28-day Repeated-dose Toxicity Research Articles

Evaluation of acute and 28-day repeated dose toxicity of Tolypocladium sinense soft capsule in Sprague-Dawley rats

Tolypocladium sinense is a new asexual strain isolated from natural Cordyceps sinensis. The mycelium produced by its fermentation culture has similar chemical components and pharmacological effects to C. sinensis. T. sinense soft capsule is primarily prepared from T. sinense mycelium, which is mainly used for the treatment of body damage induced by low-dose ionizing radiation. However, its potential toxicity remains unclear. This study was designed to assess the toxicological characteristics of T. sinense soft capsules through acute and 28-day repeated dose toxicity studies. In the acute toxicity study, no toxic symptoms or mortality were observed in rats following a single oral administration of 10 000 mg/kg of T. sinense soft capsules. The maximum tolerated dose for a single oral dose of T. sinense soft capsules in rats was over 10 000 mg/kg. During the repeated dose toxicity test, oral administration of 90, 360, and 1440 mg/kg/day of T. sinense soft capsules for 28 consecutive days did not lead to significant toxic effects in rats. The no observed adverse effect level in rats surpassed 1440 mg/kg/day. These results provide preliminary evidence that T. sinense soft capsules are relatively safe.

Safety assessment of (S)-Equol: Subchronic toxicity study in Sprague Dawley Rats

(S)-Equol is a chemically synthesized nutraceutical compound and its consumption provides several health benefits for humans. The new nutraceutical, enantiopure (S)-Equol was studied for acute and sub-chronic toxicity in Sprague Dawley Rats. The oral acute toxicity study showed that (S)-Equol is safe > 2000–5000 mg/kg body weight and it classified into GHS category 5/Unclassified. The repeated dose administration of (S)-Equol at dose levels of 20, 60, and 160 mg/kg body weight for 14 days and 250, 500, and 1000 mg/kg body weight for 90-consecitve days. The 14 days repeated-dose toxicity study showed no adverse effects in Sprague Dawley rats. The 90-day repeated dose toxicity study showed a reduction in body weight gain than that of control group. No treatment-related contrary effects were perceived on haematology, clinical chemistry, coagulation, urine parameters analysed, organ weights (absolute and relative), neurological and ophthalmological examination. No treatment-related abnormal gross pathological findings were obtained from gross necropsy. However, as a treatment-related effect, a significant decrease in cholesterol levels for 14 and 90 days of repeated dose administration was observed, which is considered as a pharmacological class effect of the (S)-Equol. In comparison to the corresponding vehicle control group, the high dose treatment group for both sexes showed no treatment-related histopathological abnormalities. For female rats, the no-observed-adverse-effect-level (NOAEL) was 250 mg/kg/day and for male rats, the lowest-observed-adverse-effect level (LOAEL) was 250 mg/kg/day.

Safety Evaluation and antioxidant potential of new probiotic strain Bacillus subtilis (NMCC-path-14) in Balb/c mice by sub-acute repeated dose toxicity

Probiotics have recently gained significant interest for their possible therapeutic effects in treating numerous health conditions. Probiotics containing Bacillus subtilis have been shown to have several health benefits, most notably in preventing diarrhea and gastrointestinal problems. A novel probiotic strain, Bacillus subtilis (NMCC-path-14), isolated from the rumen of a Nilli Ravi Buffalo, was evaluated for 28-day repeated dose toxicity in Balb/c mice. The NMCC-path-14 in low dose (1 × 108 CFU/ml) and high dose (1 × 1010 CFU/ml) was administered to the mice through gavage regularly. After 28 days of treatment, it was discovered that the no-observed-adverse-effect level (NOAEL) for NMCC-path-14 wasgreater than 1 × 1010 CFU/animal/day. This study also revealed no treatment-related changes in clinical parameters, body weight, gross pathology, or histology. Food consumption, hemoglobin, hematocrit, red blood cell counts, and colon length increased, while total/differential leukocyte count and platelets remained unchanged. The administration of NMCC-path-14 also resulted in decreased bilirubin and creatinine levels. Furthermore, NMCC-path-14 also displayed a promising antioxidant potential by increasing the antioxidant enzymes (GST, GSH, and CAT) and decreasing oxidant enzyme (MDA and NO) levels in vital organs like the liver, kidneys, spleen, and colon. TheNMCC-path-14also decreased the pathogenic bacterial population while increasing the beneficial population. Given the lack of adverse effects observed after NMCC-path-14 treatment, this strain is safe and must be considered as a potential probiotic in humans.

