Evaluating the toxicity of the roots of Asarum heterotropoides var. mandshuricum extracted using the decoction method: Genotoxicity, single-dose toxicity, and 13-week repeated-dose toxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Asarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa (AR) is a traditional herbal medicine used across Asia, including Korea, China, and Japan. AR exhibits a range of biological activities, such as anti-inflammatory, anti-cancer, cold treatment, and anti-nociceptive effects. Various extraction methods, including decoction, which utilizes traditional knowledge and techniques. The AR decoction extract expected to contain fewer toxicants and have reduced toxicity due to the use of hot water in the extraction process. However, scientific evidence on the toxicity of AR decoction extracts is lacking, necessitating further studies for safe usage. AIM OF THE STUDY: This study aimed to evaluate the genotoxicity and toxicity of single and repeated administration of AR decoction extracts. MATERIALS AND METHODS: The genotoxicity was assessed using a bacterial reverse mutation (Ames test), an in vitro mammalian chromosome aberration test (CA test), and an in vivo micronucleus test (MN test) in Sprague-Dawley (SD) rats. The general toxicity was evaluated through single-dose and 13-week repeated-dose toxicity studies. In the single-dose toxicity study, 40 SD rats were orally administered AR decoction extract at doses of 1000, 2000, and 5000 mg/kg. In the 13-week repeated-dose toxicity study, 140 SD rats received daily oral doses of 0, 250, 500, 1000, 2000, and 5000 mg/kg of AR decoction extract. RESULTS: The genotoxicity tests revealed that AR decoction extract was not genotoxic. The single-dose toxicity study showed no changes in body weight, clinical pathology, or macroscopic findings, with the approximate lethal dose (ALD) exceeding 5000 mg/kg. The 13-week repeated-dose toxicity study demonstrated no treatment-related changes in body weight, general symptoms, hematology, clinical chemistry, or urinalysis. Histopathological findings revealed hyperplasia of squamous cells in the forestomach after AR decoction extract administration, a treatment-related effect that resolved during the recovery period. The no observed adverse effect level (NOAEL) for both male and female rats was estimated to be 2000 mg/kg. CONCLUSIONS: This study establishes the non-toxic dose of AR decoction extract, providing a foundation for further non-clinical and clinical evaluations AR safety.

Nonclinical safety assessment of an mRNA Covid-19 vaccine candidate following repeated administrations and biodistribution.

Messenger RNA (mRNA) vaccines have demonstrated efficacy against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in humans. mRNA technology holds tremendous potential for rapid control and prevention of emergencies due to its flexibility with respect to production, application, and design for an efficacious and safe use in humans. We assessed the toxicity and biodistribution of MRT5500, an mRNA vaccine encoding for the full-length of the SARS-CoV-2 spike protein and delivered by lipid nanoparticles (LNPs) containing a novel ionizable lipid, Lipid-1 in preclinical animal models. In the repeated dose toxicity study, rabbits received three intramuscular (IM) injections of MRT5500 at 3-week interval followed by a 4-week observation period. In an exploratory biodistribution study in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in an LNP containing Lipid-1, the expression of the luciferase protein was monitored in vivo and ex vivo at several time points. In the regulatory biodistribution study in rabbits receiving a single IM injection of MRT5500, the quantification of the mRNA and the ionizable Lipid-1 were monitored in the same organs and time points as in the exploratory biodistribution study. MRT5500 was safe and well-tolerated with a transient acute phase response/inflammation and an expected vaccine-related immunological response, typical of those observed following a vaccine administration. The biodistribution data demonstrated that the mRNA and Lipid-1 components of the vaccine formulations were mainly detected at the injection site and in the draining lymph nodes. These results support the use of MRT5500 and its deployment into clinical trials.

Involvement of the CYP1A1 inhibition-mediated activation of aryl hydrocarbon receptor in drug-induced hepatotoxicity.

Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.

