Alternatives to animal testing are being used to assess the safety of raw ingredients during the process of developing cosmetics. However, since cosmetic products are composed of a variety of raw ingredients, the safety of the product itself should also be tested. In this study we attempted to evaluate the skin irritation potential of skin lotions by modifying existing alternative test methods. To evaluate the skin irritation potential of commercial skin lotions in the form of an objective indicator, we calculated their Irritation Score (IS) based on the results of searches using keywords related to skin irritation in review statements posted on Japanese cosmetics and beauty websites. We then modified the reconstructed human epidermis (RhE) test method to evaluate irritation by commercial skin lotions. The results showed that exposure to skin lotions with higher ISs tended to result in lower cell viability, and that exposure to many of the lotions with lower ISs resulted in higher cell viability. Next, we tried using an in vitro short-term exposure (STE) test method to assess the skin irritation potential of skin lotions. By changing the test-substance exposure concentrations and exposure times in the STE test method, we were able to obtain results that correlated with those obtained by the modified RhE test method. In conclusion, both alternative methods were helpful for assessing the possibility of developing skin irritation of skin lotions. They may also be useful for screening formulations being developed and as means of evaluation before proceeding to human patch tests.

For the non-clinical safety evaluation of pharmaceuticals for new drug applications (NDA), various toxicity studies must be conducted at each stage, from clinical trials to NDA. For topically applied drugs, the level of exposure at the administration site is high because the drug is administered directly to the administration site. However, because systemic exposure to ophthalmic drugs is generally lower than that of systemic drugs, systemic effects may not be adequately assessed. The bone marrow and liver are generally evaluated after systemic administration in in vivo genotoxicity tests, and local genotoxicity studies are conducted on a case-by-case basis. Therefore, we surveyed packages of genotoxicity and carcinogenicity tests for ophthalmic drugs approved in Japan from 2004 to 2021 to assist in the decision of test packages for the development of ophthalmic drugs. There were no major differences in genotoxicity test packages compared to systemic drugs; however, an unscheduled DNA synthesis test using the cornea after ocular instillation was conducted in some products as a test specific to ophthalmic drugs. In the development of ophthalmic drugs, if a positive result is found in an in vitro genotoxicity test, the safety margin between the positive concentration and the clinically applicable concentration (eye drop concentration) is required for safety assessment. If the safety margin cannot be ensured, additional tests may support safety assessment.

1,2-Dichloroethane, a priority assessment chemical substance under the Japan Chemical Substances Control Law (CSCL), required a detailed human health hazard assessment under Assessment II. We evaluated its general, reproductive, and developmental toxicities, genotoxicity, and carcinogenicity, based on the hazard information provided by domestic and international risk assessment organizations, and the hazard assessment values (HAVs) for oral and inhalation exposure were proposed. For oral exposure, a 78-week gavage carcinogenicity study (US NCI, 1978) with incidence data of hemangiosarcoma in male rats was selected as a significant toxicological endpoint and the lower confidence limit of benchmark dose (BMD) at 10% benchmark response (BMDL₁₀) of 9.3 mg/kg/day was obtained as a point of departure (POD). A slope factor of 1.07 × 10⁻² (mg/kg/day)⁻¹ from which a carcinogenic 10⁻⁵ risk of 0.93 µg/kg/day was derived as an oral HAV. For inhalation exposure, a 104-week inhalation exposure carcinogenicity study (Nagano et al., 2006) with a BMDL₁₀ of 11.5 ppm based on the incidence data of mammary gland tumors (adenocarcinoma + adenoma + fibroadenoma, combined) in female rats was obtained, and the human equivalent BMDL₁₀ of 15.7 mg/m³ was calculated. Therefore, a unit risk of 6.40 × 10⁻⁶ (µg/m³)⁻¹ from which a carcinogenic 10⁻⁵ risk of 1.6 µg/m³ (0.00039 ppm) was derived as an inhalation HAV.

