Advances in Genetic Toxicology and Integration of in vivo Testing into Standard Repeat Dose Studies

EPA's pesticide program is committed to using current state-of-the-science methods to enable a more effective and efficient testing and assessment paradigm for chemical risk management. We are also committed to reducing the number of animals used in testing while still producing a reliable safety assessment of pesticide chemicals.

There have been a number of recent advances in the area of genetic toxicity testing that would reduce animal usage and still provide the necessary information for an assessment of the genotoxic potential of substances (Ref 1, 2).

We are open to new, scientifically credible approaches that address pesticide genetic toxicology data requirements. We are accepting data from the new in vitro micronucleus assays (OECD test guideline 487), and we have endorsed the new OECD test guideline (488) for the transgenic rodent gene mutation assay.

Also, we encourage the incorporation of genotoxicity endpoints into routine toxicology studies where scientifically feasible. Certain genotoxicity studies, including the micronucleus and comet assays, can effectively be incorporated into routine toxicology studies. In addition, although the Pig-A gene mutation assay does not have an OECD Test guideline, it is a promising new in vivo mutation test that is sensitive and less costly than the transgenic rodent gene mutation assay, and it can be integrated into repeat-dose standard toxicology tests.

Combining Genotoxicity Testing with Standard Repeated Dose Toxicology Testing

The integration of the cytogenetic tests into repeated dose toxicity studies can be used to satisfy the in vivo cytogenetic data requirement in CFR Part 158 and Part 161. The evaluation of micronuclei in peripheral blood or bone marrow cells covers the evaluation of structural and numerical chromosomal aberrations. The integration of the mammalian bone marrow and the rodent erythrocyte micronucleus assays is technically feasible and is a scientifically acceptable alternative to conducting independent in vivo cytogenetic assays. The integration of a cytogenetic assay into repeat-dose toxicology studies is internationally accepted, as discussed in papers by the International Workshop on Genotoxicity Testing (Ref 8) and the International Committee for Harmonization (ICH) for pharmaceuticals (Ref 9).

Guidance for conducting a cytogenetic test that is integrated into a repeat-dose toxicity study can be found in the ICH guidance (Ref 9). Additional guidance on the evaluation of micronuclei (MN) can be found in other harmonized test guidelines (e.g., OECD test guideline 474¹ ). Some general guidance is provided below:

• Rats from repeat-dose studies (7- 90 days) bled for toxicokinetic investigations or for other routine toxicological purposes can be can be used for MN analysis, or a small blood sample may be obtained specifically for the MN analysis. When studies of 28-90 days’ duration in mice are conducted, there is additional advantage in obtaining the MN frequency in mature red blood cells at steady state (i.e., after approximately 1 month of exposure) to obtain a measure of the average extent of damage over the one month period prior to sampling (Ref 10). The MN frequency in reticulocytes provides a measure of damage occurring approximately 2 days before sampling.
   
• The doses tested are generally considered appropriate when the toxicology study meets the criteria for an adequate study. Further guidance can be found in ICH and OECD test guidelines (474). Routine MN determination is at terminal sacrifice (i.e., day after the final administration) and should be sufficient in most cases, although it has been recommended that an additional early sampling at approximately 3-4 days is advantageous (Ref 11).
   
• The number of animals analyzed is determined by current test guideline recommendations for the micronucleus assay (OECD TG 474) and generally does not include all the animals treated in a guideline toxicology study. Animals used for MN analyses should be randomly selected from the group used for the toxicology study.
   
• Samples for MN analysis can be collected from both sexes, but a single sex can be scored if there is no substantial sex difference evident in toxicity/metabolism.
   
• Route of administration is generally the route used in the repeat-dose toxicity study, e.g., oral (feeding or gavage), but can be modified if appropriate in order to obtain systemic exposure, e.g., for topically applied compounds.
   
