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The comet assay – Peculiarities, pitfalls and interpretation
Abstracts / Toxicology Letters 238S (2015) S15–S29
S02-2 The comet assay – Peculiarities, pitfalls and interpretation
S02-4 Application of in vivo assays in a regulatory setting
B. Burlinson
D. Kirkland
CRS Europe, Safety Assessment, Principal Scientist, Huntingdon, United Kingdom
Kirkland Consulting, Tadcaster, United Kingdom
The comet assay is already a popular and well used assay within a wide range of regulatory testing strategies. This popularity will increase further following the publication of the OECD guideline (TG489). However, while giving good guidance on the practical aspects of the comet assay in general the guideline gives little information on how to isolate cells from different tissues, what kind of variation in the data would be expected in the different tissues, if any, and what would constitute a validated tissue. At HLS we have been doing the in vivo comet assay for many years, in a variety of tissues with a wide variety of materials. This presentation will cover some of the issues we have seen and how they were resolved, the peculiar case of 2AAF (why was it negative in the JaCVAM trial) and could cycling cells or those in “S” phase produce a “false positive” response. http://dx.doi.org/10.1016/j.toxlet.2015.08.175
S02-3 The Pig-A assay – Its potential applications in regulatory mutagenicity testing R. Froetschl BfArM Federal Institute for Drugs and Medical Devices, Genetic and Reproductive Toxicology, Bonn, Germany The Pig-A gene (phosphatidylinositol glycan, class A gene) codes for the catalytic subunit of a N-acetyl glucosamine transferase. This enzyme is involved in an early step of glycosylphosphatidyl inositol (GPI) biosynthesis. GPI anchors a variety of cell surface markers (e.g., CD59, CD24, CD55) to the exterior on the cell membrane of higher eukaryotes. In mammals, Pig-A is X-chromosome linked and present as a single functional copy in cells from males and females. The Pig-A gene forms the rationale for the assay. The assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans. However most experience exists with measuring CD59-deficient erythrocytes in the peripheral blood of rats but also others markers like HIS49 have been used. The assay is optimized for flow cytometric measuring and performed in normal laboratory rat strains. There are potential advantages compared to the current gold standard in vivo mutation assay the mutation assay in transgenic rodents (TGR-mutation assay). Expensive special transgenic animals are not needed and an endogenous gene is used as marker. Furthermore the assay has the potential to be integrated into repeated dose toxicology studies. On the other hand the assay is currently still limited to blood derived cells. Currently there is no OECD protocol available for the Pig-A assay. Nevertheless the Pig-A is already mentioned in an official guideline, the ICH M7 ‘Assessment and Control of DNA-reactive (mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk’ where it is recommended as an in vivo follow up assay for AMES positive impurities and the IWGT has recommended the assay as follow up test for in vitro mutation assay positive substances. http://dx.doi.org/10.1016/j.toxlet.2015.08.176
S17
There are now more in vivo genotoxicity tests available that measure different endpoints in different tissues than in the past. The comet and transgenic rodent mutation (TGR) assays can be performed in (almost) any tissue. Methods to measure micronuclei (MN) in liver and GI tract as well as in bone marrow and blood have been evaluated and published. For a substance negative in vitro, a bone marrow/blood MN test is usually sufficient to determine there are no unique in vivo genotoxic effects. But if positive in vitro, most regulatory guidelines do not specify which test(s) should be used in which circumstances, so how do we choose? Are the in vivo tests being used to verify mode-of-action, or as weight-of-evidence? In the absence of a likely mode of action, in vivo tests in a siteof-contact tissue and the liver would seem most appropriate, but which endpoints? Are the choices affected by whether gene mutations or chromosomal damage are predominant in vitro, or whether the positive results are mainly in the absence or presence of S9? Is the comet assay appropriate to follow up on an in vitro gene mutagen – it takes less time, uses less test material and costs less money than a TGR assay? Is the MN assay in bone marrow appropriate to follow-up on an in vitro clastogen in the absence of S9, or is it necessary to perform a comet (or MN) assay in the GI tract where higher exposures will be achieved? If the substance is an aneugen, the comet and TGR assays are not appropriate, so is it acceptable to perform a MN test in GI tract or liver when there is not yet an OECD guideline? Finally, are there circumstances when it is not necessary to follow-up in vivo even when positive results have been obtained in vitro? Attempts to answer these questions will be presented. http://dx.doi.org/10.1016/j.toxlet.2015.08.177
S02-5 New advances in vivo mutagenicity tests: Application of the guidance of the European Food Safety Authority R. Crebelli 1,∗ , M. Carfì 2 , J.M. Parra Morte 2 , A.M. Rossi 2 , M.V. Vettori 2 , D. Maurici 2 1 2
Istituto Superiore di Sanità, Rome, Italy European Food Safety Authority, Parma, Italy
Under the EU legislation no substance which is classified as mutagenic can intentionally be added to food, at any dose level. Thus, the evaluation of genotoxic potential has a key role in the safety assessment of regulated food ingredients and packaging. This task was originally performed by the Scientific Committee on Food (SCF), and following the implementation of Regulation 178/2002, by the European Food Safety Authority (EFSA) which is entitled to perform the scientific assessment required for the final risk management decision of the European Commission. The operational criteria adopted by the EFSA for genotoxic hazard identification have evolved, since the original SCF recommendations, to keep up with the progress in the field. A harmonized approach, of general applicability by Panels working in different areas of food and feed safety, has been developed by the EFSA Scientific Committee in 2011. A major novelty in the EFSA guidance document on genotoxicity testing strategies is the detailed indication given for
