We were recently asked by a regulatory agency about this possibility in a capsule for which an azo dye is sued and therefore traces of nitrites are present.
I was asked a similar question at a workshop here in US. not familiar with the particular case
For that case you should check the azo colorant synthesis. As of course is an azo coupling reaction, a nitrite salt is commonly used. What is the other "couple used, does it have a secondary amine moiety? As from what I known, not a single approved colorant for food have this characteristic. (Note apart, for coloured foils, there are some cases of colorants with amine moiety, but is a different topic with different answers).
What should also be considered is that azo colorants, and even in capsules, they are used in a very low %. If any unidentified secondary or tertiary amine is nitrosated, I would expect it to be below the 18 ng/day threshold. (I asked similar a question in a FDA workshop back in early 2021 and similar response was answered around āhow much colorant do you useā).
Regarding the nitrite, I have also seen some manufacturers to use sulfamic acid as a nitrite scavenger. If there is no scavenger I would expect quite a bit of nitrite per mg of colorant at the end.
Also other points worth mention:
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The colorants in question are normally expected to comply with FDA 21 CFR regulation for colorants subject to certification and (EU) 231/2012 regulation for food additives. Therefore, an expected acceptance criteria of purity and content of impurities is known. The āresidueā after all known deductions is quite low.
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Having said this, common impurities depending on the azo colorant are primary amines and triazene like compounds. None, would form an stable nitrosamine I would say.
The same would apply to nitrite, as the quantities of colorant per capsule are low, even lower quantities of unknown impurities will be present. Therefore, I expect more nitrite could be present in other common used excipients that the capsule itself (because of azo colorants).
Now, around the gelatin capsule itself and if there is any amine functionality. Collagen have amino acids. But most amino acids are primary amines, I think there are only a few that arenĀ“t (proline, etc.) and are already known that if nitrosated are non carcinogenic. Once again, capsule gelatin composition should be known, but other common compounds part of its composition are water and mineral salts, so nothing strange af first glance.
Finally, just some clarifications, this āstatementā works best when the capsules are not so ābigā as well as the used quantity of azo colorant are not in a high % and its known to comply with FDA or EU regulation for colorants in food. Its a case by case, with some math behind depending of the maximun daily dose of the drug.
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Many thanks all of you for the responses. I agree that the amount is critical and we can use that approach to justify the potential compound will be below the 18ng limit.
Also, pH of formulation is controlled around 7,0 from which the possibility of nitrite convert into a nitrosating agent is low. Do you agree this approach?
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I added the description of EFPIA DP workflow ver2.0.
āCapsule shells are also considered to be no identified risk for nitrosamine contamination through either colours used within the capsule coating, neutrality of the plasticisers used, or the external printing ink that may be used. Nitrosation is unlikely to occur from nitrite within the gelatin because pH is likely to be neutral, gelatin contains primary amine scavengers, the low surface to volume ratio and the printing process is conducted at room temperature.ā
As @Diego_HM explained, the risk of nitrite contamination in gelatin doesnāt seem serious in many cases.
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Have any regulatory authorities accepted these justifications for the risks associated with gelatin being low due to the lack of carcinogenicity?
Hi Diego,
Thank you for sharing your Knowledge regarding this subject. Would you have any reference to share about the carcinogenicity of amino acids that you mentioned?
I would also be interested in this subject.
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Has anybody trying NAP test on the empty capsule to assess potential formation?
Hi @brunacasanova,
Regarding your question, primary amines after nitrosation forms unstable compounds, therefore, its carcinogenicity is irrelevant if their mean life is short enough that it even do not reach the tissue of interest (e.g: liver).
For the aminoacids that have scondary amine groups or reasemble one like proline & hydroxyproline. Carcinogenic studies (a bit old nonetheless) indicates that are non-carcinogenic.
Nitroso-proline & Nitroso-hydroxypropline: Studies of the tumorigenic effect in feeding of nitrosamino acids and of low doses of amines and nitrite to rats - PubMed (nih.gov)
Nitroso-tryptophan is +tive in a bacterial test Mutagenicity of nitrosated Ī±-amino acid derivatives N-acetyl-Nā -nitrosotryptophan and its methyl ester in bacteria | Carcinogenesis | Oxford Academic (oup.com) & Mechanisms and kinetics of tryptophan N-nitrosation in a gastro-intestinal model - ScienceDirect, but gelatin do not have tryptophan as part of its chains.
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