Pharmacological effects and safety of Andrographis paniculata (Burm.f.) Nees.

Andrographis paniculata (Burm.f.) Nees (AP) is widely used in most Asian and some Western countries. However, its main effects and underlying pharmacological mechanism have not been thoroughly characterized, and its safety has not been sufficiently investigated. The present study aimed to predict and visualize the potential targets and pathways, clarify the main pharmacological effects, and investigate the toxicological properties of AP extract (APE). First, ingenuity pathway analysis (IPA) was performed to directly predict AP's therapeutic targets and pathways; main pharmacological effects of AP were speculated based on IPA results and confirmed by pharmacodynamics experiments. Rodent toxicity studies were then performed through administration of a single dose of 10 g/kg or daily doses of 2, 1, or 0.5 g/kg for 8 weeks to evaluate the safety of APE, and a similar repeated-dose study was performed using dogs with doses equal to half of the above-mentioned doses. Thus, repeated-dose toxicity studies were performed with both rodents and nonrodents. The IPA analysis and confirmatory pharmacodynamics experiments revealed that the main pharmacological effect of APE was anti-inflammation, which might be achieved by influencing various targets (e.g., AR, AKT, and BAX) and pathways (IL-8). In the single-dose toxicity test, no death or abnormal consequences were observed, and maximum tolerated dose of APE was 10 g/kg. Results from the repeated-dose toxicity tests did not reveal any obvious toxic effects from the repeated daily intragastric administration of APE at 1 g/kg for 8 weeks. In conclusion, APE at a dose of 1 g/kg did not exert any adverse effects, and administration of APE could be beneficial for the inflammatory diseases' treatment. PRACTICAL APPLICATION: Andrographis paniculata (Burm.f.) Nees is a plant that exerts clearing and detoxification effects and is widely used around the world, but a comprehensive analysis of its efficacy and safety is needed.

Preclinical safety evaluation of body protective compound-157, a potential drug for treating various wounds.

BPC157 displays protective activity in various organs and tissues. This report presents preclinical toxicity studies with BPC157 in mice, rats, rabbits and dogs. The single-dose toxicity study did not show any test-related effects that could be attributed to the test article. In repeated-dose toxicity evaluations, BPC157 was well tolerated in dogs, with no abnormal changes between the BPC157-treated groups and the solvent control group, with the exception of a decrease in creatinine level at a dose of 2 mg/kg but not at lower doses. The animals recovered spontaneously after 2 weeks of withdrawal. This may be due to the pharmacological activity of BPC157. A local tolerance test showed that the irritation caused by BPC157 was mild. BPC157 also showed no genetic or embryo-fetal toxicity. In summary, BPC157 was well tolerated and did not cause any serious toxicity in mice, rats, rabbits and dogs. These preclinical safety data contribute to the initiation of an ongoing clinical study. Based on the stability and protective effect of BPC157, which has been widely reported, BPC157 may have a better application prospect than the widely used cytokine drugs in wound therapy.

Assessment of the 4-week repeated-dose oral toxicity and genotoxicity of GHX02.

Herbal medicines are widely utilized for disease prevention and health promotion. GHX02 consists of mixtures including Gwaruin (Trichosanthes kirilowii), Haengin (Prunus armeniaca), Hwangryeon (Coptis japonica) and Hwangkeum (Scutellaria baicalensis). It has been purported to have therapeutic effectiveness in cases of severe bronchitis. Non-clinical safety testing comprised a single-dose oral toxicity study and a 28-day repeated-dose oral toxicity study with a 14-day recovery period, and genotoxicity was assessed by a bacterial reverse mutation test, in vitro chromosomal aberration test, in vivo mouse bone marrow micronucleus test and single cell gel electrophoresis assay (comet assay). In the single-dose oral toxicity study, the approximate lethal dosage is estimated to be higher than 5000 mg/kg in rats. Thus, the dosage levels were set at 0, 1250, 2500 and 5000 mg/kg/day in the 28-day repeated-dose oral toxicity study, and 10 male rats and 10 female rats/dose were administered GHX02. No clinical signs of toxicological significance were recorded in any animal during the dosing and the observation period in the single-dose study. The no-observed-adverse-effect level of GHX02 was 5000 mg/kg/day when administered orally for 28 days to male and female Sprague-Dawley rats. Despite increases in the frequencies of cells with numerical chromosomal aberration in the in vitro test, the increases were not considered relevant to the in vivo genetic risk. Except for the increase of in vitro numerical chromosomal aberration, clear negative results were obtained from other genetic toxicity studies.

