EMA发布亚硝胺杂质问答附录3，列出N-亚硝胺增强Ames试验条件

EMA 亚硝胺技术文件：Appendix 3 : Enhanced Ames Test Conditions for N-nitrosamines

官方目录首次发布：2023-11-09。本次目录更新：2024-03-26。

目录文件编号：EMA/120337/2024。

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26 March 2024
EMA/120337/2024
European Medicines Agency




Appendix 3 to Questions and answers for marketing
authorisation holders/applicants on the CHMP Opinion
for the Article 5(3) of Regulation (EC) No 726/2004
referral on nitrosamine impurities in human medicinal
products

Enhanced Ames Test Conditions for N-nitrosamines




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© European Medicines Agency, 2024. Reproduction is authorised provided the source is acknowledged.

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The Organisation for Economic Co-operation and Development (OECD) Test Guideline No. 471
“Bacterial Reverse Mutation Test” provides standard recommendations for the conduct of the
bacterial reverse mutation test (also known as the Ames assay) to assess the mutagenic potential
of a test compound. For N-nitrosamines, enhanced testing conditions for the Ames assay are
recommended due to the reported reduced sensitivity of the assay under standard conditions for
some N-nitrosamines, such as N-nitroso-dimethylamine (NDMA). Moreover, very little is known
about the sensitivity of the Ames assay to N-nitrosamine drug substance related impurities
(NDSRIs), which are a recently recognized class of N-nitrosamine impurities structurally related to
the drug substance. NDSRIs generally have a wider variety of functional groups present than
typically found in low molecular weight N-nitrosamines (such as NDMA) historically studied.

If a standard Ames assay is conducted and produces a positive result, there is no need to conduct
an additional assay using enhanced testing conditions.

The enhanced Ames assay test conditions presented below are informed by work conducted by
FDA’s National Center for Toxicological Research (NCTR) (Li et. al., 2023), as well as other groups,
and have been evaluated for a variety of N-nitrosamines including NDSRIs. Evaluation of Ames
assay test conditions for N-nitrosamines is ongoing with a goal to identify the most robust Ames
testing conditions. The enhanced Ames assay test conditions described below will be updated as
warranted. Deviations from the recommended conditions should be justified.

Tester strains: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA (pKM101)
tester strains should be included.

Type of assay and preincubation time: The pre-incubation, and not plate incorporation, method
should be used. The recommended pre-incubation time is 30 minutes.

Species and concentration of S9: Ames assays should be conducted in the absence of a post-
mitochondrial fraction (S9), and also in the presence of 30% rat liver S9, as well as 30% hamster
liver S9. The rat and hamster post-mitochondrial fractions (S9s) should be prepared from rodents
treated with inducers of cytochrome P450 enzymes (e.g., a combination of phenobarbital and β-
naphthoflavone).

Negative (solvent/vehicle) control: Solvents need to be compatible with the Ames assay as per
the OECD 471 guideline. Solvents can include, but are not limited to:

         water
         organic solvents such as acetone, methanol and DMSO


          When an organic solvent is used, the lowest possible volume should be included in the pre-
          incubation mixture with justification to indicate that the volume of solvent does not
          interfere with the metabolic activation of the N-nitrosamine.

Positive controls: Concurrent strain-specific positive controls should be included per the OECD
471 guideline.

Two N-nitrosamines that are known to be mutagenic in the presence of S9 should also be included
as positive controls.

The choice of the N-nitrosamine positive controls needs to be justified based on the anticipated
metabolism of the N-nitrosamine and the cytochrome P450 enzymes most likely involved. In
addition, if an organic solvent is used to dissolve the test compound, it is recommended that the
volume of the organic solvent employed to dissolve the N-nitrosamine positive controls, results in a
similar concentration as for the test compound in the pre-incubation mix.


Appendix 3 to Questions and answers for marketing authorisation
holders/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No
726/2004 referral on nitrosamine impurities in human medicinal products
EMA/120337/2024                                                                                   Page 2/3

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N-Nitrosamine positive controls to consider include:

         1.   NDMA (CAS # 62-75-9)
         2.   1-Cyclopentyl-4-nitrosopiperazine (CAS # 61379-66-6)
         3.   An NDSRI
All other recommendations for the Ames assay should follow the OECD 471 guideline.


A checklist of the information required for the Enhanced Ames Test (EAT) is presented below and
can be used to provide a summary of the conditions used and results obtained in the report for the
EAT.


Test substance:

Sponsor:
Test facility:

GLP compliance: Y/N
Study Initiation Date:

Experimental Start Date:
Experimental Completion Date:


                    EAT requirements                          Conditions used in EAT       Comments
    Strains: (e.g. S. typhimurium TA98, TA100,
    TA1535, TA1537 and E. coli WP2 uvrA (pKM101))
    Preincubation method, 30 min
                     rat (30%)
    S9
                     hamster (30%)
    Solvent used and volume1 (µL) (a)
    Volume of preincubation mix (µL) (b)
    Solvent concentration in preincubation (% v/v)1
Concentrations tested (µg/plate)
Nitrosamine positive control 1 (µg/plate), including
solvent concentration (% v/v)2, 3
NDSRI positive control 2 (µg/plate), including
solvent concentration (% v/v)2, 3
Non-nitrosamine positive controls2,4
Negative controls2
Result (positive/negative)
1
  When an organic solvent is used, the lowest possible volume should be included in the preincubation mixture
with justification to indicate that the volume of solvent does not interfere with metabolic activation of the N-
nitrosamine. The organic solvent concentration (% v/v) should be calculated by dividing the applied solvent
volume containing the test substance in the preincubation mix by the total volume of the preincubation mix i.e.
(a/b)*100 e.g. if the test substance is applied in 100 µL organic solvent and the total preincubation mix volume
is 700 µL, the solvent concentration is 14.3% (v/v)
2
Concurrent controls
3
  Solvent used for test substance and positive control should ideally be the same or justified if not.
4
Positive control reference substance(s) e.g. as suggested by OECD 471 for assays employing a metabolic
activation system.

References:
OECD Test Guideline No. 471 “Bacterial Reverse Mutation Test”. 2020

Li et al. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and
human in vitro assays. Regulatory Pharmacology and Toxicology. 2023




Appendix 3 to Questions and answers for marketing authorisation
holders/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No
726/2004 referral on nitrosamine impurities in human medicinal products
EMA/120337/2024                                                                                         Page 3/3