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Papers by Brian Burlinson

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2015

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative inte... more As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay, we examined p-Chloroaniline, t-butylhydroquinone, and methyl carbamate. All test materials and controls were dosed orally by gavage. p-Chloroaniline produced a statistically significant increase in the mean and median % tail intensity which was also outside of the historical control range in the liver and stomach of Sprague-Dawley rats. t-Butylhydroquinone caused a statistically significant increase in the mean % tail intensity in the liver and stomach and a statistically significant increase in the median % tail intensity in the liver; however, these results are not considered to be biologically significant as all values obtained fell within the current vehicle historical control range and within the negative control range for mean % tail intensity set by the Validation Management Team (VMT) as a requirement for an acceptable assay. Methyl carbamate did not induce a statistically significant change in the mean or median % tail intensity in either liver or stomach.

Mutation Research/Genetic Toxicology, 1983

A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test usin... more A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test using C57B1/6J mice.

Mutation Research/Environmental Mutagenesis and Related Subjects, 1989

Methods in Molecular Biology, 2011

The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromoso... more The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromosome aberration or breakage (clastogenicity), and chromosome loss or gain (aneuploidy). The current generalized strategy consists of assays capable of detecting all of these endpoints using in vitro assays such as the Ames test for detecting gene mutations in bacteria, the human peripheral lymphocyte chromosome aberration (CA) test for detecting clastogenicity, and the in vitro micronucleus test for clastogenicity and aneuploidy. The primary in vivo assay, and generally the only in vivo assay required, is the in vivo rodent bone marrow micronucleus assay. However, there are instances when these assays alone are inadequate and further testing is required, especially in vivo. Historically, the preferred second assay has been the rodent liver unscheduled DNA synthesis assay but recently this has been superseded by the rodent single cell gel electrophoresis or Comet assay. This assay has numerous advantages especially in vivo, where virtually any tissue can be examined. The status of the in vitro comet assay in regulatory testing is much less clear although a preliminary review of data from the assay has shown it to be more specific than other in vitro genotoxicity tests and less prone to false positives.Detailed here are general protocols for both the in vitro and in vivo comet assays which will form the basis of the pending OECD guideline for the assay.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 1997

. Male MutaeMice were given a single intraperitoneal dose of either 1r15 M phosphate buffer pH 6 ... more . Male MutaeMice were given a single intraperitoneal dose of either 1r15 M phosphate buffer pH 6 as the vehicle control, MMS 40 mgrkg, ENU 150 mgrkg or iPMS 200 mgrkg, at a dose volume of 20 mlrkg. Animals from each group were killed 3 or 63 days after dosing, the DNA extracted from whole testes and epididymal spermatozoa and analysed for mutation frequency. In the testes, no increase in mutation frequency was observed, at either timepoint, for the animals treated with either MMS or iPMS. A slight increase in the mutation frequency, above vehicle control values, was seen in the ENU-treated animals with a 3 day expression time. A 4-fold increase was observed in the ENU-treated animals exposed for 63 days. In the epididymal spermatozoa, all of the test chemicals induced increases in mutation frequency, at both timepoints, with the exception of a negative result for MMS after 3 days. ENU induced a 2.5 and iPMS a induced a 4-fold increase above the control mutation frequency after 3 days. For all treatments, the later sampling time of 63 days gave an approximate 2-fold increase above the results of the 3-day timepoint. These increases amounted to a 2, 4.5 and 11-fold increase above control for MMS, ENU and iPMS, respectively. The MutaeMouse positive selection system appears to be sensitive to both the premeiotic germ cell mutagen, ENU and postmeiotic germ cell mutagens, MMS and iPMS.

Mutation Research Letters, 1983

The response of 3 strains of mouse (C57Bl/6J, C3H/C57 hybrid and BALBC/CBA) to cyclophosphamide (... more The response of 3 strains of mouse (C57Bl/6J, C3H/C57 hybrid and BALBC/CBA) to cyclophosphamide (75 mg/kg) and hexamethylphosphoramide (HMPA) (1.28 ml/kg) were compared in the micronucleus test. Each compound was administered by intraperitoneal injection on two consecutive days and samples of bone marrow and blood taken for examination at 48 and 72 h after the first injection. Both test chemicals produced a statistically significant increase (P 0.001) in the incidence of micronuclei in bone marrow cells in all strains at both sampling times but the response with HMPA in C57Bl/6J mice appears to occur earlier than in the other two strains. Significant increases in micronuclei were seen in circulating erythrocytes only at 48 h in C57Bl/6J mice with both test chemicals and in C3H/C57 mice only with cyclophosphamide.