Aside from the COVID-19 pandemic, the obesity and diabetes pandemics have threatened global health. Patients with diabetes are more likely to experience serious complications from COVID-19; thus, preventing obesity-associated diabetes is of paramount important. Furthermore, the development of a method to prevent diabetes and elucidation of its pathology is a currently urgent issue. We previously reported that thiamine plays a key role in suppressing abnormal glycolipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of obesity-associated diabetes. However, whether thiamine affects only OLETF rats or other animal models including a type 2 diabetes model with a different pathology requires elucidation. In this study, leptin-receptor deficiency mice were used as a model of type 2 diabetes with a different pathology to evaluate the efficacy of thiamine. The mice had free access to water containing 0.2% thiamine for 9 weeks, and the results showed that food and water consumption decreased in db/db-homo mice. Urine output, body weight gains and blood glucose levels decreased in mice that received thiamine. There were 5 mice and 1 mouse with a fasting glucose level of ≥ 300 mg/dL in the db/db-homo control group (n = 10) and db/db-homo thiamine group (n = 10), respectively, suggesting that thiamine intake may suppress an increase in blood glucose levels. The results of the present study suggest that demand for thiamine may exceed the normal range in in vivo mouse models of diabetes and continuous thiamine intake affects diabetes onset and progression.

2-Butylbenzo[d]isothiazol-3(2H)-one (BBIT, CAS No. 4299-07-4) is widely used as an industrial antiseptic and antifungal agent. To investigate its toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral toxicological study of BBIT was conducted in Crl:CD (SD) rats at doses of 0 (vehicle control, corn oil), 30, 90, or 270 mg/kg/day. There was no mortality or abnormal clinical signs related to treatment in any group. Slightly decreased body weight and food consumption were observed in the 270 mg/kg group in females. Increased urine volume and kidney weight, increased liver weight, and thickening of the forestomach mucosa in autopsy were observed in both sexes in the 270 mg/kg group. Histopathological examination revealed that hyperplasia of the squamous epithelium of the forestomach with parakeratosis and/or hyperkeratosis was observed in both sexes in all the BBIT-treated groups. Moreover, centrilobular hypertrophy of hepatocytes was observed in both sexes of the 270 mg/kg group. Similarly, increased depositions of eosinophilic bodies and/or hyaline droplets in the proximal tubules of the kidney were observed among the male in the 270 mg/kg group. Based on the forestomach changes, NOAEL was judged to be less than 30 mg/kg/day in both sexes under this study’s conditions.

2-(2H-benzotriazol-2-yl)-6-dodecyl-4-methylphenol (BTMLP, CAS No. 125304-04-3) is widely used as a liquid ultraviolet absorber that prevents deterioration of synthetic resins and so on. To investigate its toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral toxicological study of BTMLP was conducted in Crl:CD (SD) rats at doses of 0 (vehicle control, corn oil), 100, 300, and 1000 mg/kg/day. There was no observed mortality or abnormal clinical signs related to the treatment of any group. Body weight and food consumption were not affected by BTMLP treatment. In males, significant prolongations of prothrombin time and activated partial thrombin time were observed in the BTMLP-treated groups. Histopathological examination revealed a slight increase of the eosinophilic bodies and hyaline droplets in the renal cortical tubules in the 1000 mg/kg group in males. As mentioned above, the toxic effect of the BTMLP was noted in the blood coagulation system and kidneys only in males. Based on these findings, the NOAEL was judged to be less than 100 mg/kg/day in males and 1000 mg/kg/day in females under this study’s condition.

