Repeated-dose 90-day oral toxicity study of GST in Sprague-Dawley rats

Test facility

This study was conducted in compliance with the principles of Good Laboratory Practice (GLP) at KTR (Korea Testing & Research Institute), Hwasun based on the Korea Good Laboratory Practice (KGLP) and OECD “Principle of Good Laboratory Practice, ENV/MC/CHEM (98)17 (as revised in 1997)”. The study protocol was reviewed and approved (IAC2020-2293) by the Institutional Animal Care and Use Committee (IACUC) of KTR Hwasun based on the Animal Protection Act [Enforcement Date: 2020-02-12] [No.16977 (2020-02-11, partial revision)] [14] and the Laboratory Animal Act [Enforcement Date: 2019-03-12] [No. 15944 (2018-12-11, partial revision)] [15]. The KTR Hwasun has been fully accredited by the association for assessment and accreditation of laboratory animal care (AAALAC). Also, this study was conducted in accordance with the OECD Guidelines for the Testing of Chemicals, Section 4, TG 408 “Repeated dose 90-day oral toxicity study in rodents” [16].

Animal husbandry and maintenance

For the repeated dose toxicity study (Study No. TGK-2020-000233), 55 Sprague-Dawley rats [5 weeks-old, Crl;CD(SD), SPF] of each sex were obtained from the ORIENT BIO Inc. (8, Hwaaksan-ro 124, Buk-myeon, Gapyeong-gun, Gyeonggi-do, Republic of Korea) and kept carefully following an acclimation period of 8(male) – 9(female) days to ensure their suitability for the study. These animals were kept within a well-ventilated and specific pathogen-free (SPF) facility with conditions set to a temperature of 22±3 °C (measured value: 20.7–22.9 °C), a humidity of 50±20% RH (measured value: 46.8–61.8%) with artificial lighting a 12-h light/12-h dark cycle (08:00 – 20:00/20:00 – 08:00) and 10–20 air changes per hr. For study, the healthy animals were used after examining conditions included body weight, clinical signs and then 50 rats/sex were randomly divided into four groups listed in Table 1. Animals were kept in stainless steel wire cages and allowed R/O (reverse osmosis) water via a water bottle and irradiation-sterilized pellet diet (Rodent Diet 20 5053, Labdiet, USA), ad libitum.

Table 1

Groups for 90-day repeated oral toxicity study.

Test materials and preparation

The new TiO₂ materials, GST (pale yellow powder, crystalline composition of 100% anatase) was provided by Bentec Frontier Co., Ltd (139, Nanosandan-ro, Nam-myeon, Jangseong-gun, Jeollanam-do, Republic of Korea). The characterization of GST was evaluated as Table 2 [zeta potential, particle size image (SEM), TEM (transmission electron microscopy) image, size distribution].

Table 2

Evaluation for characterization of TiO₂.

For dose selection, we considered the result of previously studied dose-range finding study (DRF) (Study No. TNK-2020-000258, 28-day repeated oral toxicity; 0, 500, 1000, 2000 mg/kg B.W/day) with no toxicological effects were found at any dose group. In clinical sign, compound-colored feces were observed at 1000, 2000 mg/kg B.W/day in both males and females on day 15 up to the end of administration (day 28). Based on the DRF study, the dose 2000 mg/kg B.W was selected as the high dose and two-fold intervals were used for the middle dose (1000 mg/kg B.W) and low dose (500 mg/kg B.W), respectively. The test substance was suspended in vehicle (sterile distilled water for injection, DAI HAN PHARM. CO., LTD.) for concentrations of 50, 100, and 200 mg/mL (administration volume: 10 mL/kg) based on the most recently measured body weight (1 ~ 2 times/week). And then, the preparations were administered once/day, 7 days/week for 90 days by oral gavage by stomach tube (Disposable flexible type, FUCHIGAMI). The control group was administered vehicle.

Test procedure

Properties of GST

The characterization of GST showed in Figures 1, 2 and 3, respectively. The zeta potential is the potential between droplet surface and dispersing liquid medium and can be used to estimate surface charge of the droplets in the dispersion medium. Also, it was known for indicator of the droplet stability, where values more positive than +30 mV and more negative than −30 mV indicate good stability against coalescence [17]. This oral route of exposure to nanoparticles would ensure that nanoparticles enter an environment yielding a wide range of pH values. A hypothetical model nanoparticle is known to exhibit the largest agglomerate size at the point where its zeta potential is 0 mV. GST and P-25 nanoparticles exhibited zeta potentials of −35.4±5.99 mV and 32.3±4.35 mV respectively Figure 1. Over the course of this experiment, both GST and P-25 TiO₂ nanoparticles displayed the difference between positive and negative zeta potentials, which consequently affected their agglomeration state. The key difference between positive and negative zeta potential is that the positive zeta potential indicates that the dispersed particles in a suspension are positively charged whereas the negative zeta potential indicates that the dispersed particles in the suspension are negatively charged. The suspension stability is dependent upon physical characteristics of both the suspended nanoparticles and their suspension medium. One of the major factors involved in the agglomeration process is electrostatic stabilization. Altering the zeta potential to the point at which it exhibits zero net charge, decreases stabilization forces and promotes agglomeration. Cellular viability was influenced by a variety of factors including zeta potential and agglomeration state [18]. Also, the particle size value and distribution showed that GST was experimented to have materials of various sizes and also P-25 to have materials of various sizes due to agglomeration states Figures 2 and 3.

Figure 1

Characterization of TiO₂ particles (GST, P-25 made by Evonik Corp., Germany.) analyzed by Korea TECH: (a) negative zeta potential (GST, −35.4±5.99 mV, 30 mg/mL); (b) size distribution by intensity (GST); (c) positive zeta potential (P-25, +32.3±4.35 mV, 30 mg/mL); (d) size distribution by intensity (P-25).

