Evaluation of genotoxicity of SUNACTIVE Zn-P240 in vitro and in vivo

Abstract

We evaluated the potential genotoxic effects of the nutrient supplement SUNACTIVE Zn-P240 in vitro and in vivo. Genotoxicity tests were performed at the Korea Testing and Research Institute, a GLP certification institution. A bacterial reverse mutation test was performed using the pre-incubation method, while the in vitro chromosome aberration test was performed using a cultured Chinese hamster lung cell line in the presence or absence of metabolic activation. The in vivo micronucleus test was performed using ICR mice. The bacterial reverse mutation test revealed that SUNACTIVE Zn-P240 did not induce genetic mutations at the tested doses in Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA) tester strains. Meanwhile, the results of the in vitro chromosomal aberration and in vivo micronucleus tests revealed that SUNACTIVE Zn-P240 did not induce chromosomal aberrations. These results suggest that SUNACTIVE Zn-P240 did not exhibit mutagenic or clastogenic properties in vitro and in vivo.

Introduction

Zinc deficiency, a nutritional disorder that affects 25% of the global population [1, 2], is characterized by growth retardation, appetite loss, and impaired immune function. Severe zinc deficiency causes hair loss, diarrhea, delayed sexual maturation, erectile dysfunction, hypogonadism in males, and skin and eye lesions [1, 3–5]. Additionally, zinc deficiency can lead to weight loss, delayed wound healing, impaired taste, and mental lethargy [6–11]. In contrast, excessive intake of zinc can result in adverse health effects. The adverse effects of excessive zinc intake include nausea, vomiting, appetite loss, abdominal cramps, diarrhea, and headaches [12]. The intake of 150–450 mg of zinc per day is associated with chronic effects, such as low copper levels, altered iron function, defective immune function, and reduced levels of high-density lipoproteins [13].

Food supplements, which are concentrated sources of nutrients (i.e. mineral and vitamins) or other substances with a nutritional or physiological effect, are marketed in various dosage forms (e.g. pills, tablets, capsules, liquids in measured dose) to improve the nutritional quality of the food and provide a public health benefit with minimal health risk. This is an affordable and effective strategy to prevent mineral deficiency. However, mineral fortification in food is associated with several challenges, including unexpected changes during the food preparation process and storage, low mineral bioavailability, and high cost [14]. The application of nanotechnology techniques, such as colloidal techniques, emulsification, and surface coating can be a viable strategy to overcome the challenges associated with food fortification [15].

SUNACTIVE Zn-P240, which is a novel type of zinc supplement, respectively, has been developed using nanotechnology to overcome the challenges associated with food supplements [15]. Nanotechnology provides a wide range of options to improve food quality and enhance food taste attributes. These techniques provide high dispersion stability in water-soluble media and can be used for the preparation of liquid foods or beverages without compromising the taste attributes. SUNACTIVE Zn-P240, an emulsion of zinc oxide nanoparticles with maltodextrin and glycerides, was reported to enhance zinc bioavailability in zinc-deficient rats [16]. Although the application of nanotechnology has several advantages in food supplementation and fortification, various studies have raised safety concerns on the use of nanomaterials. Thus, there is a need to perform systemic studies on the genotoxicity of SUNACTIVE Zn-P240 to promote its applications because the effect of SUNACTIVE Zn-P240 on human health has not been identified not yet.

This study aimed to determine the genotoxicity of SUNACTIVE Zn-P240. The following three tests were performed for the accurate evaluation of SUNACTIVE Zn-P240 genotoxicities: bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo bone marrow micronucleus test. These tests were performed according to the Organisation for Economic Cooperation and Development (OECD) guidelines for the testing of chemicals under the modern Good Laboratory Practice Regulations [17]. The results of this study will provide critical information on the safety profiles of zinc supplements.

Materials and methods

Preparation of test substance

SUNACTIVE Zn-P240 was purchased from Taiyo Kagaku Co., Ltd. (Yokkaichi, Japan). According to the certificate of analysis provided by the manufacturer, the components of SUNACTIVE Zn-P240 were as follows: zinc oxide (30.0%), maltodextrin (62.12%), polyglycerol esters of fatty acids (6.0%), and enzymatically hydrolyzed lecithin (1.88%). SUNACTIVE Zn-P240 was stored under moisture-free conditions at room temperature (15–25 °C) until analysis. The test materials were suspended in sterile distilled water (Daihan Pharm. Co., Ansan, Korea) for injection immediately before treatment. The high-dose solutions were serially diluted to obtain the low-dose solutions.

