Determination of compound-specific acceptable daily intakes for 11 mutagenic carcinogens used in pharmaceutical synthesis Patricia Ellis a,⇑ , Michelle Kenyon b , Krista Dobo b a Kimberly-Clark, 40 Douglas House, London Rd, Reigate, Surrey RH2 9QP, UK b Pfizer Global Research and Development, Drug Safety Research and Development, Genetic Toxicology, Eastern Point Road, MS 8274/1317, Groton, CT 06340, USA article info Article history: Received 30 April 2012 Available online 7 December 2012 Keywords: Pharmaceuticals Genotoxic impurity Synthetic intermediate Mutagen Carcinogen Genetic safety Risk assessment Acceptable daily intake abstract The synthesis of pharmaceutical products often involves the use of reactive starting materials and inter- mediates. Low levels may be present in the final product as impurities and of particular concern are impurities that have mutagenic and carcinogenic potential. Regulatory guidance documents provide a general framework to minimise human exposure to these impurities; however, compound-specific rec- ommendations are limited. Our practical experience with 11 pharmaceutical impurities is presented. The genotoxicity and carcinogenicity data are summarised and the approach used to derive an acceptable daily intake (ADI) is described for each chemical. We have highlighted the considerations and challenges associated with calculating ADIs based on available carcinogenicity data. This may provide a useful ref- erence to others in the pharmaceutical industry regarding impurity control, where the weight of evidence indicates the chemical is a mutagenic carcinogen. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Starting materials and intermediates used to synthesise phar- maceuticals may be intrinsically reactive. It is this attribute that of- ten means they may also react with cellular components such as DNA and as a consequence may be mutagenic and carcinogenic. Following the synthetic process, starting materials and intermedi- ates may reside as impurities, often at low levels, in the final active pharmaceutical ingredient (API). It is widely accepted that their presence offers no benefit to the patient and, as such, diligence is required by pharmaceutical companies to limit human exposure to such impurities during clinical trials and from commercial prod- ucts. Regulatory authorities recognise the presence of impurities in the final API is unavoidable and consequently guidance related to the control of mutagenic and/or carcinogenic impurities has evolved considerably over the last decade. Historically, guidance was limited to the International Conference on Harmonisation (ICH) who adopted a number of quality documents intended to minimise the presence of impurities whilst maintaining patient safety (ICH Q3A(R2), 2006; ICH Q3B(R2), 2006; ICH Q3C(R4), 2009). However, none of these documents specifically addresses acceptable exposure limits for impurities that are known mutagens or carcinogens. Q3C recommends avoidance of extremely toxic or known carcinogenic solvents and describes levels considered to be toxicologically acceptable for some common residual solvents. Also, mathematical risk assessment models are presented for set- ting exposure limits in cases where reliable carcinogenicity data are available. A concentration limit for a known human carcinogen, benzene, is provided, but further compound-specific recommenda- tions are limited. More recently, the European Medicine Agency’s Committee for Medicinal Products for Human Use (CHMP) acknowledged this regulatory deficiency and published a guideline describing a general framework to manage the control of muta- genic impurities in new drug products (CHMP, 2006; CHMP Q&A(R3), 2010). The US Food and Drug Administration (FDA) fol- lowed shortly and issued draft guidance for industry on recom- mended approaches for control of mutagenic and carcinogenic impurities (FDA, 2008). Most recently, this topic has been adopted for development of an ICH guideline. Both guidance documents rely on several common principles to provide the basis for establishing appropriate exposure limits. First, both acknowledge that to determine acceptable exposure lev- els to mutagenic carcinogens, considerations of the dose–response relationship and possible mechanisms of action are important. Based on this, mutagenic impurities may be distinguished into two classes: (1) DNA-reactive (mutagenic) compounds with sufficient exper- imental evidence for a threshold-related mechanism and (2) DNA-reactive (mutagenic) without sufficient experimental evidence for a threshold-related mechanism. 0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2012.11.008 ⇑ Corresponding author. Fax: +44 1737 594849. E-mail address: [email protected](P. Ellis). Regulatory Toxicology and Pharmacology 65 (2013) 201–213 Contents lists available at SciVerse ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Transcript
Regulatory Toxicology and Pharmacology 65 (2013) 201–213
Contents lists available at SciVerse ScienceDirect
Determination of compound-specific acceptable daily intakes for 11 mutageniccarcinogens used in pharmaceutical synthesis
Patricia Ellis a,⇑, Michelle Kenyon b, Krista Dobo b
a Kimberly-Clark, 40 Douglas House, London Rd, Reigate, Surrey RH2 9QP, UKb Pfizer Global Research and Development, Drug Safety Research and Development, Genetic Toxicology, Eastern Point Road, MS 8274/1317, Groton, CT 06340, USA
a r t i c l e i n f o a b s t r a c t
Article history:Received 30 April 2012Available online 7 December 2012
The synthesis of pharmaceutical products often involves the use of reactive starting materials and inter-mediates. Low levels may be present in the final product as impurities and of particular concern areimpurities that have mutagenic and carcinogenic potential. Regulatory guidance documents provide ageneral framework to minimise human exposure to these impurities; however, compound-specific rec-ommendations are limited. Our practical experience with 11 pharmaceutical impurities is presented.The genotoxicity and carcinogenicity data are summarised and the approach used to derive an acceptabledaily intake (ADI) is described for each chemical. We have highlighted the considerations and challengesassociated with calculating ADIs based on available carcinogenicity data. This may provide a useful ref-erence to others in the pharmaceutical industry regarding impurity control, where the weight of evidenceindicates the chemical is a mutagenic carcinogen.
� 2012 Elsevier Inc. All rights reserved.
1. Introduction
Starting materials and intermediates used to synthesise phar-maceuticals may be intrinsically reactive. It is this attribute that of-ten means they may also react with cellular components such asDNA and as a consequence may be mutagenic and carcinogenic.Following the synthetic process, starting materials and intermedi-ates may reside as impurities, often at low levels, in the final activepharmaceutical ingredient (API). It is widely accepted that theirpresence offers no benefit to the patient and, as such, diligence isrequired by pharmaceutical companies to limit human exposureto such impurities during clinical trials and from commercial prod-ucts. Regulatory authorities recognise the presence of impurities inthe final API is unavoidable and consequently guidance related tothe control of mutagenic and/or carcinogenic impurities hasevolved considerably over the last decade. Historically, guidancewas limited to the International Conference on Harmonisation(ICH) who adopted a number of quality documents intended tominimise the presence of impurities whilst maintaining patientsafety (ICH Q3A(R2), 2006; ICH Q3B(R2), 2006; ICH Q3C(R4),2009). However, none of these documents specifically addressesacceptable exposure limits for impurities that are known mutagensor carcinogens. Q3C recommends avoidance of extremely toxic orknown carcinogenic solvents and describes levels considered to
ll rights reserved.
be toxicologically acceptable for some common residual solvents.Also, mathematical risk assessment models are presented for set-ting exposure limits in cases where reliable carcinogenicity dataare available. A concentration limit for a known human carcinogen,benzene, is provided, but further compound-specific recommenda-tions are limited. More recently, the European Medicine Agency’sCommittee for Medicinal Products for Human Use (CHMP)acknowledged this regulatory deficiency and published a guidelinedescribing a general framework to manage the control of muta-genic impurities in new drug products (CHMP, 2006; CHMPQ&A(R3), 2010). The US Food and Drug Administration (FDA) fol-lowed shortly and issued draft guidance for industry on recom-mended approaches for control of mutagenic and carcinogenicimpurities (FDA, 2008). Most recently, this topic has been adoptedfor development of an ICH guideline.
Both guidance documents rely on several common principles toprovide the basis for establishing appropriate exposure limits.First, both acknowledge that to determine acceptable exposure lev-els to mutagenic carcinogens, considerations of the dose–responserelationship and possible mechanisms of action are important.Based on this, mutagenic impurities may be distinguished intotwo classes:
(1) DNA-reactive (mutagenic) compounds with sufficient exper-imental evidence for a threshold-related mechanism and
(2) DNA-reactive (mutagenic) without sufficient experimentalevidence for a threshold-related mechanism.
