Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT)

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a “Use of 3D Tissues in Genotoxicity Testing” working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines

DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed I day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by P-32-postlabeling and mutant frequency (MF) was determined using the lambda Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N-6-yl]aristolactam 1, 7-[deoxyadenosin-N-6-yl]-aristolactam 11 and 7-[deoxyguanosin-N-2-yl]-aristolactam 1) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10(8) nucleotides in liver and 95-4598 adducts/10(8) nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10(-6) in liver compared with the MFs of 78-1319 x 10(-6) that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T -> T:A transversion was the predominant mutation in AA-treated rats; whereas G:C -> A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney. (c) 2006 Elsevier B.V. All rights reserved

The effect of time after treatment, treatment schedule and animal age on the frequency of 6-thioguanine-resistant T-lymphocytes induced in Fischer 344 rats by N-ethyl-N-nitrosourea

The persistence of 6-thioguanine-resistant (TGr) T-lymphocytes was investigated in Fischer 344 rats treated with N-ethyl-N-nitrosourea (ENU) using two schedules. Male rats, aged 3 months, were given i.p. injections containing a total of 0, 50 or 100 mg ENU/kg either as a single treatment (single-dose group) or divided among 10 weekly treatments (split-dose group). At 1, 3, 5, 10, 20, 30 and 50 weeks after the single-dose treatment, and 10, 20, 30 and 50 weeks after beginning the split-dose regimen, animals were assayed for the frequency of TGr spleen lymphocytes. ENU produced significant dose- and time-dependent responses in the single- and the split-dose treatment groups. Although a few of the 50 mg/kg split-dose treatments were significantly higher than the comparative single-dose groups, the number of TGr lymphocytes produced by the two dosing regimens were generally similar. The frequency of TGr cells for control animals increased with the age of the animals. The mode of ENU administration did not greatly influence the percent cloning efficiency (%CE) of the non-selection cultures, although the %CE declined in animals over 10 months of age. To investigate the relationship between the frequency of TGr cells and the age of the animals at the time of ENU administration, additional rats aged 17 months were treated with a single dose of ENU and at 1, 5 and 10 weeks following exposure, the frequencies of TGr cells were determined from the isolated lymphocytes. No difference in mutagen sensitivity between rats treated at 3 months of age and those treated at 17 months of age was detected at the time points evaluated. The data demonstrate the persistence of ENU-induced TGr T-lymphocytes in the rat and suggest that the dose and possibly the treatment schedule, but not the age of the animal at the time of treatment, affect the response. © 1993