Mutagenesis, 2002

The performance of two computer programs, DEREK and TOPKAT, was examined with regard to predictin... more The performance of two computer programs, DEREK and TOPKAT, was examined with regard to predicting the outcome of the Ames bacterial mutagenicity assay. The results of over 400 Ames tests conducted at Glaxo Wellcome (now GlaxoSmithKline) during the last 15 years on a wide variety of chemical classes were compared with the mutagenicity predictions of both computer programs. DEREK was considered concordant with the Ames assay if (i) the Ames assay was negative (not mutagenic) and no structural alerts for mutagenicity were identified or (ii) the Ames assay was positive (mutagenic) and at least one structural alert was identified. Conversely, the DEREK output was considered discordant if (i) the Ames assay was negative and any structural alert was identified or (ii) the Ames assay was positive and no structural alert was identified. The overall concordance of the DEREK program with the Ames results was 65% and the overall discordance was 35%, based on over 400 compounds. About 23% of the test molecules were outside the permissible limits of the optimum prediction space of TOPKAT. Another 4% of the compounds were either not processable or had indeterminate mutagenicity predictions; these molecules were excluded from the TOPKAT analysis. If the TOPKAT probability was (i) ≥0.7 the molecule was predicted to be mutagenic, (ii) ≤0.3 the compound was predicted to be nonmutagenic and (iii) between 0.3 and 0.7 the prediction was considered indeterminate. From over 300 acceptable predictions, the overall TOPKAT concordance was 73% and the overall discordance was 27%. While the overall concordance of the TOPKAT program was higher than DEREK, TOPKAT fared more poorly than DEREK in the critical Ames-positive category, where 60% of the compounds were incorrectly predicted by TOPKAT as negative but were mutagenic in the Ames test. For DEREK, 54% of the Ames-positive molecules had no structural alerts and were predicted to be non-mutagenic. Alternative methods of analyzing the output of the programs to increase the accuracy with Ames-positive compounds are discussed.

Mutagenesis, 1996

In vitro assays for mutagenicity are an important feature of pre-clinical testing and form part o... more In vitro assays for mutagenicity are an important feature of pre-clinical testing and form part of the current regulatory testing conducted early in drug development They can also play a part in compound selection since mutagenic compounds can be eliminated from a range of potential candidates. Bacterial tests are particularly useful in this area because they generate results quickly, though their use may be limited because they can require up to 4 g of material. A scaled-down version of the Ames test has been developed which requires only ~20 mg of material. Initial experiences with this assay using a range of known mutagens and novel compounds have shown that the Miniscreen has similar sensitivity to the Ames test The major exception is for those mutagens preferentially detected with strains TA1537 and TA1535, which, because of their low spontaneous counts, are not employed in the Miniscreen.

Mutagenesis, 2013

The comet assay can be applied to virtually any tissue and it has been noted that it can be parti... more The comet assay can be applied to virtually any tissue and it has been noted that it can be particularly useful in evaluating directly acting genotoxins at their initial site of action. Consequently, it has become relatively common practice to use the stomach comet assay after oral administration to test chemicals that have given positive in vitro genotoxicity results in the absence of metabolic activation. However, to test nontoxic substances up to the limit doses of 1000/2000mg/kg formulations approaching molar concentrations must be used resulting in the stomach mucosa being exposed to excessively high levels. Evidence is beginning to accumulate which shows positive results that do not indicate that potential carcinogenicity may be associated with such high levels of exposure. For pharmaceutical agents, toxicokinetic data are usually available to demonstrate systemic exposure after oral administration. In such cases, it is proposed that exposure of any tissue to levels of the drug substance greater than those that have given positive in vitro results in the absence of metabolic activation is sufficient. However, it is recognised that toxicokinetic data are not available for all chemicals and there are also agents designed not to leave the gastrointestinal tract (GIT). Where it is necessary to examine the GIT, the dose levels selected for examination should cover the likely or intended exposure levels, not necessarily to achieve the maximum tolerated or limit doses, even if the higher doses are required for genotoxicity endpoints in other tissues to be valid. There are usually two or three dose levels in in vivo genotoxicity studies, so when both systemically exposed tissues and the stomach are being examined, it would be possible to use one of the lower doses for the latter without increasing the numbers of animals required. It is important to consider the local concentrations achieved in the stomach or other parts of the GIT in order to avoid the comet assay generating artefactual positive results and it is hoped this will be addressed in the imminent Organisation for Economic Co-operation and Development guideline.

