Module 3 - Strategic case studies in practice

The prime target organs for oral N- nitrosamine exposure in animals are liver (rat, monkey), oesophagus (rat), lung (rat), bladder (rat) and others with liver being mostly affected. Liver can also be expected as target organ with regard to its metabolic competence. These organs might also be considered as human relevant targets although sufficient data from humans are currently not available. In the single exposure carcinogen database [Calabrese & Blain (1999)] 35 N- Nitrosamines are included that gave positive results with a single dose application. However, extrapolation of risks for low dose chronic exposure using those studies is problematic and no established methods are available. In table 2.4.3-1 above, N- nitrosamines with a TD 50 below 1.5 mg/kg/day (cohort of concern chemicals) are listed according to their TD 50 as follows: NMPEA>NDEA>NDMA>NMEA>NNK>NNN>NMOR>NMA>NDPA>NDBA>NPYR>MNNG>NMBA>NPIP 2.4.4. Use of in vitro mutagenicity data for carcinogenicity potency ranking of N- nitrosamines Using in vitro mutagenicity data for potency ranking of N- nitrosamines appears problematic. Available Ames assay data are highly predictive for a qualitative prediction of carcinogenicity in rodent studies but problematic for estimation of carcinogenic potency [McCann et al. (1988), Bogen (1995)]. Major identified problems are summarised below: • N- nitrosamines need to be activated metabolically and the artificial rat liver S9 mix used for simulation of metabolism in in vitro assays only provides limited metabolic competence, • available Ames assays use different doses and also the strains used are often not the same. It is known that the quantitative results in Ames assays vary from laboratory to laboratory and also intra-laboratory variations are not negligible [Honma et al. (2019)], • the four to five Salmonella and one E. Coli strains used in standard GLP Ames assays have different sensitivities and specificities for mutagenicity, • all standard Ames strains are alkyl transferase proficient and effectively repair alkylated guanine caused by small alkyl- N- nitrosamines. A publication by Wagner et al. (2012) used the alkyl-transferase deficient Salmonella strain YG7108, which is specifically sensitive for N- nitrosamine mutagenicity. The mutagenicity ranking for NDMA, NPIP, NMOR and NPYR was NDMA>NPIP>NMOR>NPYR, NDPHA was found not to be mutagenic. Ranking based on carcinogenic potency using the TD 50 of rat carcinogenicity studies listed in table 2.4.3-1 is however NDMA>NMOR>NPYR>NPIP>NDPHA. The same authors also developed a single cell comet assay version with specifically optimized S9 mix to enhance sensitivity to N- nitrosamines in Chinese hamster ovary (CHO) cells. With this assay the ranking of mutagenicity in CHO cells was NDMA>NPIP>NMOR, NPYR was found not to be mutagenic. This demonstrates that in vitro assays in bacteria like the Ames assay or tests in mammalian cells cannot be used as a quantitative surrogate for carcinogenic potency. They only might serve as a qualitative read out for a mutagenic potential. • published Ames data are highly variable in quality,

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