202 P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213
This manuscript is focused on DNA-reactive compounds with-out sufficient experimental evidence for a threshold-related mech-anism. Therefore, DNA-reactive compounds with sufficientexperimental evidence for a threshold-related mechanism are notspecifically addressed. This topic is explored in another manuscriptcurrently in preparation. However, it is important to note thatthere is compelling experimental evidence which indicates thresh-old or sub-linear dose–response relationships exist for some muta-genic carcinogens (Dobo et al., 2011; Elhajouji et al., 2011; Gockeet al., 2009; Johnson et al., 2009; Pottenger and Gollapudi, 2010).
For mutagenic chemicals where the carcinogenic potential isknown, a compound-specific risk assessment should be the firstconsideration for determining an ADI (CHMP, 2006; FDA, 2008;Müller et al., 2006). There are numerous methods available to cal-culate compound-specific ADI values for mutagens with no thresh-old. Neither the CHMP (2006) nor FDA (2008) provides guidance ona particular technique. One cautious approach considers thetumorigenic dose evaluated in long-term cancer bioassays fromthe most sensitive species and sex. Linear extrapolation is madefrom this dose to a dose level which attains an acceptable excesscancer risk in humans.
Based on a similar principle, the Threshold of Toxicological Con-cern (TTC) was proposed as an acceptable daily intake for com-pounds with unknown carcinogenic potential (Kroes et al., 2004).The TTC is recognised to be a very conservative limit, as numerous‘worst case’ assumptions were applied to >700 carcinogens in theCarcinogenic Potency Database (CPDB) to establish the limit (Del-aney, 2007; Kroes et al., 2004). The assumptions were as follows:
(i) Establishment of dose giving 50% tumour incidence in car-cinogenicity studies (TD50) using data from the most sensi-tive species and most sensitive site (Cheeseman et al., 1999).
(ii) Use of a select subset of the CPDB which had adequate esti-mates of TD50 following oral dosages.
(iii) Simple linear extrapolation from TD50 to a one in 1,000,000incidence (daily human exposure level below which there isconsidered negligible risk to human health).
(iv) All biological processes involved in the generation oftumours at high dosages are linear over a 500,000-fold rangeof extrapolation.
(v) Possible effects of cytoprotective, DNA repair, apoptotic andcell cycle control processes are not taken into account.
As pharmaceuticals offer a benefit to patients, an acceptablecancer risk level in humans associated with exposure to a muta-genic impurity is defined as an exposure resulting in a maximumexcess cancer risk of one in 100,000 in a 70 year lifetime and ispragmatically considered as ‘virtually no risk’ to humans (CHMP,2006; Müller et al., 2006). Where the carcinogenic potential is un-known, both the EMEA and FDA recommend a TTC limit of 1.5 lg/day for lifetime exposure to a mutagenic impurity residing in API,for all but a highly potent subset of chemical classes.
During clinical trials, a key concept termed ‘staged TTC’ estab-lishes higher ADIs for impurities based upon duration of exposure(CHMP, 2006; FDA, 2008; Müller et al., 2006). Although higher lim-its for shorter exposure durations is important, this current publi-cation focuses on establishing control limits for mutageniccarcinogens in marketing applications for new chronic use drugproducts. Therefore, lifetime exposure is assumed, and consider-ation is not given to control of impurities during the clinical stageof drug development.
In this manuscript, the approach and practical experience fromcalculating compound-specific ADIs for 11 pharmaceutical impuri-ties is presented. In addition, the limitations and challenges associ-ated with relying on the available carcinogenicity data is discussed.
Given that the compounds evaluated are likely to be commonlyused for drug substance synthesis across pharmaceutical compa-nies, these cases may serve as a useful reference for industry.
2. Methods
The genotoxicity and carcinogenicity data for each chemicalwas reviewed and is summarised in Tables 1 and 2, respectively.Complete and consistent data sets were not available for all chem-icals. Where conflicting genotoxicity data existed, assessmentsconducted by organisations such as International Agency for Re-search on Cancer (IARC), National Toxicology Program (NTP), Uni-ted States Environmental Protection Agency (US EPA), ScientificCommittee on Occupational Exposure Limits (SCOEL), and theWorld Health Organisation (WHO) International Programme onChemical Safety (IPCS) and Concise International Chemical Assess-ment Documents (CICAD) were relied upon.
The Carcinogenic Potency Database (Gold and Zeiger, 1997) wasthe primary resource for carcinogenicity data. However, other dat-abases (INCHEM, http://www.inchem.org/; TOXNET, http://tox-net.nlm.nih.gov/; Expub, http://www.expub.com/) and literaturewere searched for more recent or supplementary information. Inaddition, experimental evidence available in the literature on po-tential mode of action was considered and is included in the‘‘Mechanism’’ column of Table 2.
Our approach was to evaluate existing genotoxicity and carcin-ogenicity data to determine whether each chemical was a muta-genic carcinogen or a non-genotoxic carcinogen. Next, based on areview of scientific literature available on the mechanism of actionof the chemical or structurally-related chemicals, the dose re-sponse curve was classified as threshold or non-threshold. If thecarcinogenic mechanism was unknown, or there was insufficientdata to support a threshold dose–response relationship, then com-pounds were assumed to demonstrate a linear dose response.
Within the context of the regulatory guidance on genotoxicimpurities in pharmaceuticals, DNA-reactive compounds are re-garded to be potentially trans-species and multi-organ carcino-gens. Since direct DNA reactivity is of high concern, the primaryendpoint for defining an impurity as genotoxic is mutagenicity(CHMP, 2006, 2010; FDA, 2008). The Ames assay is a sensitive as-say for mutagen detection and is one of the most common testsused for identifying DNA-reactivity of pharmaceutical impurities(Kenyon et al., 2007; Müller et al., 2006). The authors acknowledgethat no single mutagenicity test is able to detect the entire spec-trum of induced mutagenic events (US EPA, 2007) and for alterna-tive human health assessments other genotoxicity endpoints maybe more relevant. Nevertheless, the result of the Ames assay is gen-erally considered acceptable to determine the DNA-reactivity ofimpurities (CHMP, 2006, 2010; FDA, 2008). Therefore, for the pur-poses of determining whether a compound was to be classified asgenotoxic, mutagenicity results were generally utilised unlessthere was a reason to consider a compound to be DNA-reactivein the absence of a positive Ames test (e.g. the test system doesnot have appropriate metabolic components to generate a muta-genic metabolite).
ADI values were calculated using linear extrapolation from theTD50 of the most sensitive species or the harmonic mean TD50 forthe most sensitive species when there was more than one positivestudy. The TD50 provides a standard quantitative measure for com-parisons and analyses of carcinogenicity studies (Peto et al., 1984).It is a numerical description of carcinogenic potency and is esti-mated for each set of tumour incidence data reported in the CPDB(Gold and Zeiger, 1997). The TD50 is defined as dose-rate in mg/kgbody weight/day which, if administered chronically for thestandard lifespan of the species, will halve the probability of
CA – chromosome aberrations; MLA – mouse lymphoma assay; MN – micronuclei; UDS – unscheduled DNA synthesis; GM – gene mutation.1Chemical Carcinogenesis Research Information System (CCRIS).2IPCS Environmental Health Monographs 68 (1987).3Agency for Toxic Substances and Disease Registry (ATSDR), 1989, 1997.4US EPA GENE-TOX.5IPCS Environmental Health Monographs 157 (1994).
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204 P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213
remaining tumourless throughout that period. The harmonic meanTD50 includes the most sensitive endpoint for all positive studiesfor a given species. In contrast with the derivation of the TTC,where 60 kg was used for human weight adjustment (Kroeset al., 2004), a 50 kg weight adjustment was used for calculatingADIs in the cases presented here. This conservative assumption isconsistent with ICH Q3C, where 50 kg is used to derive permitteddaily exposures for some common solvents (ICH Q3C(R4), 2009).Finally, in line with the TTC for pharmaceutical impurities, cancerrisk at a probability of 1/100,000 was applied to these com-pound-specific ADI calculations (CHMP, 2006; FDA, 2008; Mülleret al., 2006).