To investigate the effects of Chlorella alga on gut microbiota dysbiosis in type-2 diabetes (T2D). The stress perception of patients was also investigated. Chlorella (3 g/day) was administered to patients with T2D (n = 10) for a period of 30 days. Gut microbiota composition was analysed by 16S rDNA gene sequencing, and stress perception was evaluated using the perceived stress scale (PSS). A total of 13 phyla, 89 families, and 185 genera were identified from all faecal samples of patients with T2D, and Firmicutes and Bacteroidetes were the most dominant phyla among all samples. Chlorella decreased Bacteroidetes and increased Firmicutes. The proportions of the Akkermansia, Coprococcus, Dorea, Lachnospira, Phascolarctobacterium, and Ruminococcus generas increased, whereas the proportion of Paraprevotella, Prevotella, Klebsiella, and Sutterella decreased in the faeces of patients with T2D after Chlorella intake. Chlorella induced a significant reduction in perceived stress in patients with T2D, and better PSS scores negatively correlated with an increase in Akkermansia, Coprococcus, Dorea, Lachnospira, Phascolarctobacterium, and Ruminococcus and positively correlated with a decrease in Paraprevotella, Prevotella, Klebsiella, and Sutterella. Altogether, these results show the ability of Chlorella to positively modulate gut dysbiosis, leading to reduced stress perception in patients with T2D. Our findings contribute to the globally increasing search for new preventive and therapeutic strategies aimed at restoring the balance of the intestinal ecosystem.

The long-term effects of Chlorella doses on the inflammatory status and quality of life (QoL) of individuals with type-2 diabetes (T2D), and prediabetes (pre-T2D), and of nondiabetic controls were investigated. Chlorella was administered for 12 months; 1.6 g/day for the first six months and 3 g/day for the following six months. The inflammatory profile was studied by quantification of cytokines, adipokines and incretins. QoL was evaluated using the Short Form-36 health survey questionnaire (SF-36). Evaluations were performed at baseline, 6 (T6) and 12 (T12) months after initiating Chlorella intake. At baseline, QoL was more deeply impacted in T2D, a similar proinflammatory profile was observed in T2D and pre-T2D. In both, at T6 and T12, Chlorella modulated the altered levels of adipocytokines and incretins towards healthy values, and significantly improved QoL. Moderate correlations between the modulation by the alga and enhancement in QoL were observed only in the T2D group. In the nondiabetic control group, Chlorella improved QoL vitality and mental health scores. No differences were found between the two doses. Our results illustrate Chlorella adaptogen activity on inflammatory pathways and suggest its promising use as a complementary alternative in treating diseases related to insulin resistance in a wide range of chronic low-grade systemic inflammation-related diseases. Moreover, Chlorella increased QoL in all groups, the ultimate goal of all healthy interventions. Altogether, our findings suggest that one core mechanism involved in the homeostatic response produced by Chlorella is related to its rich content of carotenoids, operating mainly through inhibition of the NF-κB signalling pathway.

Multiwalled carbon nanotubes (MWCNT) are fiber-shaped nanomaterials that have a potential risk for cancer due to properties that are similar to asbestos. One type of nanotube called MWCNT-7 was categorized in Group 2B as possibly carcinogenic to humans by the International Agency for Research on Cancer. MWCNT-N, which is similar to MWCNT-7, is carcinogenic to the lung and pleura when administered to rats via the respiratory tract using intra-tracheal intra‐pulmonary spraying. Macrophages have an important role in the MWCNT induced pulmonary carcinogenicity. In this study, rat primary alveolar macrophages were employed to examine possible mechanism of carcinogenic effects of the MWCNT-N. MWCNT-N was fractionated into flow-through and retained fraction by passing it through a sieve with a pore size of 25 μm. Microarray analysis showed up-regulation of various cytokines in macrophages exposed to MWCNT-N. MWCNT-N strongly induced the expression of cytokines such as interleukin-6 (IL-6) in macrophages. In contrast, few cytokines were prominently down-regulated in macrophages treated with MWCNT-N. The sieve fractions did not have a significant effect on mRNA expression of cytokines in macrophages. These results provide useful information for understanding MWCNT-induced carcinogenicity via cytokine expression by macrophages.

