Investigational

NOVARTIS 

Impurities – 

Issues in regard to quality and safety 

Lutz Müller 

Novartis Pharma AG 

Preclinical Safety EU 

Investigational and in silico Safety & Metabolism (ISIS) 

L. Müller, Swissmedic, 21-Oct-2003

Relevant guidelines 

NOVARTIS 

ICH Guidelines 

ICH Q3A(R) Impurities in New Drug Substances 

ICH Q3B(R) Impurities in New Drug Products 

ICH Q3C Residual Solvents (including maintencance) 

CPMP draft NfG (2002) 

Specification limits for residues of metal catalysts (on hold) 

CPMP draft position paper (2002) 

Limits for genotoxic impurities (on hold) 


General approach for identification and 

qualification of impurities 

NOVARTIS 

Impurities are normally qualified in appropriate toxicological studies as part 

of a drug substance or drug product 

‣ It is assumed that their structure is normally close to that of the drug substance 

The toxicological qualification threshold for drug substance impurities is 

0.15% (0.5% for daily dose of more than 2g) = 1500ppm (or 500ppm) 

The (structure) identification threshold is 0.1% (or 1mg/day, 

whichever is lower) 

‣ (0.5% for daily dose of more than 2g) = 1000ppm (or 500ppm) 

Essential qualification studies are 

‣ Genotoxicity in vitro 

‣ A (sub) chronic toxicity study (in rodents) 

Specifications/levels in clinical batches should be covered by tox 

investigations 

‣ Either in acutal percentage 

or 

‣ with appropriate safety factors 


What about highly toxic impurities? 

NOVARTIS 

“Identification of impurities present at an apparent level of not more 

than (≤) the identification threshold is generally not considered 

necessary. However, analytical procedures should be developed 

for those potential impurities that are expected to be unusually 

potent, producing toxic or pharmacological effects at a level not 

more than (≤) the identification threshold. All impurities should be 

qualified as described later in this guideline.” 

“For impurities known to be unusually potent or to produce toxic or 

unexpected pharmacological effects, the quantitation/detection limit 

of the analytical procedures should be commensurate with the 

level at which the impurities should be controlled.” 

‣ Issue: How to control, if you do not know the structure? 


Potential presence of reactive impurities 

in drug substances 

NOVARTIS 

Process-related 

‣ Starting materials 

e.g. alkylating agents and similar reactive species 

‣ Intermediates / by-products 

e.g. strong acid/alcohol interactions 

‣ Catalysts, ligands 

Long-term storage 

‣ Degradation products (by oxidation?) 

Complete elimination of such impurities is often not feasible 


Why to address the concern regarding high 

toxicity/non-reversible toxicity of impurities? 

NOVARTIS 

We do not want to put patients at undue risk of exposure to toxic agents if 

this is avoidable according to best technical procedures 

The presence of a genotoxic/carcinogenic impurity may falsely stigmatize an 

otherwise safe drug substance if there are no efficient means to eliminate 

influences of genotoxic impurities on the test results obtained with batches 

of “poor purity 

‣ e.g. testing of such batches would be a risk for two year carcinogenicity studies in 

rodents 

ICH guidances on impurities in new drug substances and new drug products 

state that: “analytical procedures should be developed for those potential 

impurities that are expected to be unusually potent, producing toxic or 

pharmacological effects at a level of not more than the identification 

threshold.” 

It is agreed that compounds of high acute or potentially non-reversible 

toxicity would fall into this category. 

‣ Genotoxic compounds are generally believed to have the potential to exert nonreversible 

changes in the genetic material 


Genotoxic impurities – how to detect? 

NOVARTIS 

The structure of impurities present at levels above the identification threshold is 

known, their impact on safety of the drug substance can be appropriately assessed 

But: the ICH guidance on drug substance impurities does not stipulate identification 

of the structure of impurities below a level of 0.1% =1000 ppm (0.05% or 500 ppm if 

daily dose is above 2 g). 

1000 ppm (500 ppm) may be unacceptably high for an impurity if it is genotoxic. 

‣ A 2 g daily dose of a pharmaceutical may contain 2 mg of a potential toxin 

‣ E.g. 2 mg residues of an alkylating starting material would not be desired 

What would happen if you spike drug substance X with 1000ppm of an alkylating 

agent? 

