28-day Repeated-dose Toxicity Research Articles

Safety Assessment of Streptococcus salivarius UBSS-01 in Rats and Double-Blind Placebo-Controlled Study in Healthy Individuals.

Streptococcus salivarius is a common, harmless, and prevalent member of the oral microbiota in humans. In the present study, the safety of S. salivarius UBSS-01 was evaluated using in silico methods and preclinical and clinical studies. In an acute toxicity study, rats were administered with 5g/kg (500 × 109CFU) S. salivarius UBSS-01. The changes in phenotypic behaviors and hematological, biochemical, electrolytes, and urine analyses were monitored. No toxicity was observed at 14days post-treatment. The no observable effects limit (NOEL) of S. salivarius UBSS-01 was >5g/kg in rats. In a 28-day repeat dose toxicity study, rats were administered S. salivarius UBSS-01 once daily at doses of 0.1, 0.5, and 1g/kg (10, 50, and 100 billion CFU/kg, respectively) body weight. S. salivarius UBSS-01 did not influence any of the hematology parameters and clinical chemistry parameters in plasma and serum samples after 28-day repeated administration. No structural abnormality was observed in the histological examination of organs. Whole genome analysis revealed the absence of virulence factors or genes that may transmit antibiotic resistance. In the double-blind study with 60 human participants (aged 18-60years), consumption of S. salivarius UBSS-01 for 30days was found to be safe and results were comparable with placebo treatment These findings indicate that S. salivarius UBSS-01 may be safe for human consumption.

Safety Evaluation of Vatari Guggulu through Acute and 90-day Repeated Dose Toxicity Study in Experimental Animals

Abstract Background: Vatari Guggulu is an Ayurvedic Medicine used in the treatment of Vata disorders. It contains Vatari taila (Castor oil), Shuddha gandhaka (purified Sulphur), Haritaki (Terminalia chebula Retz.), Vibhitaki (Terminalia bellirica [Gaertn.] Roxb.), Amla (Emblica officinalis Gaertn) and guggul (Purified Commiphora mukul Hook). As per the Ayurvedic texts, it is widely used clinically in the treatment of rheumatoid arthritis, hip pain, sciatica, gout, joint pain, and burning sensation. However, there are no systematic toxicity studies were performed to scientifically evaluate the safety of Vatari Guggulu. Objectives: The present study was designed to investigate the safety of Vatari Guggulu in acute and 90-days repeated dose oral toxicity as per Organization for Economic Cooperation and Development guidelines 423 and 408. Materials and Methods: During the acute toxicity, Vatari Guggulu was orally administered at a single limit dose of 2000 mg/kg. The mortality and clinical signs were observed for 14 days. Similarly, the 90-days repeated dose toxicity was studied by administering the Vatari Guggulu at a limit dose of 1000 mg/kg. Results: In the acute toxicity, there were no signs of toxicity, and the LD50 was calculated as >2000 mg/kg. During the 90 days of repeated dose toxicity, the Vatari Guggulu at the dose of 1000 mg/kg did not show any signs of toxicity as compared to the control. The hematological, serum biochemical, and urine analysis profile of the Vatari Guggulu at the dose of 1000 mg/kg did not show any significant difference as compared to the control (P > 0.05). Further, the histopathological data of control and Vatari Guggulu-treated rats were found to be normal. Conclusions: It was concluded that the test drug Vatari Guggulu is found to be safe upto 2000 mg/kg in acute toxicity and 1000 mg/kg in 90-days repeated dose oral toxicity study. The NOAEL of Vatari Guggulu is established as 1000 mg/kg/day.

In-vitro and preclinical testing of bacillus subtilis UBBS-14 probiotic in rats shows no toxicity.