Characterization

X-ray diffraction analysis, which was performed using an X-ray diffractometer (Bruker AXS D2 Phaser, Billerica, MA, USA), revealed that SUNACTIVE Zn-P240 exhibited a Wurtzite crystal phase. Hydrodynamic radii, which represent the secondary particle size in distilled and deionized water suspension, was analyzed using a dynamic light scattering instrument (ELSZ-1000, Otsuka, Japan). SUNACTIVE Zn-P240 exhibited hydrodynamic radii of 350 nm. The scanning electron microscopy (SEM) analysis at low magnification revealed that SUNACTIVE Zn-P240 exhibited a sphere-like morphology. The high magnification scanning electron micrograph, which was captured using a scanning electron microscope (Quanta 250 FEG, FEI Company Hillsboro, OR, USA), of the surface revealed that the primary particle size of SUNACTIVE Zn-P240 was 112 ± 42 nm.

Bacterial reverse mutation test

SUNACTIVE Zn-P240 was subjected to the bacterial reverse mutation tests according to the OECD guidelines for the testing of chemicals, TG 471 Bacterial Reverse Mutation Test [18], and the MFDS Standards Guidelines for Toxicity Test of Pharmaceuticals. The following bacterial tester strains were used in this study: four histidine auxotroph Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537); one tryptophan auxotroph Escherichia coli strain (WP2uvrA). The tests were performed in the presence or absence of the S9 mixture, which was used for metabolic activation [19, 20]. The bacterial tester strains used in this study are sensitive to the mutagenicity of diverse chemicals. All bacterial tester strains were purchased from Molecular Toxicology Inc. (Boone, NC, USA). Mutagenic potentials were examined in the absence and presence of S9 mixture using histidine-requiring Salmonella typhimurium TA98, TA100, TA1535, and TA1537 tester strains, and tryptophan-requiring Escherichia coli WP2uvrA strain. The viable cell counts in the bacterial cultures used in the test were more than 1 × 10⁹ cells per milliliter.

To make a 50 mg/mL solution (final concentration: 5000 μg/plate), SUNACTIVE Zn-P240 was weighed and dissolved in sterile distilled water using a vortex. To obtain the final working concentrations of 313, 625, 1250, and 2500 g/plate, the stock solution was serially diluted with a vehicle. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (Wako Pure Chemical Industries Ltd., Osaka, Japan), sodium azide (Sigma-Aldrich Co., St Louis, MO, USA), 9-aminoacridine (Sigma-Aldrich Co.), and 2-aminoanthracene (Sigma-Aldrich Co.) were used as positive controls. The metabolic activation system was prepared by mixing the S9 mixture (Molecular Toxicology Inc.) with Cofactor 1 from Oriental Yeast Co., Ltd. (Japan) to obtain a final S9 concentration of 10% (v/v). The bacterial tester strains were cultured in 2.5% nutrient broth No. 2 (Oxoid Ltd., Basingstoke, UK) in a 37 °C shaking incubator (120 rpm) for approximately 10 h. The mutagenicity test was performed by incubating SUNACTIVE Zn-P240 with the bacterial tester strains in the presence or absence of S9 mixture in a water bath for 20 min at 37 °C. The mixture was combined with an overlay agar and a minimal amount of histidine-biotin (for S. typhimurium strains) or l-tryptophan (for E. coli strain). The overlay agar was poured onto the surface of a gamma-ray-sterilized Falcon^® Petri dish (Thermo Fisher Scientific, Waltham, MA, USA) containing approximately 15 mL of solidified bottom agar. The samples were incubated for 48 h at 37 °C and the revertant colonies were counted. All plates were prepared in triplicates. The results were considered positive if the number of revertant colonies in the test plates was two-fold higher than that in the negative control plates or if there was a dose-dependent increase in the number of revertant colonies.

