxicity. In addition,Guo et al. [26]reported that the LD50of policosanol derived from sugarcane wax was higher than 5 000 mg/kg in rats, mice, rabbits, and dogs after oral administration. On May 31, 2017, the National Health and Family Planning Commission of the People’s Republic of China passed the review and issued a public announcement on the addition of policosanol mixtures derived from rice bran wax into new resource foods [27]. Gao and Long et al. [28,29]studied the toxicity of policosanol derived from rice bran wax; they found that its LD50was higher than 10 g/kg and observed no acute oral toxicity. Meanwhile,the results of three types of genotoxicity tests were negative.In addition, Alemán and Rodriguez-Echenique et al. [30,31]reported that policosanol extracted from sugarcane wax was very safe and well tolerated during long-term oral administration in mice and monkeys. However, at present, no studies have been conducted on the toxicity and safety of policosanol derived from insect wax.

In the current study, the toxicity of insect wax and its policosanol was preliminarily investigated by performing acute oral toxicity and mammalian cell chromosomal aberration testsin vitro. This study may serve as a theoretical reference for further evaluation of the edibility and medicinal safety of insect wax and its policosanol to widen their application.

2. Materials and methods

2.1 Materials

Pharmaceutical-grade insect wax was purchased from Emeishan Institutes of Insect Wax (Emeishan, China). Policosanol was obtained from insect wax via reduction reaction, consisting of tetracosanol (5.2% ), hexacosanol (56.0% ), octacosanol (32.0% ), and triacontanol (4.0% ) [13]. Corn oil was purchased from Yihai Jiali Group. Mitomycin C with a purity/content of higher than 995 µg/mg was procured from Shanghai Siyu Chemical Technology Co., Ltd.Cyclophosphamide (purity/content, 97% ) was purchased from Shanghai Yuanye Biotechnological Co., Ltd. Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Thermo Fisher(Suzhou) Instrument Co., Ltd. Fetal bovine serum (FBS) was procured from Zhejiang Tianhang Biotechnological Co., Ltd. Colchicine was obtained from Huibaishi Biotechnological Co., Ltd. Giemsa staining solution was purchased from Xi’an Hert Biotechnological Co., Ltd.Anin vitrometabolic activation system containing an S9 mixture of rat liver homogenate (CHI SCIENTIFIC, Inc.) was immediately prepared before use.

SPF SD rats (9-week old) with the body weight range of 226.2–240.4 g before the first drug administration were provided by Liaoning Changsheng Biotechnological Co., Ltd. Experimental animal production license number was SCXK (Liao) 2015-0001, and experimental animal use license number was SYXK (Shan) 2016-007.

2.2 Instruments

BSA224S-CW analytical balance was from Sartorius (Gottingen,Germany); HH·CP-01W water-proof carbon dioxide incubator was from Shanghai Fuma Experimental Equipment Co., Ltd. (Shanghai,China). CJ-1D clean bench was from Tianjin Tester Instrument Co., Ltd. (Tianjin, China). 37XF Inverted Biomicroscope was from Shanghai No.1 Optical Instrument Factory (Shanghai, China); Ni-U Vertical Microscope was from Nikon (Tokyo, Japan).

2.3 Acute oral toxicity test

The test was conducted in accordance with the OECD Guideline for the Testing of Chemicals, 420 Acute Oral Toxicity-Fixed Dose Procedure [32].

2.3.1 Sample preparation

Insect wax and its policosanol (test samples) are insoluble in water but can be evenly dispersed in corn oil. Insect wax and its policosanol were ground with a small amount of edible corn oil for several times, prepared to a final volume of 50 mL using corn oil with a concentration of 250 mg/mL for insect wax and its policosanol, and then stored until gavage.