Probiotics made from Bacillus subtilis provide a wide spread of health benefits, particularly in the treatment of diarrhea and gastrointestinal problems. Herein, we employed in vitro and in vivo paradigms to assess the potential adverse effects and toxicity of B. subtilis UBBS-14. According to Organization for Economic Co-operation and Development (OECD) 423 and 407 requirements, a preclinical investigation was conducted in male and female Sprague-Dawley rats. Acute toxicity was examined following a single peroral (PO) administration of 5,000mg/kg body weight (bw) i.e. equivalent to 500 billion colony-forming units (CFU) per kg bw. Single administration of B. subtilis UBBS-14 showed no mortality or adverse effects until the 14-day observation period, indicating LD50 is >5,000mg/kg bw. Incubation of B. subtilis UBBS-14 with Caco2, HT29, and Raw 264.7 cell lines, showed no cytotoxic effects. This probiotic strain was also found responsive to the majority of antibiotics. For a 28-day repeated dose toxicity study, rats were administered 100, 500, and 1,000mg/kg bw daily once (10, 50, and 100 billion CFU/kg bw/day, respectively) doses of B. subtilis UBBS-14. No notable changes were seen in the morphology, weight, and histopathology of the critical internal organs. The haematological, biochemical, electrolyte (sodium, potassium, chloride, and calcium), and urine analytical results were within the normal range and equivalent to the vehicle-treated group. B. subtilis UBBS-14's no-observed-effect level (NOEL) was thus determined to be >1,000mg/kg bw/day following a 28-day oral dosing.

Safety, immunogenicity and efficacy of Relcovax®, a dual receptor binding domain (RBD) and nucleocapsid (N) subunit protein vaccine candidate against SARS-CoV-2 virus

SARS-CoV-2, severe acute respiratory syndrome coronavirus-2, causes coronavirus disease- 2019 (COVID-19). Mostly, COVID-19 causes respiratory symptoms that can resemble those of a cold, the flu, or pneumonia. COVID-19 may harm more than just lungs and respiratory systems. It may also have an impact on other parts of the body and debilitating effects on humans, necessitating the development of vaccines at an unprecedented rate in order to protect humans from infections. In response to SARS-CoV-2 infection, mRNA, viral vector-based carrier and inactivated virus-based vaccines, as well as subunit vaccines, have recently been developed. We developed Relcovax®, a dual antigen (Receptor binding domain (RBD) and Nucleocapsid (N) proteins) subunit protein vaccine candidate. Preliminary mouse preclinical studies revealed that Relcovax® stimulates cell-mediated immunity and provides broader protection against two SARS-CoV-2 variants, including the delta strain. Before conducting human studies, detailed preclinical safety assessments are required, so Relcovax® was tested for safety, and immunogenicity in 28-day repeated dose toxicity studies in rats and rabbits. In the toxicity studies, there were no mortality or morbidity, abnormal clinical signs, abnormalities in a battery of neurobehavioral observations, abnormalities in detailed clinical and ophthalmological examinations, or changes in body weights or feed consumption. In any of the studies, no abnormal changes in organ weights, haematology, clinical chemistry, urinalysis parameters, or pathological findings were observed. Immunogenicity tests on rats and rabbits revealed 100 % seroconversion. Relcovax® was therefore found to be safe in animals, with a No Observed Adverse Effect Level (NOAEL) of 20 µg/protein in rats and rabbits. In efficacy studies, Relcovax® immunised hamsters demonstrated dose-dependent protection against SARS-CoV-2 infection, with a high dose (20 µg/protein) being the most protective, while in cynomolgus macaque monkey study, lowest dose 5 µg/protein had the highest efficacy. In conclusion, Relcovax® was found to be safe, immunogenic, and efficacious in in vivo studies.

Characterization and Preclinical Toxicity Assessment of Intranasal Administration of Standardized Extract of <i>Centella asiatica</i> (L.) Urban Leaves (INDCA-NS) in Laboratory Rats

The objective of the present work was to characterize and assess the intranasal toxicity of a solution of total triterpenoids - based standardized extract of Centella asiatica (L.) Urban leaves (INDCA-NS). The acute and 28-day repeated-dose toxicity of intranasal administrated INDCA-NS in Wistar rats was evaluated. For the acute intranasal toxicity study, a single dose of 100 μg/rat/day was administered to five male and female rats and observed for mortality and signs of toxicity for 14 days. In the subacute toxicity study, the INDCA-NS (10, 30, and 100 μg/rat/day) were intranasally administered to a separate group of five male and female rats for 28 days, followed by a 14-day reversal period for behavioral, biochemical, and histological parameters. The effect of subacute administration of INDCA-NS on the ability to smell in rats was evaluated by a buried food test. Treatment with INDCA-NS did not result in mortality or treatment-related adverse signs during single or 28-day repeated-dose administration. In addition, intranasal INDCA-NS did not affect the sense of smell, as observed in the buried food test. Intranasal NDCA-NS was found safe in rats with a Maximum Tolerated Dose (MTD) of more than 100 μg/rat/day and No-Observed Adverse Effect Level (NOAEL) of more than 100 μg/rat/day in male and female rats during acute and subacute intranasal toxicity study respectively.