ADI ðlg=dayÞ ¼ ðTD50 mg=kg=day�weight adjustmentÞ=50;000
¼ TD50 mg=day� 10�3lg=mg
3. Results
3.1. Hydrazine
Hydrazine is genotoxic in vitro (bacterial mutagenicity, chro-mosome aberrations, mammalian mutagenicity and unscheduledDNA synthesis) but deemed not genotoxic in mice (micronucleus,intraperitoneal (i.p.) administration and unscheduled DNA synthe-sis in sperm cells) (Table 1). Hydrazine is negative in two mousein vivo micronucleus studies following i.p. administration but po-sitive in another study in mice by the same route. InternationalProgramme on Chemical Safety (IPCS, 1987) concluded that hydra-zine is not genotoxic in vivo in higher eukaryotes. Alkylation of li-ver DNA is reported in rats acutely exposed to hydrazine for 1–3 days (ATSDR, 1997), indicating hydrazine may be genotoxic viathe oral route (Table 1); however there are no oral in vivo genotox-icity studies available to substantiate this result.
There are seven carcinogenicity reports cited in the CPDB (Goldand Zeiger, 1997), one in hamsters, four in mice and two in rats. Inthree of the seven reports, the carcinogenicity studies are limitedin that there is only a single dose group of animals administeredtest article and in one case there was also less than 50 animals inthe treated group. One mouse study (Bor:NMRI, SPF-bred NMRI),in which hydrazine was administered in the drinking water is re-ported negative. In an inhalation study in female C57BL/6 mice,lung tumours were observed in 3% (12/400) of treated animals ver-sus 1% of controls (4/400); however, an opinion of the author onstudy outcome is not reported. Hydrazine is deemed to be carcin-ogenic in the remaining five studies. Via the inhalation route,hydrazine induced large intestine, nasal cavity, stomach and thy-roid gland tumours in male Syrian Golden hamsters and nasal cav-ity and lung tumours in male and female Fischer 344 rats with thenasal cavity being the most sensitive tumour site in both species inthese studies. Two oral Swiss CD-1 mouse studies are reported po-sitive with the induction of lung tumours in females treated by ga-vage and in both females and males treated with hydrazine indrinking water. Hydrazine also induces liver tumours in maleand female Wistar rats when administered in drinking water.Based on the available data, rats appear to be the most sensitivespecies with a harmonic mean TD50 of 0.613 mg/kg/day, comparedto 4.16 and 2.93 mg/kg/day for hamster (no harmonic mean; singlestudy) and mice (harmonic mean), respectively.
The toxicity of hydrazine is attributed to the generation of reac-tive intermediates. Hydrazine is catalysed by cytochrome P450-dependant mixed function oxidases and the flavin monooxygena-ses to carbocations, carbon-centered radicals and reactive oxygenspecies (ROS) (Choudhary and Hansen, 1998). These intermediateshave been reported to alkylate DNA (Kovacic and Jacintho, 2001;Snodin, 2010; Benigni and Bossa, 2011). Overall, the weight of
Table 2Summary of carcinogenicity data and ADI for 11 mutagenic carcinogens with a non-threshold mechanism of action. ADI calculations are based on linear extrapolation from TD50 to 1/100,000 risk level for a 50 kg human.
Chemical Name Carcinogenicity summarya Mechanism ADI calculation
Species Target organ Route IARC group TD50 in mg/kg/day (species) lg/day
Hydrazine Hamster Lgi, nas, sto, thy Inh 2B Alkylating agentGamberini et al. (1998), Kovacic and Jacintho (2001), Benigni and Bossa (2011)
0.613 (rat)b,c 0.613Mouse Lun Inh,
Gav,Wat
Rat Lun, nas InhLiv Wat
2-Hydroxyethylhydrazine Mouse Liv Wat,Oral
NC Alkylating agent (based on hydrazine functional group) 0.397(Mouse)b
0.397
Monoacetyl hydrazine Mouse Lun Gav NC Alkylating agent (based on hydrazine functional group) 9.85(Mouse)b
9.85
Hydroquinone Mouse Liv Gav 3 Alkylating agentGaskell et al. (2004, 2005), Benigni and Bossa (2011)
82.8 (Rat)b 82.8Liv, kid Feed
Rat Kid, hmoKid
Feed, Gav
p-Quinone dioxime Rat Ubl Feed 3 Alkylating agent (based on quinone functional group) 106 (Rat) 106o-Nitrotoluene Mouse Lgi, liv, vsc Feed 3 Aminoaryl DNA adduct forming
Jones et al. (2003), Benigni and Bossa (2011)4.66 (Rat)b,c 4.66
Rat Liv, lun, mgl, per, ski, sub Feedp-Nitrotoluene Mouse Lun Feed 3 Aminoaryl DNA adduct forming
Benigni and Bossa (2011)257 (Rat) 257
Rat Cli FeedBis-(chloromethyl)ether Mice Per i.p. 1 Alkylating agent
Van Duuren et al. (1975), Van Duuren (1988)0.00357 (Rat) 0.00357
Lun InhRat Lun, nas Inh
2-Chloro-1,1,1-trifluoroethane
Rat Tes, ute Gav 3 Indirect-acting alkylating agentSalmon et al. (1981), Guengerich (1991), Benigni and Bossa (2011)
87.3 (Rat)b 87.3
Acrylonitrile Mice Ezy, for, hag, lun, ova Gav 2B Oxidative pathway of metabolism critical in genotoxicity and carcinogenicityWHO (2002)
6.32 (Mouse)b 6.32Rat Ezy, for, nrv, orc, smi, sto Wat
Ezy, mgl, nas, nrv, orc, smi Inhp-Chloroaniline HCl* Mouse Liv, vsc Gav 2B Mechanism of action of splenic tumours unknown WHO (2003)
Generally aromatic amines are indirect aminoaryl DNA adduct forming.7.62 (Rat) 7.62
Rat Spl (rare tumor) Gav
TD50 – Dose (mg/kg/day) resulting in tumours in 50% of animals that would remain tumour-free in the absence of compound.Target organs: Cli – Clitoral gland; Ezy – Ear/Zymbal gland; For – Forestomach; Hmo – Hematopoetic system; Kid – Kidney; Lgi – Large intestine; Liv – Liver; Lun – Lung; Mgl – Mammary gland; Nas – Nasal cavity; Nrv – Nervoussystem; Orc – Oral cavity; Per – Peritoneum; Ski – Skin; Spl – Spleen; Smi – Small intestine; Sto – Stomach; Sub – Subcutaneous tissue; Tes – Testis; Thy – Thymus; Ubl – Urinary bladder; Ute – Uterus; Vsc – Vascular system.Route: Gav – Oral gavage; Inh – Inhalation; i.p. – Intraperitoneal injection; Wat – Drinking water.IARC Group Definitions: Group 1 – Carcinogenic to humans; Group 2A – Probably carcinogenic to humans; Group 2B – Possibly carcinogenic to humans; Group 3 – Not classifiable as to its carcinogenicity to humans.NC – Not classified.
a Unless otherwise noted, data summary is from CPDB (Gold and Zeiger, 1997).b Harmonic mean using the TD50 value from the most potent target site in each positive experiment.c Variation is greater than ten-fold among statistically significant TD50 values from different positive experiments.
* p-Chloroaniline is not carcinogenic in rats or mice when administered in the diet. This information is based on p-chloroaniline HCl (CAS 20265-96-7).
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evidence suggests that hydrazine is a DNA-reactive carcinogenwith a non-threshold mode of action.
The US EPA (04/01/1991) calculated an oral slope factor of 3.0mg/kg/day and a 1/100,000 risk level drinking water limit of0.1 lg/L, based on the occurrence of hepatomas in a multi-dose ga-vage study where hydrazine sulfate was administered to mice for25 weeks with animals observed throughout their lifetime. Thisequates to a daily dose of �0.2 lg/day for a 50 kg human. Basedon the occurrence of nasal tumors in rats observed in a study byMacEwen et al. (1981), the US EPA (04/01/1991) also calculatedan acceptable daily hydrazine inhalation dose of 2.0 E�3 lg/m3 atthe 1/100,000 risk level. This equates to a daily dose of 0.056 lg,based on an average daily air intake of 28,000 L.
Based on linear extrapolation from the rat harmonic mean TD50,we calculated a compound-specific ADI of 0.613 lg/day (Table 2).This ADI was 2.4 times lower than the TTC but �3–11 times higherthan the oral and inhalation values, respectively, calculated by theEPA at the same risk level.
