**2.1 ICH guidelines**

The International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use project represents the main group of guidelines with topics such as "Quality" topics and "Safety" topics. Quality topics relate to chemical and pharmaceutical quality assurance (stability testing, impurity testing, etc.) and

Genotoxic Impurities in Pharmaceuticals 389

whichever is lower

whichever is lower 10 – 100 mg 0.5% or 200 g TDI

<10 mg 1.0% or 50 g TDI

whichever is lower > 100 mg – 2 g 0.2% or 3 mg TDI

2Thresholds for degradation products are expressed either as a percentage of the drug substance or as a

Table 2. Thresholds for degradation products in new drug products (Jacobson-Kram and

The European Medicines Agency (EMEA) guideline describes a general framework and practical approaches on how to deal with genotoxic impurities in new active substances**.**  According to the guideline "The toxicological assessment of genotoxic impurities and the determination of acceptable limits for such impurities in active substances is a difficult issue and not addressed in sufficient detail in the existing ICH Q3X guidance". In addition, the EMEA guideline proposed a toxicological concern (TTC) threshold value of 1.5 μg/day intake of a genotoxic impurity which is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) in most pharmaceuticals. Based on the TTC value, a permitted level of an active substance can be calculated concerning the expected daily dose. Higher limits might be justified under certain conditions such as short-term exposure periods (European Medicines Agency/ Committee for Medicinal Products (CHMP) for Human Use, 2006). In the context of this guideline, the classification of a compound (impurity) as genotoxic in general indicates that there are positive findings in established *in vitro* or *in vivo* genotoxicity tests with the focus on DNA reactive substances that have a potential for direct DNA damage. In the absence of such information, *in vitro* genotoxics are usually considered as presumptive

total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the

> 2 g 0.15%

Identification Thresholds2,3 Qualification Thresholds2,3

whichever is lower

whichever is lower

whichever is lower

Maximum Daily Dose1

≤1 g 0.1 % > 1 g 0.05 %

McGovern, 2007)

**2.2 EMEA guideline** 

> 2 g 0.1%

degradation product is unusually toxic.

1 The amount of drug substance administered per day

3Higher thresholds should be scientifically justified.

*in vivo* mutagens and carcinogens (EMEA/CHMP, 2006).

Reporting Thresholds2,3

≤ 1 mg 1.0% or 5 g TDI

1 – 10 mg 0.5% or 20 g TDI

> 10 mg - 2 g 0.2% or 2 mg TDI

safety topics deal with *in vitro* and *in vivo* pre-clinical studies (carcinogenicity testing, genotoxicity testing, etc.) (ICH, 2008).

The ICH initially published guidelines on impurities of drug substances and pharmaceutical products in the late 1990s. In the guidelines, genotoxicity tests have been defined as *in vitro*  and *in vivo* tests designed for detecting compounds that induce genetic damage directly or indirectly (International Conference on Harmonization, 1997). The ICH quality guidelines Q3A(R) and Q3B(R) respectively address the topics of control of impurities in drug substances and degradants in pharmaceutical products, while the Q3C guideline deals with the residual solvents. However, several important issues have not been addressed in the guidelines, for example, the acceptable levels of impurities in drugs during development as well as the control of genotoxic impurities. Table 1 illustrates a series of thresholds described in ICH Q3A(R) that trigger reporting, identification, and qualification requirements. Subsequently, Table 2 depicts the thresholds for reporting, identification, and qualification of impurities in new drug products (ICH, 2006; Jacobson-Kram and McGovern, 2007). In addition, two options for standard test battery for genotoxicity are available in the ICH S2 (R1) guideline (ICH, 2008):


Table 1. Threshold for APIs

Option 1


Option 2


As stated by the ICH safety guidelines (S2A and S2B), "for compounds giving negative results, the completion of 3-test battery, perform and evaluate in accordance with current recommendations, will usually provide a sufficient level of safety to demonstrate the absence of genotoxic activity." Thus, any compound that produces a positive result in one or more assays in the standard battery has historically been regarded as genotoxic, which may require further testing for risk assessment (Müller *et al.*, 2006).