2.3.2 Test animal requirements

All rats were subjected to quarantine and adaptive feeding for 25 days after delivered and observed every day before grouping.Qualified rats were used for the test. In this study, all rats were fed continuously in a cage-covered trough supplemented with standard feeds and drinking water for free accessibility. The use of rats complied with the National Animal Welfare Regulations “Guidelines for Treating Animals Well”. The program for rat experiments was approved by the Ethics Committee of the Research Institute of Resources Insects, Chinese Academy of Forestry. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Experimental Animals. Endangered rats in the experiment and survived rats at the end of the experiment were euthanized.

2.3.3 Dose design

The limited-dose method was used in this test. Preliminarily results showed that 10 SD rats (half male and half female) administered with one sample (insect wax or its policosanol), respectively, at the dose of 10 g/kg, did not reveal obvious toxic symptoms; therefore, referring to the requirements of the test guidelines, the dose of 5 000 mg/kg,was designed as the limited dosage for insect wax and its policosanol,respectively. At the end of the quarantine adaptation period, 10 SD rats (half male and half female) with healthy and suitable body weight (body weight variation without exceeding 20% of average body weight) were randomly selected for formal tests of each sample including insect wax, policosanol, and corn oil as control. Parallel tests were conducted in triplicate for each sample.

2.3.4 Test methods

Insect wax and its policosanol enter the human body mainly through the digestive tract after administration. Thus, gavage was applied in this experiment. Rats were fasted overnight without water restriction before drug administration. The body weights of all rats were measured before the first drug administration after fasting. The prepared insect wax and its policosanol were separately administered by gavage to the rats to determine their acute oral toxicity, respectively. Each rat was administered by gavage twice with an interval of 4-5 h. After gavage,fasting was resumed for 3-4 h, and water was not prohibited during fasting.The volume of administered drugs was 20 mL/kg.

The health status and toxic symptoms of the rats were rigorously assessed by an observer who is familiar with the normal and abnormal characteristics of rats and compared with those of the rats in the control group in accordance with the OECD 420 Acute Oral Toxicity-Fixed Dose Procedure [32], GB 15193.3-2014 Acute Oral Toxicity Test [33], and the OECD Guidance Document on the Recognition,Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation [34]. After administration, the health status of the rats was observed once within 30 min and once within 2 h and 4 h. In addition, observation was conducted in the morning and afternoon every day for 14 consecutive days since the 2ndday. The apparent observation items of poisoning in the experimental rats are shown in Table 1. The body weights of the rats were measured before grouping, the first administration, the 7thand 14thdays of administration, and dissection, respectively. At the end of the experiment, all survived rats were fasted on the 15thday,anesthetized through 2% barbiturate, and sacrificed by bloodletting from the posterior abdominal aorta for gross pathological anatomical observation of the abdominal cavity, pelvic cavity, thoracic cavity,and other viscera. Further histopathological examination of the lesion site was performed and compared with the negative control when any lesions were visually examined.

Table 1Apparent observation items of poisoning in experimental rats [32,33].

2.4 In vitro mammalian cell chromosomal aberration test

The experiment was conducted in accordance with the OECD Guideline for the Testing of Chemicals, 473in vitroMammalian Chromosomal Aberration Test [35]. Cultured mammalian cells were exposed to the test substance with or without the addition of the metabolic activation system (S9). Cells were treated with a metaphase-arresting agent colchicine to stop metaphase phase division, harvested, and stained, and metaphase cells were analyzed microscopically for the presence of chromosomal aberrations.

2.4.1 Test sample preparation

The test samples (insect wax and its policosanol) were prepared in accordance with the dose design (Table 2). When the cells in the 3–6 h non-metabolic activation (-S9) treatment group were administered,samples were added into cell culture flask containing 5 mL of serumfree 1640 medium, and different concentrations of the samples were obtained (Table 2). When the cells in the 3–6 h metabolic activation(+S9) treatment group were administered, samples were added into cell culture flasks containing 4 mL of serum-free 1640 medium, and then each flask was added with 1 mL of fresh S9 mixture (20% ). The final concentrations of the test samples in the culture medium are shown in Table 2. When the cells in the 24 h non-metabolic activation(-S9) treatment group were administered, samples were added into the cell culture flask containing 5 mL of serum 1640 medium, and the final concentrations of the test samples are shown in Table 2.

