Recommended Acceptable Intake (AI) Limits and Testing Methods
- Recommended AI Limits for Certain NDSRIs Based on Predicted Carcinogenic Potency Categorization for APIs at Hypothetical Risk of Forming Such NDSRIs
- Recommended AI Limits for Certain NDSRIs Based on Compound-Specific Data or Read-Across Analysis from a Surrogate
- Recommended Interim AI Limits for Certain NDSRIs
- Recommended Testing Methods for Confirmatory Testing of NDSRIs
- Recommended Safety Testing Methods for NDSRIs
On 8/4/2023, FDA issued a final guidance on Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) (August 2023) (NDSRI Guidance). To reflect the evolving and highly technical nature of the relevant information, FDA is providing certain updated NDSRI-specific information on this website in connection with the guidance.
Specifically, FDA intends to include on this website updated information on: (1) recommended AI limits for certain NDSRIs based on their predicted carcinogenic potency categorization listed by APIs that hypothetically could be at risk of forming such NDSRIs; (2) recommended AI limits for certain NDSRIs based on compound-specific data or read-across analysis from a surrogate; (3) recommended interim AI limits for certain NDSRIs; (4) recommended testing methods for confirmatory testing of certain NDSRIs; and (5) recommended safety testing methods for NDSRIs.
This guidance (including the information on this associated website) represents the current thinking of the Food and Drug Administration (FDA or Agency) on this topic. It does not establish any rights for any person and is not binding on FDA or the public. You can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.
The public may comment on the information below by submitting a comment to the public docket established for the final guidance on Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) (Docket No. FDA-2020-D-1530). Comments may be submitted at any time for Agency consideration. Submit written comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852. Submit electronic comments to www.regulations.gov. All comments should be identified with the docket number FDA-2020-D-1530 and complete title of the guidance. For additional information, please refer to the Federal Register Notice of Availability for the final NDSRI Guidance, available at www.regulations.gov.
Based on Predicted Carcinogenic Potency Categorization for APIs at Hypothetical Risk of Forming Such NDSRIs
APIs that contain secondary amine or dimethyl tertiary amine centers are at risk of forming NDSRIs by nitrosation of the amine center. This can occur under conditions related to the formulation and manufacturing process for the drug product, such as by reaction with residual nitrites in excipients used to formulate the drug product. Table 1, below, presents NDSRIs that can hypothetically form by this process from a number of amine-containing APIs. Recommended AI limits for these hypothetical NDSRIs based on the predicted carcinogenic potency categorization approach are also presented. If an NDSRI currently listed in Table 1 is formed, the corresponding recommended AI limit in Table 1 based on the predicted carcinogenic potency categorization approach applies. FDA may move an NDSRI to Table 2 if compound-specific data or read-across analysis from a surrogate becomes available and an updated recommended AI limit applies. The recommended AI limits based on the predicted carcinogenic potency characterization approach should not be applied to NDSRIs in circumstances in which FDA otherwise recommends an AI limit (e.g., based on compound-specific assessments or read-across analysis from a surrogate) in Table 2.
For APIs with more than one nitrosatable amine center, the predicted carcinogenic potency category for each unique NDSRI formed by mono-nitrosation of the API is presented in Table 1, where each NDSRI from the same API is differentiated by the suffix “-1,” “-2,” etc. in the NDSRI Name column. Chemical structure images* for NDSRIs in Table 1 can be accessed by clicking on the hyperlinked NDSRI Name. This table will be updated periodically based on new information.
This is not an all-inclusive list of APIs that have the potential to form NDSRIs; there are other possible routes of NDSRI formation. In addition, the recommended AI limits for the NDSRIs on this website do not necessarily correspond to the observed level of any NDSRI(s) found in any particular drug product. Furthermore, inclusion of an API on the list is not confirmation that an NDSRI is present in a drug product containing that drug substance. A recommended AI limit is based on a safety assessment that includes evaluation of the mutagenic and carcinogenic potential of the impurity and represents the level at or below which FDA has determined that the impurity or impurities would not pose a safety concern for patients taking the drug product.
* Chemical structure images provided by the National Institutes of Health/National Center for Advancing Translational Sciences.