Figure 2

Characterization of TiO₂ particles (GST): (a) SEM (scanning electron microscope) image analyzed by KRICT.

Figure 3

Characterization of TiO₂ particles (GST): (a) A particles dispersed in 99.9% EtOH was deposited on a copper grid and analyzed using TEM (Transmission electron microscope) image by Korea Basic Science Institute; (b) Size distribution (95.8±46.3 nm, 46–270 nm,) of the imaged GST(image J software).

Clinical signs

During the experimental period, there were no abnormal clinical signs and mortality such as treatment-related moribund and dead animals in any of the treatment groups. Also, no significant differences were observed in other observations such as detailed clinical observation, ophthalmic examination, sensory reactivity to stimuli, functional observations including grip strength and motor activity by activity monitor.

However, the compound-colored feces were observed in all animals of treatment group. These signs were observed from on day 8 in middle and high dose group and on day 14 (female)-15 (male) in low dose group to the end of administration. In the degrees of signs, there were observed from slight to moderate in the middle and high dose group. This sign was observed up to on day 1 of recovery period and was not observed thereafter in the high dose group as follows Table 3. The color of the feces observed in daily was lighter in the high dose group than in the low and middle dose groups.

Table 3

Summary of clinical signs (compound-colored feces) for 90-day treatment and 28-day recovery period

Figure 4

Compound-colored stool at treatment group (①: control group, ②: low dose, ③: middle dose, ④: high dose): (a) Male; (b) Female.

Heo et al. reported that no abnormal clinical sings were observed in any of treatment group (250, 500, 1000 mg/kg B.W/day) in the study of oral repeated dose 90-day in rats for TiO₂ P25 (AEROXIDE^® from Evonik Industries, 80:20 ratio of anatase and rutile, particle size range 14–21 nm, agglomerated/aggregated size: approximately 180 nm) [19]. Also, no significant adverse TiO₂-related effects were observed on clinical signs in 90-day (100, 300, 1000 mg/kg B.W/day) in rats for synthesized TiO₂ (d₅₀=145 nm, 21% nanoparticles by particle number) via the chloride process with rutile type reported by D.B. Warheit et al [8]. It was thought that the difference between the clinical sign of previous publications and this study was observed due to difference in absorption by the various particle size.

Body weight and food consumptions

For the experimental period (90-day treatment period and 28-day recovery period), the body weight showed a normal increase and there were no treatment-related effects in food consumptions between the treatment and recovery group Figure 5.

Figure 5

Changes for body weight and food consumptions: (a) body weight (male); (b) Body weight (female); (c) Food consumptions (male); (d) food consumptions (female).

Urinalysis

There were no treatment-related effects in both sexes in any of treatment group (data not shown).

Hematology, biochemistry and hormone analysis

There were no treatment-related significant effects in both sexes in any of treatment group as Tables 4,5 and 6. In some of studies, the orally administered TiO₂ nanoparticles with size less than 100 nm may cause hepatic and renal toxicity as shown by the changes in the serum parameters (hepatic damage; total protein, glucose, AST, ALT, ALP/renal damage; blood urea nitrogen, uric acid) [4,20]. The significant changes for live or kidney were not observed in this study and it was thought to be due to difference in the particle size.

Table 4

Hematological parameters

Table 5

Blood chemical parameters.

Table 6

Hormone analysis

Necropsy and organ weight

At the end of treatment, the treatment-related gross finding (test substance retention) was observed in gastrointestinal tract for both sexes at treatment group Table 7 and no gross findings were observed in recovery group. These findings were thought to be minor absorption considering clinical sign (contaminant-colored feces). Also, in the absolute/relative organ weight, there were no treatment-related significant effects in both sexes in any of treatment group.

Table 7

Summary of gross finding (test substance retention in gastrointestinal tract) for treatment group.

Histopathological examination

The histopathological examination was performed to identify the treatment-related effects for treatment group and recovery group. In gastrointestinal tract where gross finding was observed at necropsy, the foreign materials were observed in lumen of organs such as stomach, duodenum, ileum, cecum, colon and rectum in any of treatment group. Considering clinical signs, such as contaminant-colored feces, microscopic findings was considered that some of aggregated or agglomerated TiO₂ was not absorbed and retention in digestive tract and then theses were excreted into feces Figure 6. But, this microscopic finding was not in other organ tissues.

Figure 6

Histopathological findings examined in stomach (a, b), duodenum (c, d), ileum (e, f), cecum (g, h), colon (i, j) and rectum (k, l).

The similar finding was observed in 28-day/90-day repeated oral toxicities for synthesized TiO₂ materials (173 nm and 145 nm) studied by D.B. Warheit et al. This study showed that the granular brown aggregates or clumps, likely indicative for the presence of TiO₂ were seen upon hematoxylin & eosin staining in the digestive tract, draining lymphoid tissue without evidence of an adverse response [7,8]. But there were no TiO₂-related findings (macroscopic/microscopic findings) in research for TiO₂ P25 (average particle size range 14–21 nm) studied by Heo et al. [18]. These differences were thought to be due to the difference in absorption according particle size.

Also, considering retinopathy finding (presence of nanomaterials) observed in previous research of ZnO for 90-day in rats [21], we examined the eyes carefully including harderian gland and there were no TiO₂-related effects as Figure 7 and it was considered to be due to the difference in absorption according particle size.

Figure 7

Eye photographs (Left: Slide scanner image, ZEISS Axio Scan. Z1, Right: Optical microscope, X50) including the corea, iris, lens, retina and accessory organs (optic nerve, harderian gland): (a) Control group; (b)High dose group.