Pharmacokinetics and 28-day repeated-dose toxicity of enniatin B after oral administration in mice.

Enniatins are emerging mycotoxins that contaminate foods. The present study investigated the oral pharmacokinetics and 28-day repeated-dose oral toxicity of enniatin B (ENNB) in CD1 (ICR) mice. In the pharmacokinetic study, male mice received a single oral or intravenous dose of ENNB [30mg/kg body weight (BW) and 1mg/kg BW, respectively]. After oral dosing, ENNB exhibited 139.9% bioavailability, a 5.1-h elimination half-life, 5.26% fecal excretion from 4 to 24h post-dose, and upregulation of Cyp7a1, Cyp2a12, Cyp2b10, and Cyp26a1 in the liver 2h post-dosing. In the 28-day toxicity study, ENNB was administered to male and female mice by oral gavage at 0, 7.5, 15, and 30mg/kg BW/day. Females (7.5 and 30mg/kg) showed dose-unrelated decreased food consumption without accompanying changes in clinical parameters. Males (30mg/kg) showed low red blood cell counts and high blood urea nitrogen levels and absolute kidney weights; however, other related parameters including the histopathology of systemic organs/tissues were unchanged. These results suggest that ENNB may not induce toxicity after 28 days of oral administration in mice, despite high absorption. The no-observed-adverse-effect level of ENNB after 28 days of repeated oral doses was 30mg/kg BW/day for both sexes of mice.

Neuroinflammation and hypersensitivity evidenced by the acute and 28-day repeated dose toxicity tests of ostrich oil in mice.

The ostrich oil (OO) has been topically used for decades to treat skin diseases. Its oral use has been encouraged through e-commerce advertising several health benefits to OO without scientific evidence on its safety or effectiveness. This study presents the chromatographic profile of a commercially available OO and its acute and 28-day repeated dose in vivo toxicological profiles. OO anti-inflammatory and antinociceptive effects were also investigated. Omega-9 (ω-9; oleic acid; 34.6%) and -6 (linoleic acid; 14.9%) were detected as OO main constituents. A high single dose of the OO (2g/kg of ω-9) demonstrated no or low acute toxicity. However, when orally treated with OO (30-300mg/kg of ω-9) for 28 consecutive days, mice exhibited altered locomotor and exploratory activities, hepatic damage, and increased hindpaw sensitivity accompanied by increased levels of cytokine and brain-derived neurotrophic factor in their spinal cords and brains. Lack of anti-inflammatory or antinociceptive activities was also evidenced in 15-day-OO treated mice. These results indicate that chronic consumption of OO induces hepatic injury, in addition to neuroinflammation and subsequent hypersensitivity and behavioural changes. Thus, there is no evidence to support OO use to treating illness in humans.

A 28-Day Repeated Dose Toxicity Evaluation of Creatinine Monohydrate and β-Hydroxy β-Methyl Butyrate Combination in Rodent Model

Substance abuse is a global concern, having a significant influence on society, healthcare, and the economy. Athletes are increasingly using substances to improve their performance or to cope with stress. Therefore, having a thorough awareness of the often misused substances, their effects, and potential toxicities is essential. One such chemical is the mixture of Creatinine Monohydrate (CM) and -Hydroxy Methyl Butyrate (HMB), which is widely used by athletes to increase strength and performance. According to studies, the combination can boost athletic performance, build muscle, and offer several health advantages. To determine the chemicals’ long-term impacts on human health, more research is required because their safety and toxicity are not well-established. Using TOPKAT(Computer-Aided Toxicity Prediction) and Discovery Studio Version 4.0, this study examined the toxicity and pharmacokinetic profiles of CM and HMB. The interaction of HMB and CM may significantly reduce cell viability compared to either drug alone. There were no negative effects seen in the animal models used for the acute oral toxicity study, but additional research is needed to confirm these results and determine the compounds’ long-term safety for human use. The study also performed cytotoxicity studies of individual substances and combinations on VERO and HEP-G2 cell lines and a 28-day repeated dose toxicity study in Wistar albino rats. The study results provide valuable information for healthcare professionals to understand the physiological complications of exposure to such drugs. Overall, the article emphasizes the importance of understanding the potential toxicity of chemical compounds, particularly those used as nutritional supplements.

