ICH M7 Workshop - Lhasa Limited

ICH M7 WorkshopICGM, Tokyo, November 2019

CEO

[email protected]

Chris Barber

Expert review under ICH M7 guidelines

Applying the ICH M7 guidelines

Is the impurity mutagenic? Is the impurity present in the final API?

What are the potential impurities?

Implement a control strategy

reagents,

intermediates, side

reactions, degradants…

Ames test or in

silico methods

Analytical or in

silico methods

Applying the ICH M7 guidelines

Database

searching

Expert prediction

Statistical

prediction

Expert

assessment

Classification

Test

Is the impurity mutagenic?ICH M7

classification

Tips for expert assessments

• Look for experimental data

• Vitic is a large, expert-curated database including public sources and member donations

• Read Derek’s detailed commentaries

• These are written by Lhasa’s expert scientists and contain peer-reviewed knowledge

• Use the confidence measures provided both Derek and Sarah

• These apply scientifically robust methodologies which have been published

• They indicate how trustworthy a prediction is and tell you where to focus

• Misclassified and unclassified features…

Features seen in false

negatives

Features not seen in

public datasets


• Look closely at supporting examples

• Review positive analogues

• could their activity be caused by additional features irrelevant to your compound?

• Review negative analogues

• have they been tested in 5 strains under OECD guidelines?

• Be conservative in your assessments

• If the evidence is not strong enough to convince you..

• treat as positive and consider using purge-based arguments

• run the Ames study


• You can overturn expert and statistical systems

Worked examples

Compound 1

Compound

Review high level predictions

?

Expert Review

M7 classification

Compound

Review the expert prediction

?

Expert Review

M7 classification

SAR

Alert comments highlight that certain anilines with electron withdrawing groups are excluded from the alert

Mechanism

Alert comments indicate that electron withdrawing groups are expected to reduce the rate of metabolic activation and destabilise intermediates. Therefore, electron withdrawing groups are expected to reduce the mutagenic potential of the aromatic amine.

Review the statistical prediction

?

Expert Review

M7 classification

Almost all close examples are negative

– one positive example is a bis-aniline


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Expert Review

M7 classification

The closest analogues in the Sarah Nexus training set contains non-mutagenic

(5 strain) aromatic amines which have relevant substitution patterns compared

to the query compound

Expert review

?

Expert Review

M7 classification

Predicted negative and two relevant nearest neighbours are

non-mutagenic having been tested in five strains

The compound fires an aromatic amine alert.

However, the alert comments indicate that aromatic

amines with electron withdrawing substituents are

known to be non-mutagenic and this relationship is

grounded by mechanistic rationale

Please make your selection

1. Class 3 – Alerting structure

2. Class 5 – No alerts or alerting with sufficient data

to demonstrate lack of mutagenicity

3. Unsure

M7 Classification – Compound 1

?

Expert Review

M7 classification

Class 5

No alerts or alerting with sufficient data to demonstrate lack of mutagenicity

Compound 2

Compound


?

Expert Review

M7 classification

Compound


?

Expert Review

M7 classification

Photomutagen mechanism is specific to furocoumarin structure and notrelevant to the query compound. The benzofuran moiety may intercalate;however, benzofuran is non-mutagenic2 and the carboxylic acid is notexpected to react with DNA.

Furocoumarins are known photomutagens which

intercalate with DNA then, upon near-ultraviolet

irradiation, bind pyrimidine bases1.

(1) Venturini et al, Mut. Res., 1981, 88, 17-22; (2) Weill-Thevenet et al, Mut. Res., 1981, 88, 355-362


?

Expert Review

M7 classification

Activity of analogues is associated with alternative functional

groups: aromatic amine, aromatic nitro.


?

Expert Review

M7 classification

Most similar compound is a benzofuran with a carboxylic acid at C2

and is negative in multiple strains.

Expert review

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Expert Review

M7 classification

• Inactive with misclassified features

• ..these are associated with an alternative mechanism that is

specific to furocoumarins and not the query compound

• Positive with 7% confidence

• Activity of nearest neighbours is associated with alternative functional groups

• Most similar example is a non-mutagenic benzofuran





3. Unsure


?

Expert Review

M7 classification

Class 5

Compound 3

Compound


?

Expert Review

M7 classification

Compound


?

Expert Review

M7 classification

Alert highlights that positive Ames results are observed for acid halides when DMSO isused as the solvent1

Acid halides typically undergo rapid hydrolysis to the carboxylic acid which is a knownAmes negative

(1) Amberg et al, Org. Proc. Res. Dev., 2015, 19, 1495-1506


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Expert Review

M7 classification

Activity associated with alternative functional groups: epoxide, N-

nitroso, alkylating agent. Similar acid chloride is negative.