In vitro chromosome aberration test

The chromosome aberration tests were performed using Chinese hamster lung (CHL) fibroblast cells according to the OECD guideline No. 473 for testing of chemicals [21]. The clastogenicity of SUNACTIVE Zn-P240 was evaluated based on their ability to induce chromosomal aberrations in CHL cells. The relative increase in cell count (RICC) method was used to assess cell growth and cytotoxicity. The cells were counted by cell count analyzer (Vi-cell™, Beckman Coulter, FL, USA). Based on the results of a concentration range-finding test, the concentration exceeding the RICC₅₅ was set as the highest concentration in each treatment condition. A clonal subline of CHL cells was obtained from the American Type Culture Collection (Rockville, MD, USA). The karyotype of the CHL cells comprised 25 chromosomes. The CHL cells were cultured in minimum essential Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin (Gibco BRL Life Technologies Inc., Gaithersburg, MD, USA) at 37 °C and 5% CO₂ in a humidified atmosphere. The Mitomycin C (0.1 µg/mL) was used as a positive control substance for reaction mixtures without metabolic activation, whereas cyclophosphamide monohydrate (6 µg/mL) was used for reaction mixtures with metabolic activation. Distilled water (Choongwae Pharma, Seoul, Korea) was used as the negative control. After 22 h of incubation, colcemid was added to the cultures at a final concentration of 0.2 μg/mL. The metaphase cells were harvested by trypsinization and centrifugation. The cells were incubated with hypotonic (0.075 M) KCl solution for 20 min at 37 °C to induce swelling. Next, the cells were washed thrice with an ice-cold fixative solution (methanol:glacial acetic acid; 3:1). Few drops of the cell pellet suspension were dropped onto precleaned glass microscope slides and air-dried. The samples were stained with 5% Giemsa buffer solution (Thermo Fisher Scientific). The number of cells with chromosomal aberrations among 200 well-spread metaphase cells was recorded. Structural (gaps, breakage, exchange) and numerical (diploidy, polyploidy, endoreduplication) aberrations were evaluated in each 100 metaphases. Chromosomal aberration was classified according to the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Group [22]. 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.

In vivo mouse bone marrow micronucleus test

The micronucleus tests were performed according to the OECD test guideline No. 474 for testing of chemicals [23]. Male ICR mice aged 6 weeks were obtained from a specific pathogen-free colony at Orient Bio Inc. (Gyeonggi, Korea). The animals were quarantined and allowed to acclimatize for 7 days. Next, the animals were housed in a stainless wire cage (180 (width) × 300 (length) × 140 (height) mm) in a room under the following conditions: circadian cycle, 12-h light/dark cycle; air exchange, 10–20 changes/h; temperature, 19–25 °C; relative humidity, 30–70%. The animals were fed on a Rodent Diet 5L79 (LabDiet, MA, USA) and had access to reverse osmosis water ad libitum. ICR mice were randomly assigned into 5 groups (N = 5 for each group) for each test substance and were maintained according to the Guide for the Care and Use of Laboratory Animals [24]. Mice were administered with SUNACTIVE Zn-P240 solution at doses of 500, 1000, and 2000 mg/kg bodyweight once daily through gavage for two days. A dose volume of 10 mL/kg bodyweight was used based on the most recent bodyweight data. The mice in the positive control group were intraperitoneally administered with CPA at a dose of 70 mg/kg bodyweight. All animals were monitored once daily following treatment for any clinical signs of toxicity and mortality during the study period. At the time of purchase, grouping, pre-drug administration, and autopsy, each mouse's bodyweight was measured. After the last administration, all animals were euthanized by CO₂ gas inhalation. FBS (Gibco BRL Life Technologies Inc.) was used to flush the bone marrow cells. The cells were transferred to a centrifuge tube and spun for 5 min at 1000 rpm. A clean glass slide was coated with the suspended cells. The smear was air-dried for 5 min before being fixed with methanol. The methanol-fixed smear was stained with 40 μg/mL acridine orange.

The micronuclei were counted in both polychromatic erythrocytes (PCE) and monochromatic erythrocytes. The percentage of micronucleated cells relative to the total number of PCE in the scored optic fields was determined. This method of scoring, which must always be followed in cases where the test substance markedly influences the cell proliferation rate in bone marrow, prevented the distortion of the results caused by the influx of peripheral blood into the damaged marrow. The scoring of micronucleated normocytes was used to determine the presence of artifacts (rare in mouse preparations), which provided additional information on the mode of action of SUNACTIVE Zn-P240. Generally, an incidence of more than one micronucleated normocyte per 1000 PCE indicates an effect on cell stages, especially on the post-S-phase. The result was considered positive if all the following acceptability criteria were met: the frequency of MNPCE/2000 PCEs is statistically reliable, increases dose-dependently, and/or shows a reproducible positive reaction at one or more concentrations, and any of the results are outside the distribution of historical negative control data. The animal study protocols were approved by the Institutional Animal Care and Use Committee (Approved number: IAC2018-116). The animals were maintained according to the Guide for the Care and Use of Laboratory Animals.