Abstract CT249: A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors

Abstract Background: TOS-358 is an orally available, highly selective covalent inhibitor of PI3Kα capable of achieving a deep and durable inhibition of PI3K-AKT signaling with virtually no off-target effects. In patient-derived xenograft models, TOS-358 has shown anti-cancer activity in several different tumor types including breast, colorectal, gastric, head and neck, bladder, and esophageal cancer, and its’ activity was superior to non-covalent compounds targeting PI3Kα. Administered at the equivalent dose, TOS-358 has shown a good safety profile in 28-day repeat dose toxicity studies in rats and dogs. Methods: The TOS-358-001 study is a multicenter dose escalation (phase 1) and dose expansion (phase 1b) clinical study evaluating the safety, tolerability, and preliminary efficacy of TOS-358. Eligible subjects are adults with one of protocol-defined tumor types, known PIK3CA mutation or amplification, adequate organ function, ECOG performance status 0 or 1, available archived or fresh tumor tissue for PIK3CA mutation detection and no prior treatment with PI3K, AKT or mTOR inhibitors (except subjects with breast cancer). The phase 1 portion of the study follows a 3+3 dose escalation design and is intended to determine the minimum effective dose (MED) and the maximum tolerated dose (MTD) of TOS-358 when administered orally on a once daily (QD) or a twice daily (BID) schedule, and the recommended phase 2 dose (RP2D). Cohort expansions at the MTD and MED to support the RP2D selection are planned. The phase 1b portion of the study will enroll subjects with tumor PIK3CA mutations or amplifications in several tumor-specific cohorts (colorectal, gastric, non-small cell lung, breast, squamous cell carcinoma of the head and neck, urothelial and select gynecological cancers) to further assess the safety and tolerability of TOS-358 and to evaluate the initial efficacy of the investigational agent. Enrolment to the study commences in US with European sites planned to open later in 2023. Clinical trial information: NCT05683418. Citation Format: Marwan Fakih, Davendra P. Sohal, Navid Hafez, Jordan D. Berlin, Kimberly Hari, Ovid C. Trifan. A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT249.

Preclinical safety assessment of JNJ-10450232 (NTM-006), a structural analog of acetaminophen, that does not cause hepatotoxicity at supratherapeutic doses

JNJ-10450232 (NTM-006) is a new molecular entity that is structurally related to acetaminophen. A comprehensive non-clinical safety program was conducted to support first-in-human and clinical efficacy studies based on preclinical data suggesting that the compound has comparable or enhanced antinociceptive and antipyretic efficacy without causing hepatotoxicity at supratherapeutic doses. No hepatic toxicity was noted in a mouse model sensitive to acetaminophen hepatotoxicity or in rats, dogs, and non-human primates in 28-day repeat dose toxicity studies at and above doses/exposures at which acetaminophen is known to cause hepatotoxicity. In the 28-day toxicity studies, all treatment-related findings were monitorable and reversible. Methemoglobinemia, which was observed in dogs and to a lesser extent in rats, is also observed with acetaminophen. This finding is considered not relevant to humans due to species differences in metabolism. Thyroid hypertrophy and hyperplasia were also observed in dogs and were shown to be a consequence of a species-specific UGT induction also demonstrated with increased thyroid hormone metabolism. Indirect bilirubin elevation was observed in rats as a result of UGT1A1 Inhibition. JNJ-10450232 (NTM-006) had no toxicologically relevant findings in safety pharmacology or genotoxicity studies. Together, these data supported progressing into safety and efficacy studies in humans.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Repeated dose toxicity study to assess the safety and reproductive/developmental safety of 7-methyl-2H-1,5-Benzodioxepin-3(4H)-One (Calone®) and the read across approach to its biodegradation metabolite 7-methyl-2H-1,5-benzodioxepin-3-ol (Calol).

