Letter to the Editor pubs.acs.org/OPRD
Potentially Mutagenic Impurities (PMIs): Optimizing Toxicological and Analytical Assessments
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In any case, methyl formate is nonalerting and has been shown to be nonmutagenic in multiple Ames’ assays.9 Formaldehyde is naturally present in polysaccarides such as gelling agents,10 and (inappropriate) use of the TTC limit would undoubtedly preclude the use of these materials in pharmaceuticals. A more serious concern over formaldehyde is its potential for API− adduct formation, various common excipients (such as lactose, Dmannitol, microcrystalline cellulose, low-substituted hydroxypropylcellulose, magnesium stearate, and light anhydrous silicic acid) having been shown to generate formaldehyde during storage of oral solid dosage forms.11 Phenol. Not structurally alerting; negative in multiple bacterial reverse mutation assays; equivocal positive in some mammalian-cell assays (not relevant to PMI status); noncarcinogenic in oral rodent lifetime bioassays;12 a recent assessment by EFSA (European Food Safety Authority)13 proposes a TDI (tolerable daily intake) of 25 mg (in a 50 kg patient). 1,2,4,5-Tetrafluorobenzoyl Chloride. Ames-negative 2-fluorobenzoyl chloride is considered to be a more appropriate model compound than benzoyl chloride whose mutagenic potential is considered to be equivocal14 (possibly related to use of DMSO as a solvent by some researchers). In addition, the latter is highly susceptible to hydrolytic degradation, having a half-life of 16 s at 25 °C.14 Fluorobenzoyl chlorides can be predicted to be even more rapidly hydrolyzed since the electron-withdrawing effect of multiple fluoro substituents will no doubt further destabilize the benzoyl cation, as has been shown with p-fluorobenzoyl chloride.15 3-Phenylpropanal. This is considered to be a suitable reference compound for the Boc-protected 3-amino analogue since an aliphatic amine group, as such or as a Boc-derivative, is nonalerting.16 It is agreed that the nonmutagenic status of Boc-S3-amino-3-phenylpropanal may need to be confirmed by experimental testing to gain acceptance by some sceptical regulatory reviewers. [3-Amino-3-phenylpropanal is highly likely to undergo Schiffbase formation, and so the amine group needs to be “masked” to obtain a stable molecule.] MIC (Methyl-2-(2-chloro-1-iminomethyl)hydrazine carboxylate). Most likely to be hydrolytically purged during synthetic work-up, probably forming methyl carbazate which is Ames’negative and noncarcinogenic. In Silico Assessment. In most, if not all cases, an expert assessment follow-up to any in silico prediction of mutagenicity is strongly advisible. Dobo et al., 201217 have reported that this approach can significantly improve the accuracy of negative predictions. Ensuring that the key reference compounds featured in the in silico output are truly relevant is another critical factor, and it is often possible to locate more appropriate reference compounds by literature searching. If a plausible positive prediction is obtained, as discussed below, many further steps
ear Editor:
In response to Raman and Prasad (10.1021/op500110n), I believe that it is important to consider the following two key interlinked elements when dealing with PMIs (potentially mutagenic impurities) in the context of assessing synthetic process capability and/or setting assay limits in APIs: · Toxicological classification · Analytical strategy.