3.2. 2-Hydroxyethylhydrazine
2-Hydroxyethylhydrazine is reported to be mutagenic to bacte-ria (Table 1). No additional genotoxicity data are available. Thereare three carcinogenicity reports cited in the CPDB (Gold and Zei-ger, 1997), one in hamsters and two in mice. All three carcinoge-nicity studies are limited as there is only a single dose group ofanimals administered test article and in some cases there are lessthan 50 animals per dose group. The Syrian Golden hamster studyand one of the two Swiss mice studies, in which 2-hydrox-yethlhydrazine was administered via drinking water, are negative.However, 2-hydroxyethylhydrazine induced liver tumours in maleB6C3F1 mice following oral administration. Based on the observa-tion of liver tumours in mice, the harmonic mean TD50 is definedas 0.397 mg/kg/day (Gold and Zeiger, 1997). Given that 2-hydroxy-ethylhydrazine contains the same functional group as hydrazine;its toxicity is likely to be mediated through a similar mechanism.Therefore, a conservative assumption was made that 2-hydroxy-ethylhydrazine is a DNA-reactive carcinogen with no thresholdfor carcinogenicity. A compound-specific ADI of 0.397 lg/day wasderived by linear extrapolation from the mouse harmonic meanTD50 (Table 2). The ADI was nearly 4-fold below the TTC. Therewere no relevant allowable limits for 2-hydroxyethylhydrazineidentified elsewhere for comparison to the limit calculated here.
3.3. Monoacetyl hydrazine
Consistent with hydrazine and 2-hydroxyethylhydrazine,monoacetyl hydrazine is mutagenic to bacteria. In addition, thereis one unconfirmed positive in vivo micronucleus study (Table 1).There is also only one carcinogenicity report cited in the CPDBwhere Swiss mice were treated via the oral gavage route of admin-istration (Gold and Zeiger, 1997). The study design is limited inthat only one or two dose groups received test article, and thereare less than 50 animals per dose group. Lung tumours are re-ported in both male and female mice, and the harmonic meanTD50 is defined as 9.85 mg/kg/day (Gold and Zeiger, 1997). Basedon the presence of the hydrazine functional group, it is reasonableto assume that monoacetyl hydrazine exerts mutagenic and carcin-ogenic activity through the same mechanism as hydrazine. There-fore, a conservative assumption was made that monoacetylhydrazine is a DNA-reactive carcinogen with no threshold for car-cinogenicity. An ADI of 9.85 lg/day was calculated by linearextrapolation from the mouse harmonic mean TD50 (Table 2). Thiswas approximately 6-fold above the TTC. There were no relevantallowable limits for monoacetyl hydrazine identified elsewherefor comparison to the limit calculated here.
3.4. Hydroquinone and p-quinone dioxime
Hydroquinone has shown both positive and negative results inbacterial mutagenicity tests. Hydroquinone is genotoxic in vitro(chromosome aberrations, mammalian mutagenicity, micronucleiand unscheduled DNA synthesis) and in vivo (chromosome aberra-tions). Conflicting results are reported for the micronucleus end-point in vivo (Table 1). In most of the negative in vivomicronucleus studies, the route of administration is i.p. injection.In contrast, a study in which hydroquinone is given by subcutaneousinjection reports a significant increase in micronucleus induction(Tunek et al., 1982). Additional conflicting in vivo micronucleus re-sults are reported in two studies using oral routes of administration.In one study, a significant but weak response is reported followingoral gavage dosing (Ciranni et al., 1988), whereas no effect on micro-nucleus induction is seen following dietary administration for6 days in another study (O’Donoghue et al., 1999). IARC (1999)concludes that hydroquinone is mutagenic in a number of in vitrosystems and induces structural chromosome aberrations in mousebone-marrow cells following i.p. injection. However, the IPCS(1994) concludes that, as the genotoxic effects are only observedfollowing parenteral or in vitro exposure, therefore the relevanceto human risk is unclear (EHC 157). There are two carcinogenicityreports cited in the CPDB (Gold and Zeiger, 1997) in which hydroqui-none is evaluated in rats and mice. The carcinogenicity studies arelimited in that only one or two dose groups are administered testarticle, and in some cases there are less than 50 animals per dosegroup. Hydroquinone induces liver tumours in B6C3F1 mice follow-ing administration by oral gavage, and liver and kidney tumoursfollowing in feed administration. Hydroquinone also induces hema-topoietic and/or kidney tumours in Fisher 344 and F344/DuCrj ratswhen administered by oral gavage or in feed, respectively. Basedon the available data, rats appear to be the more sensitive specieswith a harmonic mean TD50 of 82.8 mg/kg/day, compared to225 mg/kg/day in mice (Gold and Zeiger, 1997).
Both genotoxic and non-genotoxic mechanisms of action havebeen proposed to be responsible for the carcinogenic potential ofhydroquinone. In vitro, hydroquinone can undergo oxidative pro-cesses giving rise to ROS and also quinone and/or semi-quinonemetabolites that can interact with proteins involved in microtu-bule assembly, spindle formation (Dobo and Eastmond, 1994), hu-man topoisomerase II (Franz et al., 1996) and, by virtue of theMichael acceptor functional group, directly alkylate DNA (Gaskellet al., 2004, 2005; Benigni and Bossa, 2011). In addition, hydroqui-none is an established metabolite of benzene, classified as a Group1 human carcinogen (IARC, 1987). This may explain why the i.p.injection route of administration has been used in the majority ofthe in vivo genotoxicity studies. The pattern of human exposuresuggests that dermal, inhalation, and oral (dietary) routes ofadministration of hydroquinone are more relevant experimentalexposures and as the i.p. route of administration avoids first passmetabolism, the significance of the in vivo genotoxicity findingsfor human risk assessment is questioned (IPCS, 1994; McGregor,2007). Given the low power of the carcinogenicity studies andthe fact a genotoxic mode of action cannot be ruled out, a conser-vative assumption was made that hydroquinone is a DNA-reactivecarcinogen with no threshold. A compound-specific ADI of 82.8 lg/day was calculated by linear extrapolation from the rat harmonicmean TD50 (Table 2). This was approximately 55-fold above theTTC. There were no relevant allowable limits for hydroquinoneidentified elsewhere for comparison to the limit calculated here.
p-Quinone dioxime is genotoxic in vitro (bacterial and mamma-lian mutagenicity, chromosome aberrations) but did not induceunscheduled DNA synthesis in vivo (Table 1). No additionalin vivo genotoxicity data are available. There is one carcinogenicityreport cited in the CPDB (Gold and Zeiger, 1997) in which
P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213 207
p-quinone dioxime was evaluated in rats and mice. The carcinoge-nicity studies are limited in that there are only two dose groupsadministered test article, and less than 50 animals are included inthe control groups. Following in feed administration p-quinonedioxime is not carcinogenic in B6C3F1 mice. However, urinary blad-der tumours are observed in F344 rats, with a TD50 of 106 mg/kg/day (Gold and Zeiger, 1997; Table 2). Similar to hydroquinone, p-quinone dioxime also contains a Michael acceptor functional groupand DNA adducts have been reported, substantiating that DNAalkylation is a potential mechanism (Gaskell et al., 2004, 2005).Therefore, a conservative assumption was made that p-quinonedioxime is a DNA-reactive carcinogen with no threshold. The ADIcalculated by linear extrapolation from the TD50 equated to106 lg/day (Table 2); approximately 71-fold higher than the TTC.There were no relevant allowable limits for p-quinone dioximeidentified elsewhere for comparison to the limit calculated here.
3.5. o-Nitrotoluene and p-nitrotoluene
The genotoxicity profile for o-nitrotoluene is well establishedwith genotoxicity data available for most in vitro and in vivo end-points. o-nitrotoluene is not mutagenic in bacteria but inducesmicronuclei, and unscheduled DNA synthesis in vitro. Conversely,o-nitrotoluene does not induce chromosome aberrations in vitro.Micronuclei are not induced in vivo; however consistent with thein vitro findings, increases in unscheduled DNA synthesis are ob-served (Table 1). There is one carcinogenicity report cited in theCPDB (Gold and Zeiger, 1997) in which o-nitrotoluene was admin-istered to rats and mice in feed. In this report, three test articledose groups are evaluated, with greater than 50 animals includedper group. o-nitrotoluene induces tumours at multiple sites inmale and female B6C3F1 mice and F344 rats (Table 2). Based onthe available data, rats appear to be the more sensitive species witha harmonic mean TD50 of 4.66 mg/kg/day, compared to 128 mg/kg/day in mice (Gold and Zeiger, 1997).