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Expert Review

M7 classification

Activity associated with alternative functional groups:

epoxide, N-nitroso, aromatic azide.

Expert review

?

Expert Review

M7 classification

• Positive prediction with equivocal reasoning for acid halide

• Activity of acid halides in the Ames test is known to be a result of using DMSO

• Many acid halides rapidly undergo hydrolysis to the carboxylic acids which are

non-mutagenic

• Negative with 26% confidence

• Activity of nearest neighbours associated with other functional groups

• Most similar acid halide is non-mutagenic





3. Unsure


?

Expert Review

M7 classification

Class 5**alternatively use a purge argument to negate the risk of

the acid halide

Compound 4

Compound


?

Expert Review

M7 classification

Compound


?

Expert Review

M7 classification

• No alerts identified in DX

• No misclassified or unclassified features


?

Expert Review

M7 classification

All positive compounds have other features which

may cause the observed activity


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Expert Review

M7 classification

• Positive compounds have other features more likely

to cause activity

• Closest analogues fire a DX alert for alpha beta

unsaturated systems. Alkyl substituents at C3 are

excluded from this alert as they have reduced activity due to steric hinderance.


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Expert Review

M7 classification

• The closest analogues are negative

• …and have been tested in 5 strains

Expert review

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Expert Review

M7 classification

• Inactive prediction

• No misclassified features

• Negative prediction

• NN most similar to query compound are negative

• Positive compounds can be explained by presence of other groups

• The alpha beta unsaturated nitrile has shown activity when

unsubstituted (acrylonitrile) but this activates an alert which indicates activation requires a strong EWG and no steric hindrance in order to

react in 1,4 position.





3. Unsure


?

Expert Review

M7 classification

Class 5No alerts or alerting with sufficient data to demonstrate lack of mutagenicity

Compound 5

Compound


?

Expert Review

M7 classification

10


?

Expert Review

M7 classification



• Near-miss of alert 351 for aromatic amine

10


?

Expert Review

M7 classification

Similar compounds in the training set show inconsistent results (& strain profile)

Nearest examples are positive

10

Expert review

?

Expert Review

M7 classification



• Near-miss of alert 351 for aromatic amine

• Positive – 9% confidence

• The nearest neighbour is positive

• Positive compounds do not have any additional toxicophores

10





3. Unsure

10


?

Expert Review

M7 classification

Class 3Alerting structure.

Hard to dismiss close potential analogues – suspect we are

near an activity cliff. Recommend testing

10

Compound 6


?

Expert Review

M7 classification


?

Expert Review

M7 classification

Alert 307: N-Methylol compound or precursor

N-methylol group matches toxicophore 1.Identified as a DNA reactive centre through hydrolysis to formaldehyde (Ames positive).Covalent DNA binding reported for many N-methylol derivatives.


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Expert Review

M7 classification

Alert 307: N-Methylol compound or precursor

Toxicophore I positive example

To reflect the frequently low or absent activity of members of this class in

Ames tests the default likelihood has been set as equivocal for non-example

compounds.

Additional information (from Vitic)

?

Expert Review

M7 classification

Substructure search for N-methylol compounds in Vitic 2018.1.0: 20 matches toxicophore I.

Ames overall call positive for 6, negative for 12, conflicting data for 2. Some relevant examples below:

Positive in TA98, TA100 and TA102+/-S9

Positive in TA102+/-S9

Conflicting data in TA98 and TA100+/-S9

*Caution: Ames test may be unresponsive to this class of compounds,

due to low rate of conversion to formaldehyde, itself not highly potent.

Non-standard conditions may be required to observe activity for this class.

Negative examples firing alert 307


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Expert Review

M7 classification

Compounds from training set are dissimilar to the test compound.

None contains a N-methylol group matching the toxicophore 1 from the Derek alert 307. No Sarah hypothesis exists for this toxicophore.

Expert review

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Expert Review

M7 classification

• Active prediction

• Fires alert 307 for N-Methylol compound or precursor

Matches toxicophore 1: N-Methylol identified as DNA

reactive centre through hydrolysis to formaldehyde.


• Compounds from training set dissimilar to the test compound.

None matches the N-Methylol toxicophore

• No Sarah hypothesis exists for this toxicophore.





3. Unsure

M7 Classification – compound 6

?

Expert Review

M7 classification

Class 3*Alerting structure

*Caution: Ames test may be unresponsive to this class of compounds,

due to low rate of conversion to formaldehyde, itself not highly potent

Non-standard conditions may be required to observe activity for this class.

Compound 7


?