Table 2Design of experimental dose for chromosome aberration test in mammalian cells in vitro.

2.4.2 Dose design

Preliminary test results and existing toxicity information of two samples showed that the cell growth inhibitory rates of insect wax and policosanol reached 51.5% and exceeded 50% , respectively, at the dose of 5 000 µg/mL after 3–6 h non-metabolic activation. At the dose of 5 000 µg/mL for 24 h non-metabolic activation, the cell growth inhibitory rate of insect wax reached 54.8% , and almost all cells in the policosanol group died. Therefore, in accordance with the requirements of the test guidelines [35,36], this test was treated with non-metabolic activation (-S9) for 3–6 h, non-metabolic activation(-S9) for 24 h, and metabolic activation (+S9) for 3–6 h. Each treatment was divided into 5 dose groups, negative (blank) control group (culture medium), and positive control group (mitomycin C(MMC), cyclophosphamide (CP)) (Table 2). Two parallel samples were designed in each group.

2.4.3 Test methods

Chinese hamster lung (CHL) fibroblasts (Cell Bank of the Committee for the Preservation of Typical Cultures, Chinese Academy of Sciences) were used as the experimental system.

2.4.3.1 Drug administration in cells

Before drug administration, the well-growth CHL cells were washed several times with phosphate buffer solution and added with 0.35–1.00 mL of 0.25% trypsin digestion solution to prepare the cell suspension. Then, the number of cells was counted. CHL cells were seeded into cell culture flasks at 3.3 × 105–4.3 × 105cells/mL and supplemented with complete medium (1640 medium stock +10% FBS + 1% double antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL)) to a final volume of 5 mL for cell culture(37 °C, 95% relative humidity (RH), 5% CO2). Drug administration was conducted when the cell population reached 80% .

MMC treatment groups: A 2.5 µL volume of the working solution(1 mg/mL) was added into 5 mL of serum-free 1640 medium and mixed evenly as the positive treatment groups under non-metabolic activation conditions (-S9) for 3–6 h, and 24 h, respectively. The final concentration of the positive substance was 0.5 µg/mL.

CP treatment group: A 50 µL volume of the working solution(1 mg/mL) was added into 4 mL of serum-free 1640 medium and mixed evenly. When the positive control was administered,1 mL of S9 mixture (20% ) was added into each flask, and the final concentration of the positive substance was 10 µg/mL.

2.4.3.2 Cytotoxicity assay

Before cell harvesting, 10 µL of 0.4 mg/mL colchicine ( final concentration of 0.8 µg/mL) was added to all treatment groups for 2–3 h. After being harvested, the cells were digested with 0.25% trypsin solution, and the digestion was terminated by adding an appropriate amount of cell growth medium. Each culture was harvested separately and prepared as cell suspension. The cell density of each dose group was counted using the cell counting method, and the IC50was calculated according to the inhibition rate of the cells from each dose group.

2.4.3.3 Chromosomal aberration microscopic observation and evaluation

The highest dosages of insect wax ad its policosanol for chromosomal aberration tests were ascertained according to their cytotoxicity. Thus, according to the results of cytotoxicity, for the insect wax groups, selected dosages were 5 000, 2 500, and 1 200 µg/mL. For the policosanol groups, 5 000, 2 500, and 1 200 µg/mL were selected in the (-S9) 3–6 h treatment and 1 200, 600, and 300 µg/mL in the (+S9) 3–6 h and (-S9) 24 h treatments. Slides were stained with Giemsa dye and examined under a microscope. At least 200 well-dispersed metaphase cells were observed in each rat, and chromosomal structural aberration was recorded to calculate the chromosomal aberration rate. Fractures were recorded separately but not included in the aberration rate. The number of chromosomal aberrations, such as polyploidy and endoreplication, was recorded separately but not included in the aberration rate.