Genotoxicity tests may not be sensitive enough to detect the effect of genotoxic 

impurities in a batch if the impurity is present at the 0.1% level 

In other words: few genotoxic compounds have a potency e.g. in the Ames that 

would enable their detection at or below 0.1% (the same is true for rodent 

carcinogenicity studies) 

‣ Without a structure, we do not have an idea whether we have a genotoxic impurity 

problem unless we start to approach the problem from the synthesis side 


Genotoxic impurities - the limit of detection issue 

NOVARTIS 

Take EMS (ethylmethane sulfonate) and MMS (methylmethane 

sulfonate) as “model mutagenic impurities” 

‣ LOEC for EMS in Ames test is: 1500 µg/plate (TA 100) 

‣ LOEC for MMS in Ames test is: 15 µg/plate (TA 100) 

Question: at which level can you detect these as “mutagenic” in 

an Ames test of drug substance X 

Answer: assume that drug substance x is tested up to 5 mg/plate 

‣ EMS can be detected at 33.3% only (=330’000 ppm) 

‣ MMS can be detected at 0.33 % (15 µg in 5 mg =3’300 ppm) 

‣ A batch of drug substance X can be “qualified” as non genotoxic in an Ames 

test although it may contain e.g. 3’000 ppm MMS 

‣ Nevertheless, the presence of 3’000 ppm MMS is not acceptable 

Few genotoxic carcinogens have a detection limit in the Ames 

test below 5 µg/plate, which would correspond to 1000 ppm if 

expressed as “impurity” level in 5 mg drug substance 


LOEL in genotoxicity assays by structural alert class 

Salmonella Reverse 

Mutation Assay 

Anthraquinones (12) 

N-Oxides (Aromatic) (5) 

Azirine/Aziridine (5) 

Aromatic Nitro (45) 

Aromatic Amines (48) 

Chromium (10) 

Vic Dihalides (6) 

Quinolines (18) 

PAHs (K-, Bay-Region) (7) 

Alkylating Agents (15) 

Nitrosamines/N-Nitroso (32) 

Isocyanate (4) 

Aliphatic Epoxides (21) 

Hydroxamates (13) 

Heterocyclic Nitro (3) 

Halogenated Methane (3) 

Hydrazo/Hydrazine (9) 

Rodent Micronucleus Assay 

0.0 

0.001 

0.01 

0.1 

Azirine/ 

Aziridine (6) 

N-Nitroso (4) 

Alkylating 

Agent (15) 

Triazene (6) 

Aromatic 

Amine (9) 

Hydrazine (4) 

NOVARTIS 

mg/kg 

1 

100.0 

10.0 

0.0 

0.1 

1 

10.0 

100.0 

1000 

µg/ml 

0.0 

0.0001 

0.001 

0.01 

0.1 

1.0 

10.0 

Theoretical detection limit 

1 ppm 

% of drug substance if drug substance is 1mg/ml 


CPMP draft position paper on genotoxic impurities 

(as of December 2002) 

NOVARTIS 

Issue of non-consistent regulatory action and deficits in the existing 

ICH guidances regarding genotoxic impurities was realized by the 

CPMP: 

‣ “In the case of impurities with a genotoxic potential, determination of 

acceptable dose levels is generally considered as a particularly critical 

issue, which is not specifically covered by the existing guidelines.” (from 

the introductory paragraph of the position paper) 

While regulatory action appears to be executed preferentially in late 

stages of the development process, it is better for us to implement a 

process of continuous data exchange between TRD and PCS to 

improve step-wise our syntheses with regard to a potential carry 

over of toxic impurities at “unacceptably” high levels into the DS 

‣ To avoid regulatory surprises 

‣ To minimise risks for volunteers and patients 


Our Novartis policy regarding (geno)toxic impurities 

NOVARTIS 

We have introduced a new policy regarding the detection, testing, 

elimination and risk assessment of potentially (geno)toxic 

impurities in drug substances or drug products 

In general, the procedure starts with a “Computational toxicology 

assessment of every drug substance synthesis scheme” 

‣ Why?: focus on toxicological knowledge and informed decision making 

Product of this assessment and the interaction between 

chemistry and toxicology is a “Notification on the presence of 

unacceptable substances in synthesis” before Phase I trials. 

