N-Nitroso Hydrochlorothiazide in Hydrochlorothiazide drug substance

Can someone provide the reference for the acceptable criteria for the control of N-Nitroso Hydrochlorothiazide in Hydrochlorothiazide drug substance.

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Neither this impurity nor its degradation products are cohort of concern. This impurity is positive in the Ames assay with and without a metabolic activator (S9) which is not a characteristic feature of an N-Nitrosamine which requires metabolic activation for alpha-hydroxylation and subsequent generation of the alkyl diazonium cation.
To our current knowledge EDQM has accepted an interim limit of 1.5µg/day for this impurity pending in vivo mutagenicity (TGR) results.
Dr. @conudel has recently presented this information in the CPHI nitrosamine forum.


@Gaurav Gaurav, @Muzaffar ,

I can confirm that some manufacturers already obtained Hydrochlorothiazide CEPs with a limit of 15 ppm, based on the TTC of 1,5 µg/day and the MDD of HCTZ (100 mg/day).
kind regards


Do you have any reference or article for support purpose ??

This limit was accepted by EDQM on the basis of a position paper submitted by EFPIA (European Federation of Pharmaceutical Industries Associations). However, this document is strictly confidential and cannot be shared.

I can only suggest the following articles:
Mutagenicity of amine drugs and their products of nitrosation (Andrews 1984)
(which confirm the mutagenicity of N-nitroso HCTZ)
Pathologic Effects of Chronic Administration of Hydrochlorothiazide, with and without Sodium Nitrite, to F344 Rats
(which demonstrate that the addition of sodium nitrite with HCTZ in the diet of the rats do not increase the risk of cancer).


Claiming that nitrosamine is out of the cohort of concern based on the AMES test alone is overly optimistic. Especially in the case of nitrosohydrochlorothiazide, where the number of reverse mutants with S9 activation is about 10 times higher than without said activation. This rather suggests that, in addition to the carcinogenic mechanism typical of nitrosamines, there is an additional minor mutagenic effect, eg related to the hydrolysis of hydrochlorothiazide to formaldehyde.