Statistical analyses

The number of revertant colonies was expressed as mean ± standard deviation. The in vitro chromosomal aberration test results of the negative control or positive control and treatment groups were compared using the Fisher’s exact test [25]. The dose–response relationship of the frequency of aberrant metaphase was examined using the linear-by-linear association of the chi-square test [26]. The following tests were used to analyze the difference in the number of micronucleated polychromatic erythrocytes (MNPCE) in the in vivo mouse micronucleus test: Kruskal–Wallis H test [27] and Dunnett’s test [28], for comparison between treatment and negative control groups; Mann–Whitney U test [29], for comparison between the positive and negative control groups. Meanwhile, the differences in the PCE/(PCE + normochromatic erythrocyte (NCE)) ratio were analyzed using the following tests: analysis of variance and Dunnett’s test, for comparison between treatment and negative control groups; Student’s t-test, for comparison between the positive and negative control groups. The bodyweight of animals at the time of euthanasia was compared using analysis of variance and Dunnett’s test. All statistical analyses were performed using SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA). The significance of the differences between the treatment, positive control, and negative control groups was calculated at 1% and 5% probability levels.

Results

Bacterial reverse mutation test

No cellular toxicity and precipitates were observed at any concentration in either the absence or the presence of metabolic activation. All Salmonella tester strains were sensitive to crystal violet, whereas only the plasmid-containing Salmonella tester strains, TA98 and TA100, were resistant to ampicillin. Increased sensitivity to ultraviolet radiation was also demonstrated. At a dose of 5000 μg/plate, SUNACTIVE Zn-P240 did not exert growth-inhibitory effects on the S. typhimurium (TA98, TA100, TA1535, and TA1537) and E. coli (WP2uvrA) tester strains in the presence or absence of metabolic activation (data not shown). Thus, 5000 μg/plate was selected as the highest dose for SUNACTIVE Zn-P240. As shown in Table 1, the number of revertant colonies in the bacterial tester strains treated with SUNACTIVE Zn-P240 at doses of 312.5, 625, 1250, 2500, and 5000 μg/plate was similar to that of the negative control group irrespective of metabolic activation. In contrast, the number of revertant colonies in the positive control group was twofold higher than that in the respective negative control group in the presence or absence of metabolic activation.

Table 1.

Results of the bacterial reverse mutation test for SUNACTIVE Zn-P240 in Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA) with or without metabolic activation (S9 mixture)