Aromatic-nitro groups require metabolism and enzymaticesterification to produce electrophilic nitrenium ions (Rickert,1987; Benigni and Bossa, 2011). These in turn covalently bind toDNA, forming adducts. Jones et al. (2003) showed that when ratsare administered o-nitrotoluene in drinking water, deoxyguanineand deoxyadenine adducts form in a dose-dependent manner.Futhermore, Doolittle et al. (1983) demonstrated that intestinalbacteria are important in the bioactivation of o-nitrotoluene. Thespecific nitroreductase enzymes required to activate o-nitrotolu-ene may be limited or absent in the Ames test system. Despitethe lack of mutagenicity with o-nitrotoluene, there is evidence thatthe aromatic-nitro moiety is DNA-reactive as structurally similarnitrotoluenes exhibit mutagenicity (2,3-dimethylnitrobenzeneand 2,4-dimethylnitrobenzene, unpublished data; 1,4-dimethyl-2-nitrobenzene; CCRIS). Overall, the weight-of-evidence suggeststhat o-nitrotoluene is a DNA-reactive carcinogen and a conserva-tive assumption was made that there is no threshold for this effect.Therefore, a compound-specific ADI was calculated using linearextrapolation from the rat harmonic mean TD50. The resultantADI, 4.66 lg/day (Table 2), was approximately 3-fold higher thanthe TTC. There were no relevant allowable limits for o-nitrotolueneidentified elsewhere for comparison to the limit calculated here.
p-nitrotoluene is not mutagenic but does induce chromosomeaberrations and mammalian mutagenicity in vitro. Conversely, in-creases in micronuclei or unscheduled DNA synthesis are not ob-served in vitro. p-nitrotoluene is not genotoxic in vivo(micronuclei and unscheduled DNA synthesis; Table 1). There isone carcinogenicity report cited in the CPDB (Gold and Zeiger,1997) in which p-nitrotoluene was administered to rats and micein feed. In this report, three test article dose groups are evaluated,with greater than 50 animals included per group. p-nitrotoluene
induces equivocal evidence of carcinogenicity in B6C3F1 male micebased on an elevated incidence of lung tumours. In F344 rats, p-nitrotoluene induces clitoral gland tumours, with a TD50 of257 mg/kg/day (Gold and Zeiger, 1997; Table 2).
p-Nitrotoluene also contains the aromatic-nitro functionalgroup and given the structural similarity to o-nitrotoluene, a con-servative assumption was made that p-nitrotoluene is a DNA-reac-tive carcinogen with no threshold. An ADI of 257 lg/day wascalculated by linear extrapolation from the rat TD50 (Table 2).There were no relevant allowable limits for p-nitrotoluene identi-fied elsewhere for comparison to the limit calculated here.
3.6. Bis-(chloromethyl)ether
Bis-(chloromethyl)ether is mutagenic in vitro and inducesunscheduled DNA synthesis in vivo (Table 1). Increases in chromo-some aberrations are not induced in vivo (Table 1). There are twocarcinogenicity studies reported in the CPDB (Gold and Zeiger,1997). The routes of administration are inhalation and i.p. injec-tion. There are no oral carcinogenicity studies reported in theCPDB. The i.p. study is limited in that a single test article dosegroup is evaluated and there are only 30 animals per dose group.In the inhalation study, there are three test article dose groupsand over 100 animals per dose group. Via the i.p. route of admin-istration, bis-(chloromethyl)ether induces peritonium tumours inHa/ICR mice. Following inhalation exposure, lung tumours are ob-served in Ha/ICR mice and lung and nasal tumours are observed inSprague–Dawley Spartan rats. Based on these data, the TD50 valuesare 0.00357 and 0.182 mg/kg/day for rats and mice, respectively(Gold and Zeiger, 1997). It is also important to note that IARChas classified bis-(chloromethyl)ether as carcinogenic to humans,based on the observation of increased risk for lung cancer fromoccupational exposure (IARC, 1987).
The carcinogenic mechanism of bis-(chloromethyl)ether is re-lated to the formation of two species, the carbonium and oxoniumions. Resonance between these two electrophilic species results instabilization, providing the opportunity to react with DNA (VanDuuren, 1988). Based on the available genotoxicity and carcinoge-nicity data, it is reasonable to conclude that bis-(chloro-methyl)ether is a potent DNA-reactive carcinogen via theinhalation route of exposure. A compound-specific ADI of0.00357 lg/day was derived by linear extrapolation from the ratTD50 (Table 2). This is approximately 420-fold lower than the TTC.
The US EPA (01/01/1991a) has calculated an oral slope factor of2.2E2/mg/kg/day from which they have derived a drinking waterlimit of 1.6 E�3 lg/L at the 1/100,000 risk level. This equates to adaily dose of 0.0023 lg/day for a 50 kg human or 0.0032 lg/daybased on average daily drinking water consumption of 2 L. TheADI calculated by linear extrapolation from the TD50 (0.00357 lg/day) is in agreement with the previously published limit, whichwas also derived from inhalation carcinogenicity data.
3.7. 2-Chloro-1,1,1-trifluoroethane
2-Chloro-1,1,1-trifluoroethane is not mutagenic to bacteria andno other in vitro or in vivo genotoxicity data are available (Table 1).There is one carcinogenicity report cited in the CPDB (Gold and Zei-ger, 1997) in which 2-chloro-1,1,1-trifluoroethane was evaluatedin Alpk/Ap rats via oral gavage route of administration. The carcin-ogenicity study is limited in that there is only one dose groupadministered test article, and less than 50 animals are includedin the treatment group. In rat, 2-chloro-1,1,1-trifluoroethane in-duces uterine and testicular tumours, with a harmonic meanTD50 of 87.3 mg/kg/day (Gold and Zeiger, 1997).
Polyhalogenated alkanes exert adverse toxicity followingactivation by cytochrome P450 enzymes to highly reactive
NAcOH
NH2NAcHAcetyl CoA
cytochrome P450s
NHOH
NH
OAcN
OAc
AcN
Ac
OSO 3
trans-acetylases
Electrophilic metabolites
Covalent binding to DNA
Toxic effect (mutation and/or cancer)
Fig. 1. Metabolic pathway for aromatic amines.
208 P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213
electrophilic species which in turn alkylate DNA (Salmon et al.,1981; Guengerich, 1991; Benigni and Bossa, 2011). Yin et al.(1995) studied the fate of 2-chloro-1,1,1-trifluoroethane in ratsand showed that it undergoes oxidative metabolism catalysed bycytochrome P4502E1 leading to the generation of a halohydrinintermediate (CF3CHClOH), which eliminates hydrogen chlorideto afford trifluoroacetaldehyde. It is this electrophilic species thatmay react with DNA. In standard in vitro genotoxicity testing, aninduced rat liver S9 fraction is included as a metabolic activationsystem. Guengerich et al. (1982) analysed the complement of ratliver cytochrome P450 enzymes in uninduced and Aroclor-1254 in-duced rat liver, and cytochrome P4502E1 was not identified. Thecritical enzymes required to bioactivate 2-chloro-1,1,1-trifluoroe-thane may be absent in S9-mix, which may explain the lack ofmutagenicity observed in the Ames assay (Ku et al., 2007; Waskell,1979). Based the knowledge of the metabolic pathway, a conserva-tive assumption was made that 2-chloro-1,1,1-trifluoroethane isan alkylating agent and therefore a DNA-reactive carcinogen withno threshold. A compound-specific ADI of 87.3 lg/day was calcu-lated by linear extrapolation from the rat harmonic mean TD50(Table 2). This is approximately 58-fold higher than the TTC. Therewere no relevant allowable limits for 2-chloro-1,1,1-trifluoroe-thane identified elsewhere for comparison to the limit calculatedhere.