Re-evaluation of the reduced heart weights in male rats in a 28-day oral repeated-dose toxicity study of tetramethylammonium hydroxide

We recently conducted a detailed hazard assessment of tetramethylammonium hydroxide (TMAH), a priority chemical substance under the Japan Chemical Substances Control Law. During this assessment, there was debate regarding the reduced heart weight observed in the treated male groups in the 28-day rat oral repeated-dose toxicity study. This finding was not observed in females in this study and in both sexes of oral toxicity studies for tetramethylammonium chloride (TMAC) or tetramethylammonium hydrogen phthalate (TMAHP). Unpublished individual data from the oral TMAH developmental and reproductive toxicity (DART) screening study were also obtained; no effect on heart weight was observed. In addition, background data on rat heart weight from six 28-day oral toxicity studies conducted in the same facility, year, strain, age, and breeder as the TMAH study were obtained from the Japan Existing Chemical Substances Database (JECDB). These investigations suggest that the statistically significant lower heart weight in the treated males in the 28-day toxicity study is likely caused by an incidental skewing of individuals with heavier heart weights toward control male groups and is not due to TMAH treatment. Thus, it is worthwhile to include as much relevant data as possible to confirm or refute unexpected findings in toxicity studies.

JWH-182: a safe and effective synthetic cannabinoid for chemotherapy-induced neuropathic pain in preclinical models

Chemotherapy-induced neuropathic pain (CINP), a condition with unmet treatment needs, affects over half of cancer patients treated with chemotherapeutics. Researchers have recently focused on the endocannabinoid system because of its critical role in regulating our bodies' most important functions, including pain. We used in vitro and in vivo methods to determine the toxicity profile of a synthetic cannabinoid, JWH-182, and whether it could be potentially effective for CINP alleviation. In vitro, we evaluated JWH-182 general toxicity, measuring fibroblast viability treated with various concentrations of compound, and its neuroprotection on dorsal root ganglion neurons treated with paclitaxel. In vivo, we performed an evaluation of acute and 28-day repeated dose toxicity in mice, with monitoring of health status and a complete histopathological examination. Finally, we evaluated the efficacy of JWH-182 on a CINP model in mice using specific pain assessment tests. JWH-182 has an acceptable toxicity profile, in both, in vitro and in vivo studies and it was able to significantly reduce pain perception in a CINP model in mice. However, the translation of these results to the clinic needs further investigation.

28-day repeated-dose toxicity of orally administered Jinmao Jiedu granule in Sprague-Dawley rats

Jinmao Jiedu granule is a Chinese medicine preparation consisting of Actinidia valvata Dunn, Salvia chinensis Benth, Iphigenia indica Kunth, and chicken gizzard. For many years, it has been employed in adjuvant therapy for cancer, especially liver cancer. However, the potential toxicity of the granule has not been reported. The present study aimed to assess the repeated-dose toxicity of orally administered Jinmao Jiedu granules for Sprague-Dawley (SD) rats. SD rats were orally administered Jinmao Jiedu granules at doses of 2.85, 5.70, and 11.40 g/kg in a 28-day subchronic toxicity study. No adverse clinical signs associated with treatment were noted throughout the experiment. There were no treatment-related toxicity alterations in body weight, hematology, clinical biochemistry, urinalysis, necropsy, and histopathology in rats compared with the control group. The No Observed Adverse Effect Level (NOAEL) of the Jinmao Jiedu granule was higher than 11.40 g/kg/day in rats.

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 of oil from Schizochytrium sp. (strain CABIO-A-2) for use in infant and follow-on formula as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of Schizochytrium sp. (strain CABIO-A-2) oil as a novel food (NF) pursuant to Regulation (EU) 2015/2283. S. sp. is a single-cell microalga. The NF is a mixture of triglycerides in which docosahexaenoic acid (DHA) represents 38%-44% of fatty acids. The applicant proposed to use the NF in infant formulae (IF) and follow-on formulae (FOF). The use levels proposed by the applicant were derived from Regulation (EU) 2016/127, which states the mandatory addition of DHA to IF and FOF at the level of 20-50 mg/100 kcal. The evidence provided demonstrated that the strain S. sp. CABIO-A-2 is phylogenetically closely related to the strain S. sp. ATCC 20888. The assessment of some already authorised S. sp. oils in the Union list were also based on similarities with the strain ATCC 20888. The applicant provided a 90-day repeated dose toxicity study in rats with the NF. No adverse effects were observed up to the highest dose tested, i.e. 10.2 g/kg body weight (bw) per day. Taking into account the toxicity studies performed with the NF and with DHA-oils derived from strains belonging to the genus Schizochytrium, its phylogenetical profile, the production process, the composition of the NF and the absence of marine biotoxins and viable cells in the NF, the Panel considers that there are no concerns with regard to the toxicity of the NF. The Panel concludes that the NF is safe under the proposed conditions of use.

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.