Genotoxicity Assessment of Hwanglyeonhaedok-Tang: An Herbal Formula Evaluated Using the In Vivo Alkaline Comet Assay and Pig-a Gene Mutation Assay in Rats

Genotoxicity Assessment of Hwanglyeonhaedok-Tang: An Herbal Formula Evaluated Using the In Vivo Alkaline Comet Assay and Pig-a Gene Mutation Assay in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genotoxicity Assessment of Hwanglyeonhaedok-Tang: An Herbal Formula Evaluated Using the In Vivo Alkaline Comet Assay and Pig-a Gene Mutation Assay in Rats Seng-Min Back, Eun-Young Gu, Kang-Hyun Han, Chang-Seob Seo, Min Seong Jang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6303752/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background Hwanglyeonhaedok-tang (HHT), also known as Huang-lian-jie-du-tang or Oren-gedoku-to, is a popular herbal formula in traditional Asian medicine. Despite the clinical effectiveness of herbal formulas, concerns regarding the quality, safety, and efficacy persist. HHT exhibited genotoxicity in bacterial reverse mutation (Ames test) and in vitro chromosomal aberration (CA) test, warranting further investigation. This study aimed to verify HHT-induced genotoxicity through in vivo assays like alkaline comet and phosphatidylinositol glycan class A ( Pig-a ) gene mutation assay because HHT has shown positive results only in in vitro tests. Methods The in vivo comet assay was conducted on rat liver under Good Laboratory Practice standards to assess DNA migration, focusing on % tail DNA and hedgehog frequency (cells with small or absent heads and diffuse tails). The Pig-a gene mutation assay used peripheral blood to evaluate mutant red blood cells (RBCs) and reticulocytes (RETs), with cytotoxicity assessed by %RET. Doses of 0, 500, 1,000, and 2,000 mg/kg were tested, applying standard statistical methods. Results The comet assay showed no marked increases in % tail DNA or hedgehog frequency at the doses tested. No clinical signs, behavioral abnormalities, or weight changes were noted. Similarly, the Pig-a assay revealed no significant differences in mutant RBCs or RETs, and %RET cytotoxicity was comparable to controls. Positive controls validated assay sensitivity. Overall, HHT did not induce genotoxicity and cytotoxicity under tested conditions, supporting its safety at the evaluated dose levels. Conclusions The in vivo comet assay result suggest that the positive outcome observed in the in vitro CA test is likely a false positive. However, although negative result was obtained from short-term Pig-a gene mutation assay, it cannot be conclusively determined that HHT does not induce gene mutations. Therefore, long-term Pig-a studies are recommended. Hwanglyeonhaedok-tang (Huang-lian-jie-du-tang and oren-gedoku-to) in vivo alkaline comet assay in vivo Pig-a gene mutation assay genotoxicity Figures Figure 1 Figure 2 Figure 3 Introduction Traditional medicinal herbs have been utilized for centuries to prevent and treat various diseases, deeply rooted in cultural and historical practices. With a long history of human use, traditional herbal formulas have garnered attention for their therapeutic potential. However, recent decades have seen growing concerns about herb-drug interactions and the safety of these remedies, especially under modern pharmacological standards. Traditional Asian Medicine (TAM), extensively documented in ancient medical texts, commonly uses fixed herbal components with clearly defined therapeutic effects and relatively acceptable risks of adverse reactions [ 1 , 2 ]. Hwanglyeonhaedok-tang (HHT), known as Huang-Lian-Jie-Du-Tang in Chinese or Oren-gedoku-to in Japanese, is a classic multi-herb formula first detailed in Wang Tao’s Wai Tai Mi Yao. It consists of Coptidis chinensis Franch ( Hwanglyeon in Korean), Scutellaria baicalensis Georgi , Phellodendron amurense Rupr., and Gardenia jasminoides J. Ellis, traditionally prescribed for heat-clearing and detoxification. Numerous studies have reported its pharmacological benefits, including antipyretic, anti-inflammatory, gastroprotective, neuroprotective, and hypolipidemic effects [3 − 11]. Despite its benefits, concerns persist regarding the quality, safety, and efficacy of conventional herbal formulas. Specifically, HHT has shown genotoxic potential in in vitro Ames and chromosomal aberration (CA) tests conducted under Good Laboratory Practice (GLP) standards [ 12 ]. Components of HHT, such as Scutellaria baicalensis and Coptidis chinensis , have also demonstrated genotoxic effects in earlier studies [ 13 ]. Regulatory standards for genotoxicity testing typically follow a three-battery approach: the Ames test, CA test, and in vivo micronucleus test. While these tests detect diverse genetic damage, they often yield inconsistent results between in vitro and in vivo assays and are prone to false positives. To mitigate these limitations, the International Council for Harmonization (ICH) S2 guidelines recommend additional in vivo assays, such as the alkaline comet assay and Pig-a gene mutation assay, to enhance the robustness of genotoxicity assessments. The in vivo alkaline comet assay evaluates DNA damage in individual cells, including gene mutations, clastogenicity, and aneuploidy [ 14 ]. This assay is particularly suitable for liver tissue, the primary site of xenobiotic metabolism, making it ideal for assessing DNA damage. Meanwhile, the in vivo Pig-a assay, a newer in vivo mutation test, evaluates glycosylphosphatidylinositol (GPI)-anchored protein deficiencies as a biomarker for mutagenicity and serves as a comprehensive tool for regulatory safety evaluations [ 15 , 16 ]. This study aimed to investigate the potential genotoxicity of HHT using the in vivo alkaline comet assay and Pig-a gene mutation assay to address limitations of in vitro tests. The in vivo comet assay was conducted following Organization for Economic Co-operation and Development (OECD) guidelines, while the Pig-a assay employed validated methodologies. These assessments provide a comprehensive evaluation of HHT’s genotoxic profile and contribute to its safety characterization. Materials and Methods Plant materials The four crude herbs comprising HHT–Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex, and Gardeniae Fructus–were procured from Omniherb (Yeongcheon, Korea) and HMAX (Jecheon, Korea) [ 17 ]. Preparation of the Hwanglyeonhaedok-tang powder The HHT formulation itself was supplied by the Korea Institute of Oriental Medicine [ 17 ]. To ensure quality control of HHT, HPLC analysis was conducted for six marker compounds using a modified method based on previous research [ 17 ]. The analysis utilized a Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) equipped with photodiode array detection. The mobile phases consisted of solvent A, which was 10.0% (v/v) acetonitrile with 0.2% sodium dodecyl sulfate and 200 µL phosphoric acid, and solvent B, which was pure acetonitrile. Gradient elution was applied under the following conditions: 10–40% B for 0–20 min, 40–50% B for 20–40 min, 50–100% B for 40–50 min, 100–10% B for 50–55 min, and 10% B for 55–70 min. The flow rate was set at 1.0 mL/min. Using this optimized HPLC assay, the concentrations of six marker compounds— Geniposide, Baicalin, Jatrorrhizine, Coptisine, Palmatine , and Berberine —were quantified, as detailed in Table 1 . Figure 1 presents the HPLC chromatograms of HHT, with eluents detected at wavelengths of 240, 265, and 275 nm. Separation of these marker compounds was achieved using a Gemini C18 column maintained at 40°C. The injection volume was 10 µL, with a consistent flow rate of 1.0 mL/min. Table 1 Amounts of the marker compounds in the HHT sample analyzed using HPLC. Compound Amount (mg/g) ± SD RSD (%) RT (min) Source Geniposide 26.63 ± 0.32 1.18 4.77 Gardenia jasminoides J.Ellis Balcalin 22.98 ± 0.04 0.16 12.65 Scutellaria baicalensis Georgi Jatrorrhizine 2.36 ± 0.01 0.50 30.84 Coptidis chinensis Franch Phellodendron amurense Rupr. Coptisine 5.01 ± 0.01 0.24 31.87 Coptidis chinensis Franch Phellodendron amurense Rupr. Palmatine 4.24 ± 0.01 0.21 33.45 Coptidis chinensis Franch Phellodendron amurense Rupr. Berberine 14.56 ± 0.01 0.09 34.33 Coptidis chinensis Franch Phellodendron amurense Rupr. Preparation of the positive control articles For in vivo genotoxicity assays, N-ethyl-N-nitrosourea (ENU, Cas# 759-73-9) and ethyl methanesulfonate (EMS, Cas# 62-50-0) were employed as positive controls for the Pig-a and comet assays, respectively. Both ENU and EMS, obtained from Sigma-Aldrich (USA), were dissolved in distilled water (Dai Han Pharm. Co., Ltd., Korea) and administered orally on the dosing day. Animals and Maintenance Specific-pathogen-free male SD rats, approximately six weeks old, were acquired from