Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system

Clive, D.; Johnson, K.; Spector, J.F.S.; Batson, Amie; Brown, Martha McClellan

doi:10.1016/0027-5107(79)90195-7

Cited by 547 publications

(156 citation statements)

References 22 publications

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“…In mouse lymphoma L5178Y cells, although a slightly to significantly positive response has been reported at the TK+'-locus (12,30), it should be pointed out that the cell line is routinely precleansed with a medium that contains methotrexate. If the reported results in the mouse lymphoma assay are true, the mouse lymphoma assay data base becomes questionable because the tests would have been conducted in a cell line pre-treated with a "mutagen."…”

Section: Discussionmentioning

confidence: 99%

Methotrexate: Assessment of In Vivo Clastogenicity and Carcinogenicity

Hall¹,

Tnham²,

Manandhar³

et al. 1988

Toxicol Pathol

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ABSTRAC~The genotoxic and oncogenic potentials of methotrexate were studied in Sprague-Dawley rats. The rats received 0.1, 0.2, or 0.4 mg/kg of methotrexate as dietary admixtures on a 5 days on, 9 days 0% regimen for 23 months. In the females of the high-dose group, there was a significant increase in mortality starting at 18 months. Significant increases in the number of rats with focal pulmonary interstitial fibrosis were seen in both sexes at the high-dose level. At the mid-and high-dose levels of both sexes, there was a significantly increased number of rats with myeloid and erythroid bone marrow hypoplasia. There was no evidence of either early onset or increased incidence ofany tumor type in the treatment groups. Therefore, it is concluded that methotrexate does not have oncogenic potential. Also, at terminal sacrifice, bone marrow cells were harvested from selected animals on the last day of the 5-day dosing cycle and cytogenetic evaluation was performed. No significant increase in chromosomal aberrations was seen in any dose group relative to the control group. This observation further substantiates the absence of oncogenic potential due to methotrexate in rats. INTRODUC~IONMethotrexate is an antimetabolite which acts as a folic acid antagonist (1 1). It is one of the few drugs whose mechanism of action has been extensively studied and well characterized. Methotrexate, also known as amethopterin, is a potent inhibitor of dihydrofolate reductase which converts dihydrofolate to tetrahydrofolate. Tetrahydrofolate and subsequent folic acid derivatives serve as cofactors in the transfer of single carbon atoms in the synthesis of purines and thymidylic acid required for DNA synthesis.Methotrexate has been recognized for many years as one ofthe most effective chemotherapeutic agents for certain leukemias, choriocarcinoma and Burkitt's lymphoma as well as other neoplastic diseases (22). More recently, it has been successfully used for the treatment of severe recalcitrant psoriasis (3, 10) and rheumatoid arthritis (5) for durations longer than normally used in patients with cancer. In order to determine the oncogenic potential or the genetic liability of methotrexate, a 2-year lifetime bioassay in rats was performed. This paper describes the results of the carcinogenicity study as well as chromosomal analysis from rats in the lifetime bioassay. Furthermore, we have reviewed the Iiterature on the carcinogenic, co-carcinogenic and mutagenic potentials of methotrexate. Based on all these studies, an overall safety of methotrexate has been assessed. METHODS ClieniicalMethotrexateO,' N-[4-[[(2,4-diamino-6-pteridinyl)methylJmethylamino]benzoyIJ-l-glutamic acid (Fig. 1) is a bright yellow-orange, odorless, crystalline powder with a molecular weight of 454.5. AnimalsSprague-Dawley rats were obtained from Charles River Breeding Laboratories. The rats weighing approximately 75 g at the start of the study were assigned to different groups using a table ofrandorn numbers. All animals were individually identified by a system ...

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Section: Discussionmentioning

confidence: 99%

Methotrexate: Assessment of In Vivo Clastogenicity and Carcinogenicity

Hall¹,

Tnham²,

Manandhar³

et al. 1988

Toxicol Pathol

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“…Sunset Yellow FCF was tested for its mutagenic potential in the L5178Y tk +/-mouse lymphoma cell forward mutation assay (McGregor et al 1988) using a non-standard procedure (according to Clive and Spector, 1975;Clive et al, 1979). Cultures were exposed to the chemicals for 4 hours, then cultured for 2 days before plating in soft agar with or without the selective agent trifluorothymidine (TFT).…”

Section: In Vitro Studies In Mammalian Cellsmentioning

confidence: 99%

Statement on Allura Red AC and other sulphonated mono azo dyes authorised as food and feed additives

