What is the TTC threshold?

The threshold of toxicological concern is 1.5 micrograms per day under ICH M7 for a mutagenic impurity at lifetime exposure. It applies as long as no compound-specific limit exists. For shorter treatment durations ICH M7 allows higher values under the less-than-lifetime staging.

What exactly does class 3 mean?

Class 3 means alerting structure, unrelated to the structure of the drug substance, and no mutagenicity data available. ICH M7 offers two equivalent routes. Either you control the impurity permanently at the acceptable intake, usually the TTC threshold, in which case no assay is required. Or you run a bacterial mutagenicity assay: a negative result reclassifies the impurity to class 5 and it is treated as an ordinary impurity, a positive result places it in class 2 and it stays limited at TTC level. The assay is therefore not an obligation but the way out of the limit. Class 3 is explicitly not an all-clear.

Does the prediction replace the Ames test?

No. ICH M7 permits two complementary prediction methodologies in place of a test when both are negative and the rationale is documented. As soon as one of the two fires, the test or the limit applies. So the pre-screen decides which cases need the test at all.

Where do the structural alerts come from?

From a rule-based set of SMARTS patterns, each carrying a mechanism and a literature reference. For the nitrosamine alert the source is the IARC monograph series, for the aromatic acetamide alert Benigni and Bossa 2006, for the aminophenol alert Bolton et al. 2000. Every alert is therefore checkable, not merely asserted.

What happens to my structure?

The submitted structure is processed solely to handle the request, is not used for training, and is kept tenant-separated. Processing takes place in the EU, and deletion on request applies. This is anchored contractually in the data processing agreement, not only in this paragraph.

Can I screen an entire candidate list at once?

Yes. For series screening there is a batch variant that returns the same classification for many structures in one call. The difference to the single check is the call form, not the method.

Is this enough for a submission?

The output is a documented prediction with method, mechanism and source, so it is a defensible first pass. What becomes of it in a submission is your assessment. We do not deliver a validated system, we deliver the documentation you validate with.

ICH M7 pre-screening of nitrosamine impurities: what the class actually tells you

Author: Dr. Oliver Kraft, Dr. rer. nat., Organische Chemie · Updated 2026-08-03

Short and direct

An ICH M7 pre-screen answers exactly one question: does this impurity have to be treated as a mutagen or not. The four computed examples below show the result pattern. The three nitrosamines NDMA, NDEA and NMBA land in class 2, known mutagenic carcinogens requiring no further testing: structural alert and statistical model agree, and the consequence is a limit at the TTC threshold of 1.5 micrograms per day.

The fourth case, paracetamol, is the interesting one. The tool finds two structural alerts, the statistical model disagrees at 0.17, and the classification lands on class 3. Class 3 does not mean harmless. It means alerting structure with no mutagenicity data, and that is the class that forces a decision: a permanent limit at TTC level, or a bacterial mutagenicity assay that moves the impurity to class 5 or class 2 depending on the outcome. Looking only at the statistical score misses it.

The computed result

Compound	SMILES	ICH M7 class	Structural alerts	Statistical model	Confidence
NDMA (N-Nitrosodimethylamin)	CN(C)N=O	2	nitrosamine	0.6905	0.628
NDEA (N-Nitrosodiethylamin)	CCN(CC)N=O	2	nitrosamine	0.7215	0.656
NMBA (N-Nitroso-N-methyl-4-aminobuttersaeure)	CN(CCCC(=O)O)N=O	2	nitrosamine	0.8901	0.809
Paracetamol (Kontrollfall, kein Nitrosamin)	CC(=O)Nc1ccc(O)cc1	3	aromatic_amine_acetamide, aminophenol	0.1677	0.648

Computed 2026-08-03 with covatox_ich_m7 against mcp.covasyn.com. The values are verbatim tool output, not editorially rounded.

Why is the structural alert alone not enough?

