RESUMO
Perillaldehyde, a natural monocyclic terpenoid found most abundantly in the herb perilla, has a long history of use as a flavouring ingredient to add spiciness and citrus taste to foods. Previously, it was judged to be safe by several international expert panels. To confirm the safety of flavourings placed on the European Union list of flavourings, perillaldehyde was selected by the European Food Safety Authority as a representative of a subgroup of alicyclic aldehyde flavouring substances to be evaluated for genotoxic potential. Perillaldehyde was tested in a bacterial reverse mutation assay, an in vitro micronucleus assay in human lymphocytes, an HPRT assay in mouse lymphoma cells, and a micronucleus/comet assay in Han Wistar rats. In contrast to previously published results, perillaldehyde induced mutation in Salmonella typhimurium strain TA98 in the absence of metabolic activation. The comet assay was negative for duodenum and weakly positive for liver but only at a hepatotoxic dose of perillaldehyde. All other genotoxicity assays were negative. These data do not provide an indication of any genotoxic potential for perillaldehyde, and they provide the primary basis for recent scientific opinions regarding perillaldehyde genotoxicity announced by several international organizations responsible for safety assessment of food additives and flavourings.
Assuntos
Dano ao DNA/efeitos dos fármacos , Fígado/patologia , Linfócitos/patologia , Linfoma/patologia , Monoterpenos/toxicidade , Animais , Células Cultivadas , Ensaio Cometa/métodos , Relação Dose-Resposta a Droga , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Linfócitos/efeitos dos fármacos , Linfoma/tratamento farmacológico , Linfoma/genética , Camundongos , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Mutação/genética , Ratos , Ratos Wistar , Salmonella typhimuriumRESUMO
As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined sodium arsenite, thioacetamide, and diethanolamine. Using the JaCVAM approved study protocol version 14.2, each chemical was tested in male rats up to maximum tolerated dose levels and DNA damage in the liver and stomach was assessed approximately 3h after the final administration by gavage. Histopathology assessments of liver and stomach sections from the same animals were also examined for evidence of cytotoxicity or necrosis. No evidence of DNA damage was observed in the stomach of animals treated with sodium arsenite at 7.5, 15, or 30 mg/kg/day. However, equivocal findings were found in the liver, where increases in DNA migration were observed in two independent experiments, but not in all treated animals and not at the same dose levels. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 19, 38, and 75 mg/kg/day, but not in the liver, despite evidence of marked hepatotoxicity following histopathology assessments. No evidence of DNA damage was observed in the stomach or liver of animals treated with diethanolamine at 175, 350, or 700 mg/kg/day.
Assuntos
Arsenitos/toxicidade , Ensaio Cometa/métodos , Etanolaminas/toxicidade , Compostos de Sódio/toxicidade , Tioacetamida/toxicidade , Animais , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Masculino , Dose Máxima Tolerável , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design.