If the rate of chromosomal aberration induced by the test samples exhibited a dose-dependent increase, an increase in repeatability, and a statistically significant difference (P＜ 0.05), it could be judged as a positive result. If the results did not meet the above criteria, the test samples were considered as a negative result in this system.

2.5 Data processing

SPSS 19.0 software was used for the chi-square test test of all data.Each experiment consisted of three replicates. SPSS 19.0 software was used for the chi-square test of all data.P＜ 0.05 means a statistically significant difference.

3. Results and discussion

3.1 Acute oral toxicity of insect wax and its policosanol

Acute oral toxicity test was used to detect and evaluate the toxic effects of a substance. Animals’ toxic reactions, including signs of poisoning and death, are observed over a short period of time(usually lasting 14 days) after a one-time or repeated administration of the substance within 24 h, generally expressed as LD50. Acute oral toxicity can provide information on the health hazards caused by oral contact with the substance in a short period of time. LD50is the basis for the classification of acute toxicity, and it provides the basis of dose selection and observation index for further toxicity tests [32,33].

During the experimental observation period, the rats fed with insect wax and policosanol, respectively, all had normal water intake, skin, hair coating, body and limb movements, and respiratory,circulatory, autonomic nervous, and central nervous systems. Asthma,cough, abnormal secretion in five sense organs, tremor, convulsion,salivation, diarrhea, drowsiness, coma, toxic symptoms, and death were also absent. The average body weight of the rats normally increased, and the growth and development of the rats were good.The body weight and quality records are shown in Table 3. Compared with the control group, the anatomical indexes of the rats showed no abnormality, and no obvious lesions were observed in the external genitalia, nose, mouth, ear, and anus. The position, color, size, and hardness of the viscera in the abdominal, pelvic, and thoracic cavities,including the brain, the pituitary gland, the thyroid gland, the thymus,the lung, the heart, the liver, the spleen, the kidney, the adrenal gland,the stomach, the intestinal tract, and the reproductive organs, were normal. Similarly, no hemorrhage or adhesion was detected. No visceral lesions in major organs were observed.

Table 3Impact of insect wax and policosanol on the body weight of rats in the acute oral toxicity test.

Taken together, evaluation results of acute oral toxicity revealed no toxic reaction in the rats fed with insect wax and its policosanol at the dose of 5 000 mg/kg, suggesting that the LD50values of insect wax and its policosanol were higher than 5 000 mg/kg, which was equivalent to the lethal dose in human of 250 000–500 000 mg/kg body weight or 500 g/person. These results suggest that insect wax and its policosanol are non-acute oral toxic substances [37,38],providing a reference for the dose level design of sub-chronic,chronic, long-term, and other toxicity tests.

3.2 In vitro mammalian cell chromosomal aberration test of insect wax

In vitrochromosomal aberration test is used to identify substances that cause structural chromosomal aberrations in cultured mammalian cells. The selection of mammalian cells used should mainly concern the growth ability in culture, spontaneous frequency of chromosomal aberrations, and stability of the karyotype, among others. The CHL or Chinese hamster ovary (CHO) cell line, human, or other mammalian peripheral blood lymphocytes can be selected, but the CHL cell line is usually recommended [33,35].

Cytotoxicity should first be determined with and without metabolic activation in the main experiment. Then, the highest dosages of test samples for chromosomal aberration test can be ascertained according to their cytotoxicity.