Table 1: FDA Recommended AI Limits for Certain Hypothetical NDSRIs
Based on Predicted Carcinogenic Potency Categorization
(last updated 8/4/2023)
|NDSRI Name||Source*||Potency Category||Recommended AI Limit|
|N-nitroso-brilliant blue g||Brilliant Blue G||5||1500 ng/day|
|N-nitroso-colistin a hydrogen methanesulfonate-1||Colistin||2||100 ng/day|
|N-nitroso-colistin a hydrogen methanesulfonate-2||Colistin||2||100 ng/day|
|N-nitroso-colistin a hydrogen methanesulfonate-3||Colistin||2||100 ng/day|
|N-nitroso-colistin a hydrogen methanesulfonate-4||Colistin||2||100 ng/day|
|N-nitroso-colistin a hydrogen methanesulfonate-5||Colistin||2||100 ng/day|
|N-nitroso-colistin b hydrogen methanesulfonate-1||Colistin||2||100 ng/day|
|N-nitroso-colistin b hydrogen methanesulfonate-2||Colistin||2||100 ng/day|
|N-nitroso-colistin b hydrogen methanesulfonate-3||Colistin||2||100 ng/day|
|N-nitroso-colistin b hydrogen methanesulfonate-4||Colistin||2||100 ng/day|
|N-nitroso-colistin b hydrogen methanesulfonate-5||Colistin||2||100 ng/day|
|N-nitroso-dabigatran etexilate||Dabigatran Etexilate||3||400 ng/day|
|N-nitroso-desmethyl-doxepin, (e)-||Doxepin||1||26.5 ng/day|
|N-nitroso-desmethyl-erythromycin ethylsuccinate||Erythromycin Ethylsuccinate||3||400 ng/day|
|N-nitroso-florbetaben f-18||Florbetaben F-18||2||100 ng/day|
|N-nitroso-florbetapir f-18||Florbetapir F-18||2||100 ng/day|
|N-nitroso-flutemetamol f-18||Flutemetamol F-18||2||100 ng/day|
|N-nitroso-folic acid||Folic Acid||4||1500 ng/day|
|N-nitroso-levomefolic acid-1||Levomefolic Acid||4||1500 ng/day|
|N-nitroso-levomefolic acid-2||Levomefolic Acid||4||1500 ng/day|
|N-nitroso-meclofenamic acid||Meclofenamic Acid||5||1500 ng/day|
|N-nitroso-desmethyl-methylene blue||Methylene Blue||2||100 ng/day|
|N-nitroso-desmethyl-padimate o||Padimate O||2||100 ng/day|
|N-nitroso-desmethyl-spinosad factor a||Spinosad||2||100 ng/day|
|N-nitroso-desmethyl-spinosad factor d||Spinosad||2||100 ng/day|
|N-nitroso-desmethyl-ulipristal acetate||Ulipristal Acetate||2||100 ng/day|
Based on Compound-Specific Data or Read-Across Analysis from a Surrogate
As indicated in the NDSRI Guidance, a recommended AI limit is based on a safety assessment that includes evaluation of the mutagenic and carcinogenic potential of the impurity and represents the level at or below which FDA has determined that the impurity or impurities would not pose a safety concern for patients. A recommended compound-specific AI limit can be calculated based on rodent carcinogenic potency data such as TD50 values identified in the published scientific literature. When the mutagenic potential of an NDSRI is not adequately characterized, FDA has used structure activity relationships to support the identification of a robustly-tested surrogate that is similar in structure and reactivity to the NDSRI to generate an estimate of carcinogenic potency from which an AI limit can be scientifically determined. The rationale for the choice of surrogate (similar in structure and reactivity) is significant because test data from the identified surrogate are then used to generate an estimate, either quantitatively or qualitatively, for a compound that lacks robust mutagenicity and carcinogenicity data (commonly referred to as a read-across analysis.)
Table 2 presents FDA recommended AI Limits for certain NDSRIs based on compound-specific data or read-across analysis from a surrogate. If an NDSRI appears in Table 2, the recommended AI limit in Table 2 applies, rather than a limit based on the predicted carcinogenic potency categorization approach.
Table 2: FDA Recommended AI Limits for Certain NDSRIs
Based on Compound-Specific Data or Read-Across Analysis from a Surrogate
(last updated 8/4/2023)
Recommended AI Limit (ng/day)
|Date Added to Table|
*This limit was previously communicated on August 9, 2022.
