hazard assessment of mutagenic impurities

Sub Heading

Date

Hazard Assessment Of

Potentially Mutagenic Impurities

ICH M7 Indian Roadshows 2020

February 28, 2020

March 02, 2020

Muzaffar Khan, Ph.D

[email protected]

Introduction

2

• Hazard assessment of 8 example compounds at Laurus Labs

with varying expert rule-based and statistical-based results

• Derek Nexus and Sarah Nexus results will be presented

• A conclusion will be drawn for each compound based on

expert call

• You will be asked to assess each example and vote

accordingly

• Laurus/Lhasa will then present an expert call*

• Follow-up hazard assessment on the alerting compounds will

be discussed

*All expert calls on the examples are solely the decisions taken at Laurus Labs

and corroborated by Lhasa Limited.

Hazard Assessment - ICH M7

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What kind of arguments can be used?

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• Adequate Ames data available

• Ames test does not assess the hazard caused by the

compound class adequately

• Toxicophore identified by one system has not been

adequately assessed by the other

• Toxicophore identified is not causative of activity

• Nearest neighbours are not adequately similar to support

prediction

• Data available for nearest neighbours is not of sufficient

quality to support prediction

Example-1

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Review high level predictions

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Review the expert prediction

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• No alerts fired

• Contains unclassified features


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• The test compound contains an unclassified feature (Imidoyl chloride)

which does not match with any structural alerts or examples for Ames

positive compounds in Derek.

• Predicted to be inactive in Ames mutagenicity test.

• Imidoyl chlorides are electrophilic in nature with a good leaving group

and therefore potentially mutagenic.

• Imidoyl chlorides are analogous of acyl chlorides and highly reactive.

• Imidoyl chlorides react spontaneously with water to yield the amides.

Review the statistical prediction

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• Outside domain

• Training set does not include any example covering this functionality

Expert review

11

Inactive *

Outside domain

• Inactive prediction but contains unclassified features

• Potential alkylating agent (short lived as hydrolysis is highly likely)

• Out of domain

• Sarah Nexus training set does not contain examples covering

“imidoyl chloride”

Please make your selection

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1. Class 3 –Alerting structure

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

demonstrate lack of mutagenicity

3. Unsure

M7 Classification – Example 1

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• Derek Nexus

• Inactive prediction, contains unclassified features

• Potential alkylating agent; highly reactive

• Sarah Nexus

• Outside domain with no similar examples in the training set


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Class 3 Alerting structure

Hazard Assessment & Control Strategy:

Impurity introduced too early in synthesis (n-6 step) and unlikely to carryover to

the drug substance. Proposed ICH M7 Option 4 control strategy based on

commercial batch data and spike-purge studies.

Mirabilis is useful to justify Option 4 control strategy

Example-2

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CAS No. 173676-59-0


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17

• No alerts fired

• A certain aromatic amine and potentially mutagenic

• A more detailed review is necessary


18

• Positive hypothesis for aromatic amine• The most relevant parent (2-Acetylaniline) is non-mutagenic.

• The 4-chloroaniline containing compounds (2,3,4&5) are not relevant.

• A Vitic search indicates that 4-Chloroaniline has displayed activity in TA98 (albeit

not very consistently). Listed in ICH M7 R1 Addendum too.

Expert Review

19

• Inactive prediction with no misclassified or unclassified features

• Compound contains an aromatic amine

• Expert review of pAA is a subjective process and the in silico

results should be handled with care.

• The stability and physicochemical properties of a subset of

aromatic amines affords them capricious mutagenic properties

in the Ames test.

• Positive hypothesis for aromatic amine

• 2-Acetylaniline is non-mutagenic, no relevant examples in

training set for 4-Chloroaniline

• 4-Chloroaniline has displayed mutagenic activity in TA98.

Inactive

Positive


20

1. Class 3 – Alerting structure

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


3. Unsure


21

• Derek Nexus

• Inactive prediction with no misclassified or unclassified features.

• The 2-substituent (electron withdrawing group) deactivates the aromatic

amine, there is no suitable electron donating group at another position

• Disubstituted aniline bearing only halogen and/or trifluoromethyl

substituents on aromatic ring are generally Ames negative

• Sarah Nexus

• Positive prediction for aromatic amine

• The most relevant example is Ames negative.


22

Experimental results confirmed the compound was Ames negative

(Class 5)

Class 5 No alerts or alerting with sufficient data to demonstrate

lack of mutagenicity

Example-3

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CAS No. 27607-77-8


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25

• No alerts fired

• Compound contains unclassified features


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• Outside domain

• Nothing to report


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• Triflic acid predicted to be non-mutagenic

• No misclassified or unclassified features


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• TMS-OH predicted to be non-mutagenic



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• Negative prediction for triflic acid


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• TMS-OH is Ames negative

Expert Review

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• Inactive prediction

• Compound contains unclassified features

• Outside domain

• Nothing to report

Inactive *

Outside domain


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3. Unsure


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• Derek Nexus

• Inactive prediction but contains unclassified features.