Orient Bio Inc. (Korea) for the in vivo experiments. The rats were housed in stainless steel cages measuring 265 mm (W) 🞨 465 mm (L) 🞨 200 mm (H). Environmental conditions in the animal facility were meticulously controlled, maintaining a temperature of 22°C ± 3°C, relative humidity of 50 ± 10%, a 12 h light/dark cycle (lights on at 08:00 and off at 20:00) with illumination levels between 150–300 lux, and ventilation rates of 10–20 air exchanges per hour. Throughout the study, the animals were monitored daily to ensure their well-being. At the conclusion of the experiment, all animals, including any remaining subjects, were humanely euthanized by gradual exposure to carbon dioxide (CO₂) at a flow rate of 30–70% of the chamber volume per minute, following the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). Complete euthanasia was confirmed by a secondary physical method (thoracotomy) performed after loss of consciousness. In vivo alkaline comet assay As part of an international validation study of the in vivo alkaline comet assay under the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative, the assay was conducted using the standard validation protocol [ 18 , 19 ]. HHT was administered orally by gavage at doses of 500, 1,000, and 2,000 mg/kg at 0, 24, and 45 h, with a dosage volume of 10 mL/kg based on each rat’s body weight prior to administration. The positive control compound was similarly administered by oral gavage at 10 mL/kg once daily for three consecutive days at a dose of 200 mg/kg. The rats were euthanized 3 h after the final administration, and a portion of the left lateral lobe of the liver was excised. Single-cell suspensions were prepared within 1 h of euthanasia, and four slides per tissue (two slides per tissue per rat) were prepared. The slides were stained with SYBR Gold, and comet measurements were conducted using Comet Assay IV software (version 4.3.1, Perceptive Instruments Ltd., UK) under a fluorescence microscope at 200X magnification. For each sample (animal/tissue), 75 comets per slide (150 comets/tissue) were analyzed. The key comet assay endpoints–percentage tail DNA, olive tail moment, and tail length—were recorded. Additionally, the frequency of heavily damaged cells, including "hedgehogs" and "ghost" cells, was visually scored from 150 cells per sample. In vivo Pig-a gene mutation assay The in vivo Pig-a assay was performed according to the method described in the MutaFlow Basic-25R GLP kit (tube-based processing, version 140403, Litron Laboratories, USA) [ 19 ]. HHT was administered by oral gavage at doses of 500, 1,000, and, 2,000 mg/kg for three consecutive days. The dosage volume was set at 10 mL/kg body weight, adjusted based on the rats’ weights measured shortly before each administration. The positive control compound was also administered by oral gavage at 10 mL/kg once daily for three days at a dose level of 20 mg/kg. Peripheral blood samples were collected 29 days after the first treatment. Lymphocytes were removed from the peripheral blood using Lympholytes® (Cat No.: CL5120, Cedarlane Corp., Canada). Wild-type red blood cells (RBCs) were tagged with PE-conjugated anti-CD59 and anti-CD61 antibodies, while platelets were removed through magnetic separation. Mutant-type RBCs were concentrated using the same process, and reticulocytes (RETs) were stained with SYTO13® (Life Technologies, USA) to distinguish them from mutant phenotype RBCs. Flow cytometry analysis was performed using a (FACSCalibur (BD Biosciences, USA), with at least 1,000 counting beads per sample. The data were analyzed using Cell Quest Pro software (Version 5.2.1, BD Biosciences, USA). Gene mutation analysis included calculations of %RET, mutant RBCs/10 6 total RBCs, and mutant RETs/10 6 total RETs. Statistical analysis Statistical analyses were conducted using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA), with a significance threshold set at p- value < 0.01. Results In vivo alkaline comet assay To substantiate the weight of evidence regarding HHT, which exhibited genotoxicity in chromosome aberration studies [ 12 ], a comet assay was performed in liver tissue under GLP compliance. The primary endpoint for assessing DNA migration was the percentage of tail DNA, representing the proportion of fluorescence detected in the tail during image analysis, along with tail length (tail migration). The distribution of migration patterns among cells within each animal was also evaluated. The percentage of "hedgehogs" (ghost cells or heavily damaged cells) was analyzed as a function of treatment. For each specific tissue, the unit of analysis was the individual animal. Notably, hedgehogs–characterized by a small or nonexistent head accompanied by large and diffuse tails–were excluded from data collection. No significant increases in the percentage of tail DNA were observed in the liver tissues of rats administered HHT at any dose (0.12 ± 0.14, 0.11 ± 0.15, 0.07 ± 0.05, or 0.14 ± 0.15 for 0, 500, 1,000, and 2,000 mg/kg, respectively). Similarly, no significant increases were observed in the percentage of hedgehogs in liver tissues (0.80 ± 0.84, 0.60 ± 0.55, 1.20 ± 0.84 or 1.20 ± 0.84, for 0, 500, 1000, and 2,000 mg/kg, respectively) (Fig. 2 ; Table 2 ). Table 2 Comet assay results of HHT Dose (mg/kg) Vehicle control HHT (mg/kg) EMS 0 500 1,000 2,000 200 % Tail DNA in Liver Medians 0.12 ± 0.14 0.11 ± 0.15 0.07 ± 0.05 0.14 ± 0.15 15.47 ± 2.77** Means 1.61 ± 0.46 1.47 ± 0.38 1.58 ± 0.27 1.89 ± 0.63 16.41 ± 3.08 Tail Length in Liver Medians 18.49 ± 0.70 19.58 ± 0.73 18.63 ± 0.99 20.32 ± 0.90 36.41 ± 2.11 Means 19.04 ± 0.92 20.15 ± 0.62 19.08 ± 0.98 20.66 ± 0.93 36.66 ± 2.19 Olive Tail Moment in Liver Medians 0.01 ± 0.02 0.01 ± 0.01 0.00 ± 0.00 0.02 ± 0.02 2.40 ± 0.55 Means 0.15 ± 0.04 0.14 ± 0.04 0.14 ± 0.03 0.18 ± 0.07 2.52 ± 0.62 % Hedgehogs Means 0.80 ± 0.84 0.60 ± 0.55 1.20 ± 0.84 1.20 ± 0.84 1.80 ± 1.48 ** Significant differences from control group ( p < 0.01); EMS: Ethyl methanesulfonate During the study, no clinical signs and abnormal behaviors were observed in the animals treated with HHT throughout the observation period (Table 3 ). Additionally, there were no statistically significant differences in the mean body weights between the HHT-treated groups and the vehicle control group. Table 3 Clinical sign in comet assay for HHT at doses of 500, 1,000, or 2,000 mg/kg or EMS at 200 mg/kg via three consecutive days at approximately 24-hour intervals Clinical Signs Vehicle control HHT (mg/kg) EMS 0 500 1,000 2,000 200 Male Abnormalities Detected 0/6 0/6 0/6 0/6 0/6 Therefore, HHT did not induce DNA damage by oral gavage at any tested dose level compared to the vehicle control. In vivo Pig-a gene mutation assay The in vivo Pig-a assay, a mammalian gene mutation assay utilizing peripheral blood, was also conducted to confirm the genotoxicity of HHT observed in the Ames test [ 12 ]. This assay offers high integration potential due to its reliance on peripheral blood samples [ 20 ]. Frequencies of mutant phenotype erythrocytes (RBCs) and mutant phenotype immature erythrocytes (reticulocytes, RETs) showed no significant changes in the test groups treated with HHT at any dose. Specifically, mutant RBC frequencies were 0.96 ± 1.42, 1.04 ± 1.09, 0.54 ± 0.76, and 1.44 ± 1.68 at 0, 500, 1,000, and 2,000 mg/kg, respectively. Similarly, mutant RET frequencies were 1.06 ± 0.59, 1.02 ± 0.49, 0.90 ± 0.97, and 9.98 ± 19.71 at the respective doses. The percentage of reticulocytes (%RET), a cytotoxicity index, also did not differ significantly between the HHT-treated groups and the vehicle control group (2.62 ± 0.36, 2.54 ± 0.53, 2.80 ± 0.12, and 2.46 ± 0.15 at 0, 500, 1,000, and 2,000 mg/kg, respectively). In contrast, significant increases were observed in the frequencies of mutant phenotype RBCs (95.0 ± 11.09) and mutant phenotype RETs (207.80 ± 49.73) in the positive control group. However, no significant increases in the %RET were observed in the positive control group (Fig. 3 ; Table 4 ). Table 4 Pig-a assay results of HHT Dose (mg/kg) Vehicle control HHT (mg/kg) ENU 0 500 1,000 2,000 20 % RET Means 2.62 ± 0.36 2.54 ± 0.53 2.80 ± 0.12 2.46 ± 0.15 2.96 ± 0.28 Mutant RETs per 10 6 Total RETs Means 1.06 ± 0.59 1.02 ± 0.49 0.90 ± 0.97 9.98 ± 19.71 207.80 ± 49.73** Mutant RBCs per 10 6 Total RBCs Means 0.96 ± 1.42 1.04 ± 1.09 0.54 ± 0.76 1.44 ± 1.68 95.0 ± 11.09** ** Significant differences from vehicle control group ( p < 0.01); ENU: N-ethyl-N-nitrosourea No clinical signs of toxicity or abnormal behavior were observed in animals treated with the test compound throughout the observation period (Table 5 ). Additionally, there were no statistically significant differences in the mean body weights of the test