classification with respect to mutagenic and carcinogenic potential and resulting control actions.
| Class | Definition | Proposed action for control (details in Sections 7 and 8) |
|---|---|---|
| 1 | Known mutagenic carcinogens | Control at or below compound‐specific acceptable limit |
| 2 | Known mutagens with unknown carcinogenic potential (bacterial mutagenicity positive, no rodent carcinogenicity data) | Control at or below acceptable limits (appropriate TTC) |
| 3 | Alerting structure, unrelated to the structure of the DS; no mutagenicity data | Control at or below acceptable limits (appropriate TTC) or conduct bacterial mutagenicity assay; if nonmutagenic = Class 5; if mutagenic = Class 2 |
| 4 | Alerting structure, same alert in DS or compounds related to the DS (e.g. process intermediates), which have been tested and are nonmutagenic | Treat as nonmutagenic impurity |
| 5 | No structural alerts, or alerting structure with sufficient data to demonstrate lack of mutagenicity or carcinogenicity | Treat as nonmutagenic impurity |
A particular challenge with respect to Class 4 compounds is defining structural similarity. Mathematical approaches such as Tanimoto scores may be utilized; however, great care is required in their use and similarity cannot simply be defined by a score exceeding a predefined threshold. In all cases it is important to assess the environment, both steric and electronic, in close proximity to the alerting moiety within the impurity in question.
Based on the outcome of the SAR assessment, for those compounds considered a concern, in particular those classified as Class 3, further evaluation in the form of testing may be performed. The earlier scope section of the ICH M7 guideline makes clear that the emphasis is on mutagenic impurities and that the bacterial reverse mutation test (Ames) [10] should be used to follow up any SAR alert.
In addition this section provides an overview of potential in vivo follow‐up tests that can be utilized in order to investigate further a positive bacterial assay. The tests themselves are described in detail in Table 2.3 (based on Note 3 within the guideline).
Table 2.3 Tests to investigate the in vivo relevance of in vitro mutagens (positive bacterial mutagenicity).
Source: Reproduced from ICH M7.
| in vivo test | Factors to justify choice of test as fit‐for‐purpose |
|---|---|
| Transgenic mutation assays | For any bacterial mutagenicity positive. Justify selection of assay tissue/organ |
| Pig‐a assay (blood) | For directly acting mutagens (bacterial mutagenicity positive without S9a)b |
| Micronucleus test (blood or bone marrow) | For directly acting mutagens (bacterial mutagenicity positive without S9) and compounds known to be clastogenicb |
| Rat liver unscheduled DNA synthesis (UDS) test | In particular for bacterial mutagenicity positive with S9 only; responsible liver metabolite known to be generated in test species used to induce bulky adducts |
| Comet assay | Justification needed (chemical class specific mode of action to form alkaline labile sites or single‐strand breaks as preceding DNA damage that can potentially lead to mutations)Justify selection of assay tissue/organ |
| Others | With convincing justification |
a S9 – Supernatant fraction obtained from an organ (usually liver) homogenate and contains cytosol and microsomes. The microsomes component of the S9 fraction contains cytochrome P450 isoforms (Phase I metabolism) and other enzyme activities.
b For indirect acting mutagens (requiring metabolic activation), adequate exposure to metabolite(s) should be demonstrated.
The guideline states that such tests can be used to assess the in vivo relevance of the positive findings of the in vitro bacterial reverse mutation test, suggesting that the results may support the establishment of a compound‐specific limit.
2.2.8 Risk Characterization
This section, Section 7 in the guideline, outlines the risk characterization principles used to define acceptable limits for compounds classified in groups 1, 2, or 3, see Table 2.3.
2.2.8.1 Acceptable Intakes Based on Compound‐specific Risk Assessments
2.2.8.1.1 Mutagenic Impurities with Positive Carcinogenicity Data (Class 1)
It is important to note that the guideline specifically stipulates that where adequate carcinogenicity data exist it should be used to calculate a compound‐specific AI or ADI. It also outlines that the approach should mirror that of the derivation of the TTC itself, i.e. linear extrapolation to a risk value of 1 in 100 000 analogous in risk terms to the TTC. It also states that other established riskvassessment practices such as those used by international regulatory bodies may be applied either to calculate AIs or the actual values themselves used. This apparently helpful statement does in fact lead to considerable confusion: should for example the value for a particular compound specified by the US environmental protection agency (EPA) (or other agencies) be simply adopted or in such instances should the available data be evaluated using the linear extrapolation? The guideline provides no clear statement on such a point and nor does it provide any specific example. In practice it would seem appropriate to use the default approach of linear extrapolation where data are available. Dobo et al. [27] recently reported on various ADIs that can be generated for hydrazine, exemplifying the considerable ambiguity that can be found when trying to generate a compound‐specific ADI for regulatory use.
Linked to this section is Note 4 where a specific example calculation is provided. The calculation outlines the determination of an AI for ethylene oxide. It is surprising that ethylene oxide was chosen as it is a gas, with good purging potential and of little synthetic utility, making its presence in final product very unlikely. Furthermore there is strong evidence that it is also generated endogenously [28]. In terms of the calculation itself, it is relatively straight forward. Terminal dose (TD50) values are taken from the Carcinogenicity Potency Database (CPDB) for both rat and mouse, with the more conservative value being selected, 21.3 mg/kg/day (rat) and the limit calculated by dividing by 50 000 to adjust to a 1 in 100 000 risk and multiplied by the internationally accepted average human body weight (50 kg), to give an ADI of 21.3 μg/day for lifetime exposure.
On the face of it, this looks relatively straightforward; however, this is a simple example. In reality this is often far more complex. In many cases data are available for multiple carcinogenicity studies, within the CPDB these are combined and reported in terms of the harmonic mean. The studies