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Background to COM review

1. The COM and COC recently considered the available mutagenicity and carcinogenicity data on malachite green and leucomalachite green. (http://www.advisorybodies.doh.gov.uk/com/mglmg.htm) A number of mutagenicity studies using the lacI transgene in both rats and mice were reported. However one key piece of evidence came from the finding of an increased mutagen frequency in the liver in an in-vivo mutagenicity assay using the cII transgene in Big Blue mice fed a diet containing leucomalachite green. An overview of the development and use of in-vivo mutagenicity assays using the cII transgene was drafted for the February 2005 COM meeting. A general conclusion was reached on the basis of the information reviewed.

Data considered by COM

2. The cII transgenic mutation assay was developed and published by Jakubczak and colleagues in 1996 using F1 male mice (from FVB/N x C57BL/6 (Big Blue mice carrying the lacI and λ bacteriophage transgene).¹ The cI and cII genes are present on the λ bacteriophage transgene. A mutation yielding cII^- can be detected by packaging the λ transgene into E.coli G1250 which form plaques (lytic cell cycle of phage) whereas λ transgenes carrying cII⁺ will preferentially enter the lysogenic cell cycle and will not form plaques. The authors suggested that one advantage for the cII transgene assay was use selective growth of mutants and that the cII gene (300bp) was considerably smaller compared to the lacI transgene (1kb) allowing rapid sequencing indicating that the cII mutation assay had additional advantages compared to the lacI mutation assay. Thus it would be possible to easily identify the proportion of a mutagenic response that was due to clonal replication of a "jackpot mutation". A number of published studies which reported on studies using the cII transgene in Big Blue mice and Big Blue rats were reviewed by the COM.^2-8

3. The use of the cII transgene has also been developed in the Muta™Mouse system (which was originally developed using the lacZ transgene (3kb.). The COM reviewed a number of published studies.^9-16 There has been a debate in the scientific literature on the precise mechanism of the detection of mutations in the cII transgene in Muta™Mouse since the lacZ transgene insertion results in the loss of the cI transgene from the λ bacteriophage which plays an important role in the determination of lysogenic versus lytic life cycle for the λ bacteriophage.¹⁷ The determination of cII^- mutants using the Muta™Mouse relies of incubation of E.coli 1225 containing the transgene at 24⁰C which selects for the lytic (plaque forming) life cycle whereas incubation at 37⁰C results in plaque formation irrespective of cII transgene status.

4. The COM were aware that generic guidance on the conduct of mutagenicity assays in transgenic rodents had been published in the scientific literature.^18,19 Members also noted that a draft OCED consultation document on use of transgenic rodent mutation models was being published on 10 February 2005 for comment.

COM evaluation of data

5. Members commented that the cII transgene was smaller than the lacI and lacZ transgenes and noted the finding in control studies that approximately 18% of lytic cycle plaques had no detectable mutations. Members agreed this could occur with selection assays dependent on differentiating between lysogenic and lytic cell cycles of bacteriophage transgenes. It was therefore important to undertake sequencing of mutant colonies to confirm that false positive results had not been obtained.

6. The Committee agreed that an acute dosing approach to the cII assay could be potentially insensitive to the identification of mutagens. There was limited published information on the influence of repeated dosing on mutation induction in tissues which supported the conclusion that the duration of exposure necessary to obtain a plateau in the increase in mutation frequency was related predominantly to the cell turnover of target cells in tissues and organs. The current approaches used were pragmatic involving repeated administration for 5-28 days followed by multiple sampling times dependent on tissue or organ being sampled.

7. Members observed the study using PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), which had examined the induction of cII and lacI mutations in the colon of Big Blue^® F344, showed that the cII transgene was not responsive to frameshift or deletion mutations.⁸ In addition members noted the study from Monroe et al provided evidence that the cII transgene did not respond to MNU (N-nitroso-N-methylurea) mutagenicity in splenic T cells whereas a positive result could be demonstrated with lacI.⁷

8. The Committee considered that studies using AAF (2-acetylaminofluorene), a compound that induced frameshift mutations, would provide relevant information on the potential uses of the cII transgene. It was noted that no studies with AAF had been retrieved in the literature search for the COM paper.

