Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test

Bowen, Damian; Whitwell, James; Lillford, Lucinda; Henderson, Debbie; Kidd, Daniel; Garry, Sarah Mc; Pearce, G. S.; Beevers, Carol; Kirkland, David

doi:10.1016/j.mrgentox.2011.02.009

Cited by 82 publications

(58 citation statements)

References 27 publications

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“…These results were somewhat expected for this direct-acting ethylating agent and well-known multisite/multispecies carcinogen. The genotoxicity of ENU has been studied extensively and positive results were reported previously in rat and mouse peripheral blood and bone marrow micronucleus assays, in Comet assays conducted with multiple organs of rats, as well as in rodent transgenic gene mutation assays [Jansen et al, 1995;Suzuki et al, 1997;Bowen et al, 2011]. DNA-reactive chemicals, such as alkylating agents, generally were believed to exhibit linear doseresponses, but an increasing number of studies have demonstrated apparent thresholds for mutation and genotoxicity in vitro and in vivo [Doak et al, 2007;Gocke and Wall, 2009;Muller et al, 2009;Dobo et al, 2011].…”

Section: Discussionmentioning

confidence: 99%

“…Although the Comet assay did detect statistically significant increases of DNA damage in blood leukocytes at the lower doses, the responses were very low in comparison to the response in the Pig-a assay. In an article by Bowen et al [2011], ENU induced statistically significant increases in peripheral blood leukocyte, liver, and stomach Comet assays after a three day acute treatment, but at concentrations ranging from 10 to 40 mg/kg/day. These results indicate that the Comet assay, because it does not measure the accumulation of strand breakage over time, might be better suited for acute high-dose studies, especially when the major genotoxicity mechanism of the test agent is gene mutation.…”

Section: Cd592mentioning

confidence: 98%

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Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Cammerer

Bhalli

Cao

et al. 2011

Environ and Mol Mutagen

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N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

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Section: Discussionmentioning

confidence: 99%

Section: Cd592mentioning

confidence: 98%

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Cammerer

Bhalli

Cao

et al. 2011

Environ and Mol Mutagen

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“…The comet assay has also been employed in multi-end point assay (Bowen et al, 2011) as it can be conducted using cells from virtually all organs (Kirkland and Speit, 2008). This is very useful as some chemicals may not require liver activation and may produce tumours in the tissue of first contact.…”

Section: Appropriate In Vivo Follow-up Assay Of Genotoxicitymentioning

confidence: 99%

Toxicity of food colours and additives: A review

Thomas¹,

Adegoke²

2015

Afr. J. Pharm. Pharmacol.

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Majority of consumer goods are required to be presented with good aesthetics in order to improve acceptability in terms of colours and in some instances taste. When related to food, beverages and drug products, additives are usually added to mask un-inviting colours, obscure offensive odours and increase taste. Food additives therefore include colourants, sweeteners, preservatives and anti-caking agents. Admissible daily intake limits are often recommended for these additives. Being food products, the amount consumed over time may be subject to individual preferences and thus negating the desire to regulate and control the amount consumed cumulatively. There have been several concerns about the safety of food additives and several batteries of tests, and reports are available in literature. This review attempted to give an update on reports that have surfaced in literature over recent past on the use and safety of food colours and other additives. Some safety concerns have been related to three determinations; cytotoxicity, genotoxicity and induction or potential of inducing mutagenicity. In order to accomplish these targeted evaluations, several tests have been prescribed by International conference on harmonization (ICH), organization for economic co-operation and development (OECD) and European food safety authority (EFSA). It is observed that no single test can give a full proof of safety of these food colours and additives, hence minimal tests are recommended to be carried out in order to guarantee safety of these products. Survey of literature, revealed that once some approved additives or colours become a subject of safety concerns, comprehensive evaluations are carried out by researchers and these have often led to the de-classification of some hitherto reported agents as being non-genotoxic or non-carcinogenic. The declassifications of some food colors and additives as human carcinogens are regularly done following the comprehensive evaluation of results of mutagenicity and genotoxicity tests in vitro and some in vivo tests in mammalian tissues and whole animals. However, such declassifications are often done with caution and the implication is that regular and more comprehensive tests must be carried out. In addition, the requirements of testing for chronic exposures to this and other agents must be emphasized to prevent occurrence of subtle yet terrible side effects resulting from consuming sub-toxic doses of the additives over time.

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“…Although BPDE is not a potent inducer of large deletions and insertions, it also causes loss of heterozygosity and chromosome breaks, and is known as strong clastogen [Lambert et al, 1994;Mazur-Melnyk et al, 1996;Wei et al, 1996]. Besides serving as a positive control for in vitro genotoxicity assays, BaP is positive in many in vivo assays, including the transgenic Big Blue assay and the in vivo micronucleus (MN) assay [Skopek et al, 1996;Bowen et al, 2011]; however, it has been challenging to detect BaP genotoxicity using the in vivo Comet assay [Bowen et al, 2011;Rothfuss et al, 2011].…”

Section: Introductionmentioning

confidence: 99%

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Bhalli

Shaddock

Pearce

et al. 2011

Environ and Mol Mutagen

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Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.

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Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test

Cited by 82 publications

References 27 publications

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

Toxicity of food colours and additives: A review

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

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