• It is considered sufficient to treat animals with a positive control only periodically and not concurrently with every assay, after a laboratory has established competence in the use of the assay. Blinded, randomized scoring controls (standards or samples obtained from separate studies and then coded) should be included in each study.
   
• Guidance on the evaluation of test results does not differ from the evaluation of independent in vivo cytogenetic assays.

Flow cytometric methods may be used for the enumeration of micronuclei. This method provides more rapid and improved measurements and enhanced statistical power given that more cells can be analyzed and instrument calibrations standards are available (Ref 2-7). MN results scored by flow cytometric methods have been shown to be highly correlated with traditional microscopy, and provide substantially improved precision (Ref 12, 13).

When using any new or amended test protocols, the registrant is encouraged to consult or submit the proposed test protocol to the EPA for review before conducting the study.

References

1. EFSA Scientific Committee; Scientific Opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA Journal 2011; 9(9):2379. [68 pp.] doi:10.2903/j.efsa.2011.2379. Available online: https://www.efsa.europa.eu/en/efsajournal/pub/2379 
2. Lynch AM et al., 2011. New and Emerging Technologies for Genetic Toxicity Testing Environmental and Molecular Mutagenesis 52:205-223
    
3. MacGregor JT, et al., 2006. Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: II. An Efficient Method of Monitoring Chromosomal Damage in the Rat. Toxicological Sciences 94(1), 92–107.
    
4. Pfuhler S et al., 2009 Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities—Report from an ECVAM workshop Mutation Research 680 (2009) 31-42.
    
5. Dertinger SD (2010) Integration of Mutation and Chromosomal Damage Endpoints into 28-Day Repeat Dose Toxicology Studies. Toxicological Sciences 115(2), 401–411 (2010)
    
6. Shi J, Bezabhie R, and A Szkudinska (2010) Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminate apoptotic bodies. Mutagenesis 25: 33-40.
    
7. Bryce SM, Shi J, Micolett J, Diehl M. Sonders P, Avlasevich S, Raja S, Bemis JC, and SD Dertinger (2010). High Content flow cytometric micronucleus scoring method is applicable to attachment cells lines. Environmental Molecular Mutagenesis 51:260-266.
    
8. Rothfuss A, Honma M, Czich A, Aardema MJ, Burlinson B, Galloway S, Hamada S, Kirkland D, Heflich RH, Howe J, Nakajima M, O'Donovan M, Plappert-Helbig U, Priestley C, Recio L, Schuler M, Uno Y, Martus HJ. (2011) Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutat Res. Aug 16;723(2):108-20. Epub 2010 Dec 21.
    
9. The ICH Harmonized guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use. S2(R1)
    
10. MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. (1990). The in vivo Erythrocyte Micronucleus Test: Measurement at Steady State Increases Assay Efficiency and Permits Integration with Toxicity Studies. Fund. Appl. Toxicol., 14, 513-522.
     
11. Hamada S, Sutou S, Morita T, Wakata A, Asanami S, Hosoya S, Ozawa S, Kondo K, Nakajima M, Shimada H, Osawa K, Kondo Y, Asano N, Sato S, Tamura H, Yajima N, Marshall R, Moore C, Blakey DH, Schechtman LM, Weaver JL, Torous DK, Proudlock R, Ito S, Namiki C, Hayashi M. 2001. Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS). Environ Mol Mutagen. 2001;37(2):93-110.
     
12. Torous D, Hall NE, Murante FG, Gleason SE, Tometsho CR, and SD Dertinger. 2006. Comparative Scoring of Micronucleated Reticulocytes in Rat Peripheral Blood by Flow Cytometry and Microscopy. Toxicol Sci 74-309-314.
     
13. Dertinger SD, Bishop ME, McNamee JP, Hayashi M, Suzuki T, Asano N, Nakajima M, Saito J, Moore M, Torous DK, Macgregor JT. 2006. Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: I. Intra- and interlaboratory comparison with microscopic scoring. Toxicol Sci.94:83-91.