Tester Strain	Chemical	Dose (μg/plate)	Colonies/plate [Factor]a
			Without S9 mixture		With S9 mixture
TA98	SUNACTIVE Zn-P240	0	23 ± 2		27 ± 2
		313	23 ± 2	[1.0]	27 ± 4	[1.0]
		625	22 ± 4	[0.9]	26 ± 4	[1.0]
		1250	23 ± 3	[1.0]	26 ± 2	[1.0]
		2500	22 ± 1	[1.0]	29 ± 6	[1.1]
		5000	24 ± 5	[1.0]	26 ± 4	[1.0]
TA100	SUNACTIVE Zn-P240	0	116 ± 2		131 ± 3
	Zn-P240	313	118 ± 2	[1.0]	132 ± 3	[1.0]
		625	115 ± 4	[1.0]	128 ± 3	[1.0]
		1250	114 ± 3	[1.0]	132 ± 4	[1.0]
		2500	116 ± 2	[1.0]	136 ± 5	[1.0]
		5000	115 ± 4	[1.0]	126 ± 6	[1.0]
TA1535	SUNACTIVE Zn-P240	0	13 ± 1		15 ± 3
	Zn-P240	313	11 ± 1	[0.8]	15 ± 2	[1.0]
		625	11 ± 2	[0.9]	13 ± 2	[0.9]
		1250	13 ± 1	[1.0]	15 ± 3	[1.0]
		2500	14 ± 2	[1.1]	15 ± 3	[1.0]
		5000	13 ± 2	[1.0]	13 ± 2	[0.8]
TA1537	SUNACTIVE Zn-P240	0	8 ± 2		12 ± 3
	Zn-P240	313	8 ± 1	[1.0]	14 ± 2	[1.2]
		625	8 ± 2	[1.0]	13 ± 1	[1.1]
		1250	8 ± 1	[1.0]	12 ± 2	[1.0]
		2500	7 ± 2	[0.8]	11 ± 1	[0.9]
		5000	8 ± 1	[1.0]	11 ± 2	[0.9]
WP2uvrA	SUNACTIVE Zn-P240	0	41 ± 2		51 ± 1
	Zn-P240	313	45 ± 2	[1.1]	51 ± 3	[1.0]
		625	41 ± 1	[1.0]	51 ± 2	[1.0]
		1250	43 ± 2	[1.1]	48 ± 2	[0.9]
		2500	43 ± 3	[1.0]	49 ± 4	[1.0]
		5000	38 ± 2	[0.9]	53 ± 1	[1.0]
Positive controls
TA98	AF-2	0.1	473 ± 9	[20.3]
TA100	AF-2	0.01	504 ± 22	[4.4]
TA1535	NaN3	0.5	324 ± 7	[24.9]
TA1537	9-AA	40.0	172 ± 29	[20.6]
WP2uvrA	AF-2	0.01	324 ± 10	[8.0]
TA98	2-AA	0.5			281 ± 11	[10.5]
TA100	2-AA	1.0			577 ± 9	[4.4]
TA1535	2-AA	2.0			187 ± 32	[12.2]
TA1537	2-AA	2.0			141 ± 16	[12.1]
WP2uvrA	2-AA	10.0			318 ± 8	[6.2]

Values are presented as the mean ± SD

AF-2 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, NaN₃ sodium azide, 9-AA 9-aminoacridine hydrochloride monohydrate, 2-AA 2-aminoanthracene

^aNo. of colonies of treated plate/No. of colonies of negative control plate

In vitro chromosome aberration test

Preliminary test was performed to determine the concentration ranges of SUNACTIVE Zn-P240 for the in vitro chromosome aberration test (data not shown). The precipitation or turbidity/precipitation of test substance was not observed at all concentrations at both the beginning and end of the treatment. Test concentrations were limited by cytotoxicity. The highest concentrations of SUNACTIVE Zn-P240 for short-term treatment and continuous treatment (24 h exposure) assays in the absence of the S9 mixture (referred to as − S9 mix) were determined to be 50 and 25 μg/mL, respectively. The highest concentration of SUNACTIVE Zn-P240 for the short-term treatment assay in the presence of the S9 mixture (referred to as + S9 mix) was determined to be 100 μg/mL. The in vitro chromosome aberration tests were conducted using SUNACTIVE Zn-P240 at concentrations of 12.5, 25, and 50 μg/mL for short-term treatment and 6.25, 12.5, and 25 μg/mL for continuous treatment without metabolic activation. Meanwhile, the in vitro chromosome aberration tests were performed in the presence of the S9 mixture using SUNACTIVE Zn-P240 at concentrations of 25, 50, and 100 μg/mL.

The frequency of chromosomal aberrations in the cells treated with SUNACTIVE Zn-P240 at all tested doses was not significantly different from that in the negative control group (Table 2). Furthermore, in both the positive and negative groups, the frequency of cells with chromosomal abnormalities was within the range of historical control data.

Table 2.

Results of the in vitro chromosome aberration test for SUNACTIVE Zn-P240 in Chinese hamster lung fibroblast cells with and without metabolic activation (S9 mixture)

Concentration (μg/mL)	S9 mixture	Time (h)	SUNACTIVE Zn-P240
			% Numerical aberration	% Structural aberration (exclusive to gap)
0	−	6	0.0	0.0
12.5	−	6	0.0	0.5
25	−	6	0.0	1.5
50	−	6	0.0	0.0
Positive controla	−	6	0.0	22.5**
0	+	6	0.0	0.0
25	+	6	0.0	0.5
50	+	6	0.0	2.0
100	+	6	0.0	1.0
Positive controlb	+	6	0.0	21.5**
0	−	24	0.0	0.0
6.25	−	24	0.0	0.5
12.5	−	24	0.0	0.5
25	−	24	0.0	0.5
Positive controla	−	24	0.0	23.0**