‣ This “Notification” is constantly updated throughout the development process 

‣ The “Notification” includes actions and measures regarding purity, limits 

based on toxicity and scientific rationales on these limits 

The new policy is described in a new world-wide SOP: 

“GENERATION OF THE SYNTHESIS SCHEME AND COMPLETION 

OF THE SYNTHESIS CONTROL FORM AND NOTIFICATION FORM 

FOR KNOWN AND POTENTIAL IMPURITIES” 


What is the role of toxicology as described 

in this new SOP? 

NOVARTIS 

PCS assesses raw materials & intermediates for toxicity. 

If not listed in a general list of potential carcinogens, a structureactivity 

(in-silico) analysis for the presence of toxicological alerts is 

performed 

Are alerting structural elements present (primary focus on 

genotoxicity / carcinogenicity)? 

‣ Our present in silico systems give us about 80% accuracy for genotoxicity 

‣ The in silico systems are continuously improved with experimental data 

If no, then no special limits, normally no special testing needed. 

‣ Qualification according to existing ICH guidance procedures, i.e. appropriate 

(“dirty”) batches are tested 

If yes, a bacterial mutagenicity assay (Ames test) is normally 

initiated 


Verification/falsification of structural alerts 

NOVARTIS 

Toxicology will perform Ames test (or other suitable test as indicated 

by structural alert) 

‣ Why primarily the Ames test?: it detects with greatest reliability DNA reactive 

compounds (or metabolites of them) 

‣ Most DNA reactive compounds are multi-organ carcinogens 

‣ Few false positives in the Ames test (Ames positive non-carcinogens) 

‣ Few toxins are known that are DNA reactive in mammalian cells but not in 

bacteria 

( 

Test(s) confirm in-silico hypothesis? 

If no, then no special limits, no special testing 

If yes, acceptable limits proposed adapted to the phase of the clinical 

program 

! Important: chemical expert reasonning can be an alternative to 

testing (and limit setting) especially in early phases of development ! 


The new SOP applied 

to Novartis projects in 2002 

NOVARTIS 

Evaluation period: 2002 

46 syntheses evaluated for the presence of alerting structures: 

in silico prediction plus published/in house data on genotoxicity or 

carcinogenicity 

‣ 6 syntheses were “clean”, i.e. no alerting structures were present 

‣ Recommendations for limitations or genotoxicity testing given for 

139 intermediates 

‣ 65 intermediates tested for genotoxicity (usually Ames screen in 

two strains, using less than 100mg substance) 

‣ 38 tested negative: no limits given 

‣ 27 tested positive: limits discussed 

‣ 20 syntheses out of 46 (43%) involve a mutagenic intermediate, 

which is potentially present as impurity 


Setting limits based on toxicity data? 

NOVARTIS 

Toxic residual solvents/heavy metal catalysts limits are described in ICH 

or CPMP guidances (or draft guidance) 

However, there are extensive discussions in the quality and safety 

community on what are suitable/acceptable limits e.g. for genotoxins 

‣ Few compounds are sufficiently experimentally characterized (e.g. in rodent 

carcinogenicity studies) to do a reliable risk assessment 

‣ The draft position paper from the CPMP Safety Working Party is not very 

clear on this issue 

‣ Extensive comments via EFPIA and PhRMA have been submitted 

‣ It appears that the EFPIA proposal to adopt the concept of a “Threshold of 

Toxicological Concern (TTC) as applied in the food contact substances area 

may be acceptable to the CPMP 

‣ TTC lies at 1.5ug/day for lifetime exposure 

(exceptions: very potent carcinogens such as a few nitrosamines) 

‣ Proposed ICH topic? 


Normally used formula for calculating 

permitted daily exposure (PDE) based on toxicology data 

NOVARTIS 

PDE (mg/day) = 

NOEL or LOEL (mg / kg) x human bodyweight (50 kg) 

F1 x F2 x F3 x F4 x F5 

Uncertainty factors F1 - F5 to account for: 

F1: Interspecies differences (surface area : body weight ratio for man 

compared to testing species; rat = 5, mouse = 12) 

F2: Inter-individual differences (10) 

F3: Duration of exposure (1-10) 

F4: Severity of toxicity (1-10) (is this sufficient for genotoxicity/carcinogenicity?) 

F5: Quality of data (1-10) / LOEL = 10 


The dilemma of lacking data is 

a majorhindranceforusing 

NOEL‘s and uncertainty factors! 

NOVARTIS 

In vitro + in vivo genotoxicity assays are hazard identification 

tests, and thus do not provide data suitable for use in 

quantitative risk assessment 

Approach proposed (use of NOEL + UF) is unlikely to be 

feasible in most circumstances (no adequate data)! 