**This limit was previously communicated on July 16, 2021.
As indicated in the NDSRI Guidance, if drug product batches already in distribution contain NDSRIs at levels above the FDA recommended AI limit, and manufacturing changes or recalls are likely to lead to a disruption in the drug supply, then manufacturers and applicants should immediately contact CDER’s Drug Shortage Staff at firstname.lastname@example.org. When contacted about a potential disruption in the drug supply, FDA intends to evaluate each circumstance on a case-by-case basis. FDA may work directly with a specific manufacturer or applicant of the marketed drug and intends to consider whether it is appropriate or not appropriate to recommend an interim AI limit for a temporary period. If FDA recommends an interim AI limit, it generally does not intend to object, for example based on applicable underlying CGMP violations, to distribution of such drug product batches that contain NDSRI levels at or below the recommended interim AI limit during the specified period under certain circumstances on a case-by-case basis. In certain cases where FDA does not intend to object to the distribution of drug products from multiple drug manufacturers that contain NDSRI levels at or below the recommended interim AI limit, FDA intends to post such recommended interim AI limits on this website.
As indicated in the NDSRI Guidance, if manufacturers and applicants identify a risk of NDSRI formation in a drug product, then confirmatory testing of batches should be conducted using sensitive and appropriately validated methods.
FDA intends to provide information on FDA-generated testing methods to provide options for regulators and industry to detect NDSRI impurities in specific drug substances and drug products. The methods should be validated by the user if the resulting data are used to support a required quality assessment of the drug substance or drug product, or if the results are used in a regulatory submission.
As indicated in the NDSRI Guidance, a manufacturer or applicant may submit a scientifically justified rationale to pursue an AI limit higher than the FDA recommended limit associated with the predicted carcinogenic potency category for that NDSRI. Alternative approaches using safety data, such as obtaining compound-specific data or using read-across assessment to a suitable surrogate, could be used to support a higher limit.
In order to assist manufacturers and applicants in conducting safety testing, FDA is recommending that if in vitro mutagenicity testing is contemplated, an enhanced Ames assay be used to assess whether an NDSRI poses a mutagenic risk. A negative result in a valid enhanced Ames assay may be used to support a higher limit for an NDSRI; however, manufacturers and applicants should note that FDA may request additional safety data, beyond the enhanced Ames assay to support alternative AI limits. The following recommendations represent FDA’s current thinking; data gaps in the information available to support the safety of NDSRIs remain to be addressed with future research.
The Organisation for Economic Co-operation and Development (OECD)’s 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 by FDA due to the reported reduced sensitivity of the assay under standard conditions for some N-nitrosamines such as N-nitroso-dimethylamine (NDMA). FDA specifically recommends use of an enhanced Ames assay for NDSRIs because very little is known about the sensitivity of the Ames assay to NDSRIs. Additionally, NDSRIs generally have a wider variety of functional groups present than typically found in low molecular weight N-nitrosamines (such as NDMA) historically studied, and the additional testing conditions described in the enhanced Ames assay have been shown to be helpful in assessing mutagenic risk for NDSRIs.
If a standard Ames assay is conducted on an NDSRI and produces a positive result, FDA recommends 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 X 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 recommended by FDA for NDSRIs and described below will be updated as warranted. Deviations from the recommended conditions should be scientifically 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 method is recommended, and not plate incorporation. The recommended pre-incubation time is 30 minutes.
Species and concentration of S9: FDA recommends that 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. FDA also recommends that 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: FDA recommends that solvents be compatible with the Ames assay as per the OECD 471 guideline. Solvents can include, but are not limited to:
- 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 metabolic activation of the N-nitrosamine or NDSRI.
Positive controls: Concurrent strain-specific positive controls should be included per the OECD 471 guideline.
Two N-nitrosamines, including NDSRIs, 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, FDA recommends that the volume of 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, if possible.
N-Nitrosamine positive controls to consider include:
- NDMA (CAS # 62-75-9)
- 1-Cyclopentyl-4-nitrosopiperazine (CAS # 61379-66-6)
- An NDSRI
All other recommendations for the Ames assay should follow the OECD 471 guideline.
OECD Test Guideline No. 471 “Bacterial Reverse Mutation Test” 2020.
Li X, et al. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays. Regul Toxicol Pharmacol. 2023 Jun; 141:105410.