Approaches of validation of a 2-week combined repeated oral dose toxicity study with plasma micro sampling toxicokinetics (PMS-TK) in common marmosets.

We investigated the viability of a combined repeated dose toxicity study, including toxicokinetics (TK), in common marmosets according to the ICH-S4, ICH-S3A and ICH-S7A Guidelines using valsartan as test article whose non-clinical repeated dose toxicity studies had been conducted using this species for regulatory purpose. Valsartan was administered orally to 3 animals/sex at 200 mg/kg/day for 2 weeks. In addition to the routine parameters in repeated dose toxicity studies, safety pharmacology parameters (examinations of the central nervous, respiratory and cardiovascular systems) were also evaluated. The Plasma Micro Sampling Toxicokinetics (PMS-TK) method required ultrasensitive quantitation, was employed to evaluate the relationship between toxic changes and plasma concentrations as well as the effects of frequent blood sampling in individual animals. In valsartan, toxic findings (a deteriorated physical condition; moribundity of one male and one female on Day 14; sporadic vomitus; decreases in body weights and food consumption; decreases in erythrocytic parameters; and renal changes such as an increase in urea nitrogen, dilation of the tubules and hypertrophy of the tubular epithelium) were similar and plasma concentrations comparable to the results in the approval information. Furthermore, no side effects caused by frequent blood sampling were confirmed in the negative control group. Consequently, a combined repeated dose toxicity study including TK analysis using the PMS-TK method is viable in common marmosets and contributes to animal welfare.

Freeze-dried equine-derived redback spider antivenom: a local irritation study by intramuscular injection in rabbits and a repeated-dose toxicity study in rats.

The redback spider (Latrodectus hasseltii) is nonindigenous to Japan but has now spread throughout the country. Bites to humans are rare but can be fatal. We prepared freeze-dried redback spider antivenom for therapeutic use against bites in Japan by immunization of horse plasma. This study included two nonclinical tests of the antivenom: a local irritation study involving a single intramuscular administration to rabbits (with injections of physiological saline and an existing freeze-dried diphtheria antitoxin as control and comparison substances, respectively) and a 2-week repeated intermittent intravenous-dose toxicity study in rats. The irritation study showed the antivenom's irritancy to be comparable with that of the saline and the existing antitoxin preparations under the test conditions. In a repeated-dose toxicity study, no toxicity change was found in male or female rats, and the no-observed-adverse-effect level (NOAEL) was judged to be a dose volume of 20 mL/kg (1082 units/kg antivenom activity) in both male and female rats. In addition, there was no toxicological difference between proteinaceous diphtheria antitoxin and redback spider antivenom prepared to have the same protein content and the same additive composition. Based on these findings, we will further advance our research towards clinical application of the redback spider antivenom. This research was supported by the Research Program on Emerging and Re-emerging Infectious Disease of the Japan Agency for Medical Research and Development.

Repeated-dose liver and gastrointestinal tract micronucleus assays for quinoline in rats.