3.8. Acrylonitrile
The genotoxicity of acrylonitrile has been extensively investi-gated. It is mutagenic in the Ames assay and also produces evidenceof genotoxicity in other in vitro assays (chromosome aberrations,mammalian mutagenicity and gene mutation; Table 1). In vitro re-sults of unscheduled DNA synthesis are mixed but more commonlynegative in a range of cell types from rats and humans (WHO, 2002).Acrylonitrile is not genotoxic in vivo (micronuclei and chromosomeaberrations; Table 1), although there is no indication whether thecompound reaches the target site in the published accounts forthree of the four studies (WHO, 2002). There are five carcinogenic-ity studies reported in the CPDB (Gold and Zeiger, 1997). Therobustness of the study designs vary; in some cases three test arti-cle treated groups are evaluated with at least 50 animals per dosegroup, and in other cases only one or two dose groups are adminis-tered test article, with less than 50 animals per dose group. Acrylo-nitrile is carcinogenic in B6C3F1 mice following administration byoral gavage with tumours occurring at multiple sites. Multi-site tu-mours are also induced in multiple strains of rats via inhalation anddrinking water routes of exposure. One rat (Sprague–Dawley) oralgavage study with less than 50 animals in the acrylonitrile treatedgroup is negative. Mouse appears to be the more sensitive specieswith a harmonic mean TD50 of 6.32 mg/kg/day, compared to16.9 mg/kg/day in rat (Gold and Zeiger, 1997).
The WHO published CICDAS 39 in 2002, providing a thoroughrisk assessment of acrylonitrile. The reviewers indicate that oxida-tive metabolism is critical for induction of genotoxicity and carcin-ogenicity, implicating cyanoethylene oxide as the DNA-reactivemetabolite. Based on the available genotoxicity and carcinogenic-ity data, it is reasonable to conclude that acrylonitrile is a DNA-reactive carcinogen. A conservative assumption was made thatthere is no threshold for the carcinogenic effect. An ADI of6.32 lg/day was derived by linear extrapolation from the mouseharmonic mean TD50 (Table 2), which is approximately 4-foldhigher than the TTC. In comparison, the US EPA (01/01/1991) hascalculated an oral slope factor of 5.4 E�1/mg/kg/day and a drinkingwater limit of 6 E�1 lg/L at the 1/100,000 risk level, based on theoccurrence of multi-organ tumors in a drinking water study in rats.This equates to a daily dose of �1 lg/day for a 50 kg human, whichis almost equivalent to the TTC, and about 6-fold lower than the
limit that we calculated by linear extrapolation from the mouseharmonic mean TD50.
3.9. p-Chloroaniline
The WHO published CICDAS 48 in 2003, providing a thoroughreview and risk assessment of p-chloroaniline. p-Chloroaniline ispossibly mutagenic to bacteria, with inconsistent positive resultsin the presence of metabolic activation. Induction of chromosomeaberrations and mutagenicity is observed in mammalian cells,however no increases in micronuclei are observed. Conflictingin vivo micronucleus data are reported following oral administra-tion (Table 1). Oral carcinogenicity studies with p-chloroanilinein rats and mice show no increase in tumour incidence (Gold andZeiger, 1997). There is one carcinogenicity report cited in the CPDBin which the hydrochloride salt form of p-chloroaniline (Gold andZeiger, 1997) is evaluated in rats and mice via the oral gavage routeof administration. In male F344 rats p-chloroaniline HCl inducesrare splenic tumours. The hydrochloride salt also causes liver andvascular system tumours in male B6C3F1 mice, a finding that is alsoconsistent with the chemical class (WHO, 2003). Rat appears to bethe more sensitive species with a TD50 of 7.62 mg/kg/day, com-pared to 89.5 mg/kg/day in mouse (Gold and Zeiger, 1997).
The mechanism of action related to splenic tumours is unclear.Generally, aromatic amines require metabolic activation to yieldthe ultimate carcinogenic species and the principal pathway of bio-activation involves formation of a hydroxylamine, which decom-poses to a reactive nitrenium ion intermediate (Fig. 1.).
The bioactivation process of aromatic amines is believed to bethe same in carcinogenesis and mutagenesis (Benigni et al.,2000). Based on the available information for both p-chloroanilineand p-chloroaniline HCl a conservative assumption was made thatp-chloroaniline is a DNA-reactive carcinogen with no threshold. A
P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213 209
compound-specific ADI of 7.62 lg/day was derived by linearextrapolation from the rat TD50 for p-chloroaniline HCl (Table 2).There were no relevant allowable limits for p-chloroaniline orthe HCl salt identified elsewhere for comparison to the limit wecalculated here.
4. Discussion
Pharmaceutical development involves managing the potentialbenefits and risks of new medicines for patients. Among the risksto be considered are those arising from low level impurities thatmay reside in the API following synthesis. Risks associated withexposure to impurities are managed by identifying the chemicalsand the associated hazards, characterising the dose–response rela-tionship (when possible) and then determining a human exposurelimit expected to pose negligible risk. Of particular concern areimpurities that have a mutagenic and/or carcinogenic potential,and regulatory guidance documents provide a general frameworkto minimise risk to patients (CHMP, 2006, 2010; FDA, 2008).
To determine acceptable exposure levels for known mutageniccarcinogens, considerations of the dose–response relationshipand mechanism of action are critical. It is widely accepted that ifadequate carcinogenicity data is available, it should be used toestablish compound-specific control limits rather than default tothe TTC, which is intended for the control of impurities with un-known carcinogenic potential. The quality of the carcinogenicitydata is important. Studies should be reviewed and considerationgiven to appropriateness for use. This point is discussed furtherin the limitations section.
Methods to calculate ADIs for carcinogens use different param-eters from carcinogenicity or chronic toxicology studies, includingthe use of cancer slope factors, TD50 or harmonic mean TD50 values,LTD10 (defined as the lower 95% confidence limit on the dose esti-mated to produce an excess of tumours in 10% of the animals),benchmark dose (BMD) or maximum tolerated dose (MTD) levelsto extrapolate the animal data to a level considered as virtuallyno cancer risk for humans (Gaylor and Gold, 1995). The resultingADI limits may differ by orders of magnitude, depending on howthe value is derived and the data utilised.
This work reports practical experience associated with usingexisting carcinogenicity data to derive acceptable exposure levelsfor 11 pharmaceutically relevant impurities, the majority of whichhave no published limits. This work can serve as a starting point forestablishing limits that should be associated with negligible excesscancer risk when these chemicals are discovered to be pharmaceu-tical impurities. However, it is always important to review the lit-erature for new information and to consider whether the ADIreported in this paper or other sources is appropriate for the situ-ation of interest.
Reviewing the available genotoxicity and carcinogenicity dataand scientific literature about the mechanism of action was impor-tant for determining DNA reactivity of each compound. It is wellestablished that the risk assessment approach for chemicals witha linear dose–response is different from those that have a thresh-old dose–response. Therefore, classification was fundamental tothe derivation of an appropriate exposure level.
Four of 11 chemicals (o-nitrotoluene, p-nitrotoluene, 2-chloro-1,1,1-trifluoroethane and p-chloroaniline) were either weaklymutagenic or not mutagenic in the Ames assay and were classifiedas DNA-reactive based on other genotoxicity or mode of actiondata. Therefore, for the purposes of selecting an approach for calcu-lating an ADI, it is important to review all the available data in or-der to make a scientific judgment about DNA-reactivity and shapeof the dose–response curve. As emphasised in the US EPA (2007)Framework for Determining a Mutagenic Mode of Action for
Carcinogenicity, classification can often be enhanced by data otherthan those from mutagenicity tests, including structure–activityrelationships (SAR) with recognised mutagenic carcinogen(s). Thisapproach was employed for evaluating hydrazine, 2-hydroxyethyl-hydrazine and monoacetylhydrazine. In addition, consideration ofmutagenicity and carcinogenicity data for structural aromatic nitroanalogues was used for o- and p-nitrotoluene. Information on themetabolic pathway can influence the judgement of mode of actionfor some compounds, and this was the case for the majority ofchemicals we evaluated.