• No alerts fired for the potential hydrolysis products, triflic acid and TMS-OH

• Sarah Nexus

• Out of domain

• The hydrolysis product triflic acid is predicted negative

• TMS-OH is Ames negative


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Example- 4

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CAS NO：17933-03-8


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• Plausible mutagen

• Alert – 746: Arylboronic acid or derivative


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Exact match -Positive Ames Call reported from Vitic Nexus

• 4 most relevant nearest neighbours are all positive

• Negative nearest neighbours are dissimilar.

Expert Review

40



• No reason to over-rule Derek prediction

• Predicted positive -100% confidence (exact match)


• Ames data is reliable

Positive

Plausible


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3. Unsure


42

• Derek Nexus



• Sarah Nexus

• Predicted positive -100% confidence (exact match)


• Negative nearest neighbours are not very relevant


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Class 2 Mutagenic

Hazard Assessment & Control Strategy

Introduced in n-2 synthetic step; proposed Option-3 control strategy in

the starting material

Example- 5

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CAS No. 25487-66-5


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46

• Predicted to be inactive

• Arylboronic acid or derivative?


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Aryl boronic acids - A mutagenic response is seen both with and

without metabolic activation, most commonly with Salmonella

typhimurium TA100 and TA1537 and Escherichia coli WP2 uvrA

strains. The mechanism by which these compounds exert their

mutagenic effect remains unclear.

Electron donating and sterically hindering groups are thought to

modulate reactivity of the boron centre, which may relate to mutagenic

activity. Strong correlation with 11B NMR shifts.

Following deactivating substituents on phenylboronic acids excluded.

- Carboxylic acid or tetrazole

- 2,6-Disubstitution

- Bulky alkyl para-substitution

- 3,4-Carbocyclic aliphatic fused ring substitution


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• Equivocal

• Positive for Aryl boronic acid, however;

• Both the examples in training set are not very relevant as they do not

contain carboxylic acid moiety.

Expert Review

49

Arylboronic acid genotoxicity: Summary of data and knowledge

NMR shifts of arylboronic acids

Dr. Richard Williams, Lhasa Limitedhttps://www.lhasalimited.org/Public/Library/2015/Arylboronic%20acid%20genotoxicity%20-%20summary%20of%20data%20and%20knowledge.pdf

https://www.lhasalimited.org/Public/Library/2015/Arylboronic%20acid%20genotoxicity%20-%20summary%20of%20data%20and%20knowledge.pdf

3-Carboxyphenylboronic acid was non-mutagenic in AMES assay

Stannard, L., Giddings, A., Lad, A., Munoz-Muriedas, J., Harvey, J., and Kenny, J. (2012)

Can lowest unoccupied molecular orbital (LUMO) energies predict the mutagenicity of

boronic acids? Mutagenesis 27, 807−808.

Expert Review

Expert Review

51

• Inactive prediction with no misclassified or unclassified features

• Compounds containing carboxylic acid substitution are

excluded from the alerts list

• Equivocal

• Positive hypothesis for the aryl boronic acid

• Nearest neighbours with positive results are not very relevant

• Training set does not contain phenylboronic acids with

carboxylic acid substitution

Inactive

Equivocal


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3. Unsure


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• Derek Nexus


• No reason to over-rule Derek Nexus prediction

• Sarah Nexus

• Equivocal

• Positive examples not very relevant

• Compound is Ames negative (data available elsewhere)


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Example- 6

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56


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• Contains unclassified features


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• Positive hypothesis for Thiophene driven by presence of unrelated

toxicophores

Expert Review

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• Inactive prediction, contains unclassified features

• Thiophene itself is not an alerting structure for mutagenicity

• Thiophene was non-mutagenic in Ames test*. *AESCHBACHER,HU, et.al CONTRIBUTION OF COFFEE AROMA CONSTITUENTS TO THE MUTAGENICITY OF COFFEE; FOOD CHEM. TOXICOL. 27(4):227-232, 1989.*ZEIGER,E, ANDERSON,B et.al. SALMONELLA MUTAGENICITY TESTS: III. RESULTS FROM THE TESTING OF 255 CHEMICALS; ENVIRON. MOL. MUTAGEN. 9(SUPPL.9):1-110, 1987.

• Aryl fused thiophene not necessarily Ames negative.

• Equivocal prediction

• All positive examples were confounded• All the Ames positive compounds in training set containing thiopene

fragment contained other known mutagenic functionalities (e.g.

aromatic nitro compounds).