7-Methyl-2H-1,5-benzodioxepin-3(4H)-one (Calone®) is used in fragrances to impart a marine note. It is produced industrially at volumes requiring repeated dose and developmental/reproductive toxicology data (OECD TG 422) under European chemicals legislation (i.e., REACH). Additionally, Japanese chemicals legislation requires evaluation of Calone® biodegradability and identification of metabolites in an environmental biodegradation test. 7-Methyl-2H-1,5-benzodioxepin-3-ol (Calol) was the sole metabolite identified following biodegradation and a 28-day repeated dose toxicity study (OECD TG 407) would normally be required to support registration in Japan. The current paper presents results showing no adverse effects in the parental, reproductive, or developmental phases of an OECD TG 422 study following dietary administration of Calone® to rats at targeted doses of up to 1000mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was the highest administered dose of 791 and 922mg/kg/day for males and females, respectively. An in vitro metabolism study conducted with rat and human liver microsomes demonstrated that greater than 90% of Calone® was metabolically reduced into Calol, the same metabolite observed in the environmental biodegradation test. Accordingly, the results from the OECD TG 422 study with Calone® are directly applicable to Calol and it would be expected to have the same NOAEL.

Safety assessment of useing Ba Kich dried extract for 28 days

The “Ba kich” (Extractum Morinda ofcinalis) dried extract was evaluated for repeated dose toxicity at 28 days in rabbits. In a repeat dose toxicity test, rabbits were given oral solution of dry extract of Ba kich at the level of 0.2 g/kg and 1.0 g/kg for 28 days. Blood samples were taken on days 0, 14 and 28 to check biochemical and hematological parameters. Liver and kidney samples were obtained on day 28 for histopathology. The results of the 28-day repeated dose toxicity assessment showed that the dried Ba kich extract did not have a statistically significant effect on the biochemical and hematological indices, and did not cause damage to the histopathology of the kidney. However, dried Ba kich extract at both dose levels caused fatty degeneration and congestion in the liver of experimental rabbits.

Evaluation of acute toxicity, 28-day repeated dose toxicity, and genotoxicity of Moringa oleifera leaves infusion and powder

Ethnopharmacological relevanceMoringa oleifera Lam. leaves infusion and powder are widely used by population due the nutritional and medicinal potentials, however data regarding safety of use are still inconclusive, leading to prohibition of this plant in some countries. Aim of the studyThe present work investigated the nutritional and phytochemical composition, acute and 28-day repeated dose toxicity, and genotoxicity of M. oleifera leaves infusion and powder. Materials and methodsFor nutritional characterization of leaf powder, it was determined: humidity; mineral residue (ash); total lipid, protein, carbohydrate, and crude fiber contents; and total caloric value. Phytochemical composition was determined by high performance liquid chromatography (HPLC). The acute toxicity assay used Swiss female albino mice and oral administration in a single dose at 2000 and 5000 mg/kg of infusion or powder. The 28-day repeated dose toxicity assay employed female and male mice, with oral administration of infusion or powder at the doses 250, 500 and 1000 mg/kg. The animals were evaluated for body weight, water and feed consumption, biochemical and hematological parameters, and histology of the liver, spleen, and kidneys. In vivo genotoxicity and mutagenicity (2000 mg/kg) were evaluated by the comet assay and the micronucleus test, respectively. ResultsNutritional characterization confirmed that M. oleifera leaves are rich in proteins, carbohydrates, lipids, minerals, and fiber. HPLC indicated the presence of flavonoids and cinnamic derivatives as major polyphenols. Acute toxicity did not reveal alterations in weight gain and water and feed consumptions and no change in biochemical, hematological, and histological parameters. Behavior alterations was observed in the first 2 h after administration at 5000 mg/kg in both treatments. Infusion did not present toxicity when administered for 28 days. Conversely, the powder at 500 and 1000 mg/kg promoted liver and kidney damages observed through biochemical parameters and histopathology. Genotoxicity and mutagenicity were not detected at 2000 mg/kg. ConclusionsThe present study reveals that M. oleifera leaves are an important source of polyphenols and nutrients. Indiscriminate use of both infusion and crude leaf powder above 2000 mg/kg and powder at 500 and 1000 mg/kg are not recommended. Chronic toxicological studies and establishment of preparation protocols are suggested aiming to guarantee the safety in the use of M. oleifera leaves as nutraceutical by population.