Carcinogenic potency was quantified using an approach similarto that used to derive the TTC, which used only oral data from themost sensitive species, sex and site (Kroes et al., 2004). In contrastto this method, we considered all routes of administration andwhere more than one positive carcinogenicity study for a givencompound existed, the harmonic mean TD50 from the most sensi-tive species was utilised to calculate the ADI. This approach is stillconservative because the harmonic mean is weighted more to-wards the lowest TD50 while taking into account all of the availabledata, which often varies significantly in regard to the robustness ofthe carcinogenicity study design and degree of certainty associatedwith the TD50 calculation. An alternative method could utilise theTD50 from the most sensitive species, sex and site for a relevantroute of administration, similar to the TTC; however, this doesnot take into account other studies that may be more robust basedon the number of treatment groups and number of animals pergroup. One could also consider use of only the most robust carcin-ogenicity studies and either the most sensitive or harmonic meanTD50; however, what defines the ‘‘most robust carcinogenicitystudy’’ is subject to interpretation. Therefore, it might be usefulto develop criteria for inclusion or exclusion of carcinogenicitystudies in the calculation of compound-specific ADIs. In the currentwork, a pragmatic approach was taken and the harmonic meanTD50 for the most sensitive species reported by Gold and Zeiger(1997) was utilised.
In contrast to the US EPA (2007) Framework but similar to theTTC derivation, we did not apply an age-dependant adjustmentfactor. We considered linear extrapolation from the dose giving a50% tumour incidence (TD50) to a one in 100,000 incidence, usingharmonic mean TD50 data from the most sensitive species orTD50 data from the most sensitive species, site and sex (whereapplicable) to encompass several ‘worst-case’ assumptions in-tended to exaggerate the predicted lifetime cancer risk and ac-count for extrapolation between animals and humans.Additionally, carcinogenicity studies are typically lifetime studieswhere dosing of animals begins at an early age so adjustment fac-tors should not be necessary.
In contrast to the TTC derivation which used only oral data, car-cinogenicity data from all routes of exposure were used to deter-mine the harmonic mean TD50 in order to derive a limit thatcould be appropriate for any route of administration. To providean exposure limit for a specific route of administration, it may beacceptable to utilise only carcinogenicity data from the most rele-vant route. For example, when exposure is by the oral route ofadministration, it may be more appropriate to use only oral dataif data from another route of exposure results in a much more orless conservative ADI (see discussion about hydrazine). When datafor the route of interest is not available, data from other routes ofadministration can be considered as an alternative to a TTC default(see discussion about bis-(chloromethyl)ether).
Only three of the chemicals reviewed in this publication (hydra-zine, bis-(chloromethyl)ether and acrylonitrile) were found to havepreviously published limits intended to minimize excess cancerrisk (US EPA, 01/01/1991a, 01/01/1991b, 04/01/1991). When com-pared to the published limits, the ADIs that we derived are lessconservative for two of three compounds, which are not surprising
210 P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213
since the US EPA limits were calculated from the cancer slope fac-tor, a more conservative extrapolation method. The ADI we derivedfrom both inhalation and oral TD50 data for hydrazine was �3 and�11-fold higher than the published US EPA (04/01/1991) oral andinhalation limits, respectively. The ADI we derived from acryloni-trile TD50 data was �6-fold higher than the published US EPA(01/01/1991b) limit. The ADI we derived from inhalation data forbis-(chloromethyl)ether was nearly identical to the limit publishedby the US EPA (01/01/1991a). Based on this small number of com-pounds, it appears that use of TD50 data is likely to result in an ADIup to �1 order of magnitude greater than that derived from use ofa cancer slope factor. However, given that the TTC itself was de-rived from the distribution of TD50s for known genotoxic carcino-gens, it seems appropriate to apply this slightly less conservativemethod when establishing ADIs for pharmaceutical impurities.
Based on a weight of evidence approach, all of the chemicalspresented here were classified as DNA-reactive carcinogens witha linear dose–response, including hydrazines (3), quinones (2), aro-matic nitros (2), a chloroalkyl ether, a polyhalogenated alkane, a ni-trile and an aromatic amine. The ADIs ranged from 0.00357 to257 lg/day. Eight of 11 compounds had ADIs above the TTC.Although only a small number of compounds were analysed, it isconsistent with Delaney (2007) observation that chemicals usedas synthetic intermediates are in many instances not the most po-tent mutagenic carcinogens.
Three of the impurities (hydrazine, 2-hydroxyethylhydrazineand bis-(chloromethyl)ether) had ADIs that were below the TTC.For hydrazine, rats were the most sensitive species, with dataavailable from inhalation and oral studies. The harmonic meanTD50 (0.613 mg/kg/day) is largely influenced by the observationof nasal tumours in male and female rats following inhalationadministration. Therefore, the ADI (0.613 lg/day) may be overlyconservative for the oral route of administration (Table 3,Scenario A). However, the ADI derived by linear extrapolationfrom the harmonic mean TD50 is similar to the drinking waterlimit of 0.2 lg/day (for 1/100,000 risk level) that was derived bythe US EPA from a multi-dose oral carcinogenicity study ofhydrazine sulphate (Biancifiori, 1970), which is not reported inthe CPDB. The US EPA limit is derived from the oral slope factor(3 mg/kg/day).
If an acceptable exposure level of hydrazine specific to the oralroute is desired, all oral carcinogenicity studies for which TD50 val-ues have been published could be considered or perhaps just themost robust oral carcinogenicity study or studies could be usedto derive an oral ADI. However, in the absence of criteria that indi-cate minimal standards for carcinogenicity study design, the selec-tion of carcinogenicity data is highly subjective. Following oraladministration, hydrazine induces lung tumours in mice and liver
Table 3Comparison of various ADIs derived for hydrazine.
Scenario Data utilised
A Rat harmonic mean TD50, all positive hydrazine studies in CPDB, all routeB Mouse harmonic mean TD50, all positive oral hydrazine studies in CPDBC Rat harmonic mean TD50, all positive hydrazine oral studies in CPDBD Mouse harmonic mean TD50, all positive hydrazine sulphate oral studies i
E Rat harmonic mean TD50, all positive hydrazine sulphate oral studies in C
F Mouse harmonic mean TD50, Biancifiori (1970)G Mouse harmonic mean TD50, all positive hydrazine sulphate oral studies i
CPDB + Biancifiori (1970)
a Number of individual TD50 values included in calculating the harmonic mean TD50; nseparately.
tumours in rats (Gold and Zeiger, 1997). There are two oral studiesin mice with TD50s ranging from 2.2 to 5.7 mg/kg/day for which theharmonic mean TD50 is 2.92 mg/kg/day, equating to an ADI of2.92 lg/day for a 50 kg human (Table 3, Scenario B). However, bothstudies (Roe et al., 1967; Toth, 1972) have only one treatmentgroup and the study by Toth (1972) was discounted by the USEPA (04/01/1991) when deriving oral drinking water limits be-cause there were no concurrent controls. Therefore, one might beinclined to disregard the mouse data and use the more robust oralrat study with three treatment groups (Steinhoff and Mohr, 1988),which is reported in the CPDB. In this oral rat study, the harmonicmean TD50 is 42.6 mg/kg/day, which equates to an ADI of 42.6 lg/day (Table 3; Scenario C). This oral ADI is �69 times higher thanthe ADI derived from the harmonic mean TD50 of all positive stud-ies (including inhalation data) and �15 times higher than the oralmouse ADI. This could reflect a true species difference or themouse data may over-estimate risk. The small number of treat-ment groups makes the estimation of the TD50 value less accurate.Therefore, one might also consider evaluating available oral datafor hydrazine sulphate as the US EPA did in calculating the drinkingwater limit. The most sensitive harmonic mean TD50 for hydrazinesulphate in the CPDB (7.59 mg/kg/day) is from oral studies in mice(Gold and Zeiger, 1997). This equates to an ADI of 7.59 lg/day (Ta-ble 3, Scenario D), which is greater than the TTC but nearly fivetimes lower than the ADI derived from the oral rat data for hydra-zine (42.6 lg/day; Table 3, Scenario C). Interestingly, the ADI forhydrazine sulphate derived from rat oral carcinogenicity data,40.8 lg/day (Table 3, Scenario E), is similar to that derived fromrat oral hydrazine data. However, the ADIs for hydrazine sulphateare both derived from studies with single treatment groups and/orlow animal numbers, which would be expected to reduce the con-fidence in the TD50 value.