3.2.1 Cytotoxicity of insect wax to CHL cells cultured in vitro

Cytotoxicity is a factor determining the maximum concentration of a sample for chromosome aberration experiments. As shown in Table 4, when the cells were exposed to insect wax under nonmetabolic activation conditions for 3-6 h, its cell growth inhibitory rates at the doses of 5 000, 2 500, 1 200, 600, and 300 µg/mL were 52.1% , 48.7% , 39.6% , 40.7% , and 27.0% , respectively, and the IC50was 3 342.3 µg/mL. When the cells were exposed to insect wax for 24 h, its cell growth inhibitory rates at the above doses were 54.0% ,51.2% , 31.7% , 19.3% , and 13.6% , respectively, and the IC50was 3 242.0 µg/mL. Under metabolic activation conditions, when the cells were incubated with insect wax for 3-6 h, the cell growth inhibitory rates of insect wax at the above doses were 50.6% , 39.0% , 34.6% ,24.4% , and 9.0% , respectively, and the IC50was 4 288.1 µg/mL.The cytotoxicity of the chemicals after metabolic activation in the metabolic activation system may be different from that of the original compounds, which was also verified from the data in Table 4.Thus, determining the cytotoxicity of chemicals with different action times under metabolic activation/non-metabolic activation conditions is important, and this concentration can be considered as the highest dosage of chromosomal aberration test. It can be seen that insect wax showed low cytotoxicity with approximately 50% cell growth inhibition at 5 000 µg/mL under metabolic and non-metabolic activation conditions. Thus, the maximum concentration (5 000 µg/mL)was selected for the following chromosome aberration tests of insect wax regardless of metabolic activation conditions,considering that the cell growth inhibition rate should be less than or close to 50% , thus avoiding the false positives caused by cytotoxicity [35,36].

Table 4Cytotoxicity of insect wax to cultured CHL cells in vitro.

3.2.2 Chromosomal aberration test of insect wax in CHL cells cultured in vitro

On the basis of the cytotoxicity of insect wax showed in section 3.2.1, the cells incubated with insect wax at the doses of 5 000,2 500, and 1 200 µg/mL were selected for slicing and microscopic examination to evaluate whether or not insect wax could induce chromosomal aberrations in cultured cellsin vitro. When the CHL cells were treated under non-metabolic activation conditions for 3–6 h, metabolic activation conditions for 3–6 h, and non-metabolic activation conditions for 24 h, the chromosomal aberration rates of the cells were lower than 5% in the negative control group and 22.0% , 14.8% , and 17.9% in the positive control group, respectively,which significantly differed from those in the negative control group(P＜ 0.05). Under the condition of the non-metabolic activation system,the chromosomal aberration rates of the cells incubated for 3–6 h with insect wax at the doses of 5 000, 2 500, and 1 200 µg/mL were 4.3% , 4.4% , and 4.3% , respectively, and those of the cells incubated for 24 h were 2.5% , 4.1% , and 4.2% , respectively. Under metabolic activation conditions, the chromosomal aberration rates of the cells incubated for 3–6 h with insect wax at the above dosages were 3.8% ,3.5% and 4.8% , respectively. The chromosomal aberration rate of the cells in each dosage group was lower than 5% , no significant difference was found when compared with the negative control group (P＞ 0.05), and a significant difference was noted when compared with the positive control group (P＜ 0.05) (Table 5).

Table 5Results of the chromosome aberrations test in CHL cells treated with insect wax in vitro.

In accordance with the evaluation criteria of mammalian cell chromosomal aberration in OECD 473, the results of chromosomal aberration test in CHL cells culturedin vitrowith or without metabolic activation conditions were negative. Therefore, insect wax exerted no effect on chromosomal aberration of CHL cells within the tested dose range.