Acute and 28-day repeated dose toxicity evaluations of cold pressed Pinus halepensis Mill. seed oil in mice and rats

Pinus halepensis Mill. seed (Pinaceae), popularly known as ‘Zgougou’, is widely consumed in the Mediterranean countries and used traditionally in the treatment of some diseases such as bronchitis, rheumatism, infection, and inflammation. The present study aimed to evaluate the oral safety of cold pressed oil of Pinus halepensis Mill. seeds (COPHS) by acute and 28-day repeated dose toxicities studies in Wistar mice and rats, respectively. In the acute toxicity study, oral administration of COPHS to mice did not provoke mortality or any toxic signs at doses up to 5000 mg/kg bw. After administration of COPHS at doses of 250, 500, and 1000 mg/kg bw/day for 28 days, no abnormal changes were observed in body weight, water intake, food consumption, organ weight, blood haematological, serum biochemistry parameters, and histology profile. Furthermore, there was no animal death or any symptom of toxicity in any group during sub-acute toxicity test period. Our findings demonstrate that COPHS is relatively non-toxic and has a large safety margin (>5000 mg/kg). The results of the present research provide basic reference data for food consumption and for future in vivo screening of biological and pharmacological properties of cold pressed oil of Pinus halepensis Mill. seeds.

28-day repeated dose toxicological evaluation of Coronil in Sprague Dawley rats: Behavioral, hematological, biochemical and histopathological assessments under GLP compliance

Ayurvedic medicines are widely employed globally for prophylaxis and treatment of a variety of diseases. Coronil is a tri-herbal medicine, constituted with the traditional herbs, Tinospora cordifolia, Withania somnifera and Ocimum sanctum, with known immunomodulatory activities. Based on its proven in-vitro activity and in-vivo efficacy, Coronil has been approved as a ‘Supporting Measure for COVID-19’ by the Ministry of AYUSH, Government of India. The current study was aimed to assess the non-clinical safety of Coronil in a 28-day repeated dose toxicity study along with a 14-day recovery period in Sprague Dawley rats. This toxicity study was conducted in accordance with OECD test guideline 407, under GLP-compliance. Specific-Pathogen-Free animals of either sex, housed in Individually-Ventilated-Cages were particularly used in the study. The tested Coronil dose levels were 0, 100, 300 and 1000 mg/kg/day, orally administered to 5 males and 5 female rats per test group. In the current study, no mortality was observed in any group and in addition, Coronil did not elicit any finding of toxicological relevance with respect to clinical signs, ocular effects, hematology, urinalysis and clinical chemistry parameters, as well as macro- or microscopical changes in any organs, when compared to the control group. Accordingly, the No-Observed-Adverse-Effect-Level (NOAEL) of Coronil was ascertained to be 1000 mg/kg/day, subsequent to its 28-day oral administration to male and female rats. The acceptable safety profile of Coronil paves the way further toxicity assessments in rodents for a longer duration as well as in higher animals, and towards its clinical investigation.

Early detection of urinary bladder carcinogens in rats by immunohistochemistry for γ-H2AX: a review from analyses of 100 chemicals.

In safety evaluations of chemicals, there is an urgent need to develop short-term methods to replace long-term carcinogenicity tests. We have reported that immunohistochemistry for γ-H2AX, a well-established biomarker of DNA damage, can detect bladder carcinogens at an early stage using histopathological specimens from 28-day repeated-dose oral toxicity studies in rats. Given the markedly low level of γ-H2AX formation in the bladder urothelium of untreated rats, an increase in γ-H2AX-positive cells following chemical exposure can be relatively easy to identify. Among the 100 compounds examined to date, bladder carcinogens can be detected with high sensitivity (33/39; 84.6%) and specificity (58/61; 95.1%). As expected, γ-H2AX formation levels tended to be high following exposure to genotoxic bladder carcinogens, whereas nongenotoxic bladder carcinogens also increased the number of γ-H2AX-positive cells, probably through secondary DNA damage associated with sustained proliferative stimulation. γ-H2AX formation in the bladder urothelium reflects species differences in susceptibility to bladder carcinogenesis between rats and mice and shows a clear dose-dependency associated with the intensity of tumor development as well as high reproducibility. Some of the bladder carcinogens that showed false-negative results in the evaluation of γ-H2AX alone could be detected by combined evaluation with immunostaining for bladder stem cell markers, including aldehyde dehydrogenase 1A1. This method may be useful for the early detection of bladder carcinogens, as it can be performed by simple addition of conventional immunostaining using formalin-fixed paraffin-embedded tissues from 28-day repeated-dose toxicity studies in rodents, which are commonly used in safety evaluations of chemical substances.