Pragmatic approach is needed (amount of impurity in µg/day 

considered as generally acceptable intake level) 

‣ The NOEL / UF approach can be used for toxicologically well 

characterized compounds, a condition, which is normally not 

met for impurities 


Alternative to PDE calculation: 

the Threshold of Toxicological Concern (TTC) 

NOVARTIS 

This concept acknowledges a human exposure threshold value for chemicals 

(including those of unknown toxicity) below which there is no significant risk to 

human health. 

Established by the FDA (“Threshold of Regulation“) as a level “low enough to 

ensure that public health is protected, even in the event that a substance 

exempted from regulation .... is later found to be carcinogenic.“ 

Examined for broader applications by ILSI Europe Task Group 

Based on an analysis of over 700 chemical carcinogens from the Gold et al. 

carcinogenic potency database 

‣ estimate of a human exposure threshold value for a high probability of not 

exceeding a 1x10 -6 cancer lifetime risk : 

‣ 1.5 µg/person/day (= no safety concern!) 

‣ both, genotoxic and non-genotoxic carcinogens are covered in the 

database! 


Translate Threshold of Toxicological 

Concern (TTC) in ppm? 

NOVARTIS 

Maximum levels of impurity in a chronic use drug 

(scenario for “PDE’s” between 0.05 and 50 ug/day) 

PDE* for 

impurity 

(µg/day) 

Max. content (in ppm) of impurity in drug substance if 

daily dose of drug (g/day) is: 

0.1 

1 

2 

5 

0.05 

0.5 

0.05 0.025 0.01 

0.5 

5 

0.5 

0.25 

0.1 

1.5 

15 

1.5 

0.75 

0.3 

5 

50 

5 

2.5 

1 

50 

500 

50 

25 

10 

* Denotes “Permitted Daily Exposure 

To be used if Threshold of Toxicological Concern will be generally accepted 


Deviations from TTC possible (if justified) 

NOVARTIS 

route of administration 

target population / proposed indication 

toxicology of the impurity in relation to the active substance 

high potency carcinogens excluded from TTC 

(“the dirty four“) 

‣ aflatoxin-like compounds 

‣ azoxy compounds 

‣ N-nitrosamines 

‣ tetrasubstituted dibenzodioxins and dibenzofurans 


Advantages of Using the TTC Concept 

NOVARTIS 

scientifically (databases) derived threshold 

(in contrast to the arbitrary use of uncertainty factors) 

conservative approach resulting in low ppm values 

can be applied even without preclinical data! 

will provide more consistency in controlling genotoxic 

impurities than case-by-case use of NOEL+UF-derived limits 

already accepted for regulatory purposes 


Regulatory Applications of the TTC Concept 

NOVARTIS 

FDA (“Threshold of Regulation“): for indirect food additives = 

food contact material 

Joint FAO/WHO Expert Committee on Food Additives 

(JECFA): for flavouring substances 

Could this concept be applied more widely than just to food 

toxicology? 


Are there alternative concepts for acceptable limits? 

NOVARTIS 

Acceptable level of intake of genotoxic impurities should be 

set in relation to daily intake of food mutagens (1g/day) 

‣ “no risk“ level for a genotoxin: 10 - 100 µg/day 

(“safety margin“ of 4 -5 orders of magnitude below the “daily 

mutagenic burden“!) 

Fiori & Meyerhoff (Eli Lilly): “Extending the Threshold of 

Regulation Concept: De Minimis Limits for Carcinogens and 

Mutagens“ 

(Regul. Toxicol. Pharmacol. 35, 2002, 209-216) 

‣ “no risk“ level for a genotoxin: 90 ng/day 

‣ “no risk“ level for a carcinogen: 9 ng/day 

These are no real alternatives! 


Case study I 

4-hydrazinobenzoic acid in a life saving oncology product 

NOVARTIS 

Synthesis involves 4-hydrazinobenozoic acid (4-HBA) 

‣ Hydrazine is IARC Group 2B, 1,2-dimethylhydrazine is in Group 2A 

‣ 4-HBA is a weak Salmonella mutagen 

‣ 4-HBA does not induce chromosome aberrations in vitro 

‣ Limited published data indicate tumorigenic effects in rats (one dose level only tested 

in drinking water, low survival) 

but: Agaricus bisporus, the major cultivated mushroom of the Western 

Hemisphere (=champignon), contains 4-HBA at a level of 10 µg/g 

(10ppm) wet mushroom weight 

‣ One champignon meal (200 g) contains 2000 µg 4-HBA 

‣ Does exposure to 4-HBA really constitute a genotoxic/tumorigenic risk? 