2013

EFSA Journal

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The ANS Panel has been asked by EFSA to assess the new scientific information that has become available since the adoption of the opinion on the re-evaluation of the food colouring agent Allura Red AC in 2009, in particular the positive findings from an in vivo comet assay in mice. The findings from this study have been interpreted in conjunction with all the available relevant data from genotoxicity testing, metabolism and carcinogenicity, and in consideration of possible species differences between mouse and rat. These new data have been considered in the context of the overall relevant data available not only for Allura Red AC but also for a number of other structurally related sulphonated mono azo dyes authorised as food additives, namely: Amaranth, Ponceau 4R, Sunset Yellow FCF, Tartrazine and Azorubine/Carmoisine. The Panel concluded that the new data by themselves were insufficient at this time to change the conclusions of the 2009 opinion on the safety of Allura Red AC and that there is currently no reason to revise the ADI. The read-across exercise has highlighted a shared pattern of effects for this class of substances that would warrant further investigation The Panel therefore recommended a repetition of the in vivo comet assay in mice, to be performed in compliance with the most recent and internationally validated experimental protocol, using whole cells and examining a wide range of tissues. These recommendations apply to all the sulphonated mono azo dyes included in this review. This request originates from an opinion adopted by the FEEDAP Panel in April 2012 on the reevaluation of the substance for use in feedingstuffs for cats and dogs. In its opinion, the FEEDAP Panel had concluded that the genotoxic potential of Allura Red AC could not be excluded and that the data available were insufficient to demonstrate the safety of the substance for its proposed use.In the context of the re-evaluation of Allura Red AC for use as a food additive, a first positive in vivo comet assay in mice by Tsuda et al. (2001) The ANS Panel critically reviewed the study by Shimada et al. (2010) and noted that these results were observed by a single group of researchers using an in-house developed protocol that uses isolated nuclei from homogenised tissues. Although this protocol was considered to meet the minimum criteria for acceptance of the in vivo comet assay (EFSA, 2012), the Panel noted that such protocol had not been included in the international validation exercise. Under the same experimental conditions, no effect could be observed in the rat. The Panel noted that the same findings observed for Allura Red AC were also reported by Shimada et al. (2010) for the two other authorised food dyes tested in this study, Amaranth and Ponceau 4R.The Panel acknowledged the role of the comet assay as an indicator test for the detection of DNA lesions and/or DNA repair activity and concluded that the findings reported cannot be considered conclusive evidence of mutagenicity, i.e. of induced permanent genetic cha...

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“…In a double logarithmic plot of mutagenic and carcinogenic activity, most of the compounds showed a linear correlation, with the notable exception of several N-nitroso compounds. Clive et al (44) reported correlation studies on 25 chemicals. Carcinogenic activity in rats and mice was expressed as the frequency of tumor-bearing animals per pumole of compound administered per kilogram body weight.…”

Section: Quantitative Correlations Between the Carcinogenic And Mutagmentioning

confidence: 99%

Comparison between Carcinogenicity and Mutagenicity Based on Chemicals Evaluated in the IARC Monographs

Bartsch¹,

Tomatis²

1983

Environmental Health Perspectives

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The qualitative relationship between carcinogenicity and mutagenicity (DNA-damaging activity), based on chemicals which are known to be or suspected of being carcinogenic to man and/or to experimental animals, is analyzed using 532 chemicals evaluated in Volumes 1-25 of the IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. About 40 compounds (industrial processes) were found to be either definitely or probably carcinogenic to man, and 130 chemicals have been adequately tested in rodents and most of them also in various short-term assays. For a comparison between the carcinogenicity of a chemical and its behavior in short-term tests, systems were selected that have a value for predicting carcinogenicity. These were divided into mutagenicity in (A) the S. typhimurium/microsome assay, (B) other submammalian systems and (C) cultured mammalian cells; (D) chromosomal abnormalities in mammalian cells; (E) DNA damage and repair; (F) cell transformation (or altered growth properties) in vitro.The following conclusions can be drawn. In the absence of studies in man, long-term animal tests are still today the only ones capable of providing evidence of the carcinogenic effect of a chemical. The development and application of an appropriate combination of short-term tests (despite current limitations) can significantly contribute to the prediction/confirmation of the carcinogenic effects of chemicals in animals/man. Confidence in positive tests results is increased when they are confirmed in multiple short-term tests using nonrepetitive end points and different activation systems. Assays to detect carcinogens which do not act via electrophiles (promoters) need to be developed. The results of a given short-term test should be interpreted in the context of other toxicological data. Increasing demand for quantitative carcinogenicity data requires further examination of whether or not there is a quantitative relationship between the potency of a carcinogen in experimental animals/man, and its genotoxic activity in short-term tests. At present, such a relationship is not sufficiently established for it to be used for the prediction of the carcinogenic potency of new compounds.There is increasing evidence to suggest that DNA damage (expressed mainly as mutations) is involved in the induction of many cancers; however, the relevance of the various biological end points used in short-term assays to mechanisms of tumor induction is not known precisely. All test procedures must therefore be validated before they can be used to predict the carcinogenicity of chemicals. Ideally, such validations would be based on correlations between responses in short-term tests and data from epidemiological studies in humans.

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Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system

Cited by 547 publications

References 22 publications

Methotrexate: Assessment of In Vivo Clastogenicity and Carcinogenicity

Methotrexate: Assessment of In Vivo Clastogenicity and Carcinogenicity

Statement on Allura Red AC and other sulphonated mono azo dyes authorised as food and feed additives

Comparison between Carcinogenicity and Mutagenicity Based on Chemicals Evaluated in the IARC Monographs

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