Another approach for calculating an oral hydrazine ADI could beto derive a TD50 from the Biancifiori (1970) oral mouse study ofhydrazine sulphate utilised by the US EPA. For this comparison, aTD50 was calculated (Table 4) for each sex from the Biancifiori(1970) study based on the methods used by Gold and Zeiger(1997) and previously described by Peto et al. (1984) and Sawyeret al. (1984). The harmonic mean TD50 from this study is22.8 mg/kg/day, resulting in an ADI of 22.8 lg/day for a 50 kg indi-vidual based on linear extrapolation (Table 3, Scenario F). This va-lue is less than 2-fold lower than derived from the rat hydrazineoral data (42.6 lg/day). When the Biancifiori (1970) mouse datais combined with the oral mouse hydrazine sulphate data in theCPDB, the harmonic mean TD50 is 9.54 mg/kg/day, which equatesto an ADI of 9.54 lg/day (Table 3, Scenario G). This illustrates thevariation in exposure limits that may be calculated for a singlechemical, depending on the data utilised. It also exemplifies the
No.a Considerations/limitations ADI(lg/day)
s: 4 Inhalation and oral TD50s are disparate 0.6133 Only one treatment group (3/3) 2.922 No major limitations identified 42.6
n CPDB 9 Only one treatment group (9/9); <50 animalsin at least one group (8/9)
7.59
PDB 2 Only one treatment group (2/2); 28 or lessanimals per group (2/2)
40.8
2 30 or less animal per group (2/2) 22.8n 11 Only one treatment group (9/11); <50 animals in at
least one group (10/11)9.54
ot necessarily the number of studies as male and female TD50 values are calculated
Table 4TD50 values calculated from oral mouse cancer bioassay for hydrazine sulphate (Biancifiori, 1970).
P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213 211
difficulties that may be encountered when attempting to include orexclude specific cancer bioassay data when calculating ADIs.
The ADI we derived for 2-hydroxyethylhydrazine (0.397 lg/day) was also below the TTC. 2-Hydroxyethylhydrazine has con-flicting carcinogenicity data, inducing liver tumours in one of twostudies in mice but no tumours in hamsters. Generally, to exerttoxicity hydrazines require metabolic activation by cytochromeP450’s, which are present in high numbers in the liver. Therefore,it seems reasonable that the liver would be a target organ. As high-lighted by Kroes et al. (2004) and summarised by Snodin (2010),there is a strong association between the hydrazine structural moi-ety, mutagenicity and carcinogenicity. The positive carcinogenicitystudy did not have an ideal study design as only one treatmentgroup was included and group size was small (<20). Interestingly,the negative studies in hamsters and mice, although also limited toone dose group, had 50 animals/treated group and 100 animals/control group, which should increase the power for detecting atreatment-related response.
Based on the most sensitive species and site from the rat inha-lation carcinogenicity data, the ADI for bis-(chloromethyl)ether wederived was 420 times below the TTC. An important aspect of thecarcinogenicity of bis-(chloromethyl)ether is that chronic exposureis not required for tumourigenesis (Van Duuren et al., 1975; VanDuuren, 1988). Nasal and lung tumours have been observed in ani-mals following both intermediate and chronic exposure to bis-(chloromethyl)ether vapour (Leong et al., 1981) and epidemiolog-ical studies in exposed workers strongly suggest that bis-(chloro-methyl)ether causes lung tumours in humans (IARC, 1987). Thefirst report of the carcinogenicity of bis-(chloromethyl)ether wasthat of Goldschmidt et al. (1968). Following dermal exposure (skinpainting), bis-(chloromethyl)ether was found to produce skin pap-illomas and carcinomas in over 50% of mice tested after 325 days oftreatment. The carcinomas appeared early, with the first visibleafter only 196 days of skin application and subsequent reports con-firmed these findings (Van Duuren et al., 1972, 1975; Zajdela et al.,1980). Regarding the carcinogenicity data used to derive the ADI,rat is the most sensitive species for which only inhalation studieswere available (Leong et al., 1981). In this study, bis-(chloro-methyl)ether was administered for 6 months followed by a life-time observation period, 28 months for rats. During the course ofthis study, 86.5% of rats in the high dose group, 100 ppb, developedrare nasal tumours, esthesioneuroepitheliomas. As a result of thehigh carcinogenic potency and lack of sufficient studies from otherroutes of administration, we used the rat inhalation carcinogenic-ity to calculate an ADI. Given that bis-(chloromethyl)ether is aknown human carcinogen and the time to appearance of tumoursin animals is short compared to other carcinogens, it may not beappropriate to assume that the TTC should be used as a limit forthe oral route of exposure in the absence of oral data. Interestingly,the reactive nature of bis-(chloromethyl)ether, which makes it apotent carcinogen, also means it will readily react during the syn-thetic process and therefore is unlikely to survive as an API residue.
4.1. Limitations
The ADIs calculated here were based on available experimentaldata and in some cases there were limitations to the data that wereused. These were highlighted for each compound evaluated. The
use of high-dose extrapolation of dose–response data from animalbioassays to levels of human exposure involves considerableuncertainty, due to limited statistical power of the bioassay andminimal data points in the low-dose region. Extrapolation fromanimal data is necessary for risk assessment, but it is possible toidentify many different dose–response curves that fit a given data-set, resulting in very different implications in the low-dose region(Lutz et al., 2005; Hsu and Stedeford, 2010). Linear extrapolation isrecognised as conservative because it does not account for non-lin-ear responses that may, and are likely, to occur due to cytoprotec-tive mechanisms, DNA repair, etc. Therefore, it is likely to over-estimate carcinogenic risk in humans.
For DNA-reactive compounds assumed to have no threshold, wetook a pragmatic approach and utilised the TD50 or harmonic meanTD50 from the most sensitive species to derive ADIs since this waseasily identified in the CPDB (Gold and Zeiger, 1997). There weredefinite limitations to this approach because all studies that metthe criteria to be included in the CPDB were considered, regardlessof differences in the robustness of study design. For more than halfof the compounds (six of 11), ADIs were calculated from dataincluding only one treatment group, in addition to the control, orfrom studies with low power due to small animal numbers. Inthese cases, there was greater uncertainty related to the accuracyof the TD50, and hence the ADI. This is evident from the wide con-fidence intervals reported for some of the TD50 values and aninability to calculate an upper bound TD50 for others (Gold and Zei-ger, 1997). However, as was demonstrated by the example of cal-culating an ADI specifically for oral hydrazine exposure, choosingthe appropriate study for calculations can also be difficult.
Based on our experience, it can be challenging to select appro-priate data for ADI calculations without significant knowledge ofcarcinogenicity study design and limitations. Industry could bene-fit by developing some standards for selection of the most robustcarcinogenicity data when there is more than one study available.For example, when Cheeseman et al. (1999) re-evaluated and ex-tended the data used to calculate the FDA Threshold of Regulationto >700 compounds, they included only TD50s with statistical sig-nificance of p 6 0.01. This does not necessarily exclude studieswith study design limitations. It may be valuable to define addi-tional criteria, such as number of treatment groups and animalnumber, to help in the selection of the most robust study for ADIcalculation when there are multiple positive studies. This wouldhelp to ensure consistent selection of data for ADI derivation andensure that new data is only used if it is appropriately robust.
5. Conclusions
Diligence is required by pharmaceutical companies to controlthe presence of DNA-reactive (mutagenic) carcinogenic impurities,thereby ensuring human safety and meeting regulatory obliga-tions. By publishing our practical experience establishing ADIs for11 DNA-reactive carcinogens, we hope to provide a useful refer-ence to the pharmaceutical industry for establishing impurity lim-its in drug substances. In addition, some of the more complicatedexamples illustrate that it would be useful to develop some guidingprinciples, particularly with regard to selection of carcinogenicitystudy data that can be followed when calculating ADIs for impuri-ties that are known mutagenic carcinogens.
212 P. Ellis et al. / Regulatory Toxicology and Pharmacology 65 (2013) 201–213
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors acknowledge Andrew Fleetwood, Dave DeAntonisand Ron Ogilvie for reviewing the manuscript and providing valu-able feedback. Additionally, we acknowledge the hard work ofDingzhou Li in calculating the TD50 data for hydrazine sulphatefrom the Biancifiori (1970) study that was not included in the car-cinogenic potency database but used by the US EPA for establishingdrinking water and inhalation limits for hydrazine.
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