9. Members agreed that no conclusion could be reached on the limited published information available regarding the response of the cII transgene in cancer target organs in experimental animals.²⁰ The Committee agreed that sequencing of the cII transgene in mutant plagues was essential for determining a positive result and also for evaluating equivocal mutagenic response based on fold increase in mutation frequency.²¹

COM conclusion

10 The COM concluded that the cII transgene was a useful in-vivo mutation assay to investigate potential mutagenic effects in cancer target organs in rodents. It was necessary to routinely sequence mutant colonies in order to evaluate the significance of results obtained with the cII transgene in rodents. The COM felt that the currently data available in the published literature did not support the routine use of the cII transgene for screening chemicals but suggested it was of value in targeted studies of mutagenicity in specific organs in rodents. The COM agreed to keep the use of the cII transgene under review along with other developments of in-vivo mutagenicity testing.

June 2005
COM/05/S2

References

1. Jakubczak JL et al (1996). Analysis of genetic stability during mammary tumour progression using a novel selection-based assay for in-vivo mutations in a bacteriophage λ transgene target. Proc Natl Acad Sci, 93, 9073-78.

2. Harbach PR et al (1999). Spontaneous mutation spectrum at the lambda cII locus in liver, lung and spleen tissue of Big Blue transgenic mice. Env Mol Mut, 33, 132-143.

3. Wang J et al (2004). Time course of cII Gene mutant manifestation in the liver, spleen, and bone-marrow of N-Ethyl-N-Nitrosourea-treated Big Blue transgenic mice. Toxicological Science, 82, 124-128.

4. Zimmer DM et al (1998). Effect of plating medium and phage storage on mutant frequency and titer in the lamda cII transgenic mutation assay. Env Mol Mut, 32, 325-330.

5. Zimmer DM et al (1999). Comparison of mutant frequencies at the transgenic lamda LacI and cII/cI loci in control and ENU-treated Big Blue mice. Enviorn Mol Mut, 33, 249-256.

6. Gollapudi BB et al (1998). Hepatic lacI and cII mutation in transgenic (lambdaLIZ) rats treated with dimethylnitrosamine, Mut Res, 419, 131-5.

7. Monroe JJ et al (1998). A comparative study of in-vivo mutation assays: analysis of hprt, lacI, cII as mutational targets for N-nitroso-N-methylurea and benzo(a)pyrene in Big Blue mice. Mutation Research, 421, 121-136.

8. Stuart GR et al (2000). Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats. Mutation Research, 452, 101-121.

9 Kohara A et al (2002). Mutagenicity of Aristocholic acid in lamda/lacZ transgenic mouse (Muta™Mouse). Mutation Research, 515, 63-72. (A)

10. Noda Y et al (2002). In-vivo genotoxicity evaluationj of dimethylarsenic acid in Muta™Mouse. Mutation Research, 513, 205-212. (A)

11. Kohara A et al (2002). Dinitropyrene induce gene mutations in multiple organs of the lamda/lacZ transgenic mouse (Muta™Mouse). Mutation Research, 515, 73-83.

12. Itoh T et al (2003). Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice. Mutation Research, 539, 99-108. (A)

13. Hashimoto K et al (2004). Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™Mouse. Mutation Research, 560, 119-131.

14. Suzuki T et al (2000). Hepatocarcinogen quinoline induces GC to CG transversions in the cII gene in the liver of lambda/lacZ transgenic mice (Muta™Mouse). Mutation Research, 456, 73-81. (A)

15. Kohara A et al (2001). Mutation spectrum of o-aminoazotoluene in the cII gene of lamda/lacZ transgenic mice (Muta™Mouse). Mutation Research, 491, 211-220. (A)

16. Hakura A et al (1998). Comparison between in vivo mutagenicity and carcinogenicity in multiple organs by benzo(a)pyrene in the lacZ transgenic mouse (Muta™Mouse). Mutation Research, 398, 123-130.

17. Swiger RR (2001). Just how does the cII selection system work in Muta™Mouse?. Env Mol Mutagen, 37, 290-296.

18. Heddle JA et al (2000) . In-vivo Transgenic mutation assays. Env Mol Mut, 35, 253-259.

19. Heddle JA et al (2003). Treatment and sampling protocols for transgenic mutation assays. Env Mol Mut, 41, 1-6.

20. Nagao M et al (1998). No direct correlation between mutant frequencies and cancer incidence induced by MeIQ in various organs of Big Blue mice. Mutation Research, 400, 251-257.

21. Mei N et al (2004). Differential mutagenicity of riddelliine in liver endothelial and parenchymal cells of transgenic big blue rats. Cancer letters, 215, 151-158.

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