compound-treated groups compared to the vehicle control group (Table 6 ). Table 5 Clinical sign in Pig-a assay Clinical Signs Vehicle control HHT (mg/kg) ENU 0 500 1,000 2,000 20 Male Abnormalities Detected 0/5 0/5 0/5 0/5 0/5 Table 6 Body weight changes of male SD rats treated with HHT during Pia-a assay. Test item was administered by oral gavage at doses of 500, 1,000, and 2,000 mg/kg for three consecutive days (10 mL/kg body weight of rats based on the animal weight shortly before administration). The positive control (ENU) was administered at a dose level of 20 mg/kg. Dose (mg/kg) Vehicle control HHT (mg/kg) ENU 0 500 1,000 2,000 200 Body Weight Means Day1 252.8 ± 8.84 254.3 ± 9.07 251.1 ± 6.76 251.1 ± 7.18 251.4 ± 3.88 Day2 266.3 ± 7.55 266.6 ± 11.56 263.0 ± 8.18 261.8 ± 7.40 261.8 ± 4.43 Day3 274.2 ± 9.68 275.6 ± 10.35 271.4 ± 8.35 273.9 ± 8.17 266.0 ± 5.31 Day4 286.9 ± 10.39 287.7 ± 11.72 281.5 ± 9.62 284.6 ± 10.76 268.6 ± 7.39 Day7 314.1 ± 18.59 321.2 ± 15.29 310.0 ± 12.8 316.9 ± 15.15 309.1 ± 4.21 Day10 347.4 ± 21.24 348.1 ± 18.72 336.9 ± 14.28 345.8 ± 17.08 336.7 ± 5.42 Day15 382.4 ± 25.51 385.1 ± 26.47 372.6 ± 19.17 383.0 ± 21.03 377.9 ± 9.47 Day20 409.5 ± 28.42 419.5 ± 29.76 402.4 ± 24.62 415.2 ± 27.62 409.9 ± 13.51 Day25 433.1 ± 29.48 452.3 ± 39.3 429.7 ± 27.12 444.1 ± 30.41 442.1 ± 15.54 Discussion This study provides compelling evidence that HHT does not induce genotoxicity and cytotoxicity in vivo . The comet assay showed no significant increases in % tail DNA or hedgehog frequency, whereas the Pig-a gene mutation assay revealed no significant changes in mutant RBC or RET frequencies. These results indicate that HHT does not cause DNA damage or mutagenesis in rat liver or peripheral blood at doses up to 2,000 mg/kg. These findings contrast with previous in vitro results, such as the CA and Ames tests [ 12 ], which reported genotoxic activity. This discrepancy highlights the importance of in vivo studies, which account for metabolic and detoxification pathways absent in vitro . These physiological processes are critical in modulating genotoxic effects and provide a more accurate representation of a compound’s biological impact [21−23]. Furthermore, the GLP-compliant study design and the use of validated positive controls enhance the reliability and sensitivity of the assays, reinforcing the robustness of these findings. The absence of clinical signs of toxicity, behavioral abnormalities, or significant body weight changes in HHT-treated animals further supports its favorable safety profile. The Pig-a gene mutation assay, recognized as a reliable in vivo tool for evaluating mutagenic potential [ 20 ], confirmed that HHT does not induce significant mutational changes. Combined with data from the micronucleus test, these results provide comprehensive evidence that HHT does not pose genotoxic or cytotoxic risks under the tested conditions. The short-term exposure duration in the Pig-a gene mutation assay (three days) limits its ability to detect potential mutagenic effects that may result from chronic or cumulative exposure. To address this limitation, future research should include long-term in vivo genotoxicity studies, such as transgenic rodent mutation assays or an extended 4-week Pig-a assay. These studies would complement the current findings and provide a more comprehensive understanding of HHT’s safety profile across various exposure scenarios. Collectively, these findings demonstrate that HHT is unlikely to pose genotoxic or cytotoxic risks in vivo . However, ongoing investigation through long-term studies and advanced genotoxicity assays is essential to fully confirm its safety and strengthen its potential for therapeutic use with greater confidence. Conclusion The results of this study suggest that HHT does not exhibit genotoxicity and cytotoxicity in vivo under the tested conditions. The alkaline comet assay and Pig-a gene mutation assay showed no evidence of DNA damage or mutagenesis in rat liver or peripheral blood at doses up to 2,000 mg/kg. Additionally, the absence of clinical signs of toxicity, behavioral abnormalities, or significant body weight changes further supports HHT’s favorable safety profile. Although these findings contrast with earlier in vitro genotoxicity reports, they emphasize the importance of in vivo studies in accurate risk assessment. Further long-term evaluations are recommended to confirm the safety of HHT for broader applications. Declarations Ethics approval This animal study was approved independently by the appropriate Institutional Animal Care and Use Committees (IACUC) and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) at each participating institute and carried out in accordance with the most recent version of the Korean Association of Laboratory Animal Science. Experimental animals were handled under a protocol approved by the Institutional Animal Care and Use Committee of Korea Institute of Toxicology (Approval Nos. 19-1-0240 and 19-1-0347 for the in vivo comet and Pig-a assays, respectively) and conducted in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification No. 2017-71, issued by the Korea Food and Drug Administration on August 30, 2017). The study protocol complied with the Organization for Economic Co-operation and Development Principles of Good Laboratory Practice (1997) and was reviewed by the IACUC of the Korea Institute of Toxicology. Competing interest Not applicable. Competing interest No conflicting financial interests are disclosed by the authors. Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding This study was supported by the Korea Institute of Toxicology [KK-2504, KK-2506] and National Research Council of Science & Technology (NST) by the Korea government (MSIT) (No. CAP-21023-000). Acknowledgment We would like to thank Professor Ozden Ozkan (Kafkas University, Turkiye) for English language editing and scientific comments. Author information Authors and Affliations Division of Next Generation Non-Clinical Research, Korea Institute of Toxicology, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34114, Republic of Korea Seng-Min Back, Eun-Young Gu, Kang-Hyun Han, Min Seong Jang, Na-Young Jeung, Nan-Young Kim, Da Yeon Kim, Soyoung Jung, Yong-Bum Kim, Seong‑Hoon Park Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 483 Expo-ro, Yuseong-gu, Daejeon, 34054, Republic of Korea Chang-Seob Seo Contributions SB Writing-original draft, Visualization, Data curation, Investigation, EG Writing-original draft, Visualization, Investigation, KH Project administration, Funding acquisition, CS Investigation, MSJ Investigation, NJ Investigation, NK Investigation, DYK Investigation, SJ Investigation, YK Review & editing, Project administration, Funding acquisition, SP Writing-original draft, review & editing, Project administration, Data curation, supervision, Methodology, Funding acquisition, Conceptualization. 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Genes Environ. 2014;36(4). https://doi.org/10.3123/jemsge.2014.027 . ,169 – 73. He Y, Zhu L, Ma J, Lin G. Metabolism-mediated cytotoxicity and genotoxicity of pyrrolizidine alkaloids. Arch Toxicol. 2021;95(6). https://doi.org/10.1007/s00204-021-03060-w . ,1917-42. Shi J, Lian H, Huang Y, Zhao D, Wang H, Wang C, Li J, Ke L. In vitro genotoxicity evaluation and metabolic study of residual glutaraldehyde in animal-derived biomaterials. Regen Biomater. 2020;7(6):619–25. https://doi.org/10.1093/rb/rbaa041 . Park IH, Geum SW, Yeo MK. Reduced cellular process and developmental process genotoxicity of polystyrene nanoplastics in zebrafish embryogenesis using Aurelia aurita proteins. Mol Cell Toxicol. 2023. https://doi.org/10.1007/s13273-023-00356-z . 19,829 – 42. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6303752","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496665753,"identity":"c2b298d6-8bf5-47a2-982e-338e075fca21","order_by":0,"name":"Seng-Min Back","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Seng-Min","middleName":"","lastName":"Back","suffix":""},{"id":496665754,"identity":"b1a0158b-63ac-41ce-8af8-97aaa6603338","order_by":1,"name":"Eun-Young Gu","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Eun-Young","middleName":"","lastName":"Gu","suffix":""},{"id":496665755,"identity":"596d1461-bfa7-461a-ba24-5e5569fa178a","order_by":2,"name":"Kang-Hyun Han","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Kang-Hyun","middleName":"","lastName":"Han","suffix":""},{"id":496665756,"identity":"cb0af78f-0641-4224-8fc3-d746668fc2ea","order_by":3,"name":"Chang-Seob Seo","email":"","orcid":"","institution":"Korea Institute of Oriental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chang-Seob","middleName":"","lastName":"Seo","suffix":""},{"id":496665757,"identity":"a69ac801-b3b5-4d17-9d36-8f78f6060c7e","order_by":4,"name":"Min Seong Jang","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"Seong","lastName":"Jang","suffix":""},{"id":496665758,"identity":"c4027d48-e980-43cb-bef5-e7438be5387a","order_by":5,"name":"Na-Young Jeung","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Na-Young","middleName":"","lastName":"Jeung","suffix":""},{"id":496665759,"identity":"3457c860-b742-4755-b6b8-e1aea81a8f9f","order_by":6,"name":"Nan-Young Kim","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Nan-Young","middleName":"","lastName":"Kim","suffix":""},{"id":496665760,"identity":"7e4206cc-1889-413d-88d4-194a89614fbf","order_by":7,"name":"Da Yeon Kim","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Da","middleName":"Yeon","lastName":"Kim","suffix":""},{"id":496665761,"identity":"f975097b-9745-430e-aaf3-cba8c4280c80","order_by":8,"name":"Soyoung Jung","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Soyoung","middleName":"","lastName":"Jung","suffix":""},{"id":496665762,"identity":"5332d700-57c4-44c4-b5c3-ad6ad479ea17","order_by":9,"name":"Yong-Bum Kim","email":"","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":false,"prefix":"","firstName":"Yong-Bum","middleName":"","lastName":"Kim","suffix":""},{"id":496665763,"identity":"0f4dbcdd-02d4-4e61-9eb1-68d0c443ea46","order_by":10,"name":"Seong‑Hoon Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2QsUoDMRjHv0PIFG9OOLy+wlcCJ4XQZzk5iIsP4Bg4OBd9Aoe+QkcHh0ggLkHXky66ODmciGAnrbWnS1M7CuY35PsP+ZF/PoBI5K+S6D4ZiaTPbEtFIemdbRWL8JsyOKndw+uFzPdh9+rl2N+KdFA7llxK4OdmrYLeHYo9r8RIpxX37awghFQIjwqytFyvsKMi4409mBqKXHczSQgd3i8aQk4DxSZP34qY6+7mU0ED5j2oQEsL/vylFFy3pli9YiALKOiVypJGCbSpGmlfCUJUhaWpKD8Nbcw6Pm9kjtdn9k678XBSW8c6M86ZDxRbsEN/zhUlQOgnS5K35dh4JxKJRP4zHz2yV+FKIlE5AAAAAElFTkSuQmCC","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":true,"prefix":"","firstName":"Seong‑Hoon","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-03-25 12:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6303752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6303752/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88658096,"identity":"d028cd6e-84fa-43e1-bee5-960e286e85e2","added_by":"auto","created_at":"2025-08-08 20:04:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245146,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatograms of the standard solution (A) and HHT (B). \u003cem\u003eGeniposide\u003c/em\u003e(\u003cstrong\u003e1\u003c/strong\u003e), \u003cem\u003eBaicalin\u003c/em\u003e (\u003cstrong\u003e2\u003c/strong\u003e), \u003cem\u003eJatrorrhizine\u003c/em\u003e (\u003cstrong\u003e3\u003c/strong\u003e), \u003cem\u003eCoptisine\u003c/em\u003e (\u003cstrong\u003e4\u003c/strong\u003e), \u003cem\u003ePalmatine \u003c/em\u003e(\u003cstrong\u003e5\u003c/strong\u003e), and \u003cem\u003eBerberine\u003c/em\u003e (\u003cstrong\u003e6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe six marker components were separated using a Gemini C18 column maintained to 40°C. The injection volume and flow rate were 10 mL and 1.0 mL/min, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6303752/v1/86ac95f920a0cc86f394e5c9.jpeg"},{"id":88658095,"identity":"4aa59b2e-d12a-472e-bab9-af076c5a2f98","added_by":"auto","created_at":"2025-08-08 20:04:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42770,"visible":true,"origin":"","legend":"\u003cp\u003e(A-D) \u003cem\u003eIn vivo\u003c/em\u003e alkaline comet assay results for HHT. All the values are shown as median or mean ± standard deviation (SD). (**significantly different from the control at \u003cem\u003ep\u0026lt;\u003c/em\u003e0.01). Vehicle versus test compound-treated groups: Dunnett’s test (two-side) and linear trend test (two-side). Vehicle versus positive control groups: Student's t-test (one-side). Vehicle: Distilled water (DW), T1: 500 mg/kg, T2: 1,000 mg/kg, T3: 2,000 mg/kg, Positive: Ethyl methanesulfonate (EMS), 200 mg/kg)\u003c/p\u003e","description":"","filename":"floatimage35.png","url":"https://assets-eu.researchsquare.com/files/rs-6303752/v1/70a62eb386c4264933b00b44.png"},{"id":88658098,"identity":"0b529839-ab96-40a6-9938-f13dc72bfe9f","added_by":"auto","created_at":"2025-08-08 20:04:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":397379,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003ePig-a \u003c/em\u003eassay results and representative data of flow cytometer plots. (A-C) \u003cem\u003ePig-a\u003c/em\u003e assays were conducted after three administrations of HHT. (D-F) Typical plots measuring \u003cem\u003ePig-a\u003c/em\u003e mutant cells from vehicle control group, ENU treatment group (positive) and HHT treatment groups (n=5). All the values are shown as mean ± standard deviation (SD). (**significantly different from the control at \u003cem\u003ep\u0026lt;\u003c/em\u003e0.01). Vehicle versus test compound-treated groups: Dunnett’s test (two-side) and linear trend test (two-side). Vehicle versus positive control groups: Student's t-test (one-side). Vehicle: Distilled water (DW), T1: 500 mg/kg, T2: 1,000 mg/kg, T3: 2,000 mg/kg, Positive: N-ethyl-N-nitrosourea (ENU), 20 mg/kg)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6303752/v1/7f331397baa10e1fdadc1d7e.jpeg"},{"id":88659060,"identity":"706495b1-c762-40fb-86a6-ddf025c4ad43","added_by":"auto","created_at":"2025-08-08 20:20:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1796521,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6303752/v1/b5cec8cb-ee43-42ed-a90c-a214f8892320.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotoxicity Assessment of Hwanglyeonhaedok-Tang: An Herbal Formula Evaluated Using the In Vivo Alkaline Comet Assay and Pig-a Gene Mutation Assay in Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraditional medicinal herbs have been utilized for centuries to prevent and treat various diseases, deeply rooted in cultural and historical practices. With a long history of human use, traditional herbal formulas have garnered attention for their therapeutic potential. However, recent decades have seen growing concerns about herb-drug interactions and the safety of these remedies, especially under modern pharmacological standards. Traditional Asian Medicine (TAM), extensively documented in ancient medical texts, commonly uses fixed herbal components with clearly defined therapeutic effects and relatively acceptable risks of adverse reactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHwanglyeonhaedok-tang (HHT), known as Huang-Lian-Jie-Du-Tang in Chinese or Oren-gedoku-to in Japanese, is a classic multi-herb formula first detailed in Wang Tao\u0026rsquo;s Wai Tai Mi Yao. It consists of \u003cem\u003eCoptidis chinensis\u003c/em\u003e Franch (\u003cem\u003eHwanglyeon\u003c/em\u003e in Korean), \u003cem\u003eScutellaria baicalensis Georgi\u003c/em\u003e, \u003cem\u003ePhellodendron amurense\u003c/em\u003e Rupr., and \u003cem\u003eGardenia jasminoides\u003c/em\u003e J. Ellis, traditionally prescribed for heat-clearing and detoxification. Numerous studies have reported its pharmacological benefits, including antipyretic, anti-inflammatory, gastroprotective, neuroprotective, and hypolipidemic effects [3 \u0026minus; 11].\u003c/p\u003e\u003cp\u003eDespite its benefits, concerns persist regarding the quality, safety, and efficacy of conventional herbal formulas. Specifically, HHT has shown genotoxic potential in \u003cem\u003ein vitro\u003c/em\u003e Ames and chromosomal aberration (CA) tests conducted under Good Laboratory Practice (GLP) standards [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Components of HHT, such as \u003cem\u003eScutellaria baicalensis\u003c/em\u003e and \u003cem\u003eCoptidis chinensis\u003c/em\u003e, have also demonstrated genotoxic effects in earlier studies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegulatory standards for genotoxicity testing typically follow a three-battery approach: the Ames test, CA test, and \u003cem\u003ein vivo\u003c/em\u003e micronucleus test. While these tests detect diverse genetic damage, they often yield inconsistent results between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays and are prone to false positives. To mitigate these limitations, the International Council for Harmonization (ICH) S2 guidelines recommend additional \u003cem\u003ein vivo\u003c/em\u003e assays, such as the alkaline comet assay and \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay, to enhance the robustness of genotoxicity assessments.