safety topics deal with *in vitro* and *in vivo* pre-clinical studies (carcinogenicity testing,

The ICH initially published guidelines on impurities of drug substances and pharmaceutical products in the late 1990s. In the guidelines, genotoxicity tests have been defined as *in vitro*  and *in vivo* tests designed for detecting compounds that induce genetic damage directly or indirectly (International Conference on Harmonization, 1997). The ICH quality guidelines Q3A(R) and Q3B(R) respectively address the topics of control of impurities in drug substances and degradants in pharmaceutical products, while the Q3C guideline deals with the residual solvents. However, several important issues have not been addressed in the guidelines, for example, the acceptable levels of impurities in drugs during development as well as the control of genotoxic impurities. Table 1 illustrates a series of thresholds described in ICH Q3A(R) that trigger reporting, identification, and qualification requirements. Subsequently, Table 2 depicts the thresholds for reporting, identification, and qualification of impurities in new drug products (ICH, 2006; Jacobson-Kram and McGovern, 2007). In addition, two options for standard test battery for genotoxicity are available in the ICH S2

Reporting threshold 0.05% 0.03%

ii. A cytogenetic test for chromosomal damage (the *in vitro* metaphase chromosome aberration test or *in vitro* micronucleus test), or an *in vitro* mouse lymphoma *tk* gene

iii. An *in vivo* test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase

ii. An *in vivo* assessment of genotoxicity with two tissues, usually an assay for micronuclei

As stated by the ICH safety guidelines (S2A and S2B), "for compounds giving negative results, the completion of 3-test battery, perform and evaluate in accordance with current recommendations, will usually provide a sufficient level of safety to demonstrate the absence of genotoxic activity." Thus, any compound that produces a positive result in one or more assays in the standard battery has historically been regarded as genotoxic, which may

Identification threshold 0.10% or 1.0 mg per day intake

Qualification threshold 0.15% or 1.0 mg per day intake

using rodent hematopoietic cells and a second *in vivo* assay.

require further testing for risk assessment (Müller *et al.*, 2006).

Maximum daily dose

≤2 g/day >2 g/day

(whichever is lower) 0.05%

(whichever is lower) 0.05%

genotoxicity testing, etc.) (ICH, 2008).

(R1) guideline (ICH, 2008):

Thresholds

Table 1. Threshold for APIs

mutation assay;

i. A test for gene mutation in bacteria;

i. A test for gene mutation in bacteria;

Option 1

cells. Option 2


1 The amount of drug substance administered per day

2Thresholds for degradation products are expressed either as a percentage of the drug substance or as a total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degradation product is unusually toxic.

3Higher thresholds should be scientifically justified.

Table 2. Thresholds for degradation products in new drug products (Jacobson-Kram and McGovern, 2007)

#### **2.2 EMEA guideline**

The European Medicines Agency (EMEA) guideline describes a general framework and practical approaches on how to deal with genotoxic impurities in new active substances**.**  According to the guideline "The toxicological assessment of genotoxic impurities and the determination of acceptable limits for such impurities in active substances is a difficult issue and not addressed in sufficient detail in the existing ICH Q3X guidance". In addition, the EMEA guideline proposed a toxicological concern (TTC) threshold value of 1.5 μg/day intake of a genotoxic impurity which is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) in most pharmaceuticals. Based on the TTC value, a permitted level of an active substance can be calculated concerning the expected daily dose. Higher limits might be justified under certain conditions such as short-term exposure periods (European Medicines Agency/ Committee for Medicinal Products (CHMP) for Human Use, 2006). In the context of this guideline, the classification of a compound (impurity) as genotoxic in general indicates that there are positive findings in established *in vitro* or *in vivo* genotoxicity tests with the focus on DNA reactive substances that have a potential for direct DNA damage. In the absence of such information, *in vitro* genotoxics are usually considered as presumptive *in vivo* mutagens and carcinogens (EMEA/CHMP, 2006).

Genotoxic Impurities in Pharmaceuticals 391

salt form for secondary processing, especially wet granulation. Another benefit of these salts is their high melting point because APIs with low melting points often exhibit plastic deformation during processing which can cause both caking and aggregation. Typically, an increase in the melting point has an adverse effect on aqueous solubility owing to an increase in the crystal lattice energies. Sulfonic acid salts tend to be an exception to this rule, since they exhibit both high melting points as well as good solubility. In addition, as mentioned in the literature, the high solubility and high surface area of haloperidol mesylate result in enhanced dissolution rates (<2 min in pH 2 simulated gastric media), which are more rapid than the competing common ion formation (Elder and Snodin, 2009; Elder *et al.*,

On the other hand, sulfonic acids can react with low molecular weight alcohols such as methanol, ethanol, or isopropanol to form the corresponding sulfonate esters. In general, sulfonic acid esters are considered as potential alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems and possibly carcinogenic effects *in vivo*; thus, these compounds have raised safety concerns in recent times (Snodin, 2006; Teasdale *et* 

Mesyla Tosylate Besylate

besylate in UK-369,003-26, a novel PDE5 inhibitor (Hajikarimian *et al.*, 2010).

Sulfonate impurities comprise the most investigated group of genotoxic impurities (GIs). Initially in 2007, sulfonate impurities raised major concern when over a period of three months (March to May 2007), several thousand HIV patients in Europe were exposed to ViraceptR (nelfinavir mesylate) tablets containing the contaminant ethyl methane sulfonate (EMS). However, the available *in vitro* and animal data indicated that the levels at which HIV patients were exposed to EMS (maximal dose of 0.055 mg/kg/d) did not induce any risk; nevertheless, any further level was of significant concern to their safety (Elder and Snodin, 2009). Since 2007 other drugs have been reported for contamination by sulfonate impurities, such as alkyl benzene sulfonates in amlodipine besylate (Raman *et al.*, 2008), dimethyl sulfate (DMS) in pazopanib hydrochloride (Liu *et al.*, 2009), EMS and methyl methane sulfonate (MMS) in imatinib mesylate (Ramakrishna *et al.*, 2008), EMS in zugrastat (Schülé *et al.*, 2010), alkyl sulfonates in flouroaryl-amine (Cimarosti *et al.*, 2010), and ethyl

EMS is a well-established genotoxic agent in this group which reacts with DNA producing alkylated (specifically ethylated) nucleotides. MMS, an analog of EMS, is a genotoxic compound both *in vitro* and *in vivo*. The international agency for research on cancer (IARC) has classified EMS and MMS in group 2B and 2A, respectively (Snodin, 2006; Gocke *et al.*,

Fig. 1. Structures of common sulfonate salts

**3.1.1 Genotoxicity profile** 

2010a).

*al.*, 2009).

2009a).

Based on the importance of the mechanism of action and the dose-response relationship in the assessment of genotoxic compounds, the EMEA guideline presents two classes of genotoxic compounds:


Those genotoxic compounds with sufficient evidence would be regulated according to the procedure as outlined for class 2 solvents in the "Q3C Note for Guidance on Impurities: Residual Solvents". For genotoxic compounds without sufficient evidence for a thresholdrelated mechanism, the guideline proposes a policy of controlling levels to "as low as reasonably practicable" (ALARP) principle, where avoiding is not possible.

On the other hand, this guideline provides no advice on acceptable TTCs for drugs during development, especially for trials of short duration (Jacobson-Kram and McGovern, 2007).

The pharmaceutical research and manufacturing association (PhRMA) has established a procedure for the testing, classification, qualification, toxicological risk assessment, and control of impurities processing genotoxic potential in pharmaceutical products. As most medicines are given for a limited period of time, this procedure proposes a staged TTC to adjust the limits for shorter exposure time during clinical trials (Table 3). Thus, the staged TTC can be used for genotoxic compounds having genotoxicity data that are normally not suitable for a quantitative risk assessment (Muller *et al.*, 2006).


Table 3. PhRMA genotoxic impurity task force proposal – allowable daily intake (µg/day) for genotoxic impurities during clinical development using the staged TTC approach
