Genotoxic Impurities: From Structural Alerts to Qualification

Abstract

The presence of an in cerebro structural alert in a potential or actual impurity, most likely arising as a byproduct or carriedover reagent or starting material, in a drug substance or drug product is merely an indication that the compound may be a DNAreactive genotoxin. The correlation between structural alerts for direct or indirect electrophilic characteristics and relevant biological activity is highly imperfect. For virtually all actual or potential impurities that are structurally alerting there is likely to be a variety of possibilities for clarifying their genotoxicity status based on published data, in silico assessments or de noWo testing in bacterial reverse mutation assays. Even for compounds that test positive a number of options are available to enable a compoundspecific qualification to be made involving the use of pre-existing toxicological data (particularly from lifetime rodent bioassays) and/ or using information from appropriate additional studies. A review of representative compounds from several classes of structurally alerting substances (epoxides, hydrazines, aromatic amines, halides and aldehydes) provides examples of different types of qualification strategies Overall, it seems prudent to obtain maximum "leverage" from toxicological approaches, which are likely to be relatively low cost, before making any significant process-related changes to an active pharmaceutical ingredient (API) synthesis.

Genotoxic Impurities in Pharmaceuticals

“…Hydralazine (1-hydrazinylphthalazine) and its hydrochloride salt are Ames-positive. In another study, 20 hydrazine-derivatives were found to induce a direct DNA damage in Escherichia coli and 16 of them (80%) were Ames positive as well (Flora et al, 1984; Agency for Toxic Substance and Disease Registry, 1997;Snodin, 2010).…”

“…The SN1 mechanism leads to O-alkylation (O-6-methylguanine) which is mutagenic but not clastogenic, whereas the SN2 mechanism leads to N-methylation which is clastogenic and not mutagenic. In this group, it seems that bromo compounds are more reactive as compared to chloro compounds (Sobol et al, 2007;Snodin, 2010). Various tests have been performed to study DNA damage and mutation in alkyl halides.…”

“…As chloro-and bromobenzene are not alkylating agents, these compounds are Ames-negative. In Yeast deletion recombination (DEL) and micronucleus assays, alkyl chlorides such as n-propyl chloride are found to be negative (Sobol et al, 2007;Snodin, 2010). It was observed that alkyl chlorides in the NBP [4-(p nitrobenzyl) pyridine] alkylation assay are not reactive and that allyl chloride has minimal activity.…”

“…According to in vivo tests, chloroethane and alkyl bromides seem to be nongenotoxic carcinogens rather than genotoxic carcinogens. Based on the available data, the United States environmental protection agency (USEPA), considers tert-butyl chloride to be a group D compound or ''not classifiable as to human carcinogenicity'' (Bercu et al, 2009;Snodin, 2010).…”

“…In addition, it seems that hydrazine reacts with endogenous formaldehyde to produce formaldehyde hydrazone. Subsequent to some other reactions, alkylating compounds like diazomethane as the genotoxic moiety are produced (Bercu et al, 2009;Snodin, 2010).…”