methodology with which to determine levels.
3.2.6.9 Safety Testing
For any impurity identified as being potentially mutagenic (based on SAR evaluation) and assessed as having a high likelihood of carryover into the API, the next step is often to carry out in vitro safety testing.
If in vitro testing is selected, it is recommended that the synthesized or isolated impurity is tested for mutagenicity as an individual impurity. However, where this is impractical, then spiked samples or batches of material that contain elevated levels of the impurity of concern may be tested. The latter approach is not generally encouraged by regulatory authorities, and in such cases, an early dialogue with the relevant regulatory authority is recommended. In relation to the material quality, it is recommended to ensure the sample tested is as pure as is practically possible and is fully characterized in terms of the impurities present with the test sample. It is important to ensure that any result from subsequent testing is not confounded by the presence of trace impurities that may themselves elicit a response in the assay but would otherwise not be relevant to the route in which the (P)MI is being assessed. It is therefore important to fully characterize the test sample in terms of impurity profile with a specific emphasis on other potential mutagenic species that could be present, especially any used to synthesize the test sample.
ICH M7, Section 3 general principles, makes clear that the focus of the guideline is on deoxyribonucleic acid (DNA)‐reactive substances that have a potential to directly cause DNA damage when present at low levels leading to mutations and therefore, potentially causing cancer. It then states that to detect this type of mutagenic carcinogen a bacterial reverse mutation (mutagenicity), i.e. Ames test, has the necessary sensitivity and specificity.
A positive result in one or more of these tests is generally sufficient evidence to define the impurity as mutagenic, in which case it will be then necessary to adopt the appropriate TTC approach. Occasionally, a thresholded mechanism can be argued based on available safety data; this concept is examined in detail in Chapter 8. If an impurity is found to be negative, it is considered nonmutagenic (qualified for mutagenicity) and can then be treated as a normal impurity under ICH Q3A/B [13, 14].
The mutagenic potential of in vitro positive materials may be further evaluated in vivo, in order to look to establish the biological relevance of the in vitro findings (Table 3.5); this is highlighted in ICH M7, Note 3; and this is examined in detail in Chapter 6.
3.2.7 Quantification of Level Present
For PMIs that have been assessed as having a reasonable likelihood of being present in API at levels of concern, it may be appropriate to attempt to determine the level in parallel with, or in lieu of, the safety testing described above. The level of concern will be set by the appropriate limit (ADI, PDE, or TTC), which itself is impacted by factors such as maximum clinical dose and the maximum duration of the proposed trial(s). This in turn will have an effect on the choice of analytical technique.
Choice of technique?The nature of the impurity (analyte), the characteristics of the API or intermediate (matrix), and the level to be determined will influence the detection technique employed. Many organizations have developed specific strategies for refining such selections; this is examined in detail in Chapters 12 and 13.
Where in the process to test?Testing may be performed on upstream intermediates, API, or drug product as appropriate. It is often desirable to test as close as possible to the point of introduction of a PMI into the process. This approach may permit standard techniques, such as high‐performance liquid chromatography (HPLC) with ultraviolet (UV) detection, to be used, if this is allied to spiking experiments demonstrating the removal in the downstream process. Indeed, such an approach aligns with one of the control concepts defined within ICH M7, Section 8.1 of ICH M7 (specifically control Option 3). While development laboratories may be equipped with more sensitive techniques suitable for analysis at the low ppm level, manufacturing quality control laboratories are unlikely to have such facilities.
Quantitative assay or limit test.Both types of methods are used in the analysis of MIs. Quantitative tests are useful to furnish data for process development and to support further process modifications to reduce or more consistently control levels of a PMI. Having established a validated process, limit tests are likely to be favored for routine quality control (QC) testing.Limit tests are also more likely to be applied to upstream testing at an intermediate stage where they are used in conjunction with demonstrated evidence of further reduction through processing (control Option 3 – ICH M7) [8].Quantitative assays are usually applied at the final isolated API, as they provide a measure of true levels of the PMI/MI that would be present in the drug product, and the material would not be administered to patients if measured levels are found to be too high. Since the staged TTC concept for acceptable levels of PMIs/MIs is routinely applied during clinical stages of development, a quantitative test is generally desirable since acceptable levels vary as the clinical program develops. However, limit tests may be appropriate at the API or DP stage if this figure is well below the staged TTC control level.
3.3 Step 6 – Overall Risk Assessment
Once analytical and/or safety test data are available, these are used to finalize the risk assessment.
Possible outcomes include:
A PMI returns a negative Ames test result and thus no longer requires control as an MI but defaults to ICH Q3 levels of control [13, 14].
A PMI returns a positive Ames test result, but analytical testing demonstrates adequate process control over levels, i.e. level well below appropriate TTC limit.
Analytical data demonstrates that a PMI/MI is below a current staged TTC but above future permitted dose duration levels (i.e. where studies are of longer duration or higher doses are needed). In such circumstances this may necessitate a modification of the process to reduce or eliminate the impurity in question. If the material is potentially mutagenic rather than a known genotoxin, expediting safety testing, with the possibility of a negative Ames test result, would either remove the need for further process development and analytical control at trace levels, or confirm that additional control is required.
Analytical and safety data reveal an Ames positive material above a staged TTC level for a planned clinical study.4 In such a scenario, it is likely that the material in question would need to be reprocessed, unless a compelling case could be made for the benefit of the treatment over the risk posed (see ICH S9 [20] for example). In most cases, the process would need to be redeveloped to bring levels of the mutagen in question within the TTC for the envisaged marketed product dose and duration. For this reason it is important that an appropriate procedure to link the assessment to the formal release of material for clinical trial use sits alongside the risk assessment. Similarly, it is also important to understand whether an impurity is above the TTC prior to manufacture of drug product given that once formulated, any reprocessing becomes very difficult and secondary processing equipment could become compromised. For this reason, most companies will have an analytical release process for the drug substance that includes having a completed MI risk assessment and that any described controls for any PMIs have been met. Additionally, conducting the MI risk assessment prior to the drug substance manufacturing campaign will help identify PMIs that may be introduced late within the process, and therefore may not efficiently purge. This will allow additional processing to be engineered into the drug substance manufacturing campaign to reduce the likelihood of failed drug substance batches and reprocessing.