S02-2 The comet assay – Peculiarities, pitfalls and interpretation
S02-4 Application of in vivo assays in a regulatory setting
B. Burlinson
D. Kirkland
CRS Europe, Safety Assessment, Principal Scientist, Huntingdon, United Kingdom
Kirkland Consulting, Tadcaster, United Kingdom
The comet assay is already a popular and well used assay within a wide range of regulatory testing strategies. This popularity will increase further following the publication of the OECD guideline (TG489). However, while giving good guidance on the practical aspects of the comet assay in general the guideline gives little information on how to isolate cells from different tissues, what kind of variation in the data would be expected in the different tissues, if any, and what would constitute a validated tissue. At HLS we have been doing the in vivo comet assay for many years, in a variety of tissues with a wide variety of materials. This presentation will cover some of the issues we have seen and how they were resolved, the peculiar case of 2AAF (why was it negative in the JaCVAM trial) and could cycling cells or those in “S” phase produce a “false positive” response. http://dx.doi.org/10.1016/j.toxlet.2015.08.175
S02-3 The Pig-A assay – Its potential applications in regulatory mutagenicity testing R. Froetschl BfArM Federal Institute for Drugs and Medical Devices, Genetic and Reproductive Toxicology, Bonn, Germany The Pig-A gene (phosphatidylinositol glycan, class A gene) codes for the catalytic subunit of a N-acetyl glucosamine transferase. This enzyme is involved in an early step of glycosylphosphatidyl inositol (GPI) biosynthesis. GPI anchors a variety of cell surface markers (e.g., CD59, CD24, CD55) to the exterior on the cell membrane of higher eukaryotes. In mammals, Pig-A is X-chromosome linked and present as a single functional copy in cells from males and females. The Pig-A gene forms the rationale for the assay. The assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans. However most experience exists with measuring CD59-deficient erythrocytes in the peripheral blood of rats but also others markers like HIS49 have been used. The assay is optimized for flow cytometric measuring and performed in normal laboratory rat strains. There are potential advantages compared to the current gold standard in vivo mutation assay the mutation assay in transgenic rodents (TGR-mutation assay). Expensive special transgenic animals are not needed and an endogenous gene is used as marker. Furthermore the assay has the potential to be integrated into repeated dose toxicology studies. On the other hand the assay is currently still limited to blood derived cells. Currently there is no OECD protocol available for the Pig-A assay. Nevertheless the Pig-A is already mentioned in an official guideline, the ICH M7 ‘Assessment and Control of DNA-reactive (mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk’ where it is recommended as an in vivo follow up assay for AMES positive impurities and the IWGT has recommended the assay as follow up test for in vitro mutation assay positive substances. http://dx.doi.org/10.1016/j.toxlet.2015.08.176
S17
There are now more in vivo genotoxicity tests available that measure different endpoints in different tissues than in the past. The comet and transgenic rodent mutation (TGR) assays can be performed in (almost) any tissue. Methods to measure micronuclei (MN) in liver and GI tract as well as in bone marrow and blood have been evaluated and published. For a substance negative in vitro, a bone marrow/blood MN test is usually sufficient to determine there are no unique in vivo genotoxic effects. But if positive in vitro, most regulatory guidelines do not specify which test(s) should be used in which circumstances, so how do we choose? Are the in vivo tests being used to verify mode-of-action, or as weight-of-evidence? In the absence of a likely mode of action, in vivo tests in a siteof-contact tissue and the liver would seem most appropriate, but which endpoints? Are the choices affected by whether gene mutations or chromosomal damage are predominant in vitro, or whether the positive results are mainly in the absence or presence of S9? Is the comet assay appropriate to follow up on an in vitro gene mutagen – it takes less time, uses less test material and costs less money than a TGR assay? Is the MN assay in bone marrow appropriate to follow-up on an in vitro clastogen in the absence of S9, or is it necessary to perform a comet (or MN) assay in the GI tract where higher exposures will be achieved? If the substance is an aneugen, the comet and TGR assays are not appropriate, so is it acceptable to perform a MN test in GI tract or liver when there is not yet an OECD guideline? Finally, are there circumstances when it is not necessary to follow-up in vivo even when positive results have been obtained in vitro? Attempts to answer these questions will be presented. http://dx.doi.org/10.1016/j.toxlet.2015.08.177
S02-5 New advances in vivo mutagenicity tests: Application of the guidance of the European Food Safety Authority R. Crebelli 1,∗ , M. Carfì 2 , J.M. Parra Morte 2 , A.M. Rossi 2 , M.V. Vettori 2 , D. Maurici 2 1 2
Istituto Superiore di Sanità, Rome, Italy European Food Safety Authority, Parma, Italy
Under the EU legislation no substance which is classified as mutagenic can intentionally be added to food, at any dose level. Thus, the evaluation of genotoxic potential has a key role in the safety assessment of regulated food ingredients and packaging. This task was originally performed by the Scientific Committee on Food (SCF), and following the implementation of Regulation 178/2002, by the European Food Safety Authority (EFSA) which is entitled to perform the scientific assessment required for the final risk management decision of the European Commission. The operational criteria adopted by the EFSA for genotoxic hazard identification have evolved, since the original SCF recommendations, to keep up with the progress in the field. A harmonized approach, of general applicability by Panels working in different areas of food and feed safety, has been developed by the EFSA Scientific Committee in 2011. A major novelty in the EFSA guidance document on genotoxicity testing strategies is the detailed indication given for