compounds will usually occur only when the stakeholder has in‐house data that will enable an expert review. In the absence of such data, an expert review by a chemist can determine if the out of domain moiety is sufficiently unreactive (i.e. non‐electrophilic) to consider it a Class 5 impurity. Using an additional (Q)SAR software frequently does not solve the problem, because an out of domain moiety in one software is often also not within the database domain of the additional software, and adding on additional prediction tools can sometimes just add more uncertainty rather than clarity.
2.4.6.3 Question 6.3
Section 6 of the guideline further states that (emphasis added): “To follow up on a relevant structural alert (Class 3 in table 1), either adequate control measures could be applied or a bacterial mutagenicity assay with the impurity alone can be conducted. An appropriately conducted negative bacterial mutagenicity assay (Note 2) would overrule any structure‐based concern, and no further genotoxicity assessments would be recommended (Note 1). These impurities should be considered non‐mutagenic (Class 5 in table 1).”
| Question | Answer |
|---|---|
| In a case where an impurity is demonstrated to be negative in an Ames study but positive in a clastogenicity study (e.g. chromosomal aberration test), how would the impurity be classified per the ICH M7 classification system? | If an impurity tests negative in an Ames assay, it is considered a Class 5 impurity. Addressing positive results in a clastogenicity assay is out of scope of ICH M7. |
As discussed above in Question 1.1, it is made explicitly clear that if an impurity is negative in the Ames test and even if it is positive in the chromosomal aberrations assay (clastogenic), this impurity is considered a Class 5 impurity.
2.4.6.4 Question 6.4
The continuation of the text quoted above in Question 6.3 says the following (emphasis added): “A positive bacterial mutagenicity result would warrant further hazard assessment and/or control measures (Class 2 in table 1). For instance, when levels of the impurity cannot be controlled at an appropriate acceptable limit, it is recommended that the impurity be tested in an in vivo gene mutation assay in order to understand the relevance of the bacterial mutagenicity assay result under in vivo conditions. The selection of other in vivo genotoxicity assays should be scientifically justified based on knowledge of the mechanism of action of the impurity and expected target tissue exposure (Note 3). in vivo studies should be designed taking into consideration existing ICH genotoxicity Guidelines. Results in the appropriate in vivo assay may support setting compound specific impurity limits.”
| Question | Answer |
|---|---|
| Please clarify the rationale for the tests included under Note 3 as a follow‐up to investigate the in vivo relevance of Ames mutagen. | If an impurity is positive in the Ames test, an in vivo follow‐up test with mutagenic end point (mutagenicity) should be used. The other follow‐up tests outlined in Note 3 are also acceptable when scientific rationale is provided to support their use. For any of the above tests, adequate exposure should be demonstrated in line with ICH S2. |
Regulatory agencies expect a strong argument to overrule a positive Ames test. It is pertinent that the in vivo test(s) used to further investigate the relevance of the in vitro results be chosen correctly and that adequate exposure be demonstrated. A negative result in a transgenic mutation assay would normally be the strongest evidence to overrule a positive in vitro result, but this assay is relatively expensive and lengthy. Other assays are also acceptable, and often times a stakeholder will chose to combine several assays (e.g. Pig‐a assay, in vivo micronucleus assay, and Comet assay) to be absolutely certain that the impurity is nonmutagenic.
2.4.7 Section 7 – Risk Characterization
This section has five Q&As that start with a direct continuation to Q&A 6.4, which relates to Ames positive impurities. Then an interesting question is presented regarding the application of the LTL to AIs derived by linear extrapolation from TD50 values but not to PDEs derived for impurities. Then a Q&A relates to the explanations and implications of moving HIV disease from treatment duration of <10 years to lifetime treatment, and finally a Q&A relates to the application of limits for individual impurities when three or more Class 2 and Class 3 impurities are present.
2.4.7.1 Question 7.1
This question relates to the same paragraph from the guideline that was stated above in Question 6.4.
| Question | Answer |
|---|---|
| If an Ames positive impurity is subsequently tested in an appropriate in vivo assay and the results are clearly negative, is that sufficient to demonstrate lack of in vivo relevance? | Yes. A well‐conducted and scientifically justified in vivo study (see Question 6.4 in this document) is sufficient to demonstrate lack of in vivo mutagenic relevance. If the results of the in vivo study are clearly negative, the impurity can be assigned to ICH M7, Class 5. |
This Q&A almost states the obvious. The entire intention of Note 3 in the guideline is to provide adequate in vivo tests that can overrule a positive Ames test, and if an impurity is determined to be nonmutagenic, then it can be considered a Class 5 impurity.
2.4.7.2 Question 7.2
With regard to mutagenic impurities with positive carcinogenicity data (Class 1 impurities), the guideline states that: “Compound‐specific risk assessments to derive acceptable intakes should be applied instead of the TTC‐based acceptable intakes where sufficient carcinogenicity data exist. For a known mutagenic carcinogen, a compound‐specific acceptable intake can be calculated based on carcinogenic potency and linear extrapolation as a default approach. Alternatively, other established risk assessment practices such as those used by international regulatory bodies may be applied either to calculate acceptable intakes or to use already existing values published by regulatory authorities (Note 4).”
| Question | Answer |
|---|---|
| If an Ames positive impurity is subsequently tested in an appropriate in vivo assay and the results are positive, does that support setting compound‐specific impurity limits? | No. in vivo gene mutation assays are currently not validated to directly assess cancer risk because the end point is mutation and not carcinogenicity (i.e. they are used for hazard identification). Results from these tests could identify mode of action and/or direct further testing strategy to complement the available data for a weight of evidence approach. |
The Q&A here clarifies that in vivo mutagenicity data cannot be used to set compound‐specific impurity limits and that the proper methods to determine AIs for mutagenic carcinogens is the linear extrapolation from the TD50 or from the BMDL10 (Benchmark Dose Lower Confidence Limit 10%), as described in Note 4 of the guideline.
2.4.7.3 Question 7.3
The LTL concept, previously known as “staged TTC,” is based on the assumption that cancer risk of known carcinogens increases as a function of cumulative dose. The guideline explains that the LTL approach is applied to mutagenic impurities in which the acceptable cumulative lifetime dose is uniformly distributed over the total number of exposure days during LTL exposure. This would allow higher daily intake of mutagenic impurities than would be the case for lifetime exposure and still maintain comparable