‣ Which level would be acceptable in a pharmaceutical (life saving) preparation with 

life-long high daily dose 

• Would 5 or 10 ppm be too much, can we compare the risk in XYZ123 with eating 

champignons? 

5ppm 4-HBA in XYZ123 (the current limit) translates into an exposure of 

10µg/day 

‣ This level is considered appropriate based on the life-threatening indication and the 

relatively weak genotoxic/carcinogenic potential of this hydrazine 


Case study II 

epoxide degradation product in mometasone (published case) 

NOVARTIS 

Mometasone (corticosteroid): 

‣ Daily dose ~1600 µg 

Epoxide degradation product in 

aequeous systems 

The epoxide is a toxicological 

concern 

Limit setting approach according to 

TTC: 

‣ 1.5 µg < 1000 ppm! 

Alternative: toxicological testing 

‣ Limited to 1000ppm 

‣ Below threshold of toxicological 

concern (not published with that 

reasoning) 

‣ Apparently no specific toxicity 

studies 

‣ Proactive limitation in the absence of 

detailed tox data? 


Case study III: Imatinib (Gleevec) 

Maximum daily dose of Imatinib is 800 mg 

STID6 bears structural alerts, and is positive in several 

genotoxicity systems as well as yielded toxicological findings 

(hyperplasia, necrosis) in various organs in a 4-week study in 

rats 

Drug substance batches spiked with up to 280 ppm yielded 

negative genotoxicity test results (could have been predicted by 

calculation) 

Limit based on technical feasibility is 20 ppm 

‣ Highest human daily uptake/exposure would be 8 and 16 µg 

(equivalent to 1.6 and 3.2 ng/ml blood assuming total absorption) 

‣ Human exposure is clearly below in vitro genotoxic concentrations 

‣ But: D6 (CGP53715) is formed as major metabolite in mice and 

rabbits 

‣ No major pathway in rats, dogs, humans 

‣ But: are analytical methods capable of excluding D6 as a minor 

human metabolite 

‣ It does not make sense to be very strict with applying low ppm 

limits for impurities, but to be not able to exclude formation by 

metabolism at higher levels 

‣ Problem not considered important for the current life saving 

indications of Gleevec 

Gleevec 

N 

D6 (507-00) 

N 

NOVARTIS 

N N H NH 

N 

L. Müller, Swissmedic, 21-Oct-2003 

N 

N 

N 

O 

N N H NH 2

Decision tree for assessment of 

acceptability of genotoxic impurities 

(according to publication draft by Peter Kasper, the relevant 

drafting “regulator“ on the CPMP side) 

NOVARTIS 

Established carcinogenicity 

from long-term studies? 

NO 

YES 

Genotoxicant with 

unknown carcinogenicity 

Genotoxic 

carcinogen 

Threshold-related 

mechanism? 

Eliminate? 

YES 

NO 

NO 

Determine 

Permitted 

Daily Exposure 

(NOEL + UF) 

Control below 

“Threshold of 

Toxicological 

Concern“ 

Quantitative 

risk assessment: 

Determine 

acceptable dose 


Proposal by Novartis/EFPIA 

group for a flow chart on 

genotoxic impurities 

NOVARTIS 


Next Activities on genotoxic impurities 

NOVARTIS 

Scientific fora (experts from Industry, SWP & QWP) 

‣ DIA case study workshop in London on October 28-29) 

‣ EFPIA/CPMP round table to discuss & judge the implications of 

the various suggestions (also from a pharmaceutical point of 

view) 

Finalization of the EU Position Paper in 2004? 

Need of international harmonization within the ICH 

framework? 


Some conclusions, but not necessarily all inclusive 

NOVARTIS 

A genotoxic (mutagenic) impurity is potentially present in ~40% of all 

drug substances although batch testing for genotoxicity yielded 

mostly negative results 

For most of the genotoxic structures that show up in drug substance 

syntheses, there is no preexisting knowledge from published 

literature (i.e. we would have to generate the data) 

Organic chemistry involves the use of reactive materials, which may 

be genotoxic. Of course, it would be naïve to assume that this could 

be totally avoidable

Investigational

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