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e alkaline comet assay evaluates DNA damage in individual cells, including gene mutations, clastogenicity, and aneuploidy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This assay is particularly suitable for liver tissue, the primary site of xenobiotic metabolism, making it ideal for assessing DNA damage. Meanwhile, the \u003cem\u003ein vivo Pig-a\u003c/em\u003e assay, a newer \u003cem\u003ein vivo\u003c/em\u003e mutation test, evaluates glycosylphosphatidylinositol (GPI)-anchored protein deficiencies as a biomarker for mutagenicity and serves as a comprehensive tool for regulatory safety evaluations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the potential genotoxicity of HHT using the \u003cem\u003ein vivo\u003c/em\u003e alkaline comet assay and \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay to address limitations of \u003cem\u003ein vitro\u003c/em\u003e tests. The \u003cem\u003ein vivo\u003c/em\u003e comet assay was conducted following Organization for Economic Co-operation and Development (OECD) guidelines, while the \u003cem\u003ePig-a\u003c/em\u003e assay employed validated methodologies. These assessments provide a comprehensive evaluation of HHT\u0026rsquo;s genotoxic profile and contribute to its safety characterization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003eThe four crude herbs comprising HHT\u0026ndash;Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex, and Gardeniae Fructus\u0026ndash;were procured from Omniherb (Yeongcheon, Korea) and HMAX (Jecheon, Korea) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of the Hwanglyeonhaedok-tang powder\u003c/h3\u003e\n\u003cp\u003eThe HHT formulation itself was supplied by the Korea Institute of Oriental Medicine [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To ensure quality control of HHT, HPLC analysis was conducted for six marker compounds using a modified method based on previous research [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The analysis utilized a Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) equipped with photodiode array detection. The mobile phases consisted of solvent A, which was 10.0% (v/v) acetonitrile with 0.2% sodium dodecyl sulfate and 200 \u0026micro;L phosphoric acid, and solvent B, which was pure acetonitrile. Gradient elution was applied under the following conditions: 10\u0026ndash;40% B for 0\u0026ndash;20 min, 40\u0026ndash;50% B for 20\u0026ndash;40 min, 50\u0026ndash;100% B for 40\u0026ndash;50 min, 100\u0026ndash;10% B for 50\u0026ndash;55 min, and 10% B for 55\u0026ndash;70 min. The flow rate was set at 1.0 mL/min. Using this optimized HPLC assay, the concentrations of six marker compounds\u0026mdash;\u003cem\u003eGeniposide, Baicalin, Jatrorrhizine, Coptisine, Palmatine\u003c/em\u003e, and \u003cem\u003eBerberine\u003c/em\u003e\u0026mdash;were quantified, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the HPLC chromatograms of HHT, with eluents detected at wavelengths of 240, 265, and 275 nm. Separation of these marker compounds was achieved using a Gemini C18 column maintained at 40\u0026deg;C. The injection volume was 10 \u0026micro;L, with a consistent flow rate of 1.0 mL/min.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAmounts of the marker compounds in the HHT sample analyzed using HPLC.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmount (mg/g)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRSD (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRT (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGeniposide\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e26.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eGardenia jasminoides\u003c/em\u003e J.Ellis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBalcalin\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e22.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eScutellaria baicalensis\u003c/em\u003e Georgi\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eJatrorrhizine\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eCoptidis chinensis\u003c/em\u003e Franch\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhellodendron amurense\u003c/em\u003e Rupr.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCoptisine\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eCoptidis chinensis\u003c/em\u003e Franch\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhellodendron amurense\u003c/em\u003e Rupr.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePalmatine\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eCoptidis chinensis\u003c/em\u003e Franch\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhellodendron amurense\u003c/em\u003e Rupr.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBerberine\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eCoptidis chinensis\u003c/em\u003e Franch\u003c/p\u003e\u003cp\u003e\u003cem\u003ePhellodendron amurense\u003c/em\u003e Rupr.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of the positive control articles\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e genotoxicity assays, N-ethyl-N-nitrosourea (ENU, Cas# 759-73-9) and ethyl methanesulfonate (EMS, Cas# 62-50-0) were employed as positive controls for the \u003cem\u003ePig-a\u003c/em\u003e and comet assays, respectively. Both ENU and EMS, obtained from Sigma-Aldrich (USA), were dissolved in distilled water (Dai Han Pharm. Co., Ltd., Korea) and administered orally on the dosing day.\u003c/p\u003e\n\u003ch3\u003eAnimals and Maintenance\u003c/h3\u003e\n\u003cp\u003eSpecific-pathogen-free male SD rats, approximately six weeks old, were acquired from Orient Bio Inc. (Korea) for the \u003cem\u003ein vivo\u003c/em\u003e experiments. The rats were housed in stainless steel cages measuring 265 mm (W) \u0026#128936; 465 mm (L) \u0026#128936; 200 mm (H). Environmental conditions in the animal facility were meticulously controlled, maintaining a temperature of 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, a 12 h light/dark cycle (lights on at 08:00 and off at 20:00) with illumination levels between 150\u0026ndash;300 lux, and ventilation rates of 10\u0026ndash;20 air exchanges per hour.\u003c/p\u003e\u003cp\u003eThroughout the study, the animals were monitored daily to ensure their well-being. At the conclusion of the experiment, all animals, including any remaining subjects, were humanely euthanized by gradual exposure to carbon dioxide (CO₂) at a flow rate of 30\u0026ndash;70% of the chamber volume per minute, following the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). Complete euthanasia was confirmed by a secondary physical method (thoracotomy) performed after loss of consciousness.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ealkaline comet assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs part of an international validation study of the \u003cem\u003ein vivo\u003c/em\u003e alkaline comet assay under the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative, the assay was conducted using the standard validation protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. HHT was administered orally by gavage at doses of 500, 1,000, and 2,000 mg/kg at 0, 24, and 45 h, with a dosage volume of 10 mL/kg based on each rat\u0026rsquo;s body weight prior to administration. The positive control compound was similarly administered by oral gavage at 10 mL/kg once daily for three consecutive days at a dose of 200 mg/kg.\u003c/p\u003e\u003cp\u003eThe rats were euthanized 3 h after the final administration, and a portion of the left lateral lobe of the liver was excised. Single-cell suspensions were prepared within 1 h of euthanasia, and four slides per tissue (two slides per tissue per rat) were prepared. The slides were stained with SYBR Gold, and comet measurements were conducted using Comet Assay IV software (version 4.3.1, Perceptive Instruments Ltd., UK) under a fluorescence microscope at 200X magnification. For each sample (animal/tissue), 75 comets per slide (150 comets/tissue) were analyzed. The key comet assay endpoints\u0026ndash;percentage tail DNA, olive tail moment, and tail length\u0026mdash;were recorded. Additionally, the frequency of heavily damaged cells, including \"hedgehogs\" and \"ghost\" cells, was visually scored from 150 cells per sample.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo Pig-a\u003c/b\u003e \u003cb\u003egene mutation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo Pig-a\u003c/em\u003e assay was performed according to the method described in the MutaFlow Basic-25R GLP kit (tube-based processing, version 140403, Litron Laboratories, USA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. HHT was administered by oral gavage at doses of 500, 1,000, and, 2,000 mg/kg for three consecutive days. The dosage volume was set at 10 mL/kg body weight, adjusted based on the rats\u0026rsquo; weights measured shortly before each administration. The positive control compound was also administered by oral gavage at 10 mL/kg once daily for three days at a dose level of 20 mg/kg.