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Genotoxicity of flavor and fragrance materials was assessed in Turkey Egg Genotoxicity Assay (TEGA) using 32 P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks. Twenty materials having results in GADD45a-Gluc 'BlueScreen HC' genotoxicity assay, and standard in vitro and in vivo tests, were selected to evaluate the accuracy of TEGA. Quinoline (QUI) and 2-acetylaminofluorene (AAF) served as positive comparators. Two materials, p-tert-butyldihydrocinnamaldehyde (BDHCA) and methyl eugenol (MEU) produced DNA adducts. BDHCA, p-t-butyl-α-methylhydrocinnamic aldehyde (BMHCA), trans-2-hexenal (HEX) and maltol (MAL) produced DNA strand breaks. Fifteen other materials were negative in both assays. Based on reports of oxidative DNA damage induction by MAL and 4-hydroxy-2.5-dimethyl-3(2H) furanone (HDMF), modified comet assays were conducted. Positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Accordingly, MAL was judged to have equivocal genotoxicity in TEGA. HDMF was positive in modified comet assay, indicating an ability to produce oxidative DNA damage. TEGA showed modest concordance with results in regulatory in vitro assays. Findings in TEGA, with few exceptions, were concordant with the results of in vivo genotoxicity and carcinogenicity testing. Thus, TEGA is an attractive alternative model for the assessment of genotoxic potential of chemicals in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal's glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14 C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7- 14 C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14 C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32 P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal's gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidine-DNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal's gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal's gland may exist.

Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague-Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.

Propoxur (PPX) is a carbamate insecticide which induced urinary bladder cancer in Wistar rats when fed at 5000ppm in Altromin 1321 diet (1321). In the present investigation, PPX was studied for induction of several key events related to modes of action (MOA) of carcinogenicity in urinary bladders (UBs). Wistar rats were administered the compound for 28 days at 8000ppm in Provini Liba SA 3883 diet, which is similar to the 1321 diet. o-Anisidine HCl (AH) was used as a genotoxic UB carcinogenic comparator, and trisodium nitrilotriacetate (NTA) as an epigenetic UB carcinogen comparator. Along with the non-dosed control and three test substance groups (PPX, AH, NTA), four more groups were additionally fed 2% ammonium chloride (AC) in the diet to acidify the urine, since 1321 was reported to increase urinary pH. AC did acidify the urine, as expected, although the 3883 diet itself did not increase pH values above 8. In the alkaline comet assay, AH produced DNA single strand breaks (SSBs) in the UB urothelium (UBU) irrespective of AC administration, whereas PPX and NTA did not. In the nucleotide (32)P-postlabeling assay (NPL), AH produced DNA adducts irrespective of AC administration, whereas PPX and NTA did not. Routine (H&E) histopathology evaluation of the UBU did not reveal any hyperplasia or evidence of luminal microprecipitates or calculi in any of the groups. Assessment of UBU proliferation as measured by immunohistochemistry of proliferating cell nuclear antigen, revealed that NTA and NTA plus AC increased the replicating fraction (RF). Also AH plus AC, but not AH alone, increased the RF of UBU, whereas PPX groups were not significantly different from controls. Thus, the results reveal no evidence for DNA SSBs, binding, or alteration of DNA synthesis in the UBU by PPX, while demonstrating UBU DNA damage by AH and showing that NTA does not damage DNA, but causes increased UBU proliferation. The findings are in accord with a genotoxic MOA for AH, and an epigenetic MOA for NTA. The MOA of PPX does not involve genotoxicity and may be specific to the 1321 diet., (Copyright © 2015. Published by Elsevier GmbH.)

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The exposure-responses for several effects of limited exposures to diethylnitrosamine (DEN) in the livers of male Fischer 344 rats were measured and phenobarbital promotion was used to enhance expression of initiation of carcinogenesis. Five doses ranging from a cumulative total of 0.5 to 4 mmol DEN per kg body weight were given as weekly i.p. injections for 10 weeks. This was followed by 4 weeks recovery, after which the groups were maintained on either a basal diet or 0.05% phenobarbital (PB) to promote liver tumor development. All doses of DEN produced ethylation in liver DNA, which increased with dose. Indicative of toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was reduced in relationship to exposure up to a cumulative exposure of 3 mmol/kg. The two lower exposures to DEN produced no increase in cell proliferation, whereas higher exposures resulted in marked increases, approximately 4-fold between 1.0 and 2.0 mmol/kg cumulative. At the end of the recovery period (14 weeks), hepatocellular altered foci (HAF), which expressed the placental form of glutathione S-transferase, were induced by all exposures, with an increase of approximately 4-fold between the exposures of 1.0 and 2.0 mmol/kg being the greatest. In rats maintained on basal diet or PB for 24 weeks after exposure, HAF increased further and with exposures of 2.0 mmol/kg and above, all rats developed hepatocellular carcinomas. With 1.0 mmol/kg, no liver tumor occurred in 12 rats without promotion, whereas in those given PB, two adenomas and two carcinomas were present in 12 rats. At the lowest exposure of 0.5 mmol/ kg, no tumor occurred in rats on basal diet, although HAF increased approximately 7-fold. With PB promotion, only one adenoma developed in 12 rats and HAF increased another 2-fold. Thus, the findings document non-linearity for some of the effects of DEN and a near no-effect level for initiation of promotable liver neoplasms at the lowest exposure in spite of a substantial induction of HAF.