ICH M7 explicitly requires two complementary prediction methodologies, one expert rule-based and one statistical. The reason is visible in the paracetamol result: the aromatic acetamide alert fires on any acetylated aromatic amine, regardless of whether that specific compound is Ames-positive. The statistical model contributes the counter-evidence. A tool that delivers only one of the two either produces false alarms or misses cases.

The confidence column in the result below is therefore not decoration. For NMBA it sits at 0.81 because both methods point the same way. For paracetamol it sits at 0.65 despite two alerts, because the statistical model pulls against them. That number is the most honest figure on this page.

What does class 2 mean in practice?

Class 2 means known mutagenic carcinogen. No further mutagenicity testing is needed, and the impurity is limited to the TTC threshold of 1.5 micrograms per day unless a compound-specific limit exists. For the three nitrosamines shown, regulators publish compound-specific limits that are lower for some. The pre-screen does not replace checking the current regulatory list, it only tells you that you need to.

The mechanism the tool returns is not an extra. CYP2E1 activation to the diazonium ion followed by DNA alkylation is the reason the whole nitrosamine class is treated this way. Anyone who has to defend the classification to a regulator needs that sentence, not just the class number.

How fast is this in daily work?

The four runs below took under a minute in total. The point is not the speed as such, it is that a pre-screen moves to the start of the decision rather than the end. A candidate list of twenty possible degradation products can be reviewed before the first synthesis, not once the analytical result is in.

Frequent questions

What is the TTC threshold?: The threshold of toxicological concern is 1.5 micrograms per day under ICH M7 for a mutagenic impurity at lifetime exposure. It applies as long as no compound-specific limit exists. For shorter treatment durations ICH M7 allows higher values under the less-than-lifetime staging.
What exactly does class 3 mean?: Class 3 means alerting structure, unrelated to the structure of the drug substance, and no mutagenicity data available. ICH M7 offers two equivalent routes. Either you control the impurity permanently at the acceptable intake, usually the TTC threshold, in which case no assay is required. Or you run a bacterial mutagenicity assay: a negative result reclassifies the impurity to class 5 and it is treated as an ordinary impurity, a positive result places it in class 2 and it stays limited at TTC level. The assay is therefore not an obligation but the way out of the limit. Class 3 is explicitly not an all-clear.
Does the prediction replace the Ames test?: No. ICH M7 permits two complementary prediction methodologies in place of a test when both are negative and the rationale is documented. As soon as one of the two fires, the test or the limit applies. So the pre-screen decides which cases need the test at all.
Where do the structural alerts come from?: From a rule-based set of SMARTS patterns, each carrying a mechanism and a literature reference. For the nitrosamine alert the source is the IARC monograph series, for the aromatic acetamide alert Benigni and Bossa 2006, for the aminophenol alert Bolton et al. 2000. Every alert is therefore checkable, not merely asserted.
What happens to my structure?: The submitted structure is processed solely to handle the request, is not used for training, and is kept tenant-separated. Processing takes place in the EU, and deletion on request applies. This is anchored contractually in the data processing agreement, not only in this paragraph.
Can I screen an entire candidate list at once?: Yes. For series screening there is a batch variant that returns the same classification for many structures in one call. The difference to the single check is the call form, not the method.
Is this enough for a submission?: The output is a documented prediction with method, mechanism and source, so it is a defensible first pass. What becomes of it in a submission is your assessment. We do not deliver a validated system, we deliver the documentation you validate with.

Sources

ICH M7(R2): Assessment and control of DNA reactive mutagenic impurities in pharmaceuticals to limit potential carcinogenic risk. Source of the class 1 to 5 scheme and the TTC threshold.
IARC Monographs: Source of the nitrosamine structural alert and the stated mechanism.
Benigni R, Bossa C (2006): Structure-activity relationships for aromatic amines and amides, source of the acetamide alert.

The same calculation on your structures

The results above come from the same tools your language model can call over the Model Context Protocol. Test them free of charge, at your own pace.

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