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TOXICOLOGICAL CLASSIFICATION Regulatory Aspects. In ICH M7 (step 3)1 PMIs are classified into five categories, as shown below in Table 1. Table 1. ICH M7 classification scheme for PMIs class 1 2
3
4 5
definition
proposed action for control
known mutagenic carcinogens
control at or below compoundspecific acceptable level known bacterial mutagens with unknown control at or below acceptable carcinogenic potential (i.e. no rodent limits (generic or adjusted TTC) carcinogenicity data) alerting structure, unrelated to structure control at or below generic or of drug substance; no mutagenicity data adjusted TTC, or undertake bacterial mutagenicity assay. if nonmutagen = Class 5 if mutagen = Class 2 alerting structure, same alert as in drug treat as nonmutagenic impurity substance that tests as a nonmutagen no structural alerts, or alerting structure treat as nonmutagenic impurity with sufficient data to demonstrate lack of mutagenicity
Additional relevant considerations from ICH M7 are: · A mutagenic compound that is noncarcinogenic in an adequately conducted rodent bioassay is not associated with an increased cancer risk. · The TTC approach is inappropriate in various circumstances, for example: ◦ a PMI that is a confirmed metabolite of the drug substance; ◦ a PMI to which human exposure will be much greater from other sources such as food, or endogenous metabolism (e.g., formaldehyde); ◦ mutagenic impurities with evidence for a practical threshold. Compound-Specific Assessments. Taking account of the aspects of ICH M7 guidance shown above, some of the compounds mentioned by Raman et al. can be classified unequivocally as shown in Table 2. Further comments on formaldehyde and on other substances mentioned by Raman et al. are as follows: Formaldehyde. Linking formaldehyde with methyl formate formation is puzzling since there is no evidence for this in vivo and it seems highly unlikely to occur during chemical synthesis. © XXXX American Chemical Society
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Table 2. Toxicological Classification of Some PMIs Under Discussion
NOAEL = No Observed Adverse Effect Level; PDE = Permitted Daily Exposure [determined using ICH Q3C (R5) criteria].
Table 3. Toxicological Evaluation of Some Simple Haloalkanes
QT = qualification threshold from ICH Q3A (R2), or compound-specific limit.
structurally alerting intermediate or a mutagenic reagent) and/or can be designed to obtain an impurity profile of the API (or a key isolated intermediate). In the former case it seems strongly advisible to ensure that safety concerns over a tracked impurity are in fact valid before embarking on method development. In this respect, toxicological categorization of a structurally alerting
in the evaluation process are possible, often leading to a limit much greater than the default TTC of 1.5 μg/day.
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ANALYTICAL STRATEGY Analytical work-up of a synthetic route can track a particular actual or potential impurity that may be of concern (eg a B
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(if available), considering the indication (APIs for treating advanced cancer being outside the scope of ICH M7), applying the LTL (lifetime limit) principle, and evaluating dietary exposure via food (crotonaldehyde for example23). Making use of the full range of impurity qualification strategies impacting on process capability and/or API specification is considered infinitely preferable to the reductionist paradigm involving use of the TTC limit based on a structural alert and/or a single in silico assessment recommended by Raman et al. The cost of performing an Ames’ assay and/or an-depth toxicological review is likely to be much lower than that incurred by the (unnecessary) development of a highly sensitive assay combined with the possibility of having to operate a QC method to demonstrate long-term control. In some unusual cases where the maximum daily dose (MDD) of API is rather low (as in the case of an opthalmic product), it may not be necessary to undertake the above type of “triaging” activity for a structurally alerting impurity; assuming that the impurity is mutagenic may still yield an acceptable limit. For instance, a structurally alerting impurity in an API with MDD 2 mg would need to be controlled at ≤750 ppm for chronic (lifetime) treatment, and at ≤5000 ppm if treatment duration is ≤10 years. More generally, the ICH M7 LTL concept can be used to circumvent the need for toxicological qualification of impurities present at a level greater than the ICH Q3A (R2) qualification threshold in drug substances with low MDDs, Table 4
impurity using ICH M7 criteria and appropriate limits is recommended, from which several outcomes are possible: · Nonmutagenic impurity: treat as normal impurity; no pressing need for impurity tracking and probably no need for analytical method development; · Predicted mutagenic impurity using in silico evaluation: accept as mutagen if reference compounds are appropriate; otherwise consider Ames’ testing; if necessary, develop assay method with suitable limit of detection; · Mutagenic impurity (based on literature or in-house experimental data): ◦ TTC limit not appropriate (for example if carcinogenicity data available); develop assay method with suitable limit of detection (gas chromatography rather than GC/MS) ◦ TTC limit appropriate; highly sensitive GC/MS assay method possibly necessary
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· Effectively purged impurity; use of predicted18,19 or experimental purge factors to demonstrate absence of impurity, or reduction to a toxicologically insignificant level, in intermediate or drug substance; highly sensitive assay method probably needed to evaluate experimental purging, but no ongoing commitment to batch analysis.