Repeated-dose liver, bone marrow, and gastrointestinal tract micronucleus assays that use young adult rats were evaluated in a collaborative study that was organized by the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. A genotoxic hepatocarcinogen quinoline was orally administered to independent groups of five Crl:CD (SD) male rats at doses of 30, 60 and 120mg/kg for 14 days and at doses of 15, 30 and 60mg/kg for 28 days. After treatment, the livers were harvested and hepatocytes were isolated by collagenase treatment. The frequency of micronucleated hepatocytes (MNHEPs) increased significantly in both the 14- and 28-day repeated dose studies. However, the frequency of micronucleated cells did not increase in the bone marrow, stomach or colon cells, which were not quinoline-induced carcinogenic target organs in the rats. These results indicate that a repeated-dose liver micronucleus (RDLMN) assay using young adult rats is capable of detecting the genotoxicity of quinoline at the target organ of carcinogenicity. The protocol may also permit the integration of the genotoxic endpoint into general repeated-dose toxicity studies. Furthermore, we elucidated that conducting the micronucleus assay in multiple organs could potentially assess organ specificity.

Evaluation of the repeated-dose liver micronucleus assay with p-dimethylaminoazobenzene.

The micronucleus induction by p-dimethylaminoazobenzene (DAB), a genotoxic rat liver carcinogen, was assessed in the liver and bone marrow of young adult rats after the repeated administration of DAB for 14 (Lab. 1) and 28 (Lab. 2) days. Three dose levels, 25, 50 and 100mg/kg/day, were used for the investigations in both labs. The frequency of micronucleated hepatocytes was significantly increased in a dose-dependent manner after the repeated administration of DAB at 50mg/kg/day or more for 14 and 28 days. Similarly, the frequency of micronucleated immature erythrocytes in the bone marrow was increased after the repeated administration of DAB at 100mg/kg/day for 14 and 28 days. These results indicate that the repeated-dose liver micronucleus assay allowed for the detection of micronucleus induction by DAB, and that the lowest detectable dose for micronucleus induction in the liver was lower than in the bone marrow. Thus, the repeated-dose liver micronucleus assay using young adult rats is considered suitable for the detection of micronucleus induction by liver carcinogens, such as DAB.

Repeated-dose liver micronucleus assay: an investigation with 2-nitropropane, a hepatocarcinogen.

The utility of the repeated-dose liver micronucleus (RDLMN) assay in the detection of a genotoxic hepatocarcinogen was evaluated. In this paper, a rat hepatocarcinogen, 2-nitropropane (2-NP), was administered orally to young adult rats for 14 and 28 days without a partial hepatectomy or a mitogen, and the micronucleus induction in liver was examined using a simple method to isolate hepatocytes. In addition, a bone marrow micronucleus assay was conducted concomitantly. The frequency of micronucleated hepatocytes induced by 2-NP increased significantly in both the 14- and 28-day repeated-dose studies, while the bone marrow micronucleus assays were negative in each study. These results indicate that the RDLMN assay is useful for detecting a genotoxic hepatocarcinogen that is negative in bone marrow micronucleus assays and is a suitable in vivo genotoxicity test method for integration into a repeated-dose general toxicity study.

Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats.

The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action.

Repeated-dose liver micronucleus assay of mitomycin C in young adult rats.

The repeated-dose liver micronucleus (RDLMN) assay has been previously reported to be effective for the detection of hepatocarcinogens and suitable for general toxicology studies. A collaborative study was conducted to evaluate whether this RDLMN assay using young adult rats without collagenase perfusion of the liver can be used to detect genotoxic carcinogens. In this study, we performed the RDLMN assay in young adult rats that received intraperitoneal injections of 0.25, 0.5 or 1.0mg/kg/day of mitomycin C (MMC) for 14 and 28 days. The micronucleus induction in the bone marrow was concurrently measured, and a histopathological examination of the liver was conducted. The results revealed that the frequency of micronucleated hepatocytes (MNHEPs) was significantly increased in all of the treatment groups. However, the highest occurrence of MNHEPs was observed in the low-dose treatment group in both the 14- and the 28-day study periods. In addition, histopathological changes indicating hepatotoxicity were not observed even in the group that received the highest dose of MMC. There was no change in the frequency of metaphase hepatocytes in any of the treatment groups compared with our facility's background data. However, the frequency of proliferating hepatocytes, as assessed by Ki-67 positivity, was decreased at the highest dose, as was the frequency of MNHEPs. Therefore, the decreased induction of MNHEPs in the high-dose groups might be explained by suppression of hepatocyte cell division. In contrast, the frequency of micronucleated immature erythrocytes in the bone marrow significantly increased in a dose-dependent manner in all of the treatment groups in both study periods. Repeated treatment of MMC induced micronuclei in the liver. These results suggest that the novel RDLMN assay can be used to detect MMC genotoxicity in the liver.