Long term toxicity study of a Siddha formulation Vetpalai thailam (VT) in Rats

Vetpalai thailam, a Siddha formulation is used topically as well as orally in the treatment of Psoriasis. Vetpalai thailam is prepared from the leaves of Wrightia tinctoria by insolation with coconut oil as the base. The adverse effect profile of VT has not been addressed and hence this study was taken up to assess the long term toxicity of VT in rats. A 90-day repeated dose toxicity study was performed by modifying the protocol described by OECD guideline 408 for testing chemicals. The study constituted of six groups consisting of six rats in each group. There was no mortality among the six study groups. No signs of toxicity and behavioural changes were observed. The histopathological examination of vital organs did not show any change in the architecture. Our study demonstrates that Vetpalai thailam does not cause any toxicity on long term use in rats.

Acute toxicity, 28-day repeated-dose toxicity and toxicokinetic study of timosaponin BII in beagle dogs

The safety evaluation of timosaponin BII (TBII) in beagle dogs with toxicokinetic study was performed. For the acute oral toxicity study, the minimum lethal dose (MLD) of TBII was more than 2000 mg/kg and suggested the characteristics of absorption saturation. For the 28-day repeated dose oral toxicity and toxicokinetic studies, there was no significant effect on all test parameters except for prolonged APTT in the 60 and 180 mg/kg groups, which recovered after withdrawal. The increase of drug exposure of 180 mg/kg group was not proportional to the increase of administration dose, showing the characteristics of absorption saturation.

Abstract P39: Pre-clinical evaluation of NEOS-223, an (S)-valine-thiazole derived peptidomimetic N-heterocycle, as an anticancer agent and P-glycoprotein inhibitor

Abstract The ability to control the proliferation and cell death by inhibiting specific target kinase offers the opportunity to apply targeted therapies in the treatment of cancer. It has been found that (S)-valine-thiazole-derived compounds such as NEOS-223 are effective inhibitors of one or more of these kinases. NEOS 223 was developed, synthesized, and tested in the NCI 60 human tumor cell-screening panel demonstrating inhibition of colon (-53%), melanoma (-41%), and breast cancers (-9%). Microsomal clearance was determined in mouse, rat, dog, and human, and analyzed by LC-MS/MS by percent of parent material. IC<sub>50</sub> values for CYP inhibition of >10 μM were calculated for 1A2, 2C19, and 3A4 with IC<sub>50</sub> values of 4.86, 4.31, and 7.84 μM for 2C9 and 2D6. Microsomal clearance was high in all species with clearance rates ranging from 69-136 mL/min/kg. Plasma protein binding was determined by Rapid Equilibrium Dialysis in mice, rats, dogs, and humans. High plasma protein binding (>70%) was observed across all species. Based on the NCI results several cell lines were assayed in an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) to determine cell viability in the presence of NEOS-223 resulting in <20% viability in colon, breast, melanoma, pancreatic and prostate human cancer cell lines at a 10 uM concentration. Maximum tolerated dose studies were conducted by both intraperitoneal and oral administration in mice. NEOS-223 delivered up to 80 mg/kg was well tolerated. Minimal or no toxicity was observed in acute and repeat dose animal studies. Pharmacokinetics of oral administration demonstrated adequate systemic exposure at therapeutic levels in mice, rats, and dogs. Preliminary in vivo mouse xenograft studies were performed on colon (COLO 205, HT-29 red FLUC), breast (MDA-MB-468), melanoma (M-14), pancreatic (PANC-1), and prostate (PC3) human cancer cells with significant tumor inhibition observed compared to positive control agent groups with twice daily dosing of NEOS-223. In addition, a five-day pilot oral toxicity study in rats with dose range-finding studies and a 28-day repeat dose toxicity study performed in both rats and dogs provided favorable results. NEOS-223 has demonstrated active in vitro activity along with a favorable safety profile. <i>in vivo efficacy resulted in inhibition of growth of multiple cell line. As a novel effective structure possibly targeting multiple kinases and transporters in one hybrid molecule, NEOS-223 may be a preferred monotherapy or combined therapy for multiple cancers. If upon further development, this drug is effective in humans, it would advance clinical practice and could improve current therapy significantly. Citation Format: Sumathi Chittamuru, Timothy M. Murphy, Sara A. Little, Andrew A. Taylor, Roseanne Wexler, Laxman Desai. Pre-clinical evaluation of NEOS-223, an (S)-valine-thiazole derived peptidomimetic N-heterocycle, as an anticancer agent and P-glycoprotein inhibitor [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P39.