DNA damage may be induced by both intrinsic and extrinsic factors, the latter involving chemical exposure at workplaces. Upon DNA damage induction, checkpoint kinases such as ATM and ATR, phosphorylate serine 139 of the histone H2AX generating γH2AX, to initiate the damage response pathway. This allows antibodies that act against post-translational modifications, such as γH2AX, to be used for detecting genotoxicity. The aromatic amine 4,4'-methylenebis(2-chloroaniline) (MOCA), which is utilized in industry to produce polyurethane resins, exhibits genotoxicity and induces cancers in animals, including rodents and humans. DNA damage, due to MOCA-induced toxicity during its metabolism, is believed to be first step towards carcinogenesis. However, we failed to detect γH2AX induction by MOCA in cellular systems via western blotting, even when electrophoresis-based methods clearly indicated that physical DNA damage had been induced. In the present study, we utilized RNA sequencing of MOCA-treated cells and controls to elucidate factors underlying the discrepancies observed between these two analytical systems. Our results indicated that H2AX mRNA expression was significantly downregulated; this finding partially clarified the inefficient induction of γH2AX. Although downregulation of γH2AX in the presence of MOCA witnessed in this study was quite unexpected, we believe that this finding prompts researchers to be cautious when screening for genotoxicity using γH2AX only, due to the possibility of genotoxicity being overlooked in some cases. Thus, it appears that a combined approach may be more suitable for detecting genotoxicity.

Methylphenidate (MPH) is used as a first-line treatment for attention-deficit/hyperactivity disorder (ADHD). Because the onset of ADHD appears in early childhood and the incidence number is increasing, more patients could become adults with long-term use of MPH. In addition, few men would discontinue the medication during a fertile period. Recently, environmental factors such as diet and drug abuse have been reported to produce changes in the sperm epigenome and affect the health of the next generation. However, the effects of long-term administration of a psychostimulant such as MPH on the next generation is unknown. In this study, we examined the effects of paternal administration of MPH on the growth, behavior, and gene expression in offspring using a mouse model. Sires were subcutaneously administered MPH for 21 days and mated with naive dams. Upon reaching 6–7 weeks of age, offspring were subjected to spontaneous locomotor, elevated plus-maze, and passive avoidance tests. Additionally, RNA-seq and RT-qPCR were performed on the striatum. Paternal MPH exposure induced increased atomoxetine-sensitive impulsivity and decreased long-term memory function in the offspring. Enrichment analysis following RNA-seq revealed significant enrichment of terms involved in the nervous system. Gene expression levels of Snap25, Syt1, Drd2, Maoa, and Comt, which are associated with ADHD pathology, are altered in the striatum. These results suggest that continuous administration of MPH to male mice induces ADHD-like behavior and changes in the expression of genes involved in the nervous system in the brain of the next generation.

TRPM8, non-selective cation channel of the transient receptor potential (TRP) superfamily, required for the transduction of moderate cold temperatures, regulates proliferation of epidermal cells in cyclin-dependent kinase inhibitor p21/Cip1-dependent manner. Given that downregulation of TRPM8 decreases p21/Cip1 level, increasing risk for carcinogenesis, and other TRP family is regulated by nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma, we examined whether TRPM8 expression was regulated by PPAR gamma. Knockdown assay and inhibition of PPAR gamma revealed that PPAR gamma negatively regulates the expression of TRPM8 in normal epidermal cells but positively regulates that in squamous carcinoma cells. Later restoration of decreased TRPM8 level in PPAR gamma antagonist-treated squamous carcinoma cells was attributed to feed-back loop regulation between TRPM8 and PPAR gamma using TRPM8 knockdown assay. Consisting with this finding, p21/Cip1 decrease by TRPM8 blocker, N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin- 2-yl) tetrahydropyrazine-1 (2H)-carbox-amide (BCTC), was restored by additional BCTC treatment in squamous carcinoma cells.