Molecular Carcinogenesis, 2000

We report here the development of the polymerase inhibition assay (PI assay), a methodology capab... more We report here the development of the polymerase inhibition assay (PI assay), a methodology capable of simultaneously identifying multiple DNA-damaging agents. The PI assay was developed in order to fulfil a requirement for the screening of new pharmaceuticals for potential DNA-damaging effects. The assay has the potential to screen hundreds of new compounds per week because of the microtiter plate format employed. We review previous descriptions of the phenomenon and provide researchers with the necessary methodology to obtain optimum polymerase inhibition effects. The assay is based on the inhibition of DNA polymerases (including those used in the polymerase chain reaction (PCR)) encountering damaged DNA bases. Hence, DNA-damaging agents can be identified by a corresponding reduction in PCR amplification after exposure. We demonstrate the detection of polymerase inhibition induced by a range of model genotoxic agents (N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, and ultraviolet (UV) C radiation), illustrating the successful application of the methodology. In addition, the PI assay is shown to be capable of detecting DNA damaging agents of biological relevance, i.e., known human carcinogens. These were N-OH-PhIP (from cooked meat) and UV-B (from sunlight). In addition to its employment in the detection of putative DNA damaging agents, the PI assay may also be applied as a research tool in carcinogenicity studies.

Carcinogenesis, 1989

A new in vivo unscheduled DNA synthesis (UDS) assay is described for the detection of genotoxic d... more A new in vivo unscheduled DNA synthesis (UDS) assay is described for the detection of genotoxic damage in the rat stomach. In this assay advantage is taken of the morphology of the gastric mucosa to enable the selective isolation, and subsequent measurement of UDS, in non-S-phase cells. The absence of replicating cells allows UDS to be measured by scintillation counting without having to use hydroxyurea. Control background responses are given, these were low and acceptably stable. The sensitivity of the assay was tested using 1-methyl-3-nitro-1-nitrosoguanidine which was found positive at doses as low as 12.5 mg/kg. The selectivity of the assay for genotoxins was tested using indomethacin, a nongenotoxic, gastric irritant. This compound was negative at dose levels and exposure times known to produce gastric lesions. Two forestomach-specific carcinogens, aristolochic acid and epichlorhydrin, were also investigated. Aristolochic acid was, surprisingly, uniformly negative. Further work on this compound is obviously required especially in the light of the strong positive response produced by epichlorhydrin. These data would suggest that this assay would be a useful complement to the current in vivo short-term test battery and a helpful research tool for investigating DNA repair in stomach tissue.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2014

Mutation Research-fundamental and Molecular Mechanisms of Mutagenesis, 1987

Mutation Research/Genetic Toxicology, 1985

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described b... more The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.

Mutation Research/Genetic Toxicology, 1986

Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wav... more Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wave of S-phase activity in the liver 36 h later, followed by a wave of mitoses at 48 h. These events were monitored by autoradiography of isolated hepatocytes and by histopathology, respectively. DNA-labelling was shown to occur following both in vivo and in vitro radiolabelling. The level of S-phases observed approached that reported following partial hepatectomy. These effects were not accompanied by unscheduled DNA synthesis (UDS) nor was any frank histopathological damage to the liver evident. 2-Acetylaminofluorene (2AAF) elicited a very weak S-phase response at a dose level of 50 mg/kg, but gave marked UDS between 12 and 48 h.

Archives of Toxicology, 1985

2,4,6-Trinitrotoluene (TNT) is structurally related to the rat liver carcinogen 2,4-dinitrotoluen... more 2,4,6-Trinitrotoluene (TNT) is structurally related to the rat liver carcinogen 2,4-dinitrotoluene (technical grade), and both compounds are known to be mutagenic to bacteria in vitro. TNT is therefore established as a potential rodent carcinogen; the present paper describes experiments designed to assess if this potential is likely to be expressed in appropriately exposed animals. TNT gave a negative response in the mouse bone marrow micronucleus assay and in an in vivo/in vitro rat liver assay for unscheduled DNA synthesis (UDS). In the latter assay animals are exposed to the test chemical in vivo and their hepatocytes subsequently evaluated for UDS in vitro. The negative response observed for TNT in the liver assay at dose-levels up to 1000 mg/kg was accompanied by a positive response for the hepatocarcinogen 2,4-dinitrotoluene at the lower dose-level of 200 mg/kg. In contrast, the dinitro compound gave a negative response in the micronucleus assay, as was also observed for TNT. It is concluded that the negative response observed for TNT in the liver assay indicates that it is unlikely to be a rat hepatocarcinogen. Nonetheless, high levels of methaemoglobin were observed in the TNT-treated rats and their urine was coloured red. These facts, together with the known toxicities of this agent suggest a possible carcinogenic hazard to the haemopoetic and urinary tissues of animals exposed chronically to it at toxic dose-levels.

Zeitschrift für Gastroenterologie

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unsched... more The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenesis

Transgenic mouse mutation assays, such as MutaMouse (lacZ, CD2F1) and Big Blue (lacI, B6C3F1), af... more Transgenic mouse mutation assays, such as MutaMouse (lacZ, CD2F1) and Big Blue (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both MutaMouse and Big Blue should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics...