Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry.

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCETM.

The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells.

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

Phototoxicity Evaluation of Pharmaceutical Substances with a Reactive Oxygen Species Assay Using Ultraviolet A.

With ultraviolet and visible light exposure, some pharmaceutical substances applied systemically or topically may cause phototoxic skin irritation. The major factor in phototoxicity is the generation of reactive oxygen species (ROS) such as singlet oxygen and superoxide anion that cause oxidative damage to DNA, lipids and proteins. Thus, measuring the generation of ROS can predict the phototoxic potential of a given substance indirectly. For this reason, a standard ROS assay (ROS assay) was developed and validated and provides an alternative method for phototoxicity evaluation. However, negative substances are over-predicted by the assay. Except for ultraviolet A (UVA), other UV ranges are not a major factor in causing phototoxicity and may lead to incorrect labeling of some non-phototoxic substances as being phototoxic in the ROS assay when using a solar simulator. A UVA stimulator is also widely used to evaluate phototoxicity in various test substances. Consequently, we identified the applicability of a UVA simulator to the ROS assay for photoreactivity. In this study, we tested 60 pharmaceutical substances including 50 phototoxins and 10 non-phototoxins to predict their phototoxic potential via the ROS assay with a UVA simulator. Following the ROS protocol, all test substances were dissolved in dimethyl sulfoxide or sodium phosphate buffer. The final concentration of the test solutions in the reaction mixture was 20 to 200 µM. The exposure was with 2.0~2.2 mW/cm2 irradiance and optimization for a relevant dose of UVA was performed. The generation of ROS was compared before and after UVA exposure and was measured by a microplate spectrophotometer. Sensitivity and specificity values were 85.7% and 100.0% respectively, and the accuracy was 88.1%. From this analysis, the ROS assay with a UVA simulator is suitable for testing the photoreactivity and estimating the phototoxic potential of various test pharmaceutical substances.

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.

Determination of hexamethylenetetramine in foods by high-performance liquid chromatography (HPLC).

A simple and rapid method was developed and validated for the determination of hexamethylenetetramine (HMT) in foods. Samples were homogenised and extracted with methanol, followed by centrifugation. The resulting solution was filtered and injected into the high-performance liquid chromatograph (HPLC). HMT was separated using a Zorbax SCX-300 column coupled to a photodiode array detector. The calibration curve was linear in the range of 1.0-100 µg ml(-1), with good correlation coefficients (r(2) = 0.9992). The recoveries of HMT from foods spiked at levels of 10, 50 and 100 mg kg(-1) ranged from 91.6% to 103.8%, with relative standard deviations (RSDs) between 0.9% and 5.3%. The limit of detection and the limit of quantification of HMT were 0.3 and 1.0 mg kg(-1) based on three food matrixes (provolone cheese, glass noodle and tofu snack), respectively. Uncertainty associated with accuracy contributed mostly to the expanded uncertainty. No detectable levels of HMT were found in any of the samples retailed in Korea. The method was successful in determining HMT in foods.