3.3 Chromosomal aberration test of mammalian cells incubated with policosanol

3.3.1 Cytotoxicity of policosanol to CHL cells cultured in vitro

The cytotoxicity of policosanol was firstly determined to provide reference for further selection of the highest dosage for chromosomal aberration test. As shown in Table 6, when the cells were incubated with policosanol under non-metabolic activation conditions, its cell growth inhibitory rates at the doses of 300, 600, 1 200, 2 500, and 5 000 µg/mL were –4.7% , 18.6% , 30.0% , 22.9% , and 22.8% for 3–6 h incubation, and the IC50was 44.1 µg/mL. The cell growth inhibitory rates of policosanol at the above doses were 34.4% , 36.5% , 32.7% ,97.2% , and 98.9% after 24 h incubation, and the IC50was 806.3 µg/mL.When the cells were incubated with policosanol under metabolic activation conditions for 3–6 h, its cell growth inhibitory rates at the above doses were 4.4% , 23.8% , 39.2% , 59.3% , and 77.8% , and the IC50was 1 806.2 µg/mL. Policosanol showed very low cytotoxicity with 22.8% cell growth inhibition at 5 000 µg/mL under non-metabolic activation conditions for 3–6 h incubation. Thus, 5 000 µg/mL was selected to be the highest dosage for chromosome aberrations test of policosanol. Dose-dependent inhibition of CHL cells was observed when they were incubated with policosanol under non-metabolic activation conditions for 24 h and metabolic activation conditions for 3–6 h, and the cell growth inhibitory rates showed a sharp increase and were more than 50% when the dosage of policosanol exceeded 2 500 µg/mL. Thus, the maximum dosage was chosen as 1 200 µg/mL for the following chromosome aberration tests for policosanol under the two conditions to avoid the false positives caused by cytotoxicity [35,36].

Table 6Cytotoxicity of policosanol to cultured CHL cells in vitro.

3.3.2 Chromosomal aberration test of policosanol in CHL cells cultured in vitro

As shown in Table 7, on the basis of the toxicity results,policosanol at the doses of 1 200, 2 500, and 5 000 µg/mL were selected for slicing under non-metabolic activation conditions for 3–6 h incubation. Policosanol at the doses of 300, 600, and 1 200 µg/mL were selected for slicing and microscopic examination at the conditions with 3–6 h metabolism and 24 h non-metabolic activation to evaluate whether or not policosanol can cause chromosomal aberration in cultured cellsin vitro. Results showed that when the CHL cells were treated with non-metabolic activation for 3–6 h, metabolic activation for 3–6 h and non-metabolic activation for 24 h, the chromosomal aberration rates of cells in the negative control group was less than 5% , and 14.8% , 16.8% , and 24.3% in the positive control group, respectively, which was significantly different from those in the negative control group (P＜ 0.01). When the cells were incubated for 3–6 h with policosanol under non-metabolic activation conditions, the chromosomal aberration rates of policosanol at the doses of 1 200, 2 500, and 5 000 µg/mL were 5.8% , 3.9% , and 2.6% ,respectively, and 9.0% , 3.8% , and 5.0% at the doses of 300, 600,and 1 200 µg/mL for 24 h incubation, respectively. When the cells were exposed to policosanol under metabolic activation conditions,the chromosomal aberration rates of policosanol at the doses of 300,600, and 1 200 µg/mL were 6.5% , 5.3% , and 6.8% , respectively.No significant difference in chromosomal aberration rate was found when compared with the negative control group (P＞ 0.05), whereas a significant difference was detected when compared with the positive control group (P＜ 0.05).

Table 7Chromosome aberration test in CHL cells treated with policosanol in vitro.

On the basis of the evaluation criteria of mammalian cell chromosomal aberration in OECD 473, the results of chromosomal aberration test in CHL cells culturedin vitrowith or without metabolic activation conditions were negative. These results indicate that policosanol exerts no effect on the chromosomal aberration of CHL cells in the tested dose range.

The LD50values of insect wax and its policosanol were higher than 5 000 mg/kg, without any lesion on organs via histopathological observation, which was equivalent to the human lethal dose of 250 000-500 000 mg/kg body weight, indicating that insect wax and its policosanol are non-toxic [37,38]. This agree with the results of the study that policosanol isolated from sugarcane wax did not show any oral toxicity in doses up to 5 000 mg/kg to SD rats for 6 months [39]but contrasts with the hepatomegalia induced by other cholesterollowering drugs, such as statins, which increase levels of serum transaminases as a consequence of the direct competitive inhibition of the enzyme, in addition, high doses of statins promote the growth of live tumors in rodents and so these drugs are contraindicated in patients with active hepatic disease [40]. In contrast, insect wax and policosanol have shown lack of hepatotoxicity. And this difference in the safety profile of policosanol compared with statins may be due to that policosanol only modulate, does not directly inhibit, the enzymatic activity of HMGCoA-reductase [39]. Furthermore, the application dosage of policosanol is very small. The total cholesterol,low density lipoprotein, and apolipoprotein B levels of 40 children suffering from hypercholesterolemia significantly reduced after oral administration of 10 mg policosanol, 3 mg monacolins, and 200 mg red yeast rice extract without any side effects [41]. In addition, previous studies reported that 5–20 mg/day of policosanol can effectively reduce total cholesterol in blood after 6–8 weeks consumption [2,42,43]. No toxicity in SD rats was observed when the oral dose was 250–500 times higher than the maximum therapeutic dose (20 mg/day) approved to date, indicating that insect wax and its policosanol are very safe even when administered at very high oral doses.

Chromosome aberration and related events cause many human genetic diseases. In fact, chromosome aberration and related events causing alterations in oncogenes and tumor suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals [44]. In the present study, no chromosomal aberrationin vitrowas observed in the short-term treatment of insect wax and its policosanol under metabolic and non-metabolic activation conditions. The principle of OECD 473 and other national standard for food safety [35,36]states that if the short-term treatment results of cells under metabolic or non-metabolic activation conditions are negative, approximately 1.5 or longer cell cycles are highly necessary for continuous processing under non-metabolic activation conditions.Hence, incubation for 3–6 and 24 h under non-metabolic activation conditions and for 3–6 h under metabolic activation conditions were chosen to monitor the chromosome aberrations. The results of treatment with incubation for 3–6 h under non-metabolic and metabolic activation conditions were both negative. Furthermore,negative results were obtained after insect wax and its policosanol were treated under non-metabolic activation conditions for 24 h incubation. These results suggest that insect wax and its policosanol exert no chromosomal aberration effect on mammalian cellsin vitro. Generally known, structural abnormalities originate from breaks in both strands of the DNA double helix (doublestrand breaks, or DSBs). Some external agents, such as chemicals,high-energy radiation, or errors during DNA replication, may lead to the random breaking of DNA. Also, chromosome aberrations can arise from homologous crossing-over between repeated sequences that are misaligned. Thus, the results obtained in this study suggest that insect wax and its policosanol do not induce DNA lesions, including the homologous crossing-over of repeated sequences, misaligning of sequences either within the same chromosome or between different chromosomes, and DNA DSBs in mammalian cells [45]. This is also the first study on the genotoxicity of insect wax and its policosanol.

On the basis of the relevant provisions of toxicological evaluation in Technical Specification for Inspection and Evaluation of Health Foods issued by the Ministry of Health of the People’s Republic of China and the above experimental results, insect wax and its policosanol possessed no acute toxicity and potential chromosomal aberration within the experimental dose range. This result suggests that it is important for the popularization and application of insect wax and its policosanol and for the development of characteristic health food additives and therapeutic drugs. This study also provides relevant data on acute toxicity and genotoxicity for the safety evaluation of insect wax and its policosanol, which can provide a reference for the dose level design of sub-chronic, chronic, long-term,and other toxicity tests.

4. Conclusion

The acute oral toxicity in SD rats and mammalian cell chromosomal aberration toxicityin vitroof insect wax and its policosanol were investigated. The LD50values of insect wax and its policosanol were higher than 5 000 mg/kg without any obvious toxic symptoms and death of all rats based on the oral acute toxicity test. The chromosomal aberration rates of CHL cells subjected to the treatment with insect wax and its policosanol were negative regardless of metabolic activation conditions. Therefore, the tested dose range of insect wax and its policosanol caused no acute toxicity and potential chromosomal aberration. This study benefits the application of insect wax and its policosanol in characteristic health food additives and therapeutic drugs.

Declaration of Competing Interest

The authors declare no competing interests.

Acknowledgements

We gratefully acknowledge the support of the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2018SY025), and the National High Technology Research and Development Program of China (863 Program)(2014AA021801).