The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats.

The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect genotoxic hepatocarcinogens that can be integrated into a general toxicity study. The assay methods were thoroughly validated by 19 Japanese facilities. Methapyrilene hydrochloride (MP), known to be a non-genotoxic hepatocarcinogen, was examined in the present study. MP was dosed orally at 10, 30 and 100mg/kg/day to 6-week-old male Crl:CD (SD) rats daily for 14 days. Treatment with MP resulted in an increase in micronucleated hepatocytes (MNHEPs) with a dosage of only 100mg/kg/day. At this dose level, cytotoxicity followed by regenerative cell growth was noted in the liver. These findings suggest that MP may induce clastogenic effects indirectly on the liver or hepatotoxicity of MP followed by regeneration may cause increase in spontaneous incidence of MNHEPs.

Determining NOEL/NOAEL in Repeated-dose Toxicity Studies, When the Low Dose Group Shows Significant Difference in Quantitative Data / 한국실험동물학회지

In repeated-dose 28-day oral toxicity study design, the low dose is fixed as the no observed effect level (NOEL). But, in practice the low dose usually shows significant difference in few measurable items in most of the studies. We investigated 109 of repeated-dose 28-day oral toxicity studies in rats conducted according to the Chemical Substance Control Law, Japan and examined the measurable items (functional observational battery, urinalysis, hematology, blood chemistry and absolute and relative organ weights) of the low dose group which showed a statistical significant difference (P<0.05) compared to the respective control groups. The investigation revealed that, 205/12,167 (1.6%) measurable items showed a significant difference in the low dose groups. The significant difference shown by urinalysis was high (3.3%), followed by clinical chemistry parameters, hematology, relative organ weights and absolute organ weights (1.8-1.1%). We conclude from the investigation that the low dose may be considered as NOEL, if the significant difference of measurable items of it is about 2% (maximum <5%), compared to the control. However, due consideration may be given to the clinical relevance of the items that showed a significant difference.

Determining NOEL/NOAEL in Repeated-dose Toxicity Studies, When the Low Dose Group Shows Significant Difference in Quantitative Data / 한국실험동물학회지

In repeated-dose 28-day oral toxicity study design, the low dose is fixed as the no observed effect level (NOEL). But, in practice the low dose usually shows significant difference in few measurable items in most of the studies. We investigated 109 of repeated-dose 28-day oral toxicity studies in rats conducted according to the Chemical Substance Control Law, Japan and examined the measurable items (functional observational battery, urinalysis, hematology, blood chemistry and absolute and relative organ weights) of the low dose group which showed a statistical significant difference (P<0.05) compared to the respective control groups. The investigation revealed that, 205/12,167 (1.6%) measurable items showed a significant difference in the low dose groups. The significant difference shown by urinalysis was high (3.3%), followed by clinical chemistry parameters, hematology, relative organ weights and absolute organ weights (1.8-1.1%). We conclude from the investigation that the low dose may be considered as NOEL, if the significant difference of measurable items of it is about 2% (maximum <5%), compared to the control. However, due consideration may be given to the clinical relevance of the items that showed a significant difference.