**p < 0.01 compared with the negative control group

^aMitomycin C 0.1 μg/mL

^bCyclophosphamide monohydrate 6 μg/mL

In vivo bone marrow micronucleus test

Preliminary tests (data not shown) revealed that the highest toxic dose of SUNACTIVE Zn-P240 was 2000 mg/kg bodyweight. The PCE/(PCE + NCE) ratios, which were used as an index of cytotoxicity in the bone marrow, were not significantly different between the SUNACTIVE Zn-P240-treated and negative control group (Table 3). The MNPCE frequencies were not significantly different between the SUNACTIVE Zn-P240-treated and negative control group. Additionally, SUNACTIVE Zn-P240 did not affect the MNPCE frequency in a dose-dependent manner. The induction range of MNPCEs in the SUNACTIVE Zn-P240-treated, negative control, and positive control groups was within the historical control data. During the study period, no treatment-related adverse clinical indications or deaths were observed. The bodyweight was not significantly different between the control and SUNACTIVE Zn-P240-treated groups (Table 4).

Table 3.

Results of the in vivo micronucleus test for SUNACTIVE Zn-P240 in the bone marrow of ICR mice

Chemical	Dose (mg/kg)	No. of animal	MNPCE/2000 PCE	PCE/(PCE + NCE)
Negative control	0	5	0.06 ± 0.04	58.13 ± 1.59
SUNACTIVE Zn-P240	500	5	0.08 ± 0.03	59.66 ± 1.52
SUNACTIVE Zn-P240	1000	5	0.07 ± 0.03	60.53 ± 1.79
SUNACTIVE Zn-P240	2000	5	0.08 ± 0.06	59.47 ± 1.53
Positive control (CPA)	70	5	4.37 ± 0.33*	42.73 ± 1.26*

CPA cyclophosphamide monohydrate, MNPCE micronucleated polychromatic erythrocyte, NCE normochromatic erythrocyte, PCE polychromatic erythrocyte

*p < 0.05 compared with the negative control group

Values are presented as the mean ± SD (%)

Table 4.

Body weight changes of ICR male mice exposed to SUNACTIVE Zn-P240

Chemical treated	Dose (mg/kg)	Body weights
		Administration		Sacrifice
		1st	2nd
Negative control	0	34.27 ± 0.95	34.24 ± 0.45	33.39 ± 0.38
SUNACTIVE Zn-P240	500	34.37 ± 1.00	34.35 ± 1.06	33.82 ± 1.47
SUNACTIVE Zn-P240	1000	34.48 ± 1.02	34.33 ± 0.80	33.68 ± 0.78
SUNACTIVE Zn-P240	2000	34.30 ± 0.87	33.99 ± 1.22	33.62 ± 1.33
Positive control (CPA)	70	34.21 ± 0.96	34.15 ± 1.13	32.92 ± 1.13

Values are presented as the mean ± SD (g)

CPA cyclophosphamide monohydrate

Discussion

Systemic toxicological study of SUNACTIVE Zn-P240 using various experimental models can predict their potential toxic effects on human health. Genotoxicity assays have been mainly used to predict in vivo genotoxicity and carcinogenicity of chemicals. Compounds that show up positive in these tests have the potential to cause cancer and/or mutation in humans [30]. In this study, the potential genotoxic effects of SUNACTIVE Zn-P240 were examined using standard in vitro and in vivo genotoxicity tests (bacterial reverse mutation assay, chromosome aberration assay, and bone marrow micronucleus assay).

The bacterial reverse mutation tests identify chemicals that have a key role in the initiation and progression of tumors. This test examines point mutations, which are the etiological factors for various human genetic diseases [31, 32]. Furthermore, this assay is a well acknowledged short-term test for detecting chemicals that can induce genetic harm, resulting in gene mutations [33, 34]. In this study, the frequency of mutations in the test strains treated with SUNACTIVE Zn-P240 was not significantly different from that in the negative control both in the presence and absence of metabolic activation.

Chromosomal aberrations are typical genetic toxic responses to tumor initiation and development processes [35]. The in vitro chromosome aberration test is used to find chemicals that cause structural chromosome aberrations in cultured mammalian cells [36]. The findings of this study demonstrated that the number of numerical and structural chromosome aberrations did not significantly increase in the cells treated with various doses of SUNACTIVE Zn-P240 irrespective of metabolic activation. These findings indicate that SUNACTIVE Zn-P240 cannot induce chromosomal aberrations in CHL cells under the experimental conditions used in this study.