Derek: Inactive *

Sarah: Equivocal


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3. Unsure


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• Derek Nexus

• Inactive prediction with unclassified features.

• Sarah Nexus

• Equivocal

• Equivocal prediction driven by examples from training set containing

different toxicophores.


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Example- 7

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64


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• Positive prediction by Derek (Alert – 028: Mono- or di-alkylhydrazine)

• Hydrazines often produce weak and inconsistent responses in the Ames test

(due to their high toxicity and tendency to oxidise, which confounds testing)

and hence the compound is fired as an Equivocal alert in Derek.


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• Predicted overall negative in Sarah Nexus

• Positive hypothesis for hydrazine

Expert Review

67

• Equivocal

• Alert – 028: Mono- or di-alkylhydrazine

• Alert as per ICH M7

• Negative prediction

• Positive hypothesis for alkyl hydrazine

• Compounds in the training set with same alerting functionality are

Ames positive

Equivocal

Negative


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Imp-1

Imp-2Atazanavir API

Hazard Assessment

69

Structure Derek

Prediction

Sarah

Prediction

QSAR

Prediction

Experimental

Data

Similarity

to API

ICH M7

Class

Comments

Imp-1

Carc: Unspecified Ames: Unspecified

All Alerts found in API

EQUIVOCAL: Alert028 -

Mono- or di-alkylhydrazine

Class 4

Imp-2

Carc: Unspecified Ames: Unspecified

All Alerts found in API EQUIVOCAL: Alert028 -

Mono- or di-alkylhydrazine

Class 4


70


2. Class 4 –Alerting structure, same alert in drug substance

or compounds related to the drug substance (e.g.,

process intermediates) which have been tested and are

non-mutagenic



Imp-1


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• Derek Nexus

• Equivocal; Alert- 028: mono- or dialkyl - hydrazine

• Predicted as ICH M7 Class 4 but chemical environment of the alert

is essentially not same as that of the API

• Sarah Nexus

• Negative prediction

• Alkyl hydrazines in training set are Ames positive


72

Class 3 Alerting structure unrelated to the structure of API

Imp-1


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Hazard Assessment & Control Strategy

Option-1: Control at TTC limit

Option-2: Perform Ames test to classify as Class 2 or 5

Imp-2: Ames positive (in Salmonella

tester strains TA1537 and TA98) !Imp-1 : Ames negative

Imp-2 was non-clastogenic in the follow-up in vivo micronucleus assay in rats.

Example- 8

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75


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• Potential aromatic amine, aryl N-oxide and acrylonitrile present


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• Positive hypothesis for aryl-N-oxide

• >=1 fused aromatic ring or second toxicophore is required for activity

Expert Review

78

• Inactive prediction, no misclassified or unclassified features

• Mutagenicity from the amine between two aromatic rings is unlikely

• Aryl-N-Oxide and Acrylonitrile alerts have not been fired for valid

SAR reasons

• Positive prediction

• Aromatic N-oxide alert

• All positive examples were confounded containing > 1 fused

aromatic rings or other alerts e.g. aromatic nitro or amine

Inactive

Positive

Expert Review – Public data

79

Myatt et al. concluded based on SARs that the general class of aromatic N-Oxide is not an alert for predicting DNA-reactive mutagenicity (except quindioxins)Mutagenesis, Volume 34, Issue 1, January 2019, Pages 67–82, https://doi.org/10.1093/mutage/gey020

https://doi.org/10.1093/mutage/gey020


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3. Unsure


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• Derek Nexus


• No reason to over-rule Derek Nexus prediction

• Sarah Nexus

• Positive

• All positive examples are confounded, contain other alerts (e.g.

aromatic nitro or amine and > 1 aromatic rings) and not very

relevant


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Conclusions

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1. ICH M7 recommends using two complementary in-silico tools;

Expert rule-based (eg. Derek Nexus) and Statistical (eg. Sarah

Nexus).

2. Both expert and statistical tools are necessary!

3. Equivocal / out of domain results should be treated further with

expert opinion to arrive at meaningful conclusion.

4. Dependence only on literature based structural alerts (e.g.

Mueller’s alerts) for the expert review process, without

considering mitigating factors, could result in many more false

positives (Mayden A, Williams RV et al. (2017), Utility of published DNA

reactivity results, https://pubmed.ncbi.nlm.nih.gov/28549899.).

5. The use of Derek Nexus and Sarah Nexus significantly reduces

the number of false positives saving significant resources in the

hazard assessment process.

https://pubmed.ncbi.nlm.nih.gov/28549899

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THANK YOU for your kind attention!

ACKNOWLEDGEMENTS

Dr. Richard Williams

LAURUS LABS LTD. and LHASA LIMITED

hazard assessment of mutagenic impurities

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