\u003c/p\u003e\u003cp\u003ePeripheral blood samples were collected 29 days after the first treatment. Lymphocytes were removed from the peripheral blood using Lympholytes\u0026reg; (Cat No.: CL5120, Cedarlane Corp., Canada). Wild-type red blood cells (RBCs) were tagged with PE-conjugated anti-CD59 and anti-CD61 antibodies, while platelets were removed through magnetic separation. Mutant-type RBCs were concentrated using the same process, and reticulocytes (RETs) were stained with SYTO13\u0026reg; (Life Technologies, USA) to distinguish them from mutant phenotype RBCs. Flow cytometry analysis was performed using a (FACSCalibur (BD Biosciences, USA), with at least 1,000 counting beads per sample. The data were analyzed using Cell Quest Pro software (Version 5.2.1, BD Biosciences, USA). Gene mutation analysis included calculations of %RET, mutant RBCs/10\u003csup\u003e6\u003c/sup\u003e total RBCs, and mutant RETs/10\u003csup\u003e6\u003c/sup\u003e total RETs.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA), with a significance threshold set at \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eIn vivo alkaline comet assay\u003c/h2\u003e\u003cp\u003eTo substantiate the weight of evidence regarding HHT, which exhibited genotoxicity in chromosome aberration studies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], a comet assay was performed in liver tissue under GLP compliance.\u003c/p\u003e\u003cp\u003eThe primary endpoint for assessing DNA migration was the percentage of tail DNA, representing the proportion of fluorescence detected in the tail during image analysis, along with tail length (tail migration). The distribution of migration patterns among cells within each animal was also evaluated. The percentage of \"hedgehogs\" (ghost cells or heavily damaged cells) was analyzed as a function of treatment. For each specific tissue, the unit of analysis was the individual animal. Notably, hedgehogs\u0026ndash;characterized by a small or nonexistent head accompanied by large and diffuse tails\u0026ndash;were excluded from data collection.\u003c/p\u003e\u003cp\u003eNo significant increases in the percentage of tail DNA were observed in the liver tissues of rats administered HHT at any dose (0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, or 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 for 0, 500, 1,000, and 2,000 mg/kg, respectively). Similarly, no significant increases were observed in the percentage of hedgehogs in liver tissues (0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84, 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55, 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 or 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84, for 0, 500, 1000, and 2,000 mg/kg, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComet assay results of HHT\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"16\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDose (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c13\" namest=\"c5\"\u003e\u003cp\u003eHHT (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e\u003cp\u003eEMS\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003e1,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e\u003cp\u003e2,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e% Tail DNA in Liver\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedians\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e15.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e2.77**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e16.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e3.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTail Length in Liver\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedians\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e18.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e20.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e36.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e2.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e20.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e36.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e2.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOlive Tail Moment in Liver\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedians\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003e% Hedgehogs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e1.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"16\"\u003e** Significant differences from control group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01); EMS: Ethyl methanesulfonate\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDuring the study, no clinical signs and abnormal behaviors were observed in the animals treated with HHT throughout the observation period (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Additionally, there were no statistically significant differences in the mean body weights between the HHT-treated groups and the vehicle control group.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical sign in comet assay for HHT at doses of 500, 1,000, or 2,000 mg/kg or EMS at 200 mg/kg via three consecutive days at approximately 24-hour intervals\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eClinical Signs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eHHT (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEMS\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbnormalities Detected\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0/6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0/6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0/6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0/6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0/6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTherefore, HHT did not induce DNA damage by oral gavage at any tested dose level compared to the vehicle control.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn vivo Pig-a gene mutation assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo Pig-a\u003c/em\u003e assay, a mammalian gene mutation assay utilizing peripheral blood, was also conducted to confirm the genotoxicity of HHT observed in the Ames test [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This assay offers high integration potential due to its reliance on peripheral blood samples [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Frequencies of mutant phenotype erythrocytes (RBCs) and mutant phenotype immature erythrocytes (reticulocytes, RETs) showed no significant changes in the test groups treated with HHT at any dose. Specifically, mutant RBC frequencies were 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42, 1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09, 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76, and 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 at 0, 500, 1,000, and 2,000 mg/kg, respectively. Similarly, mutant RET frequencies were 1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59, 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49, 0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97, and 9.98\u0026thinsp;\u0026plusmn;\u0026thinsp;19.71 at the respective doses. The percentage of reticulocytes (%RET), a cytotoxicity index, also did not differ significantly between the HHT-treated groups and the vehicle control group (2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, 2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53, 2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, and 2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 at 0, 500, 1,000, and 2,000 mg/kg, respectively). In contrast, significant increases were observed in the frequencies of mutant phenotype RBCs (95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.09) and mutant phenotype RETs (207.80\u0026thinsp;\u0026plusmn;\u0026thinsp;49.73) in the positive control group. However, no significant increases in the %RET were observed in the positive control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003ePig-a\u003c/em\u003e assay results of HHT\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"16\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDose (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c13\" namest=\"c5\"\u003e\u003cp\u003eHHT (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e\u003cp\u003eENU\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003e1,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e\u003cp\u003e2,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e% RET\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e2.