The micronucleus test detects mutagenic chemicals that disrupt chromosomal distribution during cell division [37, 38]. The micronucleus tests were carried out on bone marrow cells of specific pathogen-free male ICR mice in this study. SUNACTIVE Zn-P240 did not significantly increase the frequency of MNPCEs. Additionally, the PCE/(PCE + NCE) ratios in the SUNACTIVE Zn-P240-treated groups were not significantly different from those in the negative control group at treatment doses of up to 2000 mg/kg bodyweight. These findings indicate that SUNACTIVE Zn-P240 do not increase the frequency of micronuclei in the bone marrow cells of male ICR mice under the experimental conditions used in this study. Additionally, SUNACTIVE Zn-P240 did not exert cytotoxic effects on the mouse bone marrow cells.

The toxicity of small-sized particles is higher than that of bulk material. As nanoparticles have a small size, they can enter the human body through inhalation, intake, skin penetration, or injections. Nanoparticles have the potential to interact with intracellular structures and macromolecules for prolonged durations. The toxicity of several bulk materials, such as zinc, is well-understood. However, the concentration or size of bulk materials at which they begin to exhibit new toxicological properties due to nanoscopic dimensions [39] is unclear. Previous studies have reported that most intracellular and in vivo toxicities of nanomaterials result from the production of excess reactive oxygen species (ROS) [40, 41]. Enhanced ROS levels, which indicate oxidative stress, can damage cells by promoting lipid peroxidation, protein alteration, DNA disruption, signaling dysregulation, and gene transcription modulation, which can lead to cancer, kidney disease, neurodegeneration, cardiovascular, or lung disease [42]. ROS can capture electrons from lipids in the cell membrane, which results in the deterioration of physiological function and cell death [43].

Several studies have reported that modifying surface properties using colloidal techniques, emulsification, and surface coating can mitigate the cytotoxicity and genotoxicity of some carcinogenic substances as the modified materials exhibit antioxidant activities [44–46]. The characteristic parameters of nanoparticles, including dissolution, chemical composition, size, shape, agglomeration state, crystal structure, specific surface area, surface charge, surface energy, surface morphology, and surface coating, affect the biological interaction of nanoparticles. Thus, these properties must be evaluated to determine the toxic potential of nanomaterials. The surface coating of SUNACTIVE Zn-P240 with maltodextrin can stabilize particles and avoid agglomeration. Surface coating is also effective in preventing the dissolution and release of toxic ions [47]. Additionally, the steric hindrance of coating can delay the cellular uptake and accumulation of nanomaterials or promote endocytosis [48–51]. Surface coating can also modify the surface charge or composition, which can affect the intracellular distribution and production of toxic ROS. The results of previous studies and this study suggest that SUNACTIVE Zn-P240 with the modified surface characteristics did not exhibit mutagenic or clastogenic potential in standard in vitro and in vivo genotoxicity assays.

Thus, SUNACTIVE Zn-P240 was not mutagenic in the in vitro system or clastogenic in the in vivo system under the experimental conditions used in this study. Therefore, the risk of genotoxicity posed by zinc supplements may be insignificant.

Since the genotoxicity assays in this study were conducted according to the previous OECD test guidelines (1997), there is a limitation in that the number of samples for the chromosome aberration assay and the bone marrow micronucleus assay was smaller than the recent guidelines.

Abbreviations

2-AA

2-Aminoanthracene

9-AA

9-Aminoacridine hydrochloride monohydrate

AF-2

2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

CHL

Chinese hamster lung

CPA

Cyclophosphamide monohydrate

FBS

Fetal bovine serum

MNPCE

Micronucleated polychromatic erythrocyte

NaN₃

Sodium azide

NCE

Normochromatic erythrocyte

OECD

Organisation for Economic Cooperation and Development

PCE

Polychromatic erythrocyte

RICC

Relative increase in cell count

SEM

Scanning electron microscopy

Funding

This study was financially supported by Chonnam National University (Grant number: 2020-1891). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF- 2021R1A2C2011673).

Declarations

Conflict of interest

The authors do not have any conflicts of interests to declare.