DISCUSSION
Table 4. Highest maximum daily doses (MDDs) for impurity qualification at up to 0.30% using ICH M7 LTL criteria
The full title of ICH M7 - assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk - clearly shows that the main concern relates to the carcinogenic potential of DNA-reactive impurities. Mutagenic activity in a bacterial reverse mutation assay can be viewed as a provisional indication of carcinogenic potential. Although many bacterial mutagens test positive in rodent bioassays, a significant proportion do not, including, for example, methyl styryl ketone,20 sodium azide, allyl chloride, various phenolic aromatic amines and aromatic amines with sulfonic acid substituents.21 Even if a particular mutagenic impurity is a known carcinogen (ICH M7 Class 1) a compound-specific limit (based on linear extrapolation of carcinogenic potency data) rather than the TTC is applicable. And so it is considered axiomatic to check for the existence of carcinogenicity data on any mutagenic impurity before deciding on an appropriate limit. A formal acknowledgement of appropriate methodologies for determination of compound-specific limits seems likely to be available in due course as an annex to ICH M7 setting out definitive evaluations for common mutagenic reagents.22 Table 3 shows some simple haloalkanes which, although all structurally alerting, exhibit a remarkable diversity in toxicological properties. Several compounds, including 1,1-dichloroethane and 1-chlorobutane, are nonmutagens although they may well have been predicted to be potentially mutagenic. All of the mutagenic compounds listed can be evaluated on the basis of carcinogenicity data, for example the ICH Q3C Class 2 solvents dichloromethane and chloroform. Application of the TTC limit would be inappropriate in all cases. The examples shown above are considered to illustrate some of the many opportunities for avoiding imposition of the TTC limit for structurally alerting impurities in relation to demonstrating the absence of mutagenic potential as well as showing some of the pathways for limit-setting that are available even if the impurity is a bacterial mutagen. In relation to the latter, the main approaches are using carcinogenicity data
duration of exposure
LTL limit (μg/day)
highest MDD (mg) for applying 0.30% limit
0−1 month 1−12 months 1−10 years
120 20 10
40 6.7 3.3
showing the highest MDDs for qualifying impurities present at up to 0.30% (twice the qualification threshold).
David Snodin*
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Xiphora Biopharma Consulting, Bristol, U.K.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] Notes
The authors declare no competing financial interest.
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REFERENCES
(1) ICH M7 (step 3): http://www.ema.europa.eu/docs/en_GB/ document_library/Scientific_guideline/2013/02/WC500139217.pdf. (2) Carbadox. FDA Review: http://www.fda.gov/AnimalVeterinary/ Products/ApprovedAnimalDrugProducts/FOIADrugSummaries/ ucm064222.htm. (3) EPA AEGL on chloroformates: http://www.epa.gov/oppt/aegl/ pubs/chloroformates_interim.pdf. (4) Trobalt. EPAR: http://www.ema.europa.eu/docs/en_GB/ document_library/EPAR_-_Public_assessment_report/human/ 001245/WC500104839.pdf. (5) Endogenous formaldehyde turnover in humans compared with exogenous contribution from food sources. EFSA J. 2014, 12(2), 3550, http://www.efsa.europa.eu/en/efsajournal/doc/3550.pdf. (6) Formaldehyde. EPA IRIS Assessment: http://www.epa.gov/iris/ subst/0419.htm. C