Expert Review

M7 classification


?

Expert Review

M7 classification

Alert 027: Alkylating agent

• Chloromethanamine fragment matches alkylating toxicophore (alkyl chloride).

• Alkyl halides identified as electrophilic species capable of directly alkylating DNA.

• This alert describes the genotoxicity of alkylating agents where the carbon

bearing the functional group is a primary or secondary alkyl carbon atom.

Additional information (from Vitic)

?

Expert Review

M7 classification

2 compounds matching the (chloromethyl)dimethylamine pattern with Ames positive calls.

However 2nd toxicophore present for the 2nd structure, which also fires the aromatic nitro alert 329.

Substructure search for the (chloromethyl)dimethylamine pattern

with an Ames overall call in Vitic 2018.1.0

N-(Chloromethyl)-4-nitrophthalimide

Positive in TA98 and TA100 +/-S9

2nd toxicophore present,

fires the aromatic nitro compound alert 329.

Chloromethylphthalimide

Positive in TA98 and TA100 +/-S9


?

Expert Review

M7 classification

Positive hypothesis for alkyl chloride

Most alkyl chlorides examples are

positive except 3 out of 15

(examples 2, 7 and 9).

Positive hypothesis for alkyl

chloride, toxicophore responsible

for mutagenicity, in agreement with Derek alert 027.

Expert review

?

Expert Review

M7 classification

• Active prediction

• Fires alert 027 for alkylating agents

• Chloromethanamine fragment matches alkyl chloride toxicophore

• Active prediction with 23% confidence

• Matches 2 positive hypotheses

• Alkyl chloride positive hypothesis (most relevant hypothesis),

toxicophore responsible for mutagenicity, in agreement with

Derek alert 027





3. Unsure

M7 Classification – compound 7

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Expert Review

M7 classification

Class 3Alerting structure

Compound 8

Expert Review

M7 classification


?


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Expert Review

M7 classification

INACTIVE

• No alerts identified in Derek Nexus.

• No misclassified features identified.


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Expert Review

M7 classification

• Derek alert activated requires at least 3 fused

rings for mechanism suggested

• Other positives contained additional toxicophores


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Expert Review

M7 classification

most similar compound

• 100% negative in Sarah.

• Inactive in Derek.

• Good strain profile.

Expert review

?

Expert Review

M7 classification



• Weak positive prediction (6%)

• Activity of compounds in Sarah are associated with other

functional groups that fire alternative Derek alerts.

• Most similar compound gives negative results





3. Unsure


?

Expert Review

M7 classification

Class 5

Compound 9


?

Expert Review

M7 classification

Compound


?

Expert Review

M7 classification


?

Expert Review

M7 classification


?

Expert Review

M7 classification

Expert review

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Expert Review

M7 classification

• Active prediction with plausible reasoning

• No evidence that could negate prediction

• Ethylene sulfate (similar compound) is active in TA1535

• Out of domain prediction

• Disregarded hypothesis does not have examples that

similar to query compound





3. Unsure


?

Expert Review

M7 classification

Class 3 – Alerting structure

Resources to support your expert review

• Selecting your systems

• Distinguishing between expert and statistical systems for application under ICH M7.

Barber, C. Reg. Toxicol. Pharmacol. 2017, 84, 124

• Undertaking expert review

• Establishing best practise in the application of expert review of mutagenicity under ICH M7.

Barber, C.., Reg. Toxicol. Pharmacol. 2015, 73, 367

• Use of in silico systems and expert knowledge for structure-based assessment of potentially

mutagenic impurities. Sutter, A.., Reg. Toxicol. Pharmacol. 2013, 67, 39

• Principles and procedures for implementation of ICH M7 recommended (Q)SAR analyses.

Amberg, A.., Reg. Toxicol. Pharmacol. 2016, 77, 13

• Lhasa’s website has many papers, posters, and videos…

• https://www.lhasalimited.org/library/

https://www.lhasalimited.org/publications/distinguishing-between-expert-and-statistical-systems-for-application-under-ich-m7/4210

https://www.lhasalimited.org/publications/establishing-best-practise-in-the-application-of-expert-review-of-mutagenicity-under-ich-m7/3653

https://www.lhasalimited.org/publications/use-of-in-silico-systems-and-expert-knowledge-for-structure-based-assessment-of-potentially-mutagenic-impurities/3185

https://www.lhasalimited.org/publications/principles-and-procedures-for-handling-out-of-domain-and-indeterminate-results-as-part-of-ich-m7-recommended-qsar-analyses/5195

https://www.lhasalimited.org/library/

Thank you

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ICH M7 Workshop - Lhasa Limited