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e2.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMutant RETs per 10\u003c/b\u003e\u003csup\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eTotal RETs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e9.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e19.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e207.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e49.73**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMutant RBCs per 10\u003c/b\u003e\u003csup\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eTotal RBCs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e95.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e11.09**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"16\"\u003e** Significant differences from vehicle control group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01); ENU: N-ethyl-N-nitrosourea\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNo clinical signs of toxicity or abnormal behavior were observed in animals treated with the test compound throughout the observation period (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, there were no statistically significant differences in the mean body weights of the test compound-treated groups compared to the vehicle control group (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical sign in \u003cem\u003ePig-a\u003c/em\u003e assay\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eClinical Signs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eHHT (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eENU\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbnormalities Detected\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0/5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0/5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0/5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0/5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0/5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBody weight changes of male SD rats treated with HHT during \u003cem\u003ePia-a\u003c/em\u003e assay. Test item was administered by oral gavage at doses of 500, 1,000, and 2,000 mg/kg for three consecutive days (10 mL/kg body weight of rats based on the animal weight shortly before administration). The positive control (ENU) was administered at a dose level of 20 mg/kg.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"17\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eDose (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eVehicle control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c14\" namest=\"c6\"\u003e\u003cp\u003eHHT (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c17\" namest=\"c15\"\u003e\u003cp\u003eENU\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e1,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c14\" namest=\"c12\"\u003e\u003cp\u003e2,000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c17\" namest=\"c15\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"17\" nameend=\"c17\" namest=\"c1\"\u003e\u003cp\u003eBody Weight\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e252.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e254.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e9.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e251.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e6.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e251.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e251.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e3.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e266.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e266.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e11.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e263.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e8.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e261.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e7.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e261.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e4.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e274.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e275.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e10.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e271.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e8.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e273.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e8.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e266.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e5.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e286.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e287.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e11.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e281.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e9.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e284.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e10.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e268.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e7.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e314.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e321.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e15.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e310.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e12.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e316.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e15.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e309.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e4.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e347.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e348.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e18.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e336.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e14.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e345.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e17.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e336.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e5.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e382.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e385.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e26.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e372.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e19.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e383.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e21.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e377.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e9.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e409.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e419.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e29.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e402.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e24.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e415.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e27.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e409.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e13.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e433.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e29.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e452.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e39.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e429.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e27.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e444.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e30.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e442.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e15.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides compelling evidence that HHT does not induce genotoxicity and cytotoxicity \u003cem\u003ein vivo\u003c/em\u003e. The comet assay showed no significant increases in % tail DNA or hedgehog frequency, whereas the \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay revealed no significant changes in mutant RBC or RET frequencies. These results indicate that HHT does not cause DNA damage or mutagenesis in rat liver or peripheral blood at doses up to 2,000 mg/kg.\u003c/p\u003e\u003cp\u003eThese findings contrast with previous \u003cem\u003ein vitro\u003c/em\u003e results, such as the CA and Ames tests [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which reported genotoxic activity. This discrepancy highlights the importance of \u003cem\u003ein vivo\u003c/em\u003e studies, which account for metabolic and detoxification pathways absent \u003cem\u003ein vitro\u003c/em\u003e. These physiological processes are critical in modulating genotoxic effects and provide a more accurate representation of a compound\u0026rsquo;s biological impact [21\u0026minus;23]. Furthermore, the GLP-compliant study design and the use of validated positive controls enhance the reliability and sensitivity of the assays, reinforcing the robustness of these findings.\u003c/p\u003e\u003cp\u003eThe absence of clinical signs of toxicity, behavioral abnormalities, or significant body weight changes in HHT-treated animals further supports its favorable safety profile. The \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay, recognized as a reliable \u003cem\u003ein vivo\u003c/em\u003e tool for evaluating mutagenic potential [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], confirmed that HHT does not induce significant mutational changes. Combined with data from the micronucleus test, these results provide comprehensive evidence that HHT does not pose genotoxic or cytotoxic risks under the tested conditions.\u003c/p\u003e\u003cp\u003eThe short-term exposure duration in the \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay (three days) limits its ability to detect potential mutagenic effects that may result from chronic or cumulative exposure. To address this limitation, future research should include long-term \u003cem\u003ein vivo\u003c/em\u003e genotoxicity studies, such as transgenic rodent mutation assays or an extended 4-week \u003cem\u003ePig-a\u003c/em\u003e assay. These studies would complement the current findings and provide a more comprehensive understanding of HHT\u0026rsquo;s safety profile across various exposure scenarios.\u003c/p\u003e\u003cp\u003eCollectively, these findings demonstrate that HHT is unlikely to pose genotoxic or cytotoxic risks \u003cem\u003ein vivo\u003c/em\u003e. However, ongoing investigation through long-term studies and advanced genotoxicity assays is essential to fully confirm its safety and strengthen its potential for therapeutic use with greater confidence.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this study suggest that HHT does not exhibit genotoxicity and cytotoxicity \u003cem\u003ein vivo\u003c/em\u003e under the tested conditions. The alkaline comet assay and \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay showed no evidence of DNA damage or mutagenesis in rat liver or peripheral blood at doses up to 2,000 mg/kg. Additionally, the absence of clinical signs of toxicity, behavioral abnormalities, or significant body weight changes further supports HHT\u0026rsquo;s favorable safety profile. Although these findings contrast with earlier in vitro genotoxicity reports, they emphasize the importance of \u003cem\u003ein vivo\u003c/em\u003e studies in accurate risk assessment. Further long-term evaluations are recommended to confirm the safety of HHT for broader applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis animal study was approved independently by the appropriate Institutional Animal Care and Use Committees (IACUC) and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) at each participating institute and carried out in accordance with the most recent version of the Korean Association of Laboratory Animal Science.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental animals were handled under a protocol approved by the Institutional Animal Care and Use Committee of Korea Institute of Toxicology (Approval Nos. 19-1-0240 and 19-1-0347 for the \u003cem\u003ein vivo\u003c/em\u003e comet and \u003cem\u003ePig-a\u003c/em\u003e assays, respectively) and conducted in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification No. 2017-71, issued by the Korea Food and Drug Administration on August 30, 2017). The study protocol complied with the Organization for Economic Co-operation and Development Principles of Good Laboratory Practice (1997) and was reviewed by the IACUC of the Korea Institute of Toxicology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicting financial interests are disclosed by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Korea Institute of Toxicology [KK-2504, KK-2506] and National Research Council of Science \u0026amp; Technology (NST) by the Korea government (MSIT) (No. CAP-21023-000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Professor Ozden Ozkan (Kafkas University, Turkiye) for English language editing and scientific comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDivision of Next Generation Non-Clinical Research, Korea Institute of Toxicology, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34114, Republic of Korea\u003c/p\u003e\n\u003cp\u003eSeng-Min Back, Eun-Young Gu, Kang-Hyun Han, Min Seong Jang, Na-Young Jeung, Nan-Young Kim, Da Yeon Kim, Soyoung Jung, Yong-Bum Kim, Seong‑Hoon Park\u003c/p\u003e\n\u003cp\u003eHerbal Medicine Research Division, Korea Institute of Oriental Medicine, 483 Expo-ro, Yuseong-gu, Daejeon, 34054, Republic of Korea\u003c/p\u003e\n\u003cp\u003eChang-Seob Seo\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSB Writing-original draft, Visualization, Data curation, Investigation, EG Writing-original draft, Visualization, Investigation, KH Project administration, Funding acquisition, CS Investigation, MSJ Investigation, NJ Investigation, NK Investigation, DYK Investigation, SJ Investigation, YK Review \u0026amp; editing, Project administration, Funding acquisition, SP Writing-original draft, review \u0026amp; editing, Project administration, Data curation, supervision, Methodology, Funding acquisition, Conceptualization.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQi Y, Zhang Q, Zhu H. 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Reduced cellular process and developmental process genotoxicity of polystyrene nanoplastics in zebrafish embryogenesis using \u003cem\u003eAurelia aurita\u003c/em\u003e proteins. Mol Cell Toxicol. 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13273-023-00356-z\u003c/span\u003e\u003cspan address=\"10.1007/s13273-023-00356-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 19,829\u0026thinsp;\u0026ndash;\u0026thinsp;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hwanglyeonhaedok-tang (Huang-lian-jie-du-tang and oren-gedoku-to), in vivo alkaline comet assay, in vivo Pig-a gene mutation assay, genotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-6303752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6303752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHwanglyeonhaedok-tang (HHT), also known as Huang-lian-jie-du-tang or Oren-gedoku-to, is a popular herbal formula in traditional Asian medicine. Despite the clinical effectiveness of herbal formulas, concerns regarding the quality, safety, and efficacy persist. HHT exhibited genotoxicity in bacterial reverse mutation (Ames test) and \u003cem\u003ein vitro\u003c/em\u003e chromosomal aberration (CA) test, warranting further investigation. This study aimed to verify HHT-induced genotoxicity through \u003cem\u003ein vivo\u003c/em\u003e assays like alkaline comet and phosphatidylinositol glycan class A (\u003cem\u003ePig-a\u003c/em\u003e) gene mutation assay because HHT has shown positive results only in \u003cem\u003ein vitro\u003c/em\u003e tests.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e comet assay was conducted on rat liver under Good Laboratory Practice standards to assess DNA migration, focusing on % tail DNA and hedgehog frequency (cells with small or absent heads and diffuse tails). The \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay used peripheral blood to evaluate mutant red blood cells (RBCs) and reticulocytes (RETs), with cytotoxicity assessed by %RET. Doses of 0, 500, 1,000, and 2,000 mg/kg were tested, applying standard statistical methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe comet assay showed no marked increases in % tail DNA or hedgehog frequency at the doses tested. No clinical signs, behavioral abnormalities, or weight changes were noted. Similarly, the \u003cem\u003ePig-a\u003c/em\u003e assay revealed no significant differences in mutant RBCs or RETs, and %RET cytotoxicity was comparable to controls. Positive controls validated assay sensitivity. Overall, HHT did not induce genotoxicity and cytotoxicity under tested conditions, supporting its safety at the evaluated dose levels.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e comet assay result suggest that the positive outcome observed in the \u003cem\u003ein vitro\u003c/em\u003e CA test is likely a false positive. However, although negative result was obtained from short-term \u003cem\u003ePig-a\u003c/em\u003e gene mutation assay, it cannot be conclusively determined that HHT does not induce gene mutations. 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