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(7) 4-Chlorobutanoic acid chloride, BGRCI toxicological evaluation no. 163: http://www.bgrci.de/fileadmin/BGRCI/Downloads/DL_ Praevention/Fachwissen/Gefahrstoffe/TOXIKOLOGISCHE_ BEWERTUNGEN/Bewertungen/ToxBew163-E.pdf. (8) SIDS initial assessment profile; acid chloride category: http:// webnet.oecd.org/Hpv/ui/handler.axd?id=ed351f7c-0eb3-418f-8827b5986f74ddca. (9) CCRIS (Chemical Carcinogenesis Research Information System): http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?CCRIS. (10) EFSA Report on assessment of exposure to formaldehyde from the use of gelling additives. EFSA J. 2006, 415, 1−10, http://www.efsa. europa.eu/en/efsajournal/doc/415.pdf. (11) Fujita, M.; Ueda, T.; Handa, T. Generation of formaldehyde by pharmaceutical excipients and its absorption by meglumine. Chem. Pharm. Bull. (Tokyo) 2009, 57 (10), 1096−1099. (12) Environmental Health Criteria 161, Phenol: http://www.inchem. org/documents/ehc/ehc/ehc161.htm. (13) Scientific opinion on the toxicological evaluation of phenol. EFSA J. 2013, 11(4), 3189, http://www.efsa.europa.eu/en/efsajournal/doc/ 3189.pdf. (14) Benzoyl chloride. Toxnet: http://toxnet.nlm.nih.gov/cgi-bin/sis/ search/a?dbs+hsdb:@term+@DOCNO+383. (15) Bentley, T. W.; Harris, H. C. Solvolyses of Benzoyl Chlorides in Weakly Nucleophilic Media. Int. J. Mol. Sci. 2011, 12, 4805−4818, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179133/pdf/ijms12-04805.pdf. (16) Snodin, D. J.; McCrossen, S. D. Mutagenic impurities in pharmaceuticals: A critique of the derivation of the cancer TTC (Threshold of Toxicological Concern) and recommendations for structural-class-based limits. Regul. Toxicol. Pharmacol. 2013, 67 (2), 299−316. (17) Dobo, K. L.; et al. In silico methods combined with expert knowledge rule out mutagenic potential of pharmaceutical impurities: an industry survey. Regul. Toxicol. Pharmacol. 2012, 62 (3), 449−455. (18) Teasdale, A.; Fenner, S.; Ray, A.; Ford, A.; Phillips, A. A Tool for the Semi-quantitative Assessment of Potentially Genotoxic Impurity (PGI) Carryover into API Using Physicochemical Parameters and Process Conditions. Org. Process Res. Dev. 2010, 14, 943−945. (19) Teasdale, A.; Elder, D.; et al. Risk Assessment of Genotoxic Impurities in New Chemical Entities: Strategies to Demonstrate Control. Org. Process Res. Dev. 2013, 17, 221−230. (20) NTP. Toxicology and carcinogenesis studies of methyl transstyryl ketone (CAS no. 1896-62-4) in F344/N rats and B6C3F1 mice (feed and dermal studies). Natl. Toxicol. Program Tech. Rep. Ser. May; 2012, 572, 1−188. (21) Carcinogenic Potency Database, CPDB: http://toxnet.nlm.nih. gov/cpdb/. (22) Teasdale, A. Regulatory Highlights. Org. Process Res. Dev. 2014, 18, 468−472. (23) 3-Methyl-2-butenal. OECD SIDS: http://www.chem.unep.ch/ irptc/sids/OECDSIDS/107868.pdf. (24) ICH Q3C (R5) on residual solvent impurities: http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/ 2011/03/WC500104258.pdf. (25) 1-Chloro-1,1-difluoroethane, OECD SIDS: http://www.inchem. org/documents/sids/sids/75683.pdf. (26) Waskell, L. Lack of mutagenicity of two possible metabolites of halothane. Anesthesiology 1979, 50 (1), 9−12. (27) 1-Bromobutane mutagenicity. MHLW: http://anzeninfo.mhlw. go.jp/user/anzen/kag/pdf/B/B109-65-9.pdf. (28) 1-Bromobutane carcinogenicity in the mouse. MHLW: http:// anzeninfo.mhlw.go.jp/user/anzen/kag